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Vestigial organs are historical echoes of past phenotypes. Determining whether a specific organ constitutes a functional or vestigial structure can be a challenging task, given that distinct levels of atrophy may arise between and within lineages. The mammalian pineal gland, an endocrine organ involved in melatonin biorhythmicity, represents a classic example, often yielding contradicting anatomical observations. In Xenarthra (sloths, anteaters and armadillos), a peculiar mammalian order, the presence of a distinct pineal organ was clearly observed in some species (i.e. Linnaeus’s two-toed sloth) but undetected in other closely related species (i.e. brown-throated sloth). In the nine-banded armadillo, contradicting evidence supports either functional or vestigial scenarios. Thus, to untangle the physiological status of the pineal gland in Xenarthra, we used a genomic approach to investigate the evolution of the gene hub responsible for melatonin synthesis and signaling. We show that both synthesis and signaling compartments are eroded and were lost independently. Additionally, by expanding our analysis to 157 mammal genomes we offer a comprehensive view showing that species with very distinctive habitats and lifestyles have convergently evolved a similar phenotype: Cetacea, Pholidota, Dermoptera, Sirenia and Xenarthra. Our findings suggest that the recurrent inactivation of melatonin genes correlates with pineal atrophy, and endorse the use of genomic analyses to ascertain the physiological status of suspected vestigial structures.

The Atlantic chub mackerel, Scomber colias Gmelin, 1789, is a medium-size pelagic fish with substantial importance in the fisheries of the Atlantic Ocean and the Mediterranean Sea. Over the past decade, this species has gained special relevance being one of the main targets of pelagic fisheries in the NE Atlantic. Here, we sequenced and annotated the first high-quality draft genome assembly of S. colias, produced with Pacbio HiFi long reads and Illumina Paired-End short reads. The estimated genome size is 814 Mb distributed into 2,028 scaffolds and 2,093 contigs with an N50 length of 4,19 and 3,34 Mb, respectively. We annotated 27,675 protein-coding genes and the BUSCO analyses indicated high completeness, with 97.3 % of the single-copy orthologs in the Actinopterygii library profile. The present genome assembly represents a valuable resource to address the biology and management of this relevant fishery. Finally, this is the fourth high-quality genome assembly within the Order Scombriformes and the first in the genus Scomber.

18 Pág. Departamento de Reproducción Animal.(INIA), This research was funded by COMPETE 2020 ; Portugal 2020 ; the European Union through the ERDF, grant number 031342 ; FCT through national funds ( PTDC/CTA-AMB/31342/2017 ); and grant RTI2018-093548-BI00 from the Spanish Ministry of Science and Innovation . R.V. is funded by the FCT under the grant SFRH/BD/144786/2019 . The study was also supported by trainers and veterinarians of Oceanogràfic Valencia, especially Carlos Barros-García, for the obtaining and transfer of dolphin ejaculates. S.R.D. is supported by a “Doctorados Industriales 2018” fellowship of Comunidad de Madrid ( IND2018/BIO-9610 ).

Environmental chemicals have been reported to greatly disturb the endocrine and metabolic systems of multiple animal species. A recent example involves the exploitation of the nuclear receptor (NR) heterodimeric pair composed by PPAR/RXR (peroxisome proliferator-activated receptor/retinoid X receptor), which shows lipid perturbation in mammalian species. While gene orthologues of both of these receptors have been described outside vertebrates, no functional characterization of PPAR has been carried in protostome lineages. We provide the first functional analysis of PPAR in Patella sp. (Mollusca), using model obesogens such as tributyltin (TBT), triphenyltin (TPT), and proposed natural ligands (fatty acid molecules). To gain further insights, we used site-directed mutagenesis to PPAR and replaced the tyrosine 277 by a cysteine (the human homologous amino acid and TBT anchor residue) and an alanine. Additionally, we explored the alterations in the fatty acid profiles after an exposure to the model obesogen TBT, in vivo. Our results show that TBT and TPT behave as an antagonist of Patella sp. PPAR/RXR and that the tyrosine 277 is important, but not essential in the response to TBT. Overall, these results suggest a relation between the response of the mollusc PPAR-RXR to TBT and the lipid profile alterations reported at environmentally relevant concentrations. Our findings highlight the importance of comparative analysis between protostome and deuterostome lineages to decipher the differential impact of environmental chemicals.

