Extreme High-Elevation Mammal Surveys Reveal Unexpectedly High Upper Range Limits of Andean Mice.

AbstractIn the world's highest mountain ranges, uncertainty about the upper elevational range limits of alpine animals represents a critical knowledge gap regarding the environmental limits of life and presents a problem for detecting range shifts in response to climate change. Here we report results of mountaineering mammal surveys in the Central Andes, which led to the discovery of multiple species of mice living at extreme elevations that far surpass previously assumed range limits for mammals. We livetrapped small mammals from ecologically diverse sites spanning >6,700 m of vertical relief, from the desert coast of northern Chile to the summits of the highest volcanoes in the Andes. We used molecular sequence data and whole-genome sequence data to confirm the identities of species that represent new elevational records and to test hypotheses regarding species limits. These discoveries contribute to a new appreciation of the environmental limits of vertebrate life.

Evolution of lysine-specific demethylase 1 and REST corepressor gene families and their molecular interaction.

Lysine-specific demethylase 1A (LSD1) binds to the REST corepressor (RCOR) protein family of corepressors to erase transcriptionally active marks on histones. Functional diversity in these complexes depends on the type of RCOR included, which modulates the catalytic activity of the complex. Here, we studied the duplicative history of the RCOR and LSD gene families and analyzed the evolution of their interaction. We found that RCOR genes are the product of the two rounds of whole-genome duplications that occurred early in vertebrate evolution. In contrast, the origin of the LSD genes traces back before to the divergence of animals and plants. Using bioinformatics tools, we show that the RCOR and LSD1 interaction precedes the RCOR repertoire expansion that occurred in the last common ancestor of jawed vertebrates. Overall, we trace LSD1-RCOR complex evolution and propose that animal non-model species offer advantages in addressing questions about the molecular biology of this epigenetic complex.

Genomic insights into the mystery of mouse mummies on the summits of Atacama volcanoes.

Our understanding of the limits of animal life is continually revised by scientific exploration of extreme environments. Here we report the discovery of mummified cadavers of leaf-eared mice, Phyllotis vaccarum, from the summits of three different Andean volcanoes at elevations 6,029-6,233 m above sea level in the Puna de Atacama in Chile and Argentina. Such extreme elevations were previously assumed to be completely uninhabitable by mammals. In combination with a live-captured specimen of the same species from the nearby summit of Volcán Llullaillaco (6,739 m)1, the summit mummies represent the highest altitude physical records of mammals in the world. We also report a chromosome-level genome assembly for P. vaccarum that, in combination with a whole-genome re-sequencing analysis and radiocarbon dating analysis, provides insights into the provenance and antiquity of the summit mice. Radiocarbon data indicate that the most ancient of the mummies are, at most, a few centuries old. Genomic polymorphism data revealed a high degree of continuity between the summit mice and conspecifics from lower elevations in the surrounding Altiplano. Genomic data also revealed equal numbers of males and females among the summit mice and evidence of close kinship between some individuals from the same summits. These findings bolster evidence for resident populations of Phyllotis at elevations >6,000 m and challenge assumptions about the environmental limits of vertebrate life and the physiological tolerances of small mammals.

Discovery of BbX transcription factor in the patagonian blennie: Exploring expression changes following combined bacterial and thermal stress exposure.

