Abstract

   Few fishes have evolved elevated body temperatures compared with
   ambient temperatures, and only in opah (Lampris spp) is the entire body
   affected. To understand the molecular basis of endothermy, we analyzed
   the opah genome and identified 23 genes with convergent amino acid
   substitutions across fish, birds, and mammals, including slc8b1, which
   encodes the mitochondrial Na^+/Ca^2+ exchanger and is essential for
   heart function and metabolic heat production. Among endothermic fishes,
   44 convergent genes with suggestive metabolic functions were
   identified, such as glrx3, encoding a crucial protein for hemoglobin
   maturation. Numerous genes involved in the production and retention of
   metabolic heat were also found to be under positive selection. Analyses
   of opah's unique inner-heat-producing pectoral muscle layer (PMI), an
   evolutionary key innovation, revealed that many proteins were co-opted
   from dorsal swimming muscles for thermogenesis and oxidative
   phosphorylation. Thus, the opah genome provides valuable resources and
   opportunities to uncover the genetic basis of thermal adaptations in
   fish.

   Keywords: endothermy, opah, whole-genome sequencing, convergent
   evolution, thermogenesis

Graphical abstract

   graphic file with name fx1.jpg
   [61]Open in a new tab

Public summary

     * •
       Endothermy has evolved multiple times in fishes (teleosts and
       chondrichthyans)
     * •
       Opah genome explaining genetic changes in heat production and the
       sensory and immune system
     * •
       Convergent evolution of genes in endothermic vertebrate lineages
       was investigated
     * •
       Analyses of the pectoral muscle of opah revealed numerous highly
       expressed genes for thermogenesis

Introduction

   Endothermy, the ability of an organism to metabolically produce heat to
   achieve a high, stable body temperature, is one of the most fascinating
   traits that has evolved repeatedly.[62]^1^,[63]^2 This mechanism
   provides some independence from environmental thermal fluctuations by
   permitting high activity levels even with low or variable
   temperatures.[64]^3 Mammals and birds were long considered the only
   lineages of endothermic vertebrates, and whole-body endothermy is
   thought to have evolved independently in these two lineages.[65]^1
   Except for muscular thermogenesis, in placental mammals, metabolic heat
   is mainly produced by nonshivering thermogenesis in brown adipose
   tissue. Heat production in birds likely occurs through muscular
   thermogenesis, the production of heat through the uncoupling of calcium
   ions in the sarcoplasmic reticulum of muscle cells, activated by
   sarcolipin.[66]^1 However, endothermy has also been discovered in other
   vertebrate clades, with different types of endothermy having evolved
   independently at least five times in fishes (teleosts and
   chondrichthyans).[67]^4 The thermogenic strategies of teleosts and
   chondrichthyans are mainly attributed to contractions of the
   slow-twitch aerobic red muscles during constant swimming, a
   configuration often called “red muscle endothermy”.[68]^1^,[69]^4 For
   example, tuna (Scombridae) and mackerel sharks (Lamnidae) can elevate
   their core body temperature, whereas the brain and eye regions can
   become warmer than ambient temperatures in billfishes (Istiophoridae).
   In contrast to such regional endothermy, opahs are the only known
   teleosts that can warm their entire body by circulating locally
   warmed-up blood and are thus the only whole-body endotherm species
   aside from mammals and birds.[70]4, [71]5, [72]6

