Divergent sensory and immune gene evolution in sea turtles with contrasting demographic and life histories.

Sea turtles represent an ancient lineage of marine vertebrates that evolved from terrestrial ancestors over 100 Mya. The genomic basis of the unique physiological and ecological traits enabling these species to thrive in diverse marine habitats remains largely unknown. Additionally, many populations have drastically declined due to anthropogenic activities over the past two centuries, and their recovery is a high global conservation priority. We generated and analyzed high-quality reference genomes for the leatherback (Dermochelys coriacea) and green (Chelonia mydas) turtles, representing the two extant sea turtle families. These genomes are highly syntenic and homologous, but localized regions of noncollinearity were associated with higher copy numbers of immune, zinc-finger, and olfactory receptor (OR) genes in green turtles, with ORs related to waterborne odorants greatly expanded in green turtles. Our findings suggest that divergent evolution of these key gene families may underlie immunological and sensory adaptations assisting navigation, occupancy of neritic versus pelagic environments, and diet specialization. Reduced collinearity was especially prevalent in microchromosomes, with greater gene content, heterozygosity, and genetic distances between species, supporting their critical role in vertebrate evolutionary adaptation. Finally, diversity and demographic histories starkly contrasted between species, indicating that leatherback turtles have had a low yet stable effective population size, exhibit extremely low diversity compared with other reptiles, and harbor a higher genetic load compared with green turtles, reinforcing concern over their persistence under future climate scenarios. These genomes provide invaluable resources for advancing our understanding of evolution and conservation best practices in an imperiled vertebrate lineage.

Breathing Air and Living Underwater: Molecular Evolution of Genes Related to Antioxidant Response in Cetaceans and Pinnipeds.

Cetaceans and pinnipeds are lineages of mammals that have independently returned to the aquatic environment, acquiring varying degrees of dependence on it while sharing adaptations for underwater living. Here, we focused on one critical adaptation from both groups, their ability to withstand the ischemia and reperfusion experienced during apnea diving, which can lead to the production of reactive oxygen species (ROS) and subsequent oxidative damage. Previous studies have shown that cetaceans and pinnipeds possess efficient antioxidant enzymes that protect against ROS. In this study, we investigated the molecular evolution of key antioxidant enzyme genes (CAT, GPX3, GSR, PRDX1, PRDX3, and SOD1) and the ROS-producing gene XDH, in cetaceans and pinnipeds lineages. We used the ratio of non-synonymous (dN) to synonymous (dS) substitutions as a measure to identify signatures of adaptive molecular evolution in these genes within and between the two lineages. Additionally, we performed protein modeling and variant impact analyzes to assess the functional consequences of observed mutations. Our findings revealed distinct selective regimes between aquatic and terrestrial mammals in five of the examined genes, including divergences within cetacean and pinniped lineages, between ancestral and recent lineages and between crowns groups. We identified specific sites under positive selection unique to Cetacea and Pinnipedia, with one site showing evidence of convergent evolution in species known for their long and deep-diving capacities. Notably, many sites under adaptive selection exhibited radical changes in amino acid properties, with some being damaging mutations in human variations, but with no apparent detrimental impacts on aquatic mammals. In conclusion, our study provides insights into the adaptive changes that have occurred in the antioxidant systems of aquatic mammals throughout their evolutionary history. We observed both distinctive features within each group of Cetacea and Pinnipedia and instances of convergence. These findings highlight the dynamic nature of the antioxidant system in response to challenges of the aquatic environment and provide a foundation for further investigations into the molecular mechanisms underlying these adaptations.

Molecular Footprints on Osmoregulation-Related Genes Associated with Freshwater Colonization by Cetaceans and Sirenians.

