Background: Elucidating the process of speciation requires an in-depth understanding of the evolutionary history of the species in question. Studies that rely upon a limited number of genetic loci do not always reveal actual evolutionary history, and often confuse inferences related to phylogeny and speciation. Whole-genome data, however, can overcome this issue by providing a nearly unbiased window into the patterns and processes of speciation. In order to reveal the complexity of the speciation process, we sequenced and analyzed the genomes of 10 wild pigs, representing morphologically or geographically well-defined species and subspecies of the genus Sus from insular and mainland Southeast Asia, and one African common warthog. Results: Our data highlight the importance of past cyclical climatic fluctuations in facilitating the dispersal and isolation of populations, thus leading to the diversification of suids in one of the most species-rich regions of the world. Moreover, admixture analyses revealed extensive, intra- and inter-specific gene-flow that explains previous conflicting results obtained from a limited number of loci. We show that these multiple episodes of gene-flow resulted from both natural and human-mediated dispersal. Conclusions: Our results demonstrate the importance of past climatic fluctuations and human mediated translocations in driving and complicating the process of speciation in island Southeast Asia. This case study demonstrates that genomics is a powerful tool to decipher the evolutionary history of a genus, and reveals the complexity of the process of speciation.

Copy number variable regions (CNVRs) can result in drastic phenotypic differences and may therefore be subject to selection during domestication. Studying copy number variation in relation to domestication is highly relevant in pigs because of their very rich natural and domestication history that resulted in many different phenotypes. To investigate the evolutionary dynamic of CNVRs, we applied read depth method on next generation sequence data from 16 individuals, comprising wild boars and domestic pigs from Europe and Asia. We identified 3,118 CNVRs with an average size of 13 kilobases comprising a total of 39.2 megabases of the pig genome and 545 overlapping genes. Functional analyses revealed that CNVRs are enriched with genes related to sensory perception, neurological process and response to stimulus, suggesting their contribution to adaptation in the wild and behavioral changes during domestication. Variations of copy number (CN) of antimicrobial related genes suggest an ongoing process of evolution of these genes to combat food-borne pathogens. Likewise, some genes related to the omnivorous lifestyle of pigs, like genes involved in detoxification, were observed to be CN variable. A small portion of CNVRs was unique to domestic pigs and may have been selected during domestication. The majority of CNVRs, however, is shared between wild and domesticated individuals, indicating that domestication had minor effect on the overall diversity of CNVRs. Also, the excess of CNVRs in non-genic regions implies that a major part of these variations is likely to be (nearly) neutral. Comparison between different populations showed that larger populations have more CNVRs, highlighting that CNVRs are, like other genetic variation such as SNPs and microsatellites, reflecting demographic history rather than phenotypic diversity. CNVRs in pigs are enriched for genes related to sensory perception, neurological process, and response to stimulus. The majority of CNVRs ascertained in domestic pigs are also variable in wild boars, suggesting that the domestication of the pig did not result in a change in CNVRs in domesticated pigs. The majority of variable regions were found to reflect demographic patterns rather than phenotypic.

Unraveling the genetic mechanisms associated with reduced gene flow between genetically differentiated populations is key to understand speciation. Different types of structural variations (SVs) have been found as a source of genetic diversity in a wide range of species. Previous studies provided detailed knowledge on the potential evolutionary role of SVs, especially copy number variations (CNVs), between well diverged species of e.g. primates. However, our understanding of their significance during ongoing speciation processes is limited due to the lack of CNV data from closely related species. The genus Sus (pig and its close relatives) which started to diverge ~4 Mya presents an excellent model for studying the role of CNVs during ongoing speciation. In this study, we identified 1408 CNV regions (CNVRs) across the genus Sus. These CNVRs encompass 624 genes and were found to evolve ~2.5 times faster than single nucleotide polymorphisms (SNPs). The majority of these copy number variable genes are olfactory receptors (ORs) known to play a prominent role in food foraging and mate recognition in Sus. Phylogenetic analyses, including novel Bayesian analysis, based on CNVRs that overlap ORs retain the well-accepted topology of the genus Sus whereas CNVRs overlapping genes other than ORs show evidence for random drift and/or admixture. We hypothesize that inter-specific variation in copy number of ORs provided the means for rapid adaptation to different environments during the diversification of the genus Sus in the Pliocene. Furthermore, these regions might have acted as barriers preventing massive gene flow between these species during the multiple hybridization events that took place later in the Pleistocene suggesting a possible prominent role of ORs in the ongoing Sus speciation.

