Evolution of the Critically Endangered Green Sawfish Pristis zijsron (Rhinopristiformes, Pristidae), Inferred from the Whole Mitochondrial Genome.

The green sawfish Pristis zijsron (Bleeker, 1851), a species of sawfish in the family Pristidae (Rhinopristiformes), mainly inhabits the Indo-West Pacific region. In this study, the complete mitochondrial genome of the critically endangered green sawfish is first described. The length of the genome is 16,804 bp, with a nucleotide composition of 32.0% A, 24.8% C, 13.1% G, and 30.0% T. It contains 37 genes in the typical gene order of fish. Two start (GTG and ATG) and two stop (TAG and TAA/T-) codons are found in the thirteen protein-coding genes. The 22 tRNA genes range from 67 bp (tRNA-Ser) to 75 bp (tRNA-Leu). The ratio of nonsynonymous substitution (Ka) and synonymous substitution (Ks) indicates that the family Pristidae are suffering a purifying selection. The reconstruction of Bayesian inference and the maximum likelihood phylogenetic tree show the same topological structure, and the family Pristidae is a monophyletic group with strong posterior probability. Pristis zijsron and P. pectinata form a sister group in the terminal clade. And the divergence time of Rhinopristiformes show that P. zijsron and P. pectinata diverged as two separate species in about Paleogene 31.53 Mya. Complete mitochondrial genomes of all five sawfishes have been published and phylogenetic relationships have been analyzed. The results of our study will provide base molecular information for subsequent research (e.g., distribution, conservation, phylogenetics, etc.) on this endangered group.

The challenge of implementing environmental DNA metabarcoding to detect elasmobranchs in a resource-limited marine protected area.

Elasmobranchs are threatened and eDNA metabarcoding is a powerful tool that can help efforts to better understand and conserve them. Nevertheless, the inter-calibration between optimal methodological practices and its implementation in resource-limited situations is still an issue. Based on promising results from recent studies, the authors applied a cost-effective protocol with parameters that could be easily replicated by any conservationist. Nonetheless, the results with fewer elasmobranchs detected than expected reveal that endorsed primers and sampling strategies still require further optimization, especially for applications in resource-limited conservation programmes.

The Nursehound Scyliorhinus stellaris Mitochondrial Genome-Phylogeny, Relationships among Scyliorhinidae and Variability in Waters of the Balearic Islands.

The complete mitochondrial DNA sequence of the Nursehound Scyliorhinus stellaris has been determined for the first time and compared with congeneric species. The mitogenome sequence was 16,684 bp in length. The mitogenome is composed of 13 PCGs, 2 rRNAs, 22 transfer RNA genes and non-coding regions. The gene order of the newly sequenced mitogenome is analogous to the organization described in other vertebrate genomes. The typical conservative blocks in the control region were indicated. The phylogenetic analysis revealed a monophyletic origin of the Scyliorhininae subfamily, and within it, two subclades were identified. A significant divergence of Scyliorhinus spp. together with Poroderna patherinum in relation to the group of Cephaloscyllium spp. was observed, except for Scyliorhinus torazame, more related to this last cited clade. A hypothesis of a divergent evolution consequent to a selective pressure in different geographic areas, which lead to a global latitudinal diversity gradient, has been suggested to explain this phylogenetic reconstruction. However, convergent evolution on mitochondrial genes could also involve different species in some areas of the world.

An ancient alliance: Matching evolutionary patterns of cartilaginous fishes (Elasmobranchii) and chloromyxid parasites (Myxozoa).