The genetic basis underlying adaptive physiological mechanisms has been extensively explored in mammals after colonizing the seas. However, independent lineages of aquatic mammals exhibit complex patterns of secondary colonization in freshwater environments. This change in habitat represents new osmotic challenges, and additional changes in key systems, such as the osmoregulatory system, are expected. Here, we studied the selective regime on coding and regulatory regions of 20 genes related to the osmoregulation system in strict aquatic mammals from independent evolutionary lineages, cetaceans, and sirenians, with representatives in marine and freshwater aquatic environments. We identified positive selection signals in genes encoding the protein vasopressin (AVP) in mammalian lineages with secondary colonization in the fluvial environment and in aquaporins for lineages inhabiting the marine and fluvial environments. A greater number of sites with positive selection signals were found for the dolphin species compared to the Amazonian manatee. Only the AQP5 and AVP genes showed selection signals in more than one independent lineage of these mammals. Furthermore, the vasopressin gene tree indicates greater similarity in river dolphin sequences despite the independence of their lineages based on the species tree. Patterns of distribution and enrichment of Transcription Factors in the promoter regions of target genes were analyzed and appear to be phylogenetically conserved among sister species. We found accelerated evolution signs in genes ACE, AQP1, AQP5, AQP7, AVP, NPP4, and NPR1 for the fluvial mammals. Together, these results allow a greater understanding of the molecular bases of the evolution of genes responsible for osmotic control in aquatic mammals.

Genome-wide analyses reveal drivers of penguin diversification.

Penguins are the only extant family of flightless diving birds. They currently comprise at least 18 species, distributed from polar to tropical environments in the Southern Hemisphere. The history of their diversification and adaptation to these diverse environments remains controversial. We used 22 new genomes from 18 penguin species to reconstruct the order, timing, and location of their diversification, to track changes in their thermal niches through time, and to test for associated adaptation across the genome. Our results indicate that the penguin crown-group originated during the Miocene in New Zealand and Australia, not in Antarctica as previously thought, and that Aptenodytes is the sister group to all other extant penguin species. We show that lineage diversification in penguins was largely driven by changing climatic conditions and by the opening of the Drake Passage and associated intensification of the Antarctic Circumpolar Current (ACC). Penguin species have introgressed throughout much of their evolutionary history, following the direction of the ACC, which might have promoted dispersal and admixture. Changes in thermal niches were accompanied by adaptations in genes that govern thermoregulation and oxygen metabolism. Estimates of ancestral effective population sizes (Ne ) confirm that penguins are sensitive to climate shifts, as represented by three different demographic trajectories in deeper time, the most common (in 11 of 18 penguin species) being an increased Ne between 40 and 70 kya, followed by a precipitous decline during the Last Glacial Maximum. The latter effect is most likely a consequence of the overall decline in marine productivity following the last glaciation.

A phylogenetic review of cancer resistance highlights evolutionary solutions to Peto's Paradox.

Cancer is a genetic disease present in all complex multicellular lineages. Finding ways to eliminate it is a goal of a large part of the scientific community and nature itself. Early, scientists realized that the cancer incidence at the species level was not related to the number of cells or lifespan, a phenomenon called Peto's Paradox. The interest in resolving this paradox triggered a growing interest in investigating the natural strategies for cancer suppression hidden in the animal's genomes. Here, we gathered information on the main mechanisms that confer resistance to cancer, currently described for lineages that have representatives with extended longevity and large body sizes. Some mechanisms to reduce or evade cancer are common and shared between lineages, while others are species-specific. The diversity of paths that evolution followed to face the cancer challenge involving coding, regulatory, and structural aspects of genomes is astonishing and much yet lacks discovery. Multidisciplinary studies involving oncology, ecology, and evolutionary biology and focusing on nonmodel species can greatly expand the frontiers of knowledge about cancer resistance in animals and may guide new promising treatments and prevention that might apply to humans.

Expansions and contractions in gene families of independently-evolved blood-feeding insects.

BACKGROUND: The blood-feeding behavior evolved multiple times in Insecta lineages and it represents an excellent opportunity to study patterns of convergent molecular evolution regarding this habit. In insects the expansion of some gene families is linked with blood-feeding behavior, but a wide study comparing the evolution of these gene families among different lineages is still missing. Here we gathered genomic data from six independently-evolved hematophagous lineages, aiming to identify convergent expansions and/or contractions of gene families in hematophagous lineages of insects. RESULTS: We found four rapidly evolving gene families shared by at least two hematophagous independently-evolved lineages, including a heat-shock and a chemosensory protein. On the expression of these four rapidly evolving gene families we found more genes expressed in mated individuals compared with virgin individuals in rapidly-expanded families and more genes expressed in non-blood-feeding individuals compared with blood-feeding individuals in rapidly-contracted families. CONCLUSION: Our results reveal a new set of candidate genes to be explored in further analysis to help the development of new strategies to deal with blood-feeding vectors and also presents a new perspective to study the evolution of hematophagy identifying convergent molecular patterns.