   Opah is a large mesopelagic circumglobal species with the unique
   ability to produce large quantities of metabolic heat in its dark red,
   aerobic pectoral musculature, putatively an evolutionary key innovation
   that induces forward thrust during continuous swimming by powering
   pectoral fin oscillation, and this is insulated from the cold
   surrounding waters by a thick layer of fatty connective
   tissue.[73]^7^,[74]^8 Furthermore, to reduce the cooling of warm blood
   in the gills, where most heat is lost, opah has evolved a crucial
   second key change, a counter-current retia mirabilia system in its
   gills.[75]^7 The convoluted alternating arteries with cold-oxygenated
   and warm-deoxygenated blood form the basis of the heat exchange system.
   With whole-body endothermy, the opah has the capacity for enhanced
   physiological functions in their cold, deep, and nutrient-rich
   habitats, below the ocean thermocline. Its adaptation to whole-body
   endothermy facilitates enhanced physiological functions, such as
   increased muscle power, increased capacity for more sustained
   performance, enhanced temporal resolution and neural conductance for
   the eye and brain, and an increased rate of food digestion and
   assimilation.[76]^7 Moreover, as endothermy is thought to represent an
   optimum level in the trade-off between metabolism and fitness, the case
   of the opah can inform us of how endothermy affects organism evolution
   and survivorship.[77]^9^,[78]^10

   The ability of the opah to maintain body regions at a warmer
   temperature than the ambient water challenges the general concept of
   “cold-bloodedness” in fish.[79]^3 However, unlike mammals and birds,
   the genetic characteristics associated with the occurrence of
   endothermy in fish have rarely been investigated. In many respects, the
   features of the entirely endothermic opah provide an unparalleled model
   to explore the molecular basis of endothermy as an adaptive trait in
   the ocean environment. In this study, we generated a chromosome-level
   genome of the Smalleye Pacific Opah (Lampris incognitus)
   ([80]Figure 1A). We then conducted comparative genomic, transcriptomic,
   and proteomic analyses to identify the molecular adaptations
   accompanying endothermy. In addition, we explored the genetic patterns
   that are shared during the convergent evolution of endothermy among
   endothermic fishes, mammals, and birds.

Figure 1.

   [81]Figure 1
   [82]Open in a new tab

   Evolutionary history of opah

   (A) Evolutionary key innovations and specialized physiological features
   involved in the endothermy of the Smalleye Pacific Opah, Lampris
   incognitus, and gene family expansion (red)/contraction (blue) in opah
   and other teleosts. The bootstrap value of all nodes is 100.

   (B) Percentages of transposable elements (TEs) in the different teleost
   genomes studied. LTR, long terminal repeats; LINE, long interspersed
   element; SINE, short interspersed element.

   (C) Enriched GO functional categories that were closest to the LTR
   retrotransposons (2 kb up and downstream).

   (D) Expansion of LTR retrotransposon in opah, zebrafish, bluefin tuna,
   and takifugu.

Results

Genome features and long terminal repeat (LTR) retrotransposons

   PacBio sequencing technology was used to generate a high-quality genome
   of a male opah. We generated 183.23 Gb of PacBio clean data with
   approximately 136-fold coverage. These data were assembled into a
   1,367.47 Mb genome, which approximated the size estimated by k-mer
   distribution (1,374.37 Mb, [83]Figure S1), with a contig N50 size of
   3.819 Mb and a scaffold N50 size of 21.58 Mb ([84]Table S1). Hi-C reads
   were used to scaffold the contig-level assemblies, resulting in 22
   chromosome-level scaffolds, which comprised the opah genome
   ([85]Figures S2 and [86]S3, [87]Tables S2 and [88]S3). In total, 24,658
   genes were predicted ([89]Tables S4–S6) using a combination of de novo
   gene prediction programs and homology-based methods, along with RNA-seq
   data. The evaluation of the genome for completeness based on
   Benchmarking Universal Single-Copy Orthologs (BUSCO; database:
   vertebrata_odb9) and the Core Eukaryotic Genes Mapping Approach (CEGMA)
   resulted in values of 94.08% and 98.91%, respectively ([90]Tables S7
   and [91]S8). Genome annotation completeness was also evaluated using
   BUSCO, and the results showed that our gene set contained 95.78%
   complete and 2.28% fragmented ortholog genes ([92]Table S9), showing
   that our gene annotation was highly complete. In total, 167 unique gene
   families were identified in the opah genome compared with those in
   zebrafish, fugu, and spotted gar ([93]Figure S4).