Myxozoa is a group of endoparasitic cnidarians covering almost 2600 species but merely 53 species, mostly from the genus Chloromyxum, have been reported from sharks, rays, and skates (Elasmobranchii). Elasmobranchs play a key role in the study of evolutionary trajectories of myxozoans as they represent ancestral vertebrate hosts. Our study provides new data on Chloromyxum spp. from 57 elasmobranchs, covering 20 species from geographical regions and host groups not previously investigated, such as Lamniformes and Hexanchiformes, the most basal phylogenetic shark lineage. In total, 28% of elasmobranchs were infected with Chloromyxum spp., indicating high diversity. Of the seven distinguished species, six are formally described based on morphological, morphometric, and genetic (18S rDNA) data. Comprehensive co-phylogenetic analyses and ancestral state reconstruction revealed that parasite and host phylogenies are clearly correlated, resulting in a distinct phylogenetic separation of chloromyxids from selachid (shark) vs. batoid (ray and skate) hosts. Species infecting the most ancient elasmobranchs formed a sublineage, branching off in the middle of the Chloromyxum sensu stricto clade. Our findings indicate that chloromyxids likely invaded an ancestral elasmobranch prior the time of divergence of shark and batoid lineages. Our analyses did not show a clear phylogeographic pattern of Chloromyxum parasites, probably due to the cosmopolitan distribution and migratory behaviour of many elasmobranch hosts, but geographical sampling must be extended to confirm or refute this observation. This study provides a complex view on species diversity, phylogeny, evolution, host-parasite co-phylogeny, and the phylogeographic origin of Chloromyxum species from elasmobranchs. Our results highlight the importance of adding missing data from previously un- or undersampled geographical regions and host species which results in a more accurate estimate of myxozoan biodiversity and a better understanding of the evolution of this parasite group in their hosts and in the different oceans of our planet.

Chicken-type lysozyme is a major bacteriolytic enzyme in the blood of the banded houndshark Triakis scyllium.

We examined lysozyme activities in the serum and the leukocyte extracts of the banded houndshark Triakis scyllium. The serum exhibited lytic activity, but not the leukocyte extracts. The lytic substance in the serum was of approximately 14 kDa and the N-terminal amino acid sequence was YVYSK. cDNA cloning identified a C-type lysozyme (TsLysC) gene and two G-type lysozyme (TsLysG) cDNA clones of different lengths. The TsLysC gene encodes 149 amino acids residues, and the sequence derived from the N-terminal amino acid sequencing was displayed at position 17-21. TsLysG, on the other hand, contains two ORFs that are homologous to the N- and C-terminal regions of G-type lysozyme of other fish species. TsLysC mRNA levels were high in the liver. TsLysG mRNA level was significantly lower than TsLysC mRNA in the liver.

Evidence of multiple paternity for the endemic Xingu River stingray.

Multiple paternity (MP) is a phenomenon observed for more than 30 elasmobranch species. The Batoidea is more specious than the Selachii, but only three studies of MP have been conducted on batoids. The occurrence of MP in freshwater stingrays was tested using microsatellite markers, which were developed for Potamotrygon leopoldi. Six mothers and their litters were genotyped, providing the first evidence of MP for Potamotrygonidae, with an MP frequency of 33%.

Insights into genetic variation and demographic history of sharpnose rays: examinations of three species of Telatrygon (Elasmobranchii, Dasyatidae) from the Indo-West Pacific.

Coastal and demersal chondrichthyans (sharks, rays, and skates) are expected to exhibit high levels of genetic differentiation in areas of complex geomorphology. Population genetic studies investigating the extent to which demographic history shapes the genetic structure of these fishes are rare. Here, we combined mitochondrial DNA (Cytb and ND2) and 8 nuclear microsatellite loci from 244 individuals to examine the population genetic structure and demographic history of the 3 Indo-West Pacific species of sharpnose rays (Telatrygon zugei, Telatrygon biasa, and Trygon crozieri). High levels of genetic variation both within and between species were identified. Phylogenetic analysis partitioned haplotypes into 2 lineages supporting divergence of T. zugei from T. crozieri and T. biasa during the Pleistocene. Furthermore, microsatellite-based clustering analyses identified 4 genetic groups (i.e. T. zugei from Japan, T. zugei from coastal China, T. biasa from Gulf of Thailand, and T. crozieri from the Andaman Sea). Measurements of genetic differentiation also support these 4 groups. Additionally, Pleistocene demographic expansions were examined in all genetic groups. The climate oscillations and current hydrologic cycles in the Indo-West Pacific appear to coincide with the hypothesis regarding speciation and the observed demographic history trends of the sharpnose rays. Considering the species group has, until recently, been thought to be one species, these results are critical for defining management units and guiding conservation efforts to preserve stingray biodiversity.