Latitudinal diversity gradient and cetaceans from the perspective of MHC genes.

Pathogen diversity is a key source of selective pressure on immune system genes, shaping molecular evolution mainly on widely distributed or migratory organisms such as cetaceans. Here, we investigated the effects of latitudinal span migration, different biomes occupation, and pathogen-mediated selection on MHC DQB locus divergence on cetaceans. We applied some evolutionary genetics methods using a dataset of 15 species and 121 sequences, and we found a trend on greater MHC divergence on tropical species when compared with either temperate or migratory species. In addition, oceanic cetaceans exhibit greater MHC divergence. Here, we show that, despite there was a correlation between the diversity of MHC DQB alleles with the distribution of organisms, the pattern of diversity found is not completely explained by pathogenic pressure, suggesting that other factors must be investigated for a better understanding of the processes related to the diversity of MHC in cetaceans.

Selection on different genes with equivalent functions: the convergence story told by Hox genes along the evolution of aquatic mammalian lineages.

BACKGROUND: Convergent evolution has been a challenging topic for decades, being cetaceans, pinnipeds and sirenians textbook examples of three independent origins of equivalent phenotypes. These mammalian lineages acquired similar anatomical features correlated to an aquatic life, and remarkably differ from their terrestrial counterparts. Whether their molecular evolutionary history also involved similar genetic mechanisms underlying such morphological convergence nevertheless remained unknown. To test for the existence of convergent molecular signatures, we studied the molecular evolution of Hox genes in these three aquatic mammalian lineages, comparing their patterns to terrestrial mammals. Hox genes are transcription factors that play a pivotal role in specifying embryonic regional identity of nearly any bilateral animal, and are recognized major agents for diversification of body plans. RESULTS: We detected few signatures of positive selection on Hox genes across the three aquatic mammalian lineages and verified that purifying selection prevails in these sequences, as expected for pleiotropic genes. Genes found as being positively selected differ across the aquatic mammalian lineages, but we identified a substantial overlap of their developmental functions. Such pattern likely resides on the duplication history of Hox genes, which probably provided different possible evolutionary routes for achieving the same phenotypic solution. CONCLUSIONS: Our results indicate that convergence occurred at a functional level of Hox genes along three independent origins of aquatic mammals. This conclusion reinforces the idea that different changes in developmental genes may lead to similar phenotypes, probably due to the redundancy provided by the participation of Hox paralogous genes in several developmental functions.

Molecular evolution of HoxA13 and the multiple origins of limbless morphologies in amphibians and reptiles.

Developmental processes and their results, morphological characters, are inherited through transmission of genes regulating development. While there is ample evidence that cis-regulatory elements tend to be modular, with sequence segments dedicated to different roles, the situation for proteins is less clear, being particularly complex for transcription factors with multiple functions. Some motifs mediating protein-protein interactions may be exclusive to particular developmental roles, but it is also possible that motifs are mostly shared among different processes. Here we focus on HoxA13, a protein essential for limb development. We asked whether the HoxA13 amino acid sequence evolved similarly in three limbless clades: Gymnophiona, Amphisbaenia and Serpentes. We explored variation in ω (dN/dS) using a maximum-likelihood framework and HoxA13sequences from 47 species. Comparisons of evolutionary models provided low ω global values and no evidence that HoxA13 experienced relaxed selection in limbless clades. Branch-site models failed to detect evidence for positive selection acting on any site along branches of Amphisbaena and Gymnophiona, while three sites were identified in Serpentes. Examination of alignments did not reveal consistent sequence differences between limbed and limbless species. We conclude that HoxA13 has no modules exclusive to limb development, which may be explained by its involvement in multiple developmental processes.

Increased rate of hair keratin gene loss in the cetacean lineage.