   A phylogenomic analysis assigned the opah to a basal position within
   the advanced spiny-rayed Teleostei clade (Acanthomorpha)
   ([94]Figure S5), which is in agreement with recent
   studies.[95]^11^,[96]^12 The opah genome was found to contain a
   relatively high percentage of transposable elements (TEs) and other
   interspersed repeats (taken together, ∼50%). Specifically, the genome
   contained the highest percentage of the LTR retrotransposon superfamily
   (11%) reported in any teleost to date ([97]Figure 1B, [98]Tables S10,
   and S11). Enriched genes in spatial proximity to the LTR
   retrotransposons (2 kb up and downstream) were investigated to discover
   the evolutionary role of the extremely high LTR content in the opah
   genome. Among these genes, many gene ontology terms related to
   metabolism and the sensory system were significantly overrepresented,
   including the lipid metabolic process (GO:0006629, P = 4.2e-04), ATP
   binding (GO:0005524, P = 1.9e-08), glycerol transport (GO:0015793, P =
   7.3e-04), oxygen transport (GO:0015671, P = 2.7e-03), and structural
   constituent of the eye lens (GO:0005212, P = 5.1e-03) ([99]Figure 1C,
   [100]Table S12). The expansion of LTRs during the evolution of the opah
   might thus have affected gene expression and translocation,[101]^13
   likely contributing to the endothermic ability of the opah and its
   adaptations to the deep-sea environment. In opah, the LTR
   retrotransposons were inferred to have started expanding approximately
   15 million years ago ([102]Figure 1D), which might have coevolved with
   (and potentially facilitated) whole-body endothermy.

Genome-wide signatures of convergent evolution in endothermic lineages

   A whole-body endothermic phenotype evolved independently in mammals,
   birds, and opahs, and we analyzed these three lineages to detect
   molecular signals accompanying the convergent evolution of this
   phenotype ([103]Figure 2A). After selecting five endothermic species
   (opah, turkey, chicken, mouse, and human) as foreground branches, 204
   genes were found to have a convergent signature for positive selection,
   which were enriched in terms related to primary metabolic processes
   (GO: 0044238, P = 2.3e-05), organic substance metabolic processes (GO:
   0071704, P = 8.0e-05), and carbohydrate derivative biosynthetic
   processes (GO: 1901137, P = 1.7e-03) ([104]Table S13).

Figure 2.

   [105]Figure 2
   [106]Open in a new tab

   Genomic convergence analysis of endothermy in fishes

   (A) Phylogeny of independently evolved endothermic vertebrates.

   (B) The phylogeny consists of red muscle endothermic fishes (opah,
   Pacific bluefin tuna, and great white shark) and outgroups.

   (C) Comparison of the Ka/Ks ratios of genes involved in 10 GO terms
   related to heat production and metabolism between the endothermic
   foreground branch and ectothermic background branch of panel (B).

   Genes with convergent amino acid substitutions were further screened
   among the selected mammals, birds, and opah ([107]Table S14). Notably,
   slc8b1 (mitochondrial sodium/potassium/calcium exchanger 6) showed
   signs of convergence, and it encodes the mitochondrial Na^+/Ca^2+
   exchanger (NCLX)[108]^14 and is essential for heart function and
   metabolic heat production. A substitution site (M460I) was found to be
   located in the Na_Ca_ex domain ([109]Figure S6A, [110]Table S14), which
   is conserved among ectothermic teleosts, chondrichthyans, and reptiles.
   Because our orthologous gene sets comprised only two mammals and two
   birds, we manually expanded the scope of this comparison with 64
   species, including 10 species of mammals, birds, and reptiles, five
   amphibians, and 29 fishes ([111]Table S15). We found that the
   convergent amino acid change (M460I) was restricted to the whole-body
   endothermic mammals, birds, and opah and thus not found in the regional
   endothermic fishes, bluefin tuna, and great white shark
   ([112]Figure S6A), implying potentially different mechanisms for
   evolving whole-body and regional endothermy. Additionally, another 22
   convergent genes were detected among four of the five endothermic
   species (including opah; [113]Figure 2A, [114]Table S14).

