Miguel Carneiro

Rabbit genome analysis reveals a polygenic basis for phenotypic change during domestication

Rabbits softly swept to domestication When people domesticate animals, they select for tameness and tolerance of humans. What else do they look for? To identify the selective pressures that led to rabbit domestication, Carneiro et al. sequenced a domestic rabbit genome and compared it to that of its wild brethren (see the Perspective by Lohmueller). Domestication did not involve a single gene changing, but rather many gene alleles changing in frequency between tame and domestic rabbits, known as a soft selective sweep. Many of these alleles have changes that may affect brain development, supporting the idea that tameness involves changes at multiple loci. Science, this issue p. 1074; see also p. 1000 The domestication of rabbits primarily shifted the frequencies of alleles represented, rather than creating new genes. [Also see Perspective by Lohmueller] The genetic changes underlying the initial steps of animal domestication are still poorly understood. We generated a high-quality reference genome for the rabbit and compared it to resequencing data from populations of wild and domestic rabbits. We identified more than 100 selective sweeps specific to domestic rabbits but only a relatively small number of fixed (or nearly fixed) single-nucleotide polymorphisms (SNPs) for derived alleles. SNPs with marked allele frequency differences between wild and domestic rabbits were enriched for conserved noncoding sites. Enrichment analyses suggest that genes affecting brain and neuronal development have often been targeted during domestication. We propose that because of a truly complex genetic background, tame behavior in rabbits and other domestic animals evolved by shifts in allele frequencies at many loci, rather than by critical changes at only a few domestication loci.

Reference-Free Population Genomics from Next-Generation Transcriptome Data and the Vertebrate–Invertebrate Gap

In animals, the population genomic literature is dominated by two taxa, namely mammals and drosophilids, in which fully sequenced, well-annotated genomes have been available for years. Data from other metazoan phyla are scarce, probably because the vast majority of living species still lack a closely related reference genome. Here we achieve de novo, reference-free population genomic analysis from wild samples in five non-model animal species, based on next-generation sequencing transcriptome data. We introduce a pipe-line for cDNA assembly, read mapping, SNP/genotype calling, and data cleaning, with specific focus on the issue of hidden paralogy detection. In two species for which a reference genome is available, similar results were obtained whether the reference was used or not, demonstrating the robustness of our de novo inferences. The population genomic profile of a hare, a turtle, an oyster, a tunicate, and a termite were found to be intermediate between those of human and Drosophila, indicating that the discordant genomic diversity patterns that have been reported between these two species do not reflect a generalized vertebrate versus invertebrate gap. The genomic average diversity was generally higher in invertebrates than in vertebrates (with the notable exception of termite), in agreement with the notion that population size tends to be larger in the former than in the latter. The non-synonymous to synonymous ratio, however, did not differ significantly between vertebrates and invertebrates, even though it was negatively correlated with genetic diversity within each of the two groups. This study opens promising perspective regarding genome-wide population analyses of non-model organisms and the influence of population size on non-synonymous versus synonymous diversity.

Recombination and Speciation: Loci Near Centromeres Are More Differentiated Than Loci Near Telomeres Between Subspecies of the European Rabbit (Oryctolagus cuniculus)

Recent empirical and theoretical studies suggest that regions of restricted recombination play an important role in the formation of new species. To test this idea, we studied nucleotide variation in two parapatric subspecies of the European rabbit (Oryctolagus cuniculus). We surveyed five loci near centromeres, where recombination is expected to be suppressed, and five loci near telomeres, where recombination is expected to be higher. We analyzed this multilocus data set using a divergence-with-gene flow framework and we report three main findings. First, we estimated that these subspecies diverged ∼1.8 MYA and maintained large effective population sizes (O. c. algirus Ne ≈ 1,600,000 and O. c. cuniculus Ne ≈ 780,000). Second, we rejected a strict allopatric model of divergence without gene flow; instead, high rates of gene flow were inferred in both directions. Third, we found different patterns between loci near centromeres and loci near telomeres. Loci near centromeres exhibited higher levels of linkage disequilibrium than loci near telomeres. In addition, while all loci near telomeres showed little differentiation between subspecies, three of five loci near centromeres showed strong differentiation. These results support a view of speciation in which regions of low recombination can facilitate species divergence in the presence of gene flow.