BACKGROUND: Hair represents an evolutionary innovation that appeared early on mammalian evolutionary history, and presumably contributed significantly to the rapid radiation of the group. An interesting event in hair evolution has been its secondary loss in some mammalian groups, such as cetaceans, whose hairless phenotype appears to be an adaptive response to better meet the environmental conditions. To determine whether different repertoire of keratin genes among mammals can potentially explain the phenotypic hair features of different lineages, we characterized the type I and II clusters of alpha keratins from eight mammalian species, including the hairless dolphin and minke whale representing the order Cetacea. RESULTS: We combined the available genomic information with phylogenetic analysis to conduct a comprehensive analysis of the evolutionary patterns of keratin gene clusters. We found that both type I and II gene clusters are fairly conserved among the terrestrial mammals included in this study, with lineage specific gene duplication and gene loss. Nevertheless, there is also evidence for an increased rate of pseudogenization in the cetacean lineage when compared to their terrestrial relatives, especially among the hair type keratins. CONCLUSIONS: Here we present a comprehensive characterization of alpha-keratin genes among mammals and elucidate the mechanisms involved in the evolution of this gene family. We identified lineage-specific gene duplications and gene loss among the Laurasiatherian and Euarchontoglires species included in the study. Interestingly, cetaceans present an increased loss of hair-type keratin genes when compared to other terrestrial mammals. As suggested by the 'less-is-more' hypothesis, we do not rule out the possibility that the gene loss of hair-type keratin genes in these species might be associated to the hairless phenotype and could have been adaptive in response to new selective pressures imposed by the colonization of a new habitat. Our study provides support for the idea that pseudogenes are not simply 'genomic fossils' but instead have adaptive roles during the evolutionary process.

Whole-genome duplication and the functional diversification of teleost fish hemoglobins.

Subsequent to the two rounds of whole-genome duplication that occurred in the common ancestor of vertebrates, a third genome duplication occurred in the stem lineage of teleost fishes. This teleost-specific genome duplication (TGD) is thought to have provided genetic raw materials for the physiological, morphological, and behavioral diversification of this highly speciose group. The extreme physiological versatility of teleost fish is manifest in their diversity of blood-gas transport traits, which reflects the myriad solutions that have evolved to maintain tissue O(2) delivery in the face of changing metabolic demands and environmental O(2) availability during different ontogenetic stages. During the course of development, regulatory changes in blood-O(2) transport are mediated by the expression of multiple, functionally distinct hemoglobin (Hb) isoforms that meet the particular O(2)-transport challenges encountered by the developing embryo or fetus (in viviparous or oviparous species) and in free-swimming larvae and adults. The main objective of the present study was to assess the relative contributions of whole-genome duplication, large-scale segmental duplication, and small-scale gene duplication in producing the extraordinary functional diversity of teleost Hbs. To accomplish this, we integrated phylogenetic reconstructions with analyses of conserved synteny to characterize the genomic organization and evolutionary history of the globin gene clusters of teleosts. These results were then integrated with available experimental data on functional properties and developmental patterns of stage-specific gene expression. Our results indicate that multiple α- and ß-globin genes were present in the common ancestor of gars (order Lepisoteiformes) and teleosts. The comparative genomic analysis revealed that teleosts possess a dual set of TGD-derived globin gene clusters, each of which has undergone lineage-specific changes in gene content via repeated duplication and deletion events. Phylogenetic reconstructions revealed that paralogous genes convergently evolved similar functional properties in different teleost lineages. Consistent with other recent studies of globin gene family evolution in vertebrates, our results revealed evidence for repeated evolutionary transitions in the developmental regulation of Hb synthesis.

Accelerated evolutionary rate of the myoglobin gene in long-diving whales.

Cetaceans, early in their evolutionary history, had developed many physiological adaptations to secondarily return to the sea. Among these adaptations, changes in molecules that transport oxygen and that ultimately support large periods of acute tissue hypoxia probably represent one big step toward the conquest of aquatic environments. Myoglobin contributes to intracellular oxygen storage and transcellular diffusion of oxygen in muscle, and plays an important role in supplying oxygen in hypoxic or ischemic conditions. Here we looked for evidence of adaptive molecular evolution of myoglobin in the cetacean lineage, relative to their terrestrial counterparts. We performed a comparative analysis to examine the variation of the parameter ω (d N/d S) and infer past period of adaptive evolution during the cetacean transition from the terrestrial to the aquatic environment. We also analyzed the changes in amino acid properties. At the nucleotide level, the results showed significant differences in selective pressure between cetacean and non-cetacean myoglobin (ω value three times higher in cetaceans when compared to terrestrial mammals), and also among cetacean lineages according to their diving capacities. Interestingly, both families with long duration diving cetaceans present two parallel substitutions (on sites 4 and 12). Regarding the amino acid properties, our analysis identified four significant physicochemical amino acid changes among residues in myoglobin protein under positive destabilizing selection.