The expression pattern of genes coding for enzymes of the retinoic acid (RA) synthetic and degradation pathways was characterized in adult female zebrafish Danio rerio. Females were conditioned until maturation and post-spawn expression dynamics were determined. A striking upregulation of cyp26b1, but not cyp26a1, was observed following egg deposition, decreasing to initial levels during recovery. A similar, yet lower, fluctuation was observed for aldh1a2 and rdh10a, the enzymes participating in the two-step RA biosynthesis cascade. The present work highlights the dynamics of the adult D. rerio oogenesis and uncovers novel, yet elusive, metabolic contributors. Possible compartmentalized roles for the different gene paralogue isoforms are discussed.

Signalling molecules and their cognate receptors are central components of the Metazoa endocrine system. Defining their presence or absence in extant animal lineages is critical to accurately devise evolutionary patterns, physiological shifts and the impact of endocrine disrupting chemicals. Here, we address the evolution of retinoic acid (RA) signalling in the Priapulida worm, Priapulus caudatus Lamarck, 1816, an Ecdysozoa. RA signalling has been shown to be central to chordate endocrine homeostasis, participating in multiple developmental and physiological processes. Priapulids, with their slow rate of molecular evolution and phylogenetic position, represent a key taxon to investigate the early phases of Ecdysozoa evolution. By exploring a draft genome assembly, we show, by means of phylogenetics and functional assays, that an orthologue of the nuclear receptor retinoic acid receptor (RAR) subfamily, a central mediator of RA signalling, is present in Ecdysozoa, contrary to previous perception. We further demonstrate that the Priapulida RAR displays low-affinity for retinoids (similar to annelids), and is not responsive to common endocrine disruptors acting via RAR. Our findings provide a timeline for RA signalling evolution in the Bilateria and give support to the hypothesis that the increase in RA affinity towards RAR is a late acquisition in the evolution of the Metazoa.

Recent reports raise the concern that exposure to several environmental chemicals may induce persistent changes that go beyond the exposed organisms, being transferred to subsequent generations even in the absence of the original chemical insult. These changes in subsequent non-exposed generations have been related to epigenetic changes. Although highly relevant for hazard and risk assessment, biomarkers of epigenetic modifications that can be associated with adversity, are still not integrated into hazard assessment frameworks. Here, in order to validate new biomarkers of epigenetic modifications in a popular animal model, zebrafish embryos were exposed to different concentrations of Bisphenol A (0.01, 0.1, 1 and 10 mg/L) and Valproic Acid (0.8, 4, 20 and 100 mg/L), two chemicals reported to alter the modulation of the epigenome. Morphological abnormalities and epigenetic changes were assessed at 80 hours-post fertilization, including DNA global methylation and gene expression of both DNA and histone epigenetic modifications. Gene expression changes were detected at concentrations below those inducing morphological abnormalities. These results further support the importance of combining epigenetic biomarkers with apical endpoints to improve guidelines for chemical testing and hazard assessment, and favour the integration of new biomarkers of epigenetic modifications into the standardized OECD test guideline 236 with zebrafish embryos.

The origin and diversification of the metazoan endocrine systems represents a fundamental research issue in biology. Nuclear receptors are critical components of these systems. A particular group named VDR/PXR/CAR (NR1I/J) is central in the mediation of detoxification responses. While orthologues have been thoroughly characterized in vertebrates, a sparse representation is currently available for invertebrates. Here, we provide the first isolation and characterization of a lophotrochozoan protostome VDR/PXR/CAR nuclear receptor (NR1J), in the estuarine bivalve the peppery furrow shell (Scrobicularia plana). Using a reporter gene assay, we evaluated the xenobiotic receptor plasticity comparing the human PXR with the S. plana NR1Jβ. Our results show that the molluscan receptor responds to a natural toxin (okadaic acid) in a similar fashion to that reported for other invertebrates. In contrast, the pesticide esfenvalerate displayed a unique response, since it down regulated transactivation at higher concentrations, while for triclosan no response was observed. Additionally, we uncovered lineage specific gene duplications and gene loss in the gene group encoding NRs in protostomes with likely impacts on the complexity of detoxification mechanisms across different phyla. Our findings pave the way for the development of multi-specific sensor tools to screen xenobiotic compounds acting via the NR1I/J group.