High-Mobility Group (HMG) proteins are involved in different processes such as transcription, replication, DNA repair, and immune response. The role of HMG proteins in the immune response of fish has been studied mainly for HMGB1, where its expression can be induced by the stimulation of viral/bacterial PAMPs and can act as a proinflammatory mediator and as a global regulator of transcription in response to temperature. However, for BbX this role remains to be discovered. In this work, we identified the BbX of E. maclovinus and evaluated the temporal expression levels after simultaneous challenge with P. salmonis and thermal stress. Phylogenetic analysis does not significantly deviate from the expected organismal relationships suggesting orthologous relationships and that BbX was present in the common ancestor of the group. BbX mRNA expression levels were very high in the intestinal tissue of E. maclovinus (foregut, midgut, and hindgut). Nevertheless, the protein levels analyzed by WB showed the highest levels of BbX protein in the liver (constitutive expression). On the other hand, the mRNA expression levels of BbX in the liver of E. maclovinus injected with P. salmonis and subjected to thermal stress showed an increase at days 16 and 20 in all treatments applied at 12 °C and 18 °C. Meanwhile, the protein levels quantified by WB showed a statistically significant increase in the HMG-Bbx at all experimental times (4, 8, 12, 16, and 20 dpi). However, at 4 dpi the HMG-Bbx protein levels were much higher than the other days evaluated. The results suggest that BbX protein may be implicated in the response mechanism to temperature and bacterial stimulation in the foregut, midgut, hindgut, and liver, according to our findings at the level of mRNA and protein. Furthermore, our WB analysis suggests an effect of P. salmonis on the expression of this protein that can be observed in condition C+ 12 °C compared to C- 12 °C. Then, there is an effect of temperature that can be evidenced in the condition AM 18 °C and SM 18 °C, compared to AB 18 °C and SB 18 °C at 4, 8, and 12 dpi. We found not differences in the levels of this protein if the thermal stress is achieved through acclimatization or shock. More research is necessary to clarify the importance of this type of HMG in the immune response and thermal tolerance in fish.

Extreme high-elevation mammal surveys reveal unexpectedly high upper range limits of Andean mice.

In the world's highest mountain ranges, uncertainty about the upper elevational range limits of alpine animals represents a critical knowledge gap regarding the environmental limits of life and presents a problem for detecting range shifts in response to climate change. Here we report results of mountaineering mammal surveys in the Central Andes, which led to the discovery of multiple species of mice living at extreme elevations that far surpass previously assumed range limits for mammals. We live-trapped small mammals from ecologically diverse sites spanning >6700 m of vertical relief, from the desert coast of northern Chile to the summits of the highest volcanoes in the Andes. We used molecular sequence data and whole-genome sequence data to confirm the identities of species that represent new elevational records and to test hypotheses regarding species limits. These discoveries contribute to a new appreciation of the environmental limits of vertebrate life.

How Many Sirtuin Genes Are Out There? Evolution of Sirtuin Genes in Vertebrates With a Description of a New Family Member.

Studying the evolutionary history of gene families is a challenging and exciting task with a wide range of implications. In addition to exploring fundamental questions about the origin and evolution of genes, disentangling their evolution is also critical to those who do functional/structural studies to allow a deeper and more precise interpretation of their results in an evolutionary context. The sirtuin gene family is a group of genes that are involved in a variety of biological functions mostly related to aging. Their duplicative history is an open question, as well as the definition of the repertoire of sirtuin genes among vertebrates. Our results show a well-resolved phylogeny that represents an improvement in our understanding of the duplicative history of the sirtuin gene family. We identified a new sirtuin gene family member (SIRT3.2) that was apparently lost in the last common ancestor of amniotes but retained in all other groups of jawed vertebrates. According to our experimental analyses, elephant shark SIRT3.2 protein is located in mitochondria, the overexpression of which leads to an increase in cellular levels of ATP. Moreover, in vitro analysis demonstrated that it has deacetylase activity being modulated in a similar way to mammalian SIRT3. Our results indicate that there are at least eight sirtuin paralogs among vertebrates and that all of them can be traced back to the last common ancestor of the group that existed between 676 and 615 millions of years ago.

Sequence and structural conservation reveal fingerprint residues in TRP channels.

Transient receptor potential (TRP) proteins are a large family of cation-selective channels, surpassed in variety only by voltage-gated potassium channels. Detailed molecular mechanisms governing how membrane voltage, ligand binding, or temperature can induce conformational changes promoting the open state in TRP channels are still a matter of debate. Aiming to unveil distinctive structural features common to the transmembrane domains within the TRP family, we performed phylogenetic reconstruction, sequence statistics, and structural analysis over a large set of TRP channel genes. Here, we report an exceptionally conserved set of residues. This fingerprint is composed of twelve residues localized at equivalent three-dimensional positions in TRP channels from the different subtypes. Moreover, these amino acids are arranged in three groups, connected by a set of aromatics located at the core of the transmembrane structure. We hypothesize that differences in the connectivity between these different groups of residues harbor the apparent differences in coupling strategies used by TRP subgroups.