The molecular evolution of genes previously associated with large sizes reveals possible pathways to cetacean gigantism.

Cetaceans are a group of aquatic mammals with the largest body sizes among living animals, including giant representatives such as blue and fin whales. To understand the genetic bases of gigantism in cetaceans, we performed molecular evolutionary analyses on five genes (GHSR, IGF2, IGFBP2, IGFBP7, and EGF) from the growth hormone/insulin-like growth factor axis, and four genes (ZFAT, EGF, LCORL, and PLAG1) previously described as related to the size of species evolutionarily close to cetaceans, such as pigs, cows, and sheep. Our dataset comprised 19 species of cetaceans, seven of which are classified as giants because they exceed 10 m in length. Our results revealed signs of positive selection in genes from the growth hormone/insulin-like growth factor axis and also in those related to body increase in cetacean-related species. In addition, pseudogenization of the EGF gene was detected in the lineage of toothless cetaceans, Mysticeti. Our results suggest the action of positive selection on gigantism in genes that act both in body augmentation and in mitigating its consequences, such as cancer suppression when involved in processes such as division, migration, and cell development control.

Patterns of enrichment and acceleration in evolutionary rates of promoters suggest a role of regulatory regions in cetacean gigantism.

BACKGROUND: Cetaceans (whales, porpoises, and dolphins) are a lineage of aquatic mammals from which some species became giants. Only recently, gigantism has been investigated from the molecular point of view. Studies focused mainly on coding regions, and no data on the influence of regulatory regions on gigantism in this group was available. Accordingly, we investigated the molecular evolution of non-coding regulatory regions of genes already described in the literature for association with size in mammals, focusing mainly on the promoter regions. For this, we used Ciiider and phyloP tools. Ciiider identifies significantly enriched transcription factor binding sites, and phyloP estimates the molecular evolution rate of the promoter. RESULTS: We found evidence of enrichment of transcription binding factors related to large body size, with distinct patterns between giant and non-giant cetaceans in the IGFBP7 and NCAPG promoters, in which repressive agents are present in small cetaceans and those that stimulate transcription, in giant cetaceans. In addition, we found evidence of acceleration in the IGF2, IGFBP2, IGFBP7, and ZFAT promoters. CONCLUSION: Our results indicate that regulatory regions may also influence cetaceans' body size, providing candidate genes for future research to understand the molecular basis of the largest living animals.

Resolution of the laurasiatherian phylogeny: evidence from genomic data.

Despite great progress over the past decade, some portions of the mammalian tree of life remain unresolved. In particular, relationships among the different orders included within the supraordinal group Laurasiatheria have been proven difficult to determine, and have received poor support in the vast majority of phylogenomic studies of mammalian systematics. We estimated interordinal relationships within Laurasiatheria using sequence data from 3733 protein-coding genes. Our study included data from from 11 placental mammals, corresponding to five of the six orders of Laurasiatheria, plus five outgroup species. Ingroup and outgroup species were chosen to maximize the number single-copy ortholog genes for which sequence data was available for all species in our study. Phylogenetic analyses of the concatenated dataset using maximum likelihood and Bayesian methods resulted on an identical and well supported topology in all alignment strategies compared. Our analyses provide high support for the sister relationship between Chiroptera and Cetartiodactyla and also provide support for placing Perissodactyla as sister to Carnivora. We obtained maximal estimates of bootstrap support (100%) and posterior probability (1.00) for all nodes within Laurasiatheria. Our study provides a further demonstration of the utility of very large and conserved genomic dataset to clarify our understanding of the evolutionary relationships among mammals.

Positive selection in multiple salivary gland proteins of Anophelinae reveals potential targets for vector control.