Clupeiformes, such as sardines and herrings, represent an important share of worldwide fisheries. Among those, the European sardine (Sardina pilchardus, Walbaum 1792) exhibits significant commercial relevance. While the last decade showed a steady and sharp decline in capture levels, recent advances in culture husbandry represent promising research avenues. Yet, the complete absence of genomic resources from sardine imposes a severe bottleneck to understand its physiological and ecological requirements. We generated 69 Gbp of paired-end reads using Illumina HiSeq X Ten and assembled a draft genome assembly with an N50 scaffold length of 25579 bp and BUSCO completeness of 82.1% (Actinopterygii). The estimated size of the genome ranges between 655 and 850 Mb. Additionally, we generated a relatively high-level liver transcriptome. To deliver a proof of principle of the value of this dataset, we established the presence and function of enzymes (elovl2, elovl5 and fads2) that have pivotal roles in the biosynthesis of long chain polyunsaturated fatty acids, essential nutrients particularly abundant in oily fish such as sardines. Our study provides the first omics dataset from a valuable economic marine teleost species, the European sardine, an essential resource for their effective conservation, management and sustainable exploitation.

Nucleobase-Ascorbate Transporter (NAT) family includes ascorbic acid, nucleobases and uric acid transporters, with a broad evolutionary distribution. In vertebrates, four members have been previously recognized, the ascorbate transporters Slc23a1 and Slc3a2, the nucleobase transporter Slc23a4 and an orphan transporter SLC23A3. Here we identify a fifth member of the vertebrateslc23complement (slc23a5), expressed in neopterygians (gars and teleosts) and amphibians, and clarify the evolutionary relationships between the novel gene and knownslc23genes. Further comparative analysis puts forward uric acid as the preferred substrate for Slc23a5. Gene expression quantification suggests kidney and testis as major expression sites inXenopus tropicalis(western clawed frog) andDanio rerio(zebrafish). Additional expression in brain was detected inD. rerio, while in the NeoteleosteiOryzias latipes(medaka)slc23a5expression is restricted to brain. The biological relevance of the retention of an extra transporter in fish and amphibians is examined: with respect to the (1) antioxidant role of uric and ascorbic acid in seminal fluid and brain, (2) the ability to endogenously synthesize ascorbic acid and (3) the morphological adaptations of the male urogenital system.

[Background] Provision of long-chain polyunsaturated fatty acids (LC-PUFA) in vertebrates occurs through the diet or via endogenous production from C18 precursors through consecutive elongations and desaturations. It has been postulated that the abundance of LC-PUFA in the marine environment has remarkably modulated the gene complement and function of Fads in marine teleosts. In vertebrates two fatty acyl desaturases, namely Fads1 and Fads2, encode ∆5 and ∆6 desaturases, respectively. To fully clarify the evolutionary history of LC-PUFA biosynthesis in vertebrates, we investigated the gene repertoire and function of Fads from species placed at key evolutionary nodes., [Results] We demonstrate that functional Fads1Δ5 and Fads2∆6 arose from a tandem gene duplication in the ancestor of vertebrates, since they are present in the Arctic lamprey. Additionally, we show that a similar condition was retained in ray-finned fish such as the Senegal bichir and spotted gar, with the identification of fads1 genes in these lineages. Functional characterisation of the isolated desaturases reveals the first case of a Fads1 enzyme with ∆5 desaturase activity in the Teleostei lineage, the Elopomorpha. In contrast, in Osteoglossomorpha genomes, while no fads1 was identified, two separate fads2 duplicates with ∆6 and ∆5 desaturase activities respectively were uncovered., [Conclusions] We conclude that, while the essential genetic components involved LC-PUFA biosynthesis evolved in the vertebrate ancestor, the full completion of the LC-PUFA biosynthesis pathway arose uniquely in gnathostomes., We acknowledge Fundação para a Ciência e a Tecnologia for the support to M.L.-M (SFRH/BD/84238/2012). LFCC research is supported by Norte2020 and FEDER (Coral - Sustainable Ocean Exploitation - Norte-01-0145-FEDER-000036) a project supported by the North Portugal Regional Operational Program (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). The funding bodies played no role in the design of the study and collection, analysis, interpretation of data, or in writing the manuscript.