Recurrent Introgression of Mitochondrial DNA Among Hares (Lepus spp.) Revealed by Species-Tree Inference and Coalescent Simulations

Understanding recent speciation history requires merging phylogenetic and population genetics approaches, taking into account the persistence of ancestral polymorphism and possible introgression. The emergence of a clear phylogeny of hares (genus Lepus) has been hampered by poor genomic sampling and possible occurrence of mitochondrial DNA (mtDNA) introgression from the arctic/boreal Lepus timidus into several European temperate and possibly American boreal species. However, no formal test of introgression, taking also incomplete lineage sorting into account, has been done. Here, to clarify the yet poorly resolved species phylogeny of hares and test hypotheses of mtDNA introgression, we sequenced 14 nuclear DNA and 2 mtDNA fragments (8205 and 1113 bp, respectively) in 50 specimens from 11 hare species from Eurasia, North America, and Africa. By applying an isolation-with-migration model to the nuclear data on subsets of species, we find evidence for very limited gene flow from L. timidus into most temperate European species, and not into the American boreal ones. Using a multilocus coalescent-based method, we infer the species phylogeny, which we find highly incongruent with mtDNA phylogeny using parametric bootstrap. Simulations of mtDNA evolution under the speciation history inferred from nuclear genes did not support the hypothesis of mtDNA introgression from L. timidus into the American L. townsendii but did suggest introgression from L. timidus into 4 temperate European species. One such event likely resulted in the complete replacement of the aboriginal mtDNA of L. castroviejoi and of its sister species L. corsicanus. It is remarkable that mtDNA introgression in hares is frequent, extensive, and always from the same donor arctic species. We discuss possible explanations for the phenomenon in relation to the dynamics of range expansions and species replacements during the climatic oscillations of the Pleistocene.

A Comparison of Brain Gene Expression Levels in Domesticated and Wild Animals

Domestication has led to similar changes in morphology and behavior in several animal species, raising the question whether similarities between different domestication events also exist at the molecular level. We used mRNA sequencing to analyze genome-wide gene expression patterns in brain frontal cortex in three pairs of domesticated and wild species (dogs and wolves, pigs and wild boars, and domesticated and wild rabbits). We compared the expression differences with those between domesticated guinea pigs and a distant wild relative (Cavia aperea) as well as between two lines of rats selected for tameness or aggression towards humans. There were few gene expression differences between domesticated and wild dogs, pigs, and rabbits (30–75 genes (less than 1%) of expressed genes were differentially expressed), while guinea pigs and C. aperea differed more strongly. Almost no overlap was found between the genes with differential expression in the different domestication events. In addition, joint analyses of all domesticated and wild samples provided only suggestive evidence for the existence of a small group of genes that changed their expression in a similar fashion in different domesticated species. The most extreme of these shared expression changes include up-regulation in domesticates of SOX6 and PROM1, two modulators of brain development. There was almost no overlap between gene expression in domesticated animals and the tame and aggressive rats. However, two of the genes with the strongest expression differences between the rats (DLL3 and DHDH) were located in a genomic region associated with tameness and aggression, suggesting a role in influencing tameness. In summary, the majority of brain gene expression changes in domesticated animals are specific to the given domestication event, suggesting that the causative variants of behavioral domestication traits may likewise be different.

A ranking system for reference libraries of DNA barcodes: application to marine fish species from Portugal.

Background The increasing availability of reference libraries of DNA barcodes (RLDB) offers the opportunity to the screen the level of consistency in DNA barcode data among libraries, in order to detect possible disagreements generated from taxonomic uncertainty or operational shortcomings. We propose a ranking system to attribute a confidence level to species identifications associated with DNA barcode records from a RLDB. Here we apply the proposed ranking system to a newly generated RLDB for marine fish of Portugal. Methodology/Principal Findings Specimens (n = 659) representing 102 marine fish species were collected along the continental shelf of Portugal, morphologically identified and archived in a museum collection. Samples were sequenced at the barcode region of the cytochrome oxidase subunit I gene (COI-5P). Resultant DNA barcodes had average intra-specific and inter-specific Kimura-2-parameter distances (0.32% and 8.84%, respectively) within the range usually observed for marine fishes. All specimens were ranked in five different levels (A–E), according to the reliability of the match between their species identification and the respective diagnostic DNA barcodes. Grades A to E were attributed upon submission of individual specimen sequences to BOLD-IDS and inspection of the clustering pattern in the NJ tree generated. Overall, our study resulted in 73.5% of unambiguous species IDs (grade A), 7.8% taxonomically congruent barcode clusters within our dataset, but awaiting external confirmation (grade B), and 18.7% of species identifications with lower levels of reliability (grades C/E). Conclusion/Significance We highlight the importance of implementing a system to rank barcode records in RLDB, in order to flag taxa in need of taxonomic revision, or reduce ambiguities of discordant data. With increasing DNA barcode records publicly available, this cross-validation system would provide a metric of relative accuracy of barcodes, while enabling the continuous revision and annotation required in taxonomic work.