   Despite these similarities, teleosts and chondrichthyans generally
   generate heat mainly with their swim musculature, which differs from
   the heat production strategy of mammals and birds.[115]^15^,[116]^16 To
   account for this discrepancy, we performed another convergent evolution
   analysis comprising only fishes, namely the opah, Pacific bluefin tuna,
   and the great white shark ([117]Figure 2B). Firstly, positive selection
   analyses were conducted with three different datasets by using either
   the opah, the Pacific bluefin tuna, or the great white shark as the
   foreground branch, revealing the genetic landscape of 36 genes with
   adaptive mutations that occurred during the evolution of endothermic
   fishes ([118]Table S16). Among them, the opa1 gene is crucial for the
   maintenance of mtDNA,[119]^17 and pyroxd2 plays important roles in
   regulating mitochondrial functions.[120]^18 Then, we set all three
   endothermic fishes as foreground branches simultaneously. A total of
   112 genes were found to have convergent signatures of positive
   selection, which were enriched in gene ontology terms related to a
   citrate lyase complex (GO: 0009346, P = 4.4e-03), energy homeostasis
   (GO: 0097009, P = 5.5e-03), and mitochondrial DNA metabolic process
   (GO: 0032042, P = 5.5e-03) ([121]Table S17). In addition, we calculated
   the Ka/Ks ratio for each gene and found that four GO terms related to
   heat production and metabolism had significantly higher Ka/Ks ratios in
   the three endothermic fishes ([122]Figure 2C, [123]Table S18).

   Among the endothermic fishes, 78 convergent amino acid replacements in
   44 genes were identified ([124]Table S19). One particular convergent
   amino acid substitution was identified in all three endothermic fish
   species at the V198A site of glutaredoxin 3 (glrx3) ([125]Table S19), a
   gene that plays a crucial role in Fe/S protein biogenesis and is
   indispensable for hemoglobin maturation.[126]^19 The convergent site
   V198A was found to be located in the GRX domain, which has thiol
   oxidoreductase activity that is responsible for the reduction in
   protein disulfides or glutathione-protein mixed disulfides and is
   involved in cellular redox regulation. Convergent amino acid
   substitutions might affect the activity of GRX, which could in turn
   influence the function of Glrx3. We manually analyzed 45 bony fishes
   and six chondrichthyans and found that this convergent amino acid
   change (V198A) only existed in the three endothermic fishes
   ([127]Figure S6B). This substitution is located in the GRX domain,
   which is conserved among teleosts, chondrichthyans, and lamprey,
   indicating the ancient and conserved role of glrx3 in binding Fe/S
   clusters. In addition, three convergently changed sites (R8L, R159Q,
   and S201A) in the citrate lyase subunit β -like protein (Clybl) were
   found to be shared between opah and bluefin tuna ([128]Table S19).
   Clybl plays important roles in energy metabolism, as it encodes a
   malate/β-methylmalate synthase, which can convert glyoxylate and
   acetyl-CoA to malate or glyoxylate and propionyl-CoA to
   β-methylmalate.[129]^20 Additionally, we performed convergent analysis
   for background (nonendothermic species), and no convergent genes were
   found in either nonendothermic vertebrates (zebrafish, takifugu,
   medaka, lizard, snake, and elephant shark) or nonendothermic fish
   (takifugu, ocean sunfish, bamboo shark, whale shark, elephant shark,
   and lamprey). In summary, our genome scans provide evidence of
   convergent selective pressures and genetic responses shared by these
   endothermic fish species.

