Novel microRNAs are associated with population divergence in transcriptional response to thermal stress in an intertidal copepod

The role of gene expression in adaptation to differing thermal environments has been assayed extensively. Yet, in most natural systems, analyses of gene expression reveal only one level of the complexity of regulatory machineries. MicroRNAs (miRNAs) are small noncoding RNAs which are key components of many gene regulatory networks, and they play important roles in a variety of cellular pathways by modulating post-transcriptional quantities of mRNA available for protein synthesis. The characterization of miRNA loci and their regulatory dynamics in nonmodel systems are still largely understudied. In this study, we examine the role of miRNAs in response to high thermal stress in the intertidal copepod Tigriopus californicus. Allopatric populations of this species show varying levels of local adaptation with respect to thermal regimes, and previous studies showed divergence in gene expression between populations from very different thermal environments. We examined the transcriptional response to temperature stress in two populations separated by only 8 km by utilizing RNA-seq to quantify both mRNA and miRNA levels. Using the currently available genome sequence, we first describe the repertoire of miRNAs in T. californicus and assess the degree to which transcriptional response to temperature stress is governed by miRNA activity. The two populations showed large differences in the number of genes involved, the magnitude of change in commonly used genes and in the number of miRNAs involved in transcriptional modulation during stress. Our results suggest that an increased level of regulatory network complexity may underlie improved survivorship under thermal stress in one of the populations

Reduction of Paraoxonase Expression Followed by Inactivation across Independent Semiaquatic Mammals Suggests Stepwise Path to Pseudogenization.

Convergent adaptation to the same environment by multiple lineages frequently involves rapid evolutionary change at the same genes, implicating these genes as important for environmental adaptation. Such adaptive molecular changes may yield either change or loss of protein function; loss of function can eliminate newly deleterious proteins or reduce energy necessary for protein production. We previously found a striking case of recurrent pseudogenization of the Paraoxonase 1 (Pon1) gene among aquatic mammal lineages-Pon1 became a pseudogene with genetic lesions, such as stop codons and frameshifts, at least four times independently in aquatic and semiaquatic mammals. Here, we assess the landscape and pace of pseudogenization by studying Pon1 sequences, expression levels, and enzymatic activity across four aquatic and semiaquatic mammal lineages: pinnipeds, cetaceans, otters, and beavers. We observe in beavers and pinnipeds an unexpected reduction in expression of Pon3, a paralog with similar expression patterns but different substrate preferences. Ultimately, in all lineages with aquatic/semiaquatic members, we find that preceding any coding-level pseudogenization events in Pon1, there is a drastic decrease in expression, followed by relaxed selection, thus allowing accumulation of disrupting mutations. The recurrent loss of Pon1 function in aquatic/semiaquatic lineages is consistent with a benefit to Pon1 functional loss in aquatic environments. Accordingly, we examine diving and dietary traits across pinniped species as potential driving forces of Pon1 functional loss. We find that loss is best associated with diving activity and likely results from changes in selective pressures associated with hypoxia and hypoxia-induced inflammation

Data from: Convergent evolution on the hypoxia-inducible factor (HIF) pathway genes EGLN1 and EPAS1 in high-altitude ducks

During periods of reduced O2 supply, the most profound changes in gene expression are mediated by hypoxia-inducible factor (HIF) transcription factors that play a key role in cellular responses to low O2 tension. Using target-enrichment sequencing, we test whether variation in 26 genes in the HIF signaling pathway is associated with high-altitude and corresponding O2 availability in three duck species that colonized the Andes from ancestral low-altitude habitats in South America. We found strong support for convergent evolution in the case of two of the three duck species with the same genes (EGLN1, EPAS1), and even the same exons (exon 12, EPAS1), exhibiting extreme outliers with a high probability of directional selection in the high-altitude populations. These results mirror patterns of adaptation seen in human populations, which showed mutations in EPAS1, and transcriptional regulation differences in EGLN1, causing changes in downstream target transactivation, associated with a blunted hypoxic response

Convergent evolution on the hypoxia-inducible factor (HIF) pathway genes EGLN1 and EPAS1 in high-altitude ducks

During periods of reduced O supply, the most profound changes in gene expression are mediated by hypoxia-inducible factor (HIF) transcription factors that play a key role in cellular responses to low-O tension. Using target-enrichment sequencing, we tested whether variation in 26 genes in the HIF signaling pathway was associated with high altitude and therefore corresponding O availability in three duck species that colonized the Andes from ancestral low-altitude habitats in South America. We found strong support for convergent evolution in the case of two of the three duck species with the same genes (EGLN1, EPAS1), and even the same exons (exon 12, EPAS1), exhibiting extreme outliers with a high probability of directional selection in the high-altitude populations. These results mirror patterns of adaptation seen in human populations, which showed mutations in EPAS1, and transcriptional regulation differences in EGLN1, causing changes in downstream target transactivation, associated with a blunted hypoxic response

Independent Losses of the Hypoxia-Inducible Factor (HIF) Pathway within Crustacea