The genomic architecture of population divergence between subspecies of the european rabbit

The analysis of introgression of genomic regions between divergent populations provides an excellent opportunity to determine the genetic basis of reproductive isolation during the early stages of speciation. However, hybridization and subsequent gene flow must be relatively common in order to localize individual loci that resist introgression. In this study, we used next-generation sequencing to study genome-wide patterns of genetic differentiation between two hybridizing subspecies of rabbits (Oryctolagus cuniculus algirus and O. c. cuniculus) that are known to undergo high rates of gene exchange. Our primary objective was to identify specific genes or genomic regions that have resisted introgression and are likely to confer reproductive barriers in natural conditions. On the basis of 326,000 polymorphisms, we found low to moderate overall levels of differentiation between subspecies, and fewer than 200 genomic regions dispersed throughout the genome showing high differentiation consistent with a signature of reduced gene flow. Most differentiated regions were smaller than 200 Kb and contained very few genes. Remarkably, 30 regions were each found to contain a single gene, facilitating the identification of candidate genes underlying reproductive isolation. This gene-level resolution yielded several insights into the genetic basis and architecture of reproductive isolation in rabbits. Regions of high differentiation were enriched on the X-chromosome and near centromeres. Genes lying within differentiated regions were often associated with transcription and epigenetic activities, including chromatin organization, regulation of transcription, and DNA binding. Overall, our results from a naturally hybridizing system share important commonalities with hybrid incompatibility genes identified using laboratory crosses in mice and flies, highlighting general mechanisms underlying the maintenance of reproductive barriers.

Evidence for widespread positive and purifying selection across the European rabbit (Oryctolagus cuniculus) genome

The nearly neutral theory of molecular evolution predicts that the efficacy of both positive and purifying selection is a function of the long-term effective population size (N(e)) of a species. Under this theory, the efficacy of natural selection should increase with N(e). Here, we tested this simple prediction by surveying ~1.5 to 1.8 Mb of protein coding sequence in the two subspecies of the European rabbit (Oryctolagus cuniculus algirus and O. c. cuniculus), a mammal species characterized by high levels of nucleotide diversity and N(e) estimates for each subspecies on the order of 1 × 10(6). When the segregation of slightly deleterious mutations and demographic effects were taken into account, we inferred that >60% of amino acid substitutions on the autosomes were driven to fixation by positive selection. Moreover, we inferred that a small fraction of new amino acid mutations (<4%) are effectively neutral (defined as 0 < N(e)s < 1) and that this fraction was negatively correlated with a genes expression level. Consistent with models of recurrent adaptive evolution, we detected a negative correlation between levels of synonymous site polymorphism and the rate of protein evolution, although the correlation was weak and nonsignificant. No systematic X chromosome-autosome difference was found in the efficacy of selection. For example, the proportion of adaptive substitutions was significantly higher on the X chromosome compared with the autosomes in O. c. algirus but not in O. c. cuniculus. Our findings support widespread positive and purifying selection in rabbits and add to a growing list of examples suggesting that differences in N(e) among taxa play a substantial role in determining rates and patterns of protein evolution.

Identifying Loci Under Selection Against Gene Flow in Isolation-with-Migration Models

When divergence occurs in the presence of gene flow, there can arise an interesting dynamic in which selection against gene flow, at sites associated with population-specific adaptations or genetic incompatibilities, can cause net gene flow to vary across the genome. Loci linked to sites under selection may experience reduced gene flow and may experience genetic bottlenecks by the action of nearby selective sweeps. Data from histories such as these may be poorly fitted by conventional neutral model approaches to demographic inference, which treat all loci as equally subject to forces of genetic drift and gene flow. To allow for demographic inference in the face of such histories, as well as the identification of loci affected by selection, we developed an isolation-with-migration model that explicitly provides for variation among genomic regions in migration rates and/or rates of genetic drift. The method allows for loci to fall into any of multiple groups, each characterized by a different set of parameters, thus relaxing the assumption that all loci share the same demography. By grouping loci, the method can be applied to data with multiple loci and still have tractable dimensionality and statistical power. We studied the performance of the method using simulated data, and we applied the method to study the divergence of two subspecies of European rabbits (Oryctolagus cuniculus).

Diversity and evolutionary history of the MHC DQA gene in leporids

The European rabbit (Oryctolagus cuniculus) is used as a model for many human diseases, yet comparatively little is known of its genetics, particularly at important loci such as the major histocompatibility complex (MHC). This study investigated genetic diversity and evolutionary history of the DQA gene in a range of leporid species by analysing coding sequence diversity of exon 2 and intron 2 in 53 individuals of 16 different species. Fifty leporid DQA alleles were detected, including 13 novel European rabbit alleles. In the rabbit, the highest levels of diversity were observed in wild rabbits from Portugal, with wild rabbits from England and domestic rabbits showing less diversity. Within the sample, several recombination events were detected and trans-specific evolution of alleles was evidenced, both being general characteristics of mammalian MHC genes. Positive selection is implicated as operating on six codons within exon 2, which are also subject to positive selection in other mammals. Some of these positions are putative antigen recognition sites and underline the importance of pathogen-driven selection on these MHC genes.