Identification of multiple TAR DNA binding protein retropseudogene lineages during the evolution of primates.

The TAR DNA Binding Protein (TARDBP) gene has become relevant after the discovery of its several pathogenic mutations. The lack of evolutionary history is in contrast to the amount of studies found in the literature. This study investigated the evolutionary dynamics associated with the retrotransposition of the TARDBP gene in primates. We identified novel retropseudogenes that likely originated in the ancestors of anthropoids, catarrhines, and lemuriformes, i.e. the strepsirrhine clade that inhabit Madagascar. We also found species-specific retropseudogenes in the Philippine tarsier, Bolivian squirrel monkey, capuchin monkey and vervet. The identification of a retropseudocopy of the TARDBP gene overlapping a lncRNA that is potentially expressed opens a new avenue to investigate TARDBP gene regulation, especially in the context of TARDBP associated pathologies.

Evolution of the DAN gene family in vertebrates.

The DAN gene family (DAN, Differential screening-selected gene Aberrant in Neuroblastoma) is a group of genes that is expressed during development and plays fundamental roles in limb bud formation and digitation, kidney formation and morphogenesis and left-right axis specification. During adulthood the expression of these genes are associated with diseases, including cancer. Although most of the attention to this group of genes has been dedicated to understanding its role in physiology and development, its evolutionary history remains poorly understood. Thus, the goal of this study is to investigate the evolutionary history of the DAN gene family in vertebrates, with the objective of complementing the already abundant physiological information with an evolutionary context. Our results recovered the monophyly of all DAN gene family members and divide them into five main groups. In addition to the well-known DAN genes, our phylogenetic results revealed the presence of two new DAN gene lineages; one is only retained in cephalochordates, whereas the other one (GREM3) was only identified in cartilaginous fish, holostean fish, and coelacanth. According to the phyletic distribution of the genes, the ancestor of gnathostomes possessed a repertoire of eight DAN genes, and during the radiation of the group GREM1, GREM2, SOST, SOSTDC1, and NBL1 were retained in all major groups, whereas, GREM3, CER1, and DAND5 were differentially lost.

Evolutionary history of the vertebrate Piwi gene family.

PIWIs are regulatory proteins that belong to the Argonaute family. Piwis are primarily expressed in gonads and protect the germline against the mobilization and propagation of transposable elements (TEs) through transcriptional gene silencing. Vertebrate genomes encode up to four Piwi genes: Piwil1, Piwil2, Piwil3 and Piwil4, but their duplication history is unresolved. We leveraged phylogenetics, synteny and expression analyses to address this void. Our phylogenetic analysis suggests Piwil1 and Piwil2 were retained in all vertebrate members. Piwil4 was the result of Piwil1 duplication in the ancestor of gnathostomes, but was independently lost in ray-finned fishes and birds. Further, Piwil3 was derived from a tandem Piwil1 duplication in the common ancestor of marsupial and placental mammals, but was secondarily lost in Atlantogenata (Xenarthra and Afrotheria) and some rodents. The evolutionary rate of Piwil3 is considerably faster than any Piwi among all lineages, but an explanation is lacking. Our expression analyses suggest Piwi expression has mostly been constrained to gonads throughout vertebrate evolution. Vertebrate evolution is marked by two early rounds of whole genome duplication and many multigene families are linked to these events. However, our analyses suggest Piwi expansion was independent of whole genome duplications.

Whole-Genome Duplications and the Diversification of the Globin-X Genes of Vertebrates.