Metazoans respond to hypoxic stress via the hypoxia-inducible factor (HIF) pathway, a mechanism thought to be extremely conserved due to its importance in monitoring cellular oxygen levels and regulating responses to hypoxia. However, recent work revealed that key members of the HIF pathway have been lost in specific lineages (a tardigrade and a copepod), suggesting that this pathway is not as widespread in animals as previously assumed. Using genomic and transcriptomic data from 70 different species across 12 major crustacean groups, we assessed the degree to which the gene HIFα, the master regulator of the HIF pathway, was conserved. Mining of protein domains, followed by phylogenetic analyses of gene families, uncovered group-level losses of HIFα, including one across three orders within Cirripedia, and in three orders within Copepoda. For these groups, additional assessment showed losses of HIF repression machinery (EGLN and VHL). These results suggest the existence of alternative mechanisms for cellular response to low oxygen and highlight these taxa as models useful for probing these evolutionary outcomes

Hypoxia Inducible Factor (HIF) transcription factor family expansion, diversification, divergence and selection in eukaryotes

<div><p>Hypoxia inducible factor (HIF) transcription factors are crucial for regulating a variety of cellular activities in response to oxygen stress (hypoxia). In this study, we determine the evolutionary history of HIF genes and their associated transactivation domains, as well as perform selection and functional divergence analyses across their four characteristic domains. Here we show that the HIF genes are restricted to metazoans: At least one HIF-α homolog is found within the genomes of non-bilaterians and bilaterian invertebrates, while most vertebrate genomes contain between two and six HIF-α genes. We also find widespread purifying selection across all four characteristic domain types, bHLH, PAS, NTAD, CTAD, in HIF-α genes, and evidence for Type I functional divergence between HIF-1α, HIF-2α /EPAS, and invertebrate HIF genes. Overall, we describe the evolutionary histories of the HIF transcription factor gene family and its associated transactivation domains in eukaryotes. We show that the NTAD and CTAD domains appear <i>de novo</i>, without any appearance outside of the HIF-α subunits. Although they both appear in invertebrates as well as vertebrate HIF- α sequences, there seems to have been a substantial loss across invertebrates or were convergently acquired in these few lineages. We reaffirm that HIF-1α is phylogenetically conserved among most metazoans, whereas HIF-2α appeared later. Overall, our findings can be attributed to the substantial integration of this transcription factor family into the critical tasks associated with maintenance of oxygen homeostasis and vascularization, particularly in the vertebrate lineage.</p></div

Maximum likelihood tree showing phylogenetic relationships between eukaryotic bHLH+PAS containing proteins.

<p>The 10 major clades representing a majority of the bHLH+PAS gene families are highlighted. The names given to each clade are derived from the human gene names found within those clades. For example, bilaterian NPAS1/3 represents a highly supported clade, Bayesian posterior probability (BPP) ≥ 0.90, that contains bilaterian sequences that group with human NPAS1 and human NPAS3. The one exception is the invertebrate-specific clade that contains the <i>Drosophila melanogaster</i> methoprene-tolerant gene. The unicellular bHLH+PAS genes typically grouped together. Purple circles indicate congruent nodes between both Bayesian and Maximum likelihood trees with a Bayesian posterior probability support value ≥ 0.90.All support values for this Maximum Likelihood tree, along with Bayesian inference tree and its corresponding support values, are found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179545#pone.0179545.s002" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179545#pone.0179545.s003" target="_blank">S2</a> Files.</p

Phylogenetic distribution of the HIF-α genes and associated transactivation domains in Metazoa.

<p>Schematics for each HIF-α identified in each metazoan species are shown. Black boxes represent the bHLH domains, blue boxes represent the PAS domains, yellow boxes represent the NTAD, and red boxes represent the CTAD. Invertebrate genes duplicated to give rise to the different vertebrate paralogs, because of the vertebrate genome duplication events (green circle). Additional paralogs of HIF-1α and HIF-2α in <i>D</i>. <i>rerio</i> are due to the teleost-specific genome duplication event (blue circle). Proteins are drawn to scale. Species phylogenetic relationships are based [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179545#pone.0179545.ref035" target="_blank">35</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179545#pone.0179545.ref038" target="_blank">38</a>].</p

Phylogenetic distribution of the ARNT and ARNTL gene families in Metazoa.

<p>Schematics for each ARNT, ARNT2, ARNTL, and ARNTL2 identified in each metazoan species are shown. Black boxes represent the bHLH domains, while the blue boxes represent the PAS domains. Invertebrate genes duplicated to give rise to the different vertebrate paralogs, as a result of the vertebrate genome duplication events (green circle). <i>Danio rerio</i> has two ARNTL paralogs, and <i>Takifugu rubripes</i> has two ARNTL2 paralogs, both due to the teleost-specific genome duplication event (blue circle). Proteins are drawn to scale. Species phylogenetic relationships are based [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179545#pone.0179545.ref035" target="_blank">35</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179545#pone.0179545.ref038" target="_blank">38</a>].</p