Evolutionary basis of whole-body endothermy

   We performed comparative genomic analyses with other fish genomes to
   uncover opah-specific expanded/contracted gene families and positively
   selected genes (PSGs). We identified a total of 850 (36 gene families
   with P < 0.05) and 1,038 (38 gene families with P < 0.05) gene families
   to be expanded or contracted in opah, respectively ([130]Figure 1A,
   [131]Tables S20 and [132]S21), and identified 233 genes with signatures
   of positive selection ([133]Table S22). A notably large number of
   expanded gene families was enriched for GO terms related to the
   composition of muscle fibers (e.g., actin cytoskeleton GO: 0015629,
   myofibril GO: 0030016) and the contractile machinery of skeletal muscle
   (e.g., actin filament organization GO: 0007015, skeletal muscle thin
   filament assembly GO: 0030240) ([134]Figure 3A, [135]Table S23). In
   addition, many PSGs, such as med12, rmnd1, and alg2, are considered to
   be involved in the regulation of muscle formation and development. The
   expanded gene families of the structural proteins myosin, actin, and
   troponin encode key components of the sarcomere structure
   ([136]Figure 3B). The expanded gene families were also enriched in the
   term cardiac muscle cell development (GO: 0055013), and three PSGs,
   utp15 (U3 small nucleolar RNA-associated protein 15), utp14a (U3 small
   nucleolar RNA-associated protein 14 homolog A), and robo4 (roundabout
   homolog 4) ([137]Figures 3C and [138]S7), were related to the genesis,
   regulation, and patterning of vasculature. A mutation in utp15 disrupts
   vascular patterning in zebrafish embryos,[139]^21 utp14a promotes
   angiogenesis,[140]^22 and robo4 is expressed in emerging blood vessels
   and can neutralize signaling to maintain vessel integrity through an
   angiogenic factor.[141]^23 These expanded gene families and PSGs found
   in opah might thus be related to the evolution of the counter-current
   retia mirabilia.

Figure 3.

   [142]Figure 3
   [143]Open in a new tab

   Genomic features related to the production and retention of metabolic
   heat in opah

   (A) The top 20 significantly enriched GO terms for expanded gene
   families in the opah genome are shown for cellular components (CCs) and
   biological processes (BPs).

   (B) PSGs (blue) and expanded genes (red) related to muscle development
   and skeletal muscle contraction.

   (C) PSGs (blue) and expanded genes (red) are involved in vasculature
   genesis, regulation, and patterning.

   The identified 233 PSGs were significantly enriched in organic
   substance metabolic processes and nitrogen compound metabolic processes
   ([144]Figure 4A, [145]Table S24). Among them, agrp regulates energy
   homeostasis;[146]^24 acsf4 and acot8 are involved in lipid metabolism
   ([147]Figure 4B); and ndufa10, ndufs4, and cox15 encode core proteins
   in the mitochondrial electron transport chain ([148]Figure 4C), which
   is crucial for oxidative phosphorylation. Remarkably, ndufa10 also
   exhibited convergent amino acid changes among the fast-running ruminant
   species, which require a sufficient quantity of red muscle and
   efficient contractions to guarantee a supply of metabolic heat.[149]^25
   Moreover, gene families involved in lipid metabolism (mcadd),
   carbohydrate metabolism (gapd, enolase), and oxidative phosphorylation
   (ndufv, cytc, cox7a2, and atp1a1) were found to be expanded in the opah
   genome ([150]Figure 4C). The genetic changes detected in opah indicate
   enhanced physiological functions, including increased aerobic
   performance, food digestion, and assimilation.

Figure 4.

   [151]Figure 4
   [152]Open in a new tab

   Genomic features related to the whole-body endothermy of opah

   (A) Top 15 enriched GO terms (category: biological process) among PSGs
   in the opah genome.

   (B and C) Expanded genes (red) and PSGs (blue) in opah are involved in
   lipid metabolism, carbohydrate metabolism, and (C) oxidative
   phosphorylation.