Clupeiformes, such as sardines and herrings, represent an important share of worldwide fisheries. Among those, the European sardine (Sardina pilchardus, Walbaum 1792) exhibits significant commercial relevance. While the last decade showed a steady and sharp decline in capture levels, recent advances in culture husbandry represent promising research avenues. Yet, the complete absence of genomic resources from sardine imposes a severe bottleneck to understand its physiological and ecological requirements. We generated 69 Gbp of paired-end reads using Illumina HiSeq X Ten and assembled a draft genome assembly with an N50 scaffold length of 25,579 bp and BUSCO completeness of 82.1% (Actinopterygii). The estimated size of the genome ranges between 655 and 850 Mb. Additionally, we generated a relatively high-level liver transcriptome. To deliver a proof of principle of the value of this dataset, we established the presence and function of enzymes (Elovl2, Elovl5, and Fads2) that have pivotal roles in the biosynthesis of long chain polyunsaturated fatty acids, essential nutrients particularly abundant in oily fish such as sardines. Our study provides the first omics dataset from a valuable economic marine teleost species, the European sardine, representing an essential resource for their effective conservation, management, and sustainable exploitation., We acknowledge the North Portugal Regional Operational Program (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) that supported this research through the Coral—Sustainable Ocean Exploitation (reference NORTE-01-0145-FEDER-000036). R.R.d.F. thanks the Danish National Research Foundation for its support of the Center for Macroecology, Evolution, and Climate (grant DNRF96).

Nuclear receptors (NRs) regulate numerous aspects of the endocrine system. They mediate endogenous and exogenous cues, ensuring a homeostatic control of development and metabolism. Gene duplication, loss and mutation have shaped the repertoire and function of NRs in metazoans. Here, we examine the evolution of a pivotal orchestrator of cholesterol metabolism in vertebrates, the liver X receptors (LXRs). Previous studies suggested that LXRα and LXRβ genes emerged in themammalian ancestor. However, we show through genomean alysis and functional assay that bona fide LXRα and LXRβ orthologues are present in reptiles, coelacanth and chondrichthyans but not in cyclostomes. These findings show that LXR duplicated before gnathostome radiation, followed by asymmetric paralogue loss in some lineages. We suggest that a tighter control of cholesterol levels in vertebrates was achieved through the exploitation of a wider range of oxysterols, an ability contingent on ligand-binding pocket remodeling. © 2016 The Author. We thank Neelakanteswar Aluru and Pedro Morais for the samples of little skate and amphioxus, respectively. This work was supported by Norte2020 and FEDER (Coral-Sustainable Ocean Exploitation-Norte-01-0145-FEDER- 000036), EXPL/MAR-EST/1540/2012. SFRH/BD/79305/2011 to E.F., SFRH/BD/84238/2012 to M.L.-M. and SFRH/BPD/72519/2010 to R.R. L.F.C.C. and M.M.S. are supported by the European Regional Development Fund (ERDF) through the COMPETE-Operational Competitiveness Programme and POPH-Operational Human Potential Programme and national funds through FCT-Foundation for Science and Technology (UID/Multi/04423/2013). This work was supported by the Branchiostoma lanceolatum genome consortium that provided access to the Branchiostoma lanceolatum genome sequence. We thank three anonymous reviewers for their comments and suggestions.

