The class Chondrichthyes includes all of the cartilaginous fishes: the chimaeras, sharks, skates, and rays. The extant members of the class comprise at least 1,207 species [1], divided into two major groups: Subclasses Holocephali (chimaeras) and Elasmobranchii (sharks, skates, and rays). Recent molecular dating analyses suggest a split between holocephalans and elasmobranchs at about 420 Mya [2, 3]. Chondrichthyans as a whole, are thought to have diverged from bony vertebrates (Osteichthyes: ray-finned fishes, coelacanths, lungfishes, and tetrapods) approximately 450–475 Mya [2–4]; see however Giles et al. 2015 [5], for evidence of a chondrichthyan/osteichthyan ancestry of 415 Mya. Because of their phylogenetic position in vertebrate evolution, chondrichthyans provide an important reference for our understanding of vertebrate genome evolution. In addition to their ancient history and fundamental position in vertebrate systematics, chondrichthyans possess a wide variety of biological characteristics of note. Such traits include, but are not limited to, the presence of a primitive adaptive immune system, efficient wound healing, and the evolution of regional endothermy in several species.
One of the most rapidly expanding areas of research in elasmobranch biology is in understanding the function of the immune system [6]. Cartilaginous fishes are the most ancient group of vertebrates that possesses an adaptive immune system based on the same B and T cell receptor genes that form the foundation of adaptive immunity in higher vertebrates [7]. However, adaptive immunity in chondrichthyans differs from higher vertebrates (including teleost fishes) in the lack of bone marrow (where B cells typically develop), in the types of immunoglobulins (Ig hereinafter), and in the genomic organization of the underlying genes [8–11]. Additionally, elasmobranchs contain a novel immunoglobulin, referred to as new antigen receptor (or IgNAR), which differs from traditional immunoglobulins in that it lacks light chain molecules and is comprised entirely of heavy chain domains [12, 13]. IgNARs have received considerable interest recently, in regard to their unique structure and the possibility of adapting these molecules for future diagnostic work or drug delivery systems [14–16]. Despite this interest, transcriptomic analyses of the similarities and differences between the elasmobranch immunome and that of higher vertebrates are not currently available.
Regional (or partial) endothermy arose independently in each of the billfishes and tunas (both highly migratory, large bodied teleosts), and the highly migratory, large-bodied lamnid sharks. All three groups have convergently evolved adaptations for increased aerobic capacity, continuous swimming, elevated cruising speed, and heat production and/or retention [17–21]. Although the heart is at ambient temperature in regionally endothermic species, its function is critical to endothermic physiology because of its role in modulating blood flow and oxygen delivery to heat producing tissues (i.e. red muscle) and through the vasculature responsible for the counter-current heat exchange [18]. However, to date the genetic loci that might be associated with this remarkable example of convergent evolution in fishes remain obscure and there are few studies that specifically attempt to investigate this. A recent study of the cytochrome oxidase C subunit (COX) genes found no evidence of molecular convergence across endothermic fishes in these mitochondrial loci involved in aerobic metabolism [22]. Another recent study has shown differences in expression of genes involved in calcium storage and cycling (serca2 and ryr2) in heart tissue of tuna species with different temperature tolerance, with the greatest expression in Pacific bluefin tuna (Thunnus orientalis), the species with the greatest cold tolerance of the three tested [23]. In a comparative study of gene expression in heart tissue of Pacific bluefin tuna that were acclimated to cold and warm temperatures, Jayasundara and colleagues found upregulation of genes associated with protein turnover, lipid and carbohydrate metabolism, heat shock proteins, and genes involved in protection against oxidative stress in cold acclimated individuals [24]. This study also detected elevated levels of the SERCA enzyme in cold acclimated individuals [24]. Collectively, this suggests the importance of regulating genes involved in metabolism, control of heart contraction and function, and cellular protection against oxidative stress in heart tissue of an organism with an endothermic physiology. Our goal here was to use the heart transcriptome to examine a large repertoire of genes for possible evidence of convergent evolution in regional endothermy, in terms of either genes expressed, or shared genes with a history of molecular adaptation.
