José Luis Villanueva-Cañas

Extreme genomic erosion after recurrent demographic bottlenecks in the highly endangered Iberian lynx

BackgroundGenomic studies of endangered species provide insights into their evolution and demographic history, reveal patterns of genomic erosion that might limit their viability, and offer tools for their effective conservation. The Iberian lynx (Lynx pardinus) is the most endangered felid and a unique example of a species on the brink of extinction.ResultsWe generate the first annotated draft of the Iberian lynx genome and carry out genome-based analyses of lynx demography, evolution, and population genetics. We identify a series of severe population bottlenecks in the history of the Iberian lynx that predate its known demographic decline during the 20th century and have greatly impacted its genome evolution. We observe drastically reduced rates of weak-to-strong substitutions associated with GC-biased gene conversion and increased rates of fixation of transposable elements. We also find multiple signatures of genetic erosion in the two remnant Iberian lynx populations, including a high frequency of potentially deleterious variants and substitutions, as well as the lowest genome-wide genetic diversity reported so far in any species.ConclusionsThe genomic features observed in the Iberian lynx genome may hamper short- and long-term viability through reduced fitness and adaptive potential. The knowledge and resources developed in this study will boost the research on felid evolution and conservation genomics and will benefit the ongoing conservation and management of this emblematic species.

Improving Genome-Wide Scans of Positive Selection by Using Protein Isoforms of Similar Length

Large-scale evolutionary studies often require the automated construction of alignments of a large number of homologous gene families. The majority of eukaryotic genes can produce different transcripts due to alternative splicing or transcription initiation, and many such transcripts encode different protein isoforms. As analyses tend to be gene centered, one single-protein isoform per gene is selected for the alignment, with the de facto approach being to use the longest protein isoform per gene (Longest), presumably to avoid including partial sequences and to maximize sequence information. Here, we show that this approach is problematic because it increases the number of indels in the alignments due to the inclusion of nonhomologous regions, such as those derived from species-specific exons, increasing the number of misaligned positions. With the aim of ameliorating this problem, we have developed a novel heuristic, Protein ALignment Optimizer (PALO), which, for each gene family, selects the combination of protein isoforms that are most similar in length. We examine several evolutionary parameters inferred from alignments in which the only difference is the method used to select the protein isoform combination: Longest, PALO, the combination that results in the highest sequence conservation, and a randomly selected combination. We observe that Longest tends to overestimate both nonsynonymous and synonymous substitution rates when compared with PALO, which is most likely due to an excess of misaligned positions. The estimation of the fraction of genes that have experienced positive selection by maximum likelihood is very sensitive to the method of isoform selection employed, both when alignments are constructed with MAFFT and with Prank+F. Longest performs better than a random combination but still estimates up to 3 times more positively selected genes than the combination showing the highest conservation, indicating the presence of many false positives. We show that PALO can eliminate the majority of such false positives and thus that it is a more appropriate approach for large-scale analyses than Longest. A web server has been set up to facilitate the use of PALO given a user-defined set of gene families; it is available at http://evolutionarygenomics.imim.es/palo.

Comparative Genomics of Mammalian Hibernators Using Gene Networks

In recent years, the study of the molecular processes involved in mammalian hibernation has shifted from investigating a few carefully selected candidate genes to large-scale analysis of differential gene expression. The availability of high-throughput data provides an unprecedented opportunity to ask whether phylogenetically distant species show similar mechanisms of genetic control, and how these relate to particular genes and pathways involved in the hibernation phenotype. In order to address these questions, we compare 11 datasets of differentially expressed (DE) genes from two ground squirrel species, one bat species, and the American black bear, as well as a list of genes extracted from the literature that previously have been correlated with the drastic physiological changes associated with hibernation. We identify several genes that are DE in different species, indicating either ancestral adaptations or evolutionary convergence. When we use a network approach to expand the original datasets of DE genes to large gene networks using available interactome data, a higher agreement between datasets is achieved. This indicates that the same key pathways are important for activating and maintaining the hibernation phenotype. Functional-term-enrichment analysis identifies several important metabolic and mitochondrial processes that are critical for hibernation, such as fatty acid beta-oxidation and mitochondrial transport. We do not detect any enrichment of positive selection signatures in the coding sequences of genes from the networks of hibernation-associated genes, supporting the hypothesis that the genetic processes shaping the hibernation phenotype are driven primarily by changes in gene regulation.