Recent genomic and biochemical evidence in invertebrate species pushes back the origin of the retinoid metabolic and signaling modules to the last common ancestor of all bilaterians. However, the evolution of retinoid pathways are far from fully understood. In the majority of non-chordate invertebrate lineages, the ongoing functional characterization of retinoid-related genes (metabolism and signaling pathways), as well as the characterization of the endogenous retinoid content (precursors and active retinoids), is still incomplete. Despite limited, the available data supports the presence of biologically active retinoid pathways in invertebrates. Yet, the mechanisms controlling the spatial and temporal distribution of retinoids as well as their physiological significance share similarities and differences with vertebrates. For instance, retinol storage in the form of retinyl esters, a key feature for the maintenance of retinoid homeostatic balance in vertebrates, was only recently demonstrated in some mollusk species, suggesting that such ability is older than previously anticipated. In contrast, the enzymatic repertoire involved in this process is probably unlike that of vertebrates. The suggested ancestry of active retinoid pathways implies that many more metazoan species might be potential targets for endocrine disrupting chemicals. Here, we review the current knowledge about the occurrence and functionality of retinoid metabolic and signaling pathways in invertebrate lineages, paying special attention to the evolutionary origin of retinoid storage mechanisms. Additionally, we summarize existing information on the endocrine disruption of invertebrate retinoid modules by environmental chemicals. Research priorities in the field are highlighted.

To compare genelociconsidering a phylogenetic framework is a promising approach to uncover the genetic basis of human diseases. Imbalance of dopaminergic systems is suspected to underlie some emerging neurological disorders. The physiological functions of dopamine are transduced via G-protein-coupled receptors, including DRD5which displays a relatively higher affinity towards dopamine. Importantly, DRD5knockout mice are hypertense, a condition emerging from an increase in sympathetic tone. We investigated the evolution of DRD5, a high affinity receptor for dopamine, in mammals. Surprisingly, among 124 investigated mammalian genomes, we found that Cetacea lineages (Mysticeti and Odontoceti) have independently lost this gene, as well as the burrowingChrysochloris asiatica (Cape golden mole).We suggest that DRD5inactivation parallels hypoxia-induced adaptations, such as peripheral vasoconstriction required for deep-diving in Cetacea, in accordance with the convergent evolution of vasoconstrictor genes in hypoxia-exposed animals. Our findings indicate that Cetacea are natural knockouts for DRD5and might offer valuable insights into the mechanisms of some forms of vasoconstriction responses and hypertension in humans.

Melatonin, the hormone of darkness, is a peculiar molecule found in most living organisms. Emerging as a potent broad-spectrum antioxidant, melatonin was repurposed into extra roles such as the modulation of circadian and seasonal rhythmicity, affecting numerous aspects of physiology and behaviour, including sleep entrainment and locomotor activity. Interestingly, the pineal gland&mdash, the melatonin synthesising organ in vertebrates&mdash, was suggested to be absent or rudimentary in some mammalian lineages, including Cetacea. In Cetacea, pineal regression is paralleled by their unique bio-rhythmicity, as illustrated by the unihemispheric sleeping behaviour and long-term vigilance. Here, we examined the genes responsible for melatonin synthesis (Aanat and Asmt) and signalling (Mtnr1a and Mtnr1b) in 12 toothed and baleen whale genomes. Based on an ample genomic comparison, we deduce that melatonin-related gene modules are eroded in Cetacea.

Nucleobase-Ascorbate Transporter (NAT) family includes ascorbic acid, nucleobases, and uric acid transporters: With broad evolutionary distribution. In vertebrates, four members have been previously recognized, the ascorbate transporters Slc23a1 and Slc3a2, the nucleobase transporter Slc23a4 and an orphan transporter Slc23a3. Using phylogenetic and synteny analysis, we identify a fifth member of the vertebrate slc23 complement (slc23a5), present in neopterygians (gars and teleosts) and amphibians, and clarify the evolutionary relationships between the novel gene and known slc23 genes. Further comparative analysis puts forward uric acid as the preferred substrate for Slc23a5. Gene expression quantification, using available transcriptomic data, suggests kidney and testis as major expression sites in Xenopus tropicalis (western clawed frog) and Danio rerio (zebrafish). Additional expression in brain was detected in D. rerio, while in the Neoteleostei Oryzias latipes (medaka) slc23a5 expression is restricted to the brain. The biological relevance of the retention of an extra transporter in fish and amphibians is discussed.