Comparative genomics of chondrichthyans remains limited, with a single genome sequence available for the holocephalan, Callorhinchus milii [25, 26], and a few additional genome projects in progress (reviewed in [27], including the whale shark Rhincodon typus (http://whaleshark.georgiaaquarium.org), white shark, Carcharodon carcharias (our laboratory), catshark, Scyliorhinus canicula (Genoscope: http://www.genoscope.cns.fr), and the batoid, the little skate, Leucoraja erinacea [28]. There are a larger number of transcriptomic and RNA-seq studies, however, these genetic resources are still limited compared to those of other vertebrate taxa [27]. Transcriptome sequence examples include a heart transcriptome of the white shark [29]; brain, liver, pancreas, and embryo from the small-spotted catshark, S. canicula, [30, 31]; an embryo of cloudy catshark, Scyliorhinus torazame [32]; whole embryo from the little skate [28]; and spleen and thymus from nurse shark, Ginglymostoma cirratum [26] and spleen, thymus, testis, ovary, liver, muscle, kidney, intestine, heart, gills, and brain from elephant shark (a holocephalan), C. milii [26]. In addition, EST (expressed sequence tag) sequences exist for cell lines derived from L. erinacea and the spiny dogfish, Squalus acanthias [33].
Interspecific transcriptomic comparisons of many taxonomic groups, and in particular groups with limited genetic resources such as elasmobranchs, are confounded by both the haphazard sampling of different tissues associated with different studies as well as the different technologies used to obtain the sequence data. At present limited comparative data sets of the same tissue type and technology are available across many taxa, however, this is beginning to change and there exist a few important exceptions; see for example, [34–36].
To examine transcriptomic differences between elasmobranchs vs. teleosts and endothermic vs. ectothermic (i.e. non-endothermic) species, we sampled heart tissue since it is a metabolically active tissue, and expression of major components in innate and adaptive immunity have been demonstrated in heart and associated blood tissues [37, 38]. Compared to ectothermic fishes, regionally endothermic fishes such as tunas tend to have an elevated heart rate and this in part supports the maintenance of elevated temperature in some tissues [18, 39]. We hypothesize, therefore, that there are differences in expressed gene content of heart tissue of endothermic species relative to ectothermic species, to compensate for this increased heart rate. Our study included the following seven species: elasmobranchs – white shark (Carcharodon carcharias), shortfin mako (Isurus oxyrinchus; hereinafter termed mako), great hammerhead (Sphyrna mokarran; hereinafter termed hammerhead), and yellow stingray (Urobatis jamaicensis); teleosts – swordfish (Xiphias gladius), hogfish (Lachnolaimus maximus), and ocean surgeonfish Acanthurus bahianus; hereinafter termed surgeonfish). Both white shark and mako (Lamnidae, Lamniformes), like other lamnids, display elevated internal temperatures indicative of regional endothermy [40]; the great hammerhead (Sphyrnidae, Carcharhiniformes) and the yellow stingray (hereinafter referred to as ray) (Urotrygonidae, Myliobatiformes) represent the two ectothermic elasmobranchs included in our study. Molecular phylogenetic studies support rays and skates as the sister group to sharks and suggest that this split was approximately 300 Mya [2, 3, 41]. The swordfish (Xiphiidae, Perciformes) is a representative of a regionally endothermic teleost, while both hogfish (Labridae, Perciformes) and surgeonfish (Acanthuridae, Perciformes) are ectotherms.
In comparing these seven heart transcriptomes we had several specific aims. First, we sought to identify differences in expressed gene content that are a reflection of evolutionary taxonomy (e.g. elasmobranchs vs. teleosts). Secondly, we aimed to identify whether there were significant differences involving the comparative groups– i.e., elasmobranchs vs. teleosts and endotherms vs. ectotherms—in the types of genes (as identified by differences in Gene Ontology, or GO) that are expressed, especially in regards to particular phenomena of interest (e.g. adaptive immunity and wound healing in elasmobranchs, metabolic function in endotherms). Finally, we sought to identify genes with a history of molecular adaptation in elasmobranchs and the endothermic taxa in our data set, through the identification of genes under positive selection in the respective lineages.