Anopheles is a genus belonging to the Culicidae family, which has great medical importance due to its role as a vector of Plasmodium, the causative agent of malaria. Great focus has been given to the salivary gland proteins (SGPs) group from Anopheles' functional genomics. This class of proteins is essential to blood-feeding behavior as they have attributes such as vasodilators and anti-clotting properties. Recently, a comprehensive review on Anopheles SGPs was performed; however, the authors did not deeply explore the adaptive molecular evolution of these genes. In this context, this work aimed to perform a more detailed analysis of the adaptive molecular evolution of SGPs in Anopheles, carrying out positive selection and gene family evolution analysis on 824 SGPs. Our results show that most SGPs have positively selected codon sites that can be used as targets in developing new strategies for vector control and that younger SGPs evolve at a faster rate than older SGPs. Notably, we could not find any evidence of an accelerated shift in SGPs' rates of gene gain and loss compared with other proteins, as suggested in previous works.

Analyses of RAG1 and RAG2 genes suggest different evolutionary rates in the Cetacea lineage.

V(D)J recombination is a process of somatic recombination catalyzed by proteins encoded by RAG1 and RAG2 genes, both restricted to the genome of jawed vertebrates. Their proteins constitute the enzymatic core of V(D)J recombination machinery and are crucial for jawed vertebrate adaptive immunity. Mammals possess great ecological diversity, and their complex evolutionary history associated with radiation to different environments presented many distinct pathogenic challenges from these different habitats. Cetaceans comprise a mammalian order of fully aquatic mammals that have arisen from a complete terrestrial ancestor and, accordingly, was confronted with challenges from changing environmental pathogens while they transitioned from land to sea. In this study we undertook molecular evolutionary analyses of RAG1 and RAG2 genes, exploring the possible role of natural selection acting on these genes focusing on the cetacean lineage. We performed phylogenetic reconstructions on IQ-TREE, together with selection analyses in the codeml program of the PAML package, and in the FITMODEL program for codon evolution and switching on both the RAG1 and RAG2 genes. Our findings demonstrate that RAG1 and RAG2 remained fairly conserved among tetrapods, with purifying selection acting on both genes, with evidence for a few punctuated shifts in nucleotide substitution rates of both genes along tetrapod evolution. We demonstrate differential evolution in the closely linked genes RAG1 and RAG2 specifically in cetaceans.

The role of selection in the evolution of marine turtles mitogenomes.

Sea turtles are the only extant chelonian representatives that inhabit the marine environment. One key to successful colonization of this habitat is the adaptation to different energetic demands. Such energetic requirement is intrinsically related to the mitochondrial ability to generate energy through oxidative phosphorylation (OXPHOS) process. Here, we estimated Testudines phylogenetic relationships from 90 complete chelonian mitochondrial genomes and tested the adaptive evolution of 13 mitochondrial protein-coding genes of sea turtles to determine how natural selection shaped mitochondrial genes of the Chelonioidea clade. Complete mitogenomes showed strong support and resolution, differing at the position of the Chelonioidea clade in comparison to the turtle phylogeny based on nuclear genomic data. Codon models retrieved a relatively increased dN/dS (ω) on three OXPHOS genes for sea turtle lineages. Also, we found evidence of positive selection on at least three codon positions, encoded by NADH dehydrogenase genes (ND4 and ND5). The accelerated evolutionary rates found for sea turtles on COX2, ND1 and CYTB and the molecular footprints of positive selection found on ND4 and ND5 genes may be related to mitochondrial molecular adaptation to stress likely resulted from a more active lifestyle in sea turtles. Our study provides insight into the adaptive evolution of the mtDNA genome in sea turtles and its implications for the molecular mechanism of oxidative phosphorylation.

Gene fusion of heterophyletic gamma-globin genes in platyrrhine primates.

We performed phylogenetic analyses of HBG genes to assess its origin and interspecific variation among primates. Our analyses showed variation in HBG genes copy number ranging from one to three, some of them pseudogenes. For platyrrhines HBG genes, phylogenetic reconstructions of flanking regions recovered orthologous clades with distinct topologies for 5' and 3' flanking regions. The 5' region originated in the common ancestor of platyrrhines but the 3' region had an anthropoid origin. We hypothesize that the platyrrhine HBG genes of 5' and 3' heterophyletic origins arose from subsequent fusions of the (earlier) platyrrhine 5' portion and the (later) anthropoid 3' portion.