Transcriptomic and proteomic profiles of opah pectoral musculature

   Being the major “heat motor” for the whole-body endothermy of opah,
   further studies were conducted to evaluate the evolutionary origin of
   the dark red, aerobic pectoral musculature using its transcriptomic and
   proteomic profiles. Seven tissues were used to identify the
   tissue-specific expressed genes as follows: the inner (PMI) and
   superficial layer (PMS) of the pectoral musculature, dorsal musculature
   (DM), ventral musculature (VM), liver, kidney, and gill ([153]Table
   S25). Additionally, five tissues were used to identify the
   tissue-specific expressed proteins, namely PMI, PMS, VM, liver, and
   gill. Transcriptomic profiles revealed that the 100 genes with the
   highest expression in PMI, which included many expanded gene families
   such as cytochrome c oxidase (cytc), ATP synthase F0 subunit 6 (atp6),
   glyceraldehyde-3-phosphate dehydrogenase (gapdh), and troponin
   (troponin) ([154]Figure 5, [155]Table S26). Consistent with the
   transcriptomic profiles, the 100 most abundant proteins included Cytc,
   Atp6, Gapdh, troponin, and myosin ([156]Figure 5, [157]Table S27).
   These proteins have been reported to be involved in cytochrome heat
   production, ATP biosynthesis, glycolysis, and skeletal muscle
   contraction.[158]26, [159]27, [160]28 In addition, hemoglobins were
   also highly expressed, which might indicate a type of functional
   compensation, considering the high oxygen consumption ([161]Tables S26
   and [162]S27).[163]^29 Taken together, the PMI highly expressed
   proteins are likely involved in endothermy, providing insights into the
   genetic basis for the evolutive “whole-body endothermy” of opah.

Figure 5.

   [164]Figure 5
   [165]Open in a new tab

   Transcriptomic and proteomic profiles of the novel pectoral musculature
   that was co-opted for heat production

   Proteomics: KEGG pathway enrichment analysis of the upregulated DEPs of
   the PMI and the PMS, including thermogenesis and oxidative
   phosphorylation. Endothermic molecules: the 100 most highly expressed
   genes and proteins in the PMI contributing to whole-body endothermy.
   Transcriptomics: the number and expression profiles of genes recruited
   from other tissues for the development of the dark red, aerobic PMI.

   At both the structural and molecular levels, the PMI is a unique tissue
   that differs from muscles in other opah regions. Previous studies have
   indicated that the evolutionary origin of “new organs” or key
   innovations such as pectoral musculature often depends on the
   recruitment and co-option of genes that were originally expressed in
   other tissues.[166]^30 In this study, the comparative transcriptome
   analysis indicated that the PMI depends largely on genes recruited from
   the segmental swimming musculature, specifically the DM ([167]Table
   S28), whereas the gene expression profile of the PMS was closest to
   that of VM. To determine the disparity between the PMI and other
   musculature, we conducted further analyses and identified 1,480, 6,645,
   and 4,771 differentially expressed genes in PMI versus DM, PMI versus
   VM, and PMI versus PMS, respectively ([168]Figures S8 and [169]S9,
   [170]Tables S29, [171]30 , and [172]S31). In addition, a comparative
   label-free proteomic approach identified 470 (149 downregulated, 321
   upregulated) and 573 (402 downregulated, 171 upregulated)
   differentially expressed proteins (DEPs) in PMI versus VM and PMI
   versus PMS, respectively ([173]Tables S32 and [174]S33). A Kyoto
   Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of
   these DEPs revealed that the highly expressed proteins in the PMI were
   mainly involved in thermogenesis and oxidative phosphorylation,
   indicating that there has been an adaptive change in the heat
   production of the evolved musculature ([175]Figure S8).