Globin-X (GbX) is an enigmatic member of the vertebrate globin gene family with a wide phyletic distribution that spans protostomes and deuterostomes. Unlike canonical globins such as hemoglobins and myoglobins, functional data suggest that GbX does not have a primary respiratory function. Instead, evidence suggests that the monomeric, membrane-bound GbX may play a role in cellular signaling or protection against the oxidation of membrane lipids. Recently released genomes from key vertebrates provide an excellent opportunity to address questions about the early stages of the evolution of GbX in vertebrates. We integrate bioinformatics, synteny, and phylogenetic analyses to characterize the diversity of GbX genes in nonteleost ray-finned fishes, resolve relationships between the GbX genes of cartilaginous fish and bony vertebrates, and demonstrate that the GbX genes of cyclostomes and gnathostomes derive from independent duplications. Our study highlights the role that whole-genome duplications (WGDs) have played in expanding the repertoire of genes in vertebrate genomes. Our results indicate that GbX paralogs have a remarkably high rate of retention following WGDs relative to other globin genes and provide an evolutionary framework for interpreting results of experiments that examine functional properties of GbX and patterns of tissue-specific expression. By identifying GbX paralogs that are products of different WGDs, our results can guide the design of experimental work to explore whether gene duplicates that originate via WGDs have evolved novel functional properties or expression profiles relative to singleton or tandemly duplicated copies of GbX.

Independent duplications of the Golgi phosphoprotein 3 oncogene in birds.

Golgi phosphoprotein 3 (GOLPH3) was the first reported oncoprotein of the Golgi apparatus. It was identified as an evolutionarily conserved protein upon its discovery about 20 years ago, but its function remains puzzling in normal and cancer cells. The GOLPH3 gene is part of a group of genes that also includes the GOLPH3L gene. Because cancer has deep roots in multicellular evolution, studying the evolution of the GOLPH3 gene family in non-model species represents an opportunity to identify new model systems that could help better understand the biology behind this group of genes. The main goal of this study is to explore the evolution of the GOLPH3 gene family in birds as a starting point to understand the evolutionary history of this oncoprotein. We identified a repertoire of three GOLPH3 genes in birds. We found duplicated copies of the GOLPH3 gene in all main groups of birds other than paleognaths, and a single copy of the GOLPH3L gene. We suggest there were at least three independent origins for GOLPH3 duplicates. Amino acid divergence estimates show that most of the variation is located in the N-terminal region of the protein. Our transcript abundance estimations show that one paralog is highly and ubiquitously expressed, and the others were variable. Our results are an example of the significance of understanding the evolution of the GOLPH3 gene family, especially for unraveling its structural and functional attributes.

Positive selection and gene duplications in tumour suppressor genes reveal clues about how cetaceans resist cancer.

Cetaceans are the longest-living species of mammals and the largest in the history of the planet. They have developed mechanisms against diseases such cancer, although the underlying molecular bases of these remain unknown. The goal of this study was to investigate the role of natural selection in the evolution of 1077 tumour suppressor genes (TSGs) in cetaceans. We used a comparative genomic approach to analyse two sources of molecular variation in the form of dN/dS rates and gene copy number variation. We found a signal of positive selection in the ancestor of cetaceans within the CXCR2 gene, an important regulator of DNA damage, tumour dissemination and immune system. Further, in the ancestor of baleen whales, we found six genes exhibiting positive selection relating to diseases such as breast carcinoma, lung neoplasm (ADAMTS8) and leukaemia (ANXA1). The TSGs turnover rate (gene gain and loss) was almost 2.4-fold higher in cetaceans when compared with other mammals, and notably even faster in baleen whales. The molecular variants in TSGs found in baleen whales, combined with the faster gene turnover rate, could have favoured the evolution of their particular traits of anti-cancer resistance, gigantism and longevity. Additionally, we report 71 genes with duplications, of which 11 genes are linked to longevity (e.g. NOTCH3 and SIK1) and are important regulators of senescence, cell proliferation and metabolism. Overall, these results provide evolutionary evidence that natural selection in TSGs could act on species with large body sizes and extended lifespan, providing novel insights into the genetic basis of disease resistance.