Evolutionary developmental genetics of teeth and odontodes in jawed vertebrates: a perspective from the study of elasmobranchs.

Most extant vertebrates display a high variety of tooth and tooth-like organs (odontodes) that vary in shape, position over the body and nature of composing tissues. The development of these structures is known to involve similar genetic cascades and teeth and odontodes are believed to share a common evolutionary history. Gene expression patterns have previously been compared between mammalian and teleost tooth development but we highlight how the comparative framework was not always properly defined to deal with different tooth types or tooth developmental stages. Larger-scale comparative analyses also included cartilaginous fishes: sharks display oral teeth and dermal scales for which the gene expression during development started to be investigated in the small-spotted catshark Scyliorhinus canicula during the past decade. We report several descriptive approaches to analyse the embryonic tooth and caudal scale gene expressions in S. canicula. We compare these expressions wih the ones reported in mouse molars and teleost oral and pharyngeal teeth and highlight contributions and biases that arise from these interspecific comparisons. We finally discuss the evolutionary processes that can explain the observed intra and interspecific similarities and divergences in the genetic cascades involved in tooth and odontode development in jawed vertebrates.

Phylogeography, genetic diversity and population structure of the freshwater stingray, Paratrygon aiereba (Müller & Henle, 1841) (Myliobatiformes: Potamotrygonidae) in the Colombian Amazon and Orinoco basins.

The freshwater stingray Paratrygon aiereba have coloration, osteological and morphometric variations that could suggest the existence of more than one species in Colombia. In order to evaluate the phylogeography, population structure and genetic diversity for P. aiereba distributed in the Amazon and Orinoco basins, we amplified Cytochrome oxidase subunit 1 (COI) partial region of mitochondrial DNA (mtDNA) in 50 samples from eight different sub-basins. Our results suggest three phylogroups and a vicariance event occurred 43 million years ago proposing how Paratrygon diverged into the basins. A high population structure (ΦST = 0.692; p < 0.005) and a value of (K) of 3 were defined. A high genetic diversity within phylogroups was found: Phylogroup A (h = 0.64; π% = 2.48), Phylogroup B (h = 0.552; π% = 1.67), and Phylogroup C (h = 0.49; π% = 0.73). These results should be considered in local management plans, conservation programs and reclassification in at least Amazon and Orinoco.

The immunoglobulins of cartilaginous fishes.

Cartilaginous fishes, comprising the chimeras, sharks, skates, and rays, split from the common ancestor with other jawed vertebrates approx. 450 million years ago. Being the oldest extant taxonomic group to possess an immunoglobulin (Ig)-based adaptive immune system, examination of this group has taught us much about the evolution of adaptive immunity, as well as the conserved and taxon-specific characteristics of Igs. Significant progress has been made analyzing sequences from numerous genomic and transcriptomic data sets. These findings have been supported by additional functional studies characterizing the Igs and humoral response of sharks and their relatives. This review will summarize what we have learned about the genomic organization, protein structure, and in vivo function of these Ig isotypes in cartilaginous fishes and highlight the areas where our knowledge is still lacking.

Development of the ventricular myocardial trabeculae in Scyliorhinus canicula (Chondrichthyes): evolutionary implications.