Adaptive sensory and immune systems pave the way to the deep sea

   In opah, approximately 5.6% (13 out of 234 genes) of the identified
   PSGs play important roles in the visual system ([176]Figure 6A,
   [177]Table S22): the centrosomal protein POC5 (poc5) is a causative
   gene for retinal disease, and previous studies have shown that the loss
   of poc5 caused retinal degeneration in zebrafish;[178]^31 the mutant
   dynamin binding protein (dnmbp) and mitochondrial isoleucyl-tRNA
   synthetase 2 (iars2) are associated with cataracts in
   mammals;[179]^32^,[180]^33 tripartite motif-containing protein 44
   (trim44) is crucial for iris development, and the variants in trim44
   are considered the major cause of aniridia;[181]^34 biallelic mutations
   in the S-phase cyclin-A-associated protein in the ER (scaper) can cause
   autosomal recessive retinitis pigmentosa with
   intellectual disability;[182]^35 optic atrophy protein 1 (opa1) plays
   an important role in optic nerve development.[183]^36 In addition,
   significant expansions of the β- and γ-crystallin family have been
   found in the opah genome, which also provide support for the enhanced
   visual capability of opah ([184]Figure 6A). However, in contrast to
   their enhanced eyesight, opah might have a reduced olfactory
   performance, as they have the lowest number of olfactory receptor genes
   reported to date for teleosts (only 24, compared with 26 in Hippocampus
   comes and 156 in Danio rerio; [185]Figure 6B).[186]^37^,[187]^38
   Additionally, in the opah genome, MHCII genes were significantly
   expanded, especially in the DA group of the MHCII β-chain
   ([188]Figure S10). Compared with the DB and DE groups, the DA group
   exhibited the classical characteristics of MHCII, and these genes were
   found to be specifically and highly expressed in the immune tissues
   (liver, kidney, and gill) of opah ([189]Figure S11).

Figure 6.

   [190]Figure 6
   [191]Open in a new tab

   Genomic analysis of genes related to vision and olfaction in opah

   (A) Expanded genes (red) and PSGs (blue) in opah are involved in visual
   development.

   (B) Gene phylogeny of olfactory receptors across the genomes of opah,
   zebrafish, fugu, and cod.

Discussion

   The evolution of endothermy in mammals and birds has been intensively
   studied during the last few decades, and it has been hypothesized to
   confer numerous physiological and ecological
   advantages.[192]^15^,[193]^39 Different thermogenic strategies have
   been adopted by those vertebrate clades that evolved endothermy
   independently;[194]^1 however, the adaptive changes that accompanied
   the evolution of endothermy exhibit many similarities, including a
   higher metabolic rate, a higher capability of sustained aerobic
   performance, and enhanced neural
   conductance.[195]^7^,[196]^15^,[197]^16 Adaptive phenotypic convergence
   between distantly related taxa has occurred repeatedly in nature and
   has generally been attributed to convergent genetic changes driven by
   similar selective pressures.[198]40, [199]41, [200]42 Several lineages
   of elasmobranchs and teleosts independently evolved specific forms of
   muscular endothermy allowing them to elevate their body temperature
   relative to that of the ambient water.[201]^1 Previous studies on tuna
   and the great white shark revealed convergent genetic changes involved
   in oxidative phosphorylation, the Krebs cycle, muscle contraction, and
   lipid and fat metabolism,[202]^15^,[203]^16 and a recent study on tuna
   and billfish observed convergent amino acid replacements in proteins
   related to heat production and the visual system.[204]^43

   Fish endothermy is mainly dependent on red muscle activity, which
   produces heat as a by-product during muscle
   contraction[205]^7^,[206]^15 and is located close to the center of the
   body, in most endothermic species (e.g., tuna and great white
   sharks).[207]^44 To achieve the extraordinarily high respiratory
   metabolic rate, endothermic fishes have evolved a number of key
   adaptations. In this study, we found a convergent amino acid
   substitution at the V198A site located in the conserved GRX domain of
   glrx3 for the three endothermic fishes among the 51 fishes
   investigated. As glrx3 is indispensable for hemoglobin maturation, the
   replaced amino acid in the convergent site of the endothermic species,
   relative to that in ectothermic groups, might change the ability to
   provide oxygen to the musculature, thus affecting muscle strength and
   endurance in the opah, bluefin tuna, and great white sharks.