The Globin Gene Family in Arthropods: Evolution and Functional Diversity.

Globins are small heme-proteins that reversibly bind oxygen. Their most prominent roles in vertebrates are the transport and storage of O2 for oxidative energy metabolism, but recent research has suggested alternative, non-respiratory globin functions. In the species-rich and ecologically highly diverse taxon of arthropods, the copper-containing hemocyanin is considered the main respiratory protein. However, recent studies have suggested the presence of globin genes and their proteins in arthropod taxa, including model species like Drosophila. To systematically assess the taxonomic distribution, evolution and diversity of globins in arthropods, we systematically searched transcriptome and genome sequence data and found a conserved, widespread occurrence of three globin classes in arthropods: hemoglobin-like (HbL), globin X (GbX), and globin X-like (GbXL) protein lineages. These globin types were previously identified in protostome and deuterostome animals including vertebrates, suggesting their early ancestry in Metazoa. The HbL genes show multiple, lineage-specific gene duplications in all major arthropod clades. Some HbL genes (e.g., Glob2 and 3 of Drosophila) display particularly fast substitution rates, possibly indicating the evolution of novel functions, e.g., in spermatogenesis. In contrast, arthropod GbX and GbXL globin genes show high evolutionary stability: GbXL is represented by a single-copy gene in all arthropod groups except Brachycera, and representatives of the GbX clade are present in all examined taxa except holometabolan insects. GbX and GbXL both show a brain-specific expression. Most arthropod GbX and GbXL proteins, but also some HbL variants, include sequence motifs indicative of potential N-terminal acylation (i.e., N-myristoylation, 3C-palmitoylation). All arthropods except for the brachyceran Diptera harbor at least one such potentially acylated globin copy, confirming the hypothesis of an essential, conserved globin function associated with the cell membrane. In contrast to other animals, the fourth ancient globin lineage, represented by neuroglobin, appears to be absent in arthropods, and the putative arthropod orthologs of the fifth metazoan globin lineage, androglobin, lack a recognizable globin domain. Thus, the remarkable evolutionary stability of some globin variants is contrasted by occasional dynamic gene multiplication or even loss of otherwise strongly conserved globin lineages in arthropod phylogeny.

Discovery of the world's highest-dwelling mammal.

Environmental limits of animal life are invariably revised when the animals themselves are investigated in their natural habitats. Here we report results of a scientific mountaineering expedition to survey the high-altitude rodent fauna of Volcán Llullaillaco in the Puna de Atacama of northern Chile, an effort motivated by video documentation of mice (genus Phyllotis) at a record altitude of 6,205 m. Among numerous trapping records at altitudes of >5,000 m, we captured a specimen of the yellow-rumped leaf-eared mouse (Phyllotis xanthopygus rupestris) on the very summit of Llullaillaco at 6,739 m. This summit specimen represents an altitudinal world record for mammals, far surpassing all specimen-based records from the Himalayas and other mountain ranges. This discovery suggests that we may have generally underestimated the altitudinal range limits and physiological tolerances of small mammals simply because the world's high summits remain relatively unexplored by biologists.

Evolutionary analyses reveal independent origins of gene repertoires and structural motifs associated to fast inactivation in calcium-selective TRPV channels.

Essential for calcium homeostasis, TRPV5 and TRPV6 are calcium-selective channels belonging to the transient receptor potential (TRP) gene family. In this study, we investigated the evolutionary history of these channels to add an evolutionary context to the already available physiological information. Phylogenetic analyses revealed that paralogs found in mammals, sauropsids, amphibians, and chondrichthyes, are the product of independent duplication events in the ancestor of each group. Within amniotes, we identified a traceable signature of three amino acids located at the amino-terminal intracellular region. The signature correlates with both the duplication events and the phenotype of fast inactivation observed in mammalian TRPV6 channels. Electrophysiological recordings and mutagenesis revealed that the signature sequence modulates the phenotype of fast inactivation in all clades of vertebrates but reptiles. A transcriptome analysis showed a change in tissue expression from gills, in marine vertebrates, to kidneys in terrestrial vertebrates. Our results highlight a cytoplasmatic structural triad composed by the Helix-Loop-Helix domain, the S2-S3 linker, and the TRP domain helix that is important on modulating the activity of calcium-selective TRPV channels.