The development of the ventricular myocardial trabeculae occurs in three steps: emergence, trabeculation and remodeling. The whole process has been described in vertebrates with two different myocardial structural types, spongy (zebrafish) and compact (chicken and mouse). In this context, two alternative mechanisms of myocardial trabeculae emergence have been identified: (1) in chicken and mouse, the endocardial cells invade the two-layered myocardium; (2) in zebrafish, cardiomyocytes from the monolayered myocardium invaginate towards the endocardium. Currently, the process has not been studied in detail in vertebrates having a mixed type of ventricular myocardium, with an inner trabecular and an outer compact layer, which is presumptively the most primitive morphology in gnathostomes. We studied the formation of the mixed ventricular myocardium in the lesser spotted dogfish (Scyliorhinus canicula, Elasmobranchii), using light, scanning and transmission electron microscopy. Our results show that early formation of the mixed ventricular myocardium, specifically the emergence and the trabeculation steps, is driven by an endocardial invasion of the myocardium. The mechanism of trabeculation of the mixed ventricular myocardium in chondrichthyans is the one that best reproduces how this developmental process has been established from the beginning of the gnathostome radiation. The process has been apparently preserved throughout the entire group of sarcopterygians, including birds and mammals. In contrast, teleosts, at least those possessing a mostly spongy ventricular myocardium, seem to have introduced notable changes in their myocardial trabeculae development.

Dissecting the Interaction Deficiency of a Cartilaginous Fish Digestive Lipase with Pancreatic Colipase: Biochemical and Structural Insights.

A full-length cDNA encoding digestive lipase (SmDL) was cloned from the pancreas of the smooth-hound (Mustelus mustelus). The obtained cDNA was 1350 bp long encoding 451 amino acids. The deduced amino acid sequence has high similarity with known pancreatic lipases. Catalytic triad and disulphide bond positions are also conserved. According to the established phylogeny, the SmDL was grouped with those of tuna and Sparidae lipases into one fish digestive lipase cluster. The recently purified enzyme shows no dependence for bile salts and colipase. For this, the residue-level interactions between lipase-colipase are yet to be clearly understood. The structural model of the SmDL was built, and several dissimilarities were noticed when analyzing the SmDL amino acids corresponding to those involved in HPL binding to colipase. Interestingly, the C-terminal domain of SmDL which holds the colipase shows a significant role for colipase interaction. This is apt to prevent the interaction between fish lipase and the pancreatic colipase which and can provide more explanation on the fact that the classical colipase is unable to activate the SmDL.

Microsatellite primer development in elasmobranchs using next generation sequencing of enriched libraries.

Microsatellites are useful in studies of population genetics, sibship, and parentage. Here, we screened for microsatellites from multiple elasmobranch genomic libraries using an enrichment protocol followed by sequencing on an Illumina platform. We concurrently screened five and then nine genomes and describe the number of potential loci from each respective round of sequencing. To validate the efficacy of the protocol, we developed and tested primers for the pelagic thresher shark, Alopias pelagicus. The method described here is a cost-effective protocol to increase the pool of potential useful loci and allows the concurrent screening of multiple libraries.

Twins! Microsatellite analysis of two embryos within one egg case in oviparous elasmobranchs.

Elasmobranchs display various reproductive modes, which have been key to their evolutionary success. In recent decades there has been a rise in the number of reported cases of foetal abnormalities including fertilised, double-embryos held within one egg capsule, hereafter referred to as twins. Previously, the occurrences of twin egg cases have been reported in two batoid and one shark species. We report the first cases of twins in three species of oviparous elasmobranchs: the undulate ray (Raja undulata), the nursehound (Scyliorhinus stellaris), and the small-spotted catshark (Scyliorhinus canicula). We investigated the genetic relationships between the twins in S. stellaris, and S. canicula using microsatellite markers. Whilst the S. stellaris twins displayed the same genotypes, we found that the S. canicula twin individuals arose through heteropaternal superfecundation. This is the first reported incidence of such a paternity in elasmobranchs. The relationship between environmental change and reproductive strategy in elasmobranchs is unclear and further research is needed to determine its effect on the prevalence and mechanisms of formation of elasmobranch twins.

Conservation genetics of elasmobranchs of the Mexican Pacific Coast, trends and perspectives.