Beyond SNPs: how to detect selection on transposable element insertions

Summary Identifying the genomic basis of adaptive evolution is a growing field of research. The number of statistics and methodologies aimed at identifying adaptive loci continues to increase. Moreover, the availability of whole-genome sequences allows us to make inferences of selection on a diverse set of species. However, detecting footprints of selection has mostly been restricted to one type of genomic variation: single-nucleotide polymorphisms (SNPs). Other genomic variants such as transposable element (TE) insertions that are likely to contribute to adaptive evolution have been largely ignored. Here, we present an overview of different approaches that can be used to infer selection acting on TE insertions. We focused on five main approaches: (i) DNA sequence conservation analysis; (ii) selection on linked polymorphisms; (iii) environmental association analyses; (iv) estimation of allele age; and (v) functional assays to identify the molecular and fitness effects. For each of these five approaches, we focus on the latest developments and illustrate them with recent examples from the literature. We also identify the data requirements and the limitations associated with the different methodologies. We conclude that the availability of third-generation sequencing technologies should allow for a systematic analysis of TE insertions as sources of adaptive mutations. Incorporating the knowledge of the role of TE insertions in adaptive evolution will allow us to get a more complete picture of the adaptive process.

New Genes and Functional Innovation in Mammals

Abstract The birth of genes that encode new protein sequences is a major source of evolutionary innovation. However, we still understand relatively little about how these genes come into being and which functions they are selected for. To address these questions, we have obtained a large collection of mammalian-specific gene families that lack homologues in other eukaryotic groups. We have combined gene annotations and de novo transcript assemblies from 30 different mammalian species, obtaining ∼6,000 gene families. In general, the proteins in mammalian-specific gene families tend to be short and depleted in aromatic and negatively charged residues. Proteins which arose early in mammalian evolution include milk and skin polypeptides, immune response components, and proteins involved in reproduction. In contrast, the functions of proteins which have a more recent origin remain largely unknown, despite the fact that these proteins also have extensive proteomics support. We identify several previously described cases of genes originated de novo from noncoding genomic regions, supporting the idea that this mechanism frequently underlies the evolution of new protein-coding genes in mammals. Finally, we show that most young mammalian genes are preferentially expressed in testis, suggesting that sexual selection plays an important role in the emergence of new functional genes.

Gene Expression Profiling in the Hibernating Primate, Cheirogaleus Medius

Hibernation is a complex physiological response that some mammalian species employ to evade energetic demands. Previous work in mammalian hibernators suggests that hibernation is activated not by a set of genes unique to hibernators, but by differential expression of genes that are present in all mammals. This question of universal genetic mechanisms requires further investigation and can only be tested through additional investigations of phylogenetically dispersed species. To explore this question, we use RNA-Seq to investigate gene expression dynamics as they relate to the varying physiological states experienced throughout the year in a group of primate hibernators—Madagascar’s dwarf lemurs (genus Cheirogaleus). In a novel experimental approach, we use longitudinal sampling of biological tissues as a method for capturing gene expression profiles from the same individuals throughout their annual hibernation cycle. We identify 90 candidate genes that have variable expression patterns when comparing two active states (Active 1 and Active 2) with a torpor state. These include genes that are involved in metabolic pathways, feeding behavior, and circadian rhythms, as might be expected to correlate with seasonal physiological state changes. The identified genes appear to be critical for maintaining the health of an animal that undergoes prolonged periods of metabolic depression concurrent with the hibernation phenotype. By focusing on these differentially expressed genes in dwarf lemurs, we compare gene expression patterns in previously studied mammalian hibernators. Additionally, by employing evolutionary rate analysis, we find that hibernation-related genes do not evolve under positive selection in hibernating species relative to nonhibernators.