   As tuna, billfish, and the great white shark are only able to warm up
   specific regions of their bodies, they are not typically considered
   warm blooded. Opah exhibit “whole-body endothermy,” through the
   production of vast amounts of heat, by constantly flapping their
   enlarged pectoral fins when continuously swimming in a labrid
   fashion.[208]^7^,[209]^8 In the opah genome, a substantial portion of
   PSGs and expanded gene families have been identified as involved in not
   only skeletal muscle contraction but also lipid metabolism,
   carbohydrate metabolism, and oxidative phosphorylation, which coincides
   with expectations that endothermic species have increased metabolic
   rates and aerobic capacities.[210]^15 Furthermore, the expansion of LTR
   retrotransposons in opah was also suspected to regulate the
   transposition and expression of genes related to heat-generating
   processes. Genes involved in muscle contraction and energy metabolism
   were found to have high expression levels in the PMI, which have thick
   fibers and show a much deeper red color compared with muscles in other
   regions. Interestingly, the genes in the PMI were largely recruited
   from the DM rather than the PMS, suggesting that the PMI is more
   derived and specialized in opah. Hence, we agree with previous studies
   suggesting that the novel structure and function of PMI is the key to
   the evolution of endothermy in opah.

   Adaptive changes have been hypothesized to have arisen in the sensory
   systems of opah alongside endothermy, including enhanced temporal
   resolution and neural conductance for the eye and brain.[211]^7 As a
   high-performance predator, opah have large eyes and are thought to rely
   heavily on vision to seek and capture active prey such as fast swimming
   squid and fish.[212]^45 This was also reflected by its genome, as
   crystallin genes were duplicated, and several PSGs related to the
   visual system were identified; this enhanced visual capability is
   likely one of the benefits from endothermy, since an improvement in
   vision has also been reported in billfish and tuna, which are able to
   warm their brains and eyes.[213]^43^,[214]^46 The reduced olfactory
   performance might be compensated by the enhanced vision, which has been
   reported to be an adaptation to facilitate habitat utilization of deep
   and cold waters.[215]^38 Enhanced vision, along with an expanded
   thermal tolerance to the nutrient-poor pelagic environment, would
   enable increased access to high-energy prey and provide a strong
   selective benefit.[216]^39 The MHCII molecules are heterodimeric
   surface proteins involved in the presentation of exogenous antigens
   during the adaptive immune response.[217]^47 Since endothermy
   represents an optimum in the trade-off between metabolism and fitness
   of immunity,[218]^9^,[219]^10 the adaptive genetic changes in the
   immune system might be coordinated with the evolution of endothermy in
   opah. The unique endothermy of opah has put the species in a unique
   situation where it can exploit cold, nutrient-rich deep waters without
   the shortcomings of reduced metabolic rates.

Conclusions

   In this study, we generated a high-quality chromosome-level genome
   assembly of the Smalleye Pacific Opah (Lampris incognitus). By
   comparing the opah with mammals, birds, and other regionally
   endothermic fishes, strong genomic signatures of convergent evolution
   in the heat production process and visual system were identified. We
   identified a large number of positively selective genes and expanded
   gene families in the opah genome that are involved in the processes of
   skeletal muscle contraction, lipid metabolism, carbohydrate metabolism,
   and oxidative phosphorylation, among others, indicating that they are
   part of the genomic basis for whole-body endothermy. Our transcriptomic
   and proteomic analyses showed that genes involved in muscle contraction
   and energy metabolism have high expression levels in the PMI, which
   might be the evolutionary key innovation involved in the evolution of
   whole-body endothermy. These adaptations might explain how opahs can
   have such an active lifestyle in cold and deep-sea environments.

Acknowledgments