The biosynthesis of long-chain polyunsaturated fatty acids (LC-PUFA) provides an intriguing example on how multi-enzymatic cascades evolve. Essential LC-PUFA, such as arachidonic, eicosapentaenoic, and docosahexaenoic acids (DHA), can be acquired from the diet but are also endogenously retailored from C precursors through consecutive elongations and desaturations catalyzed, respectively, by fatty acyl elongase and desaturase enzymes. The molecular wiring of this enzymatic pathway defines the ability of a species to biosynthesize LC-PUFA. Exactly when and how in animal evolution a functional LC-PUFA pathway emerged is still elusive. Here we examine key components of the LC-PUFA cascade, the Elovl2/Elovl5 elongases, from amphioxus, an invertebrate chordate, the sea lamprey, a representative of agnathans, and the elephant shark, a basal jawed vertebrate. We show that Elovl2 and Elovl5 emerged from genome duplications in vertebrate ancestry. The single Elovl2/5 from amphioxus efficiently elongates C and C and, to a marked lesser extent, C LC-PUFA. Lamprey is incapable of elongating C substrates. The elephant shark Elovl2 showed that the ability to efficiently elongate C PUFA and thus to synthesize DHA through the Sprecher pathway, emerged in the jawed vertebrate ancestor. Our findings illustrate how non-integrated >metabolic islands> evolve into fully wired pathways upon duplication and neofunctionalization., This work was supported by the Fundação para a Ciência e a Tecnologia (FCT) (Strategic Funding UID/Multi/04423/2013, SFRH/BD/84238/2012 to M.L.-M. and SFRH/BPD/72519/2010 to R.R.) and partly by MINECO (Spanish Government - AGL2013-40986R). The access to the Institute of Aquaculture laboratories was funded by the European Union’s Seventh Framework Programme (FP7/2007-2013) under grant agreement n° 262336 (AQUAEXCEL), Transnational Access Project Number 0095/06/03/13.

Germ cell commitment and meiosis initiation are among the multitude of physiological roles of retinoic acid (RA) in vertebrates. Acting via receptor-mediated transcription, RA induces the expression of meiotic factors, triggering meiosis. Contrasting with vertebrates, invertebrate RA metabolism is scarcely understood. Still, some physiological processes appear to be conserved. Here we set to evaluate the role of retinoids in the gonad maturation process of the marine gastropod Patella vulgata. We found that retinoid concentration in gonadal tissue, namely RA, varies between breeding and resting specimens, with maxima attained in the latter. Additionally, we isolated and quantified the expression of both the retinoic acid receptor (RAR) and the retinoid X receptor (RXR) in gonads. In view of the stability of retinoid receptor expression, we suggest that the balance of RA levels operates through the enzymatic control of synthetic and catabolic processes. Overall, the reported data are supportive for a developmental role of RA during gonadal maturation in P. vulgata, which should be addressed in other protostome lineages.

The Carnitine palmitoyltransferase I (Cpt1) gene family plays a crucial role in energy homeostasis since it is required for the occurrence of fatty acid β-oxidation in the mitochondria. The exact gene repertoire in different vertebrate lineages is variable. Presently, four genes are documented: Cpt1a, also known as Cpt1a1, Cpt1a2; Cpt1b and Cpt1c. The later is considered a mammalian innovation resulting from a gene duplication event in the ancestor of mammals, after the divergence of sauropsids. In contrast, Cpt1a2 has been found exclusively in teleosts. Here, we reassess the overall evolutionary relationships of Cpt1 genes using a combination of approaches, including the survey of the gene repertoire in basal gnathostome lineages. Through molecular phylogenetics and synteny studies, we find that Cpt1c is most likely a rapidly evolving orthologue of Cpt1a2. Thus, Cpt1c is present in other lineages such as cartilaginous fish, reptiles, amphibians and the coelacanth. We show that genome duplications (2R) and variable rates of sequence evolution contribute to the history of Cpt1 genes in vertebrates. Finally, we propose that loss of Cpt1b is the likely cause for the unusual energy metabolism of elasmobranch.