Author Correction: The Reprimo gene family member, reprimo-like (rprml), is required for blood development in embryonic zebrafish.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Evolution of nodal and nodal-related genes and the putative composition of the heterodimers that trigger the nodal pathway in vertebrates.

Nodal is a signaling molecule that belongs to the transforming growth factor-ß superfamily that plays key roles during the early stages of development of animals. In vertebrates Nodal forms an heterodimer with a GDF1/3 protein to activate the Nodal pathway. Vertebrates have a paralog of nodal in their genomes labeled Nodal-related, but the evolutionary history of these genes is a matter of debate, mainly because of the presence of a variable numbers of genes in the vertebrate genomes sequenced so far. Thus, the goal of this study was to investigate the evolutionary history of the Nodal and Nodal-related genes with an emphasis in tracking changes in the number of genes among vertebrates. Our results show the presence of two gene lineages (Nodal and Nodal-related) that can be traced back to the ancestor of jawed vertebrates. These lineages have undergone processes of differential retention and lineage-specific expansions. Our results imply that Nodal and Nodal-related duplicated at the latest in the ancestor of gnathostomes, and they still retain a significant level of functional redundancy. By comparing the evolution of the Nodal/Nodal-related with GDF1/3 gene family, it is possible to infer that there are several types of heterodimers that can trigger the Nodal pathway among vertebrates.

The Reprimo gene family member, reprimo-like (rprml), is required for blood development in embryonic zebrafish.

The Reprimo gene family comprises a group of single-exon genes for which their physiological function remains poorly understood. Heretofore, mammalian Reprimo (RPRM) has been described as a putative p53-dependent tumor suppressor gene that functions at the G2/M cell cycle checkpoint. Another family member, Reprimo-like (RPRML), has not yet an established role in physiology or pathology. Importantly, RPRML expression pattern is conserved between zebrafish and human species. Here, using CRISPR-Cas9 and antisense morpholino oligonucleotides, we disrupt the expression of rprml in zebrafish and demonstrate that its loss leads to impaired definitive hematopoiesis. The formation of hemangioblasts and the primitive wave of hematopoiesis occur normally in absence of rprml. Later in development there is a significant reduction in erythroid-myeloid precursors (EMP) at the posterior blood island (PBI) and a significant decline of definitive hematopoietic stem/progenitor cells (HSPCs). Furthermore, loss of rprml also increases the activity of caspase-3 in endothelial cells within the caudal hematopoietic tissue (CHT), the first perivascular niche where HSPCs reside during zebrafish embryonic development. Herein, we report an essential role for rprml during hematovascular development in zebrafish embryos, specifically during the definitive waves of hematopoiesis, indicating for the first time a physiological role for the rprml gene.

Phylogenetic evidence for independent origins of GDF1 and GDF3 genes in anurans and mammals.

Growth differentiation factors 1 (GDF1) and 3 (GDF3) are members of the transforming growth factor superfamily (TGF-ß) that is involved in fundamental early-developmental processes that are conserved across vertebrates. The evolutionary history of these genes is still under debate due to ambiguous definitions of homologous relationships among vertebrates. Thus, the goal of this study was to unravel the evolution of the GDF1 and GDF3 genes of vertebrates, emphasizing the understanding of homologous relationships and their evolutionary origin. Our results revealed that the GDF1 and GDF3 genes found in anurans and mammals are the products of independent duplication events of an ancestral gene in the ancestor of each of these lineages. The main implication of this result is that the GDF1 and GDF3 genes of anurans and mammals are not 1:1 orthologs. In other words, genes that participate in fundamental processes during early development have been reinvented two independent times during the evolutionary history of tetrapods.