One of the most critical threats to biodiversity is the high extinction rate driven by human activities. Reducing extinction rates requires the implementation of conservation programmes based on robust scientific data. Elasmobranchs are important ecological components of the ocean, and several species sustain substantial economic activities. Unfortunately, elasmobranchs are one of the most threatened and understudied animal taxa. The Mexican Pacific Coast (MPC) is a region with high elasmobranch diversity and is the seat of major elasmobranch fisheries. But it is also a developing region with several conservation and management challenges which require national and international attention. Here, we review the conservation genetics literature of elasmobranchs from the MPC. We present a synthesis of the works using samples from the region and emphasize the main gaps and biases in these data. In addition, we discuss the benefits and challenges of generating genomic information to improve the management and conservation of an elasmobranch biodiversity hotspot in a developing country. We found 47 elasmobranch genetic articles that cover <30% of the elasmobranch diversity in the region. These studies mainly used mitochondrial DNA sequences to analyse the genetic structure of commercially important and abundant species of the order Carcharhiniformes. Some of these papers also assessed mating systems, demographic parameters, and taxonomic uncertainties, all of which are important topics for efficient management decisions. In terms of conservation genetics, elasmobranchs from the MPC remain understudied. However, high-throughput sequencing technologies have increased the power and accessibility of genomic tools, even in developing countries such as Mexico. The tools described here provide information relevant for biodiversity conservation. Therefore, we strongly suggest that investment in genomic research will assist implementation of efficient management strategies. In time, this will reduce the extinction risk of the unique elasmobranch biodiversity from the MPC.

DNA analysis of elasmobranch products originating from Bangladesh reveals unregulated elasmobranch fishery and trade on species of global conservation concern.

Trade involving elasmobranch products in Bangladesh is a four-decade-long practice in large scale and there is little understanding of its impact on species composition, population, and subsequent conservation. Capacity for monitoring and identification is lacking in landing and shark processing centres. A rapid survey and collection of tissue samples were performed in three landings and nine shark processing centres between 2016 and 2017 in the south-eastern coastal region of Bangladesh. Sequencing for a 707-bp fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene was used to assess the taxonomic status and species composition from 71 elasmobranch tissue samples collected from the shark processing centre only. Good quality COI sequences were obtained for 34 specimens representing 21 species-the majority of which are threatened with extinction. A total of ten species of sharks (Carcharhinus brevipinna, C. amboinensis, C. leucas, C. sorrah, C. amblyrhynchoides, Chiloscyllium burmensis, Galeocerdo cuvier, Rhincodon typus, Scoliodon laticaudus, and Sphyrna lewini), eleven species of rays (Aetomylaeus maculatus, Gymnura poecilura, Mobula mobular, M. kuhlii, Neotrygon indica, Pateobatis uarnacoides, Rhinoptera javanica, and R. jayakari), including three species of guitarfish (Glaucostegus granulatus, G. obtusus, and G. typus), were identified. Four species (14.7% of samples) were found to be listed in the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) in Appendix II. Sixteen species (59% of the specimens) were threatened with extinction according to IUCN Red List, whereas 41% were data deficient or not assessed. The results have important implications for the management of regional fisheries and the conservation of elasmobranchs as they 1) represent a preliminary understanding of elasmobranch diversity in trade; 2) depict a lack of awareness and monitoring; and 3) demonstrate a need for urgent monitoring and regulation of elasmobranch trade in Bangladesh.

Genetic and historical evidence of common sawsharks Pristiophorus cirratus in the waters of southern Queensland.

In 2011, a male pristiophorid was caught by a prawn trawler north east of Cape Moreton, Queensland, Australia. Molecular analyses confirmed the specimen to be the common sawshark Pristiophorus cirratus. Historical catch data indicate the occurrence of the species in the region but this is the first verified record of P. cirratus occurring in the waters of southern Queensland. Together, these records extend the recognised northern limit of P. cirratus by c. 500 km, which suggests that further investigation of its distribution is warranted.