Translation of neutrally evolving peptides provides a basis for de novo gene evolution

Accumulating evidence indicates that some protein-coding genes have originated de novo from previously non-coding genomic sequences. However, the processes underlying de novo gene birth are still enigmatic. In particular, the appearance of a new functional protein seems highly improbable unless there is already a pool of neutrally evolving peptides that are translated at significant levels and that can at some point acquire new functions. Here, we use deep ribosome-profiling sequencing data, together with proteomics and single nucleotide polymorphism information, to search for these peptides. We find hundreds of open reading frames that are translated and that show no evolutionary conservation or selective constraints. These data suggest that the translation of these neutrally evolving peptides may be facilitated by the chance occurrence of open reading frames with a favourable codon composition. We conclude that the pervasive translation of the transcriptome provides plenty of material for the evolution of new functional proteins.Genes can originate de novo from random non-coding DNA. Here, the authors identify a pool of young proteins in mice that are translated at significant levels and evolved in a neutral manner, and are putative precursors of de novo genes.

Transcriptomics in the wild: Hibernation physiology in free‐ranging dwarf lemurs

Hibernation is an adaptive strategy some mammals use to survive highly seasonal or unpredictable environments. We present the first investigation on the transcriptomics of hibernation in a natural population of primate hibernators: Crossleys dwarf lemurs (Cheirogaleus crossleyi). Using capture–mark–recapture techniques to track the same animals over a period of 7 months in Madagascar, we used RNA‐seq to compare gene expression profiles in white adipose tissue (WAT) during three distinct physiological states. We focus on pathway analysis to assess the biological significance of transcriptional changes in dwarf lemur WAT and, by comparing and contrasting what is known in other model hibernating species, contribute to a broader understanding of genomic contributions of hibernation across Mammalia. The hibernation signature is characterized by a suppression of lipid biosynthesis, pyruvate metabolism and mitochondrial‐associated functions, and an accumulation of transcripts encoding ribosomal components and iron‐storage proteins. The data support a key role of pyruvate dehydrogenase kinase isoenzyme 4 (PDK4) in regulating the shift in fuel economy during periods of severe food deprivation. This pattern of PDK4 holds true across representative hibernating species from disparate mammalian groups, suggesting that the genetic underpinnings of hibernation may be ancestral to mammals.

Functional and non-functional classes of peptides produced by long non-coding RNAs.

Cells express thousands of transcripts that show weak coding potential. Known as long non-coding RNAs (IncRNAs), they typically contain short open reading frames (ORFs) having no homology with known proteins. Recent studies have reported that a significant proportion of IncRNAs are translated, challenging the view that they are non-coding. These results are based on the selective sequencing of ribosome-protected fragments, or ribosome profiling. The present study uses ribosome profiling data from eight mouse tissues and cell types, combined with ~330,000 synonymous and non-synonymous single nucleotide variants, to dissect the patterns of purifying selection in proteins translated from IncRNAs. Using the three-nucleotide read periodicity that characterizes actively translated regions, we identify about 1,365 translated IncRNAs. About one fourth of them (350 IncRNAs) show conservation in humans; this is likely to produce functional micropeptides, including the recently discovered myoregulin. For other IncRNAs, the ORF codon usage bias distinguishes between two classes. The first has significant coding scores and evidence of purifying selection, consistent with the presence of lineage-specific functional proteins. The second large class, comprising >500 IncRNAs, produces proteins that show no significant purifying selection signatures. We show that the translation of these IncRNAs depends on the transcript expression level and the chance occurrence of ORFs with a favorable codon composition. Some of these IncRNAs may be precursors of novel protein-coding genes, filling a gap in our current understanding of de novo gene birth.