Skeletal Mineralization in Association with Type X Collagen Expression Is an Ancestral Feature for Jawed Vertebrates.

In order to characterize the molecular bases of mineralizing cell evolution, we targeted type X collagen, a nonfibrillar network forming collagen encoded by the Col10a1 gene. It is involved in the process of endochondral ossification in ray-finned fishes and tetrapods (Osteichthyes), but until now unknown in cartilaginous fishes (Chondrichthyes). We show that holocephalans and elasmobranchs have respectively five and six tandemly duplicated Col10a1 gene copies that display conserved genomic synteny with osteichthyan Col10a1 genes. All Col10a1 genes in the catshark Scyliorhinus canicula are expressed in ameloblasts and/or odontoblasts of teeth and scales, during the stages of extracellular matrix protein secretion and mineralization. Only one duplicate is expressed in the endoskeletal (vertebral) mineralizing tissues. We also show that the expression of type X collagen is present in teeth of two osteichthyans, the zebrafish Danio rerio and the western clawed frog Xenopus tropicalis, indicating an ancestral jawed vertebrate involvement of type X collagen in odontode formation. Our findings push the origin of Col10a1 gene prior to the divergence of osteichthyans and chondrichthyans, and demonstrate its ancestral association with mineralization of both the odontode skeleton and the endoskeleton.

Electroreception in marine fishes: chondrichthyans.

Electroreception in marine fishes occurs across a variety of taxa and is best understood in the chondrichthyans (sharks, skates, rays, and chimaeras). Here, we present an up-to-date review of what is known about the biology of passive electroreception and we consider how electroreceptive fishes might respond to electric and magnetic stimuli in a changing marine environment. We briefly describe the history and discovery of electroreception in marine Chondrichthyes, the current understanding of the passive mode, the morphological adaptations of receptors across phylogeny and habitat, the physiological function of the peripheral and central nervous system components, and the behaviours mediated by electroreception. Additionally, whole genome sequencing, genetic screening and molecular studies promise to yield new insights into the evolution, distribution, and function of electroreceptors across different environments. This review complements that of electroreception in freshwater fishes in this special issue, which provides a comprehensive state of knowledge regarding the evolution of electroreception. We conclude that despite our improved understanding of passive electroreception, several outstanding gaps remain which limits our full comprehension of this sensory modality. Of particular concern is how electroreceptive fishes will respond and adapt to a marine environment that is being increasingly altered by anthropogenic electric and magnetic fields.

Evolutionary Plasticity in Detoxification Gene Modules: The Preservation and Loss of the Pregnane X Receptor in Chondrichthyes Lineages.

To appraise how evolutionary processes, such as gene duplication and loss, influence an organism's xenobiotic sensitivity is a critical question in toxicology. Of particular importance are gene families involved in the mediation of detoxification responses, such as members of the nuclear receptor subfamily 1 group I (NR1I), the pregnane X receptor (PXR), and the constitutive androstane receptor (CAR). While documented in multiple vertebrate genomes, PXR and CAR display an intriguing gene distribution. PXR is absent in birds and reptiles, while CAR shows a tetrapod-specific occurrence. More elusive is the presence of PXR and CAR gene orthologs in early branching and ecologically-important Chondrichthyes (chimaeras, sharks and rays). Therefore, we investigated various genome projects and use them to provide the first identification and functional characterization of a Chondrichthyan PXR from the chimaera elephant shark (Callorhinchus milii, Holocephali). Additionally, we substantiate the targeted PXR gene loss in Elasmobranchii (sharks and rays). Compared to other vertebrate groups, the chimaera PXR ortholog displays a diverse expression pattern (skin and gills) and a unique activation profile by classical xenobiotic ligands. Our findings provide insights into the molecular landscape of detoxification mechanisms and suggest lineage-specific adaptations in response to xenobiotics in gnathostome evolution.