Pigs (Sus scrofa) provide relevant biomedical models to dissect complex diseases due to their anatomical, genetic, and physiological similarities with humans. Aberrant DNA methylation has been linked to many of these diseases and is associated with gene expression; however, the functional similarities and differences between porcine and human DNA methylation patterns are largely unknown. DNA and RNA was isolated from eight tissue samples (fat, heart, kidney, liver, lung, lymph node, muscle, and spleen) from the adult female Duroc utilized for the pig genome sequencing project. Reduced representation bisulfite sequencing (RRBS) and RNA-seq were performed on an Illumina HiSeq2000. RRBS reads were aligned using BSseeker2, and only sites with a minimum depth of 10 reads were used for methylation analysis. RNA-seq reads were aligned using Tophat, and expression analysis was performed using Cufflinks. In addition, SNP calling was performed using GATK for targeted control and whole genome sequencing reads for CpG site validation and allelic expression analysis, respectively. Analysis on the influence of DNA variation in methylation calling revealed a reduced effectiveness of WGS datasets in covering CpG rich regions, as well as the usefulness of a targeted control library for SNP detection. Analysis of over 500,000 CpG sites demonstrated genome wide methylation patterns similar to those observed in humans, including reduced methylation within CpG islands and at transcription start sites (TSS), X chromosome inactivation, and anticorrelation of TSS CpG methylation with gene expression. In addition, a positive correlation between TSS CpG density and expression, and a negative correlation between TSS TpG density and expression were demonstrated. Low but non-random non-CpG methylation (<1%) was also detected in all non-neuronal somatic tissues, with differences in tissue clustering observed based on CpG and non-CpG methylation patterns. Finally, allele specific expression analysis revealed enrichment of genes involved in metabolic and regulatory processes. These results provide transcriptional and DNA methylation datasets for the biomedical community that are directly relatable to current genomic resources. In addition, the correlation between TSS CpG density and expression suggests increased mutation rates at CpG sites play a significant role in adaptive evolution by reducing CpG density at TSS over time, resulting in higher methylation levels in these regions and more permanent changes to lower gene expression. This is proposed to occur predominantly through deamination of 5-methylcytosine to thymidine, resulting in the replacement of CpG with TpG sites in these regions, as indicated by the increased TSS TpG density observed in non-expressed genes, resulting in a negative correlation between expression and TSS TpG density. This study provides baseline methylation and gene transcription profiles for a healthy adult pig, reports similar patterns to those observed in humans, and supports future porcine studies related to human disease and development. Additionally, the observed reduced CpG and increased TpG density at TSS of lowly expressed genes suggests DNA methylation plays a significant role in adaptive evolution through more permanent changes to lower gene expression.

Reproductive traits such as number of stillborn piglets (SB) and number of teats (NT) have been evaluated in many genome-wide association studies (GWAS). Most of these GWAS were performed under the assumption that these traits were normally distributed. However, both SB and NT are discrete (e.g. count) variables. Therefore, it is necessary to test for better fit of other appropriate statistical models based on discrete distributions. In addition, although many GWAS have been performed, the biological meaning of the identified candidate genes, as well as their functional relationships still need to be better understood. Here, we performed and tested a Bayesian treatment of a GWAS model assuming a Poisson distribution for SB and NT in a commercial pig line. To explore the biological role of the genes that underlie SB and NT and identify the most likely candidate genes, we used the most significant single nucleotide polymorphisms (SNPs), to collect related genes and generated gene-transcription factor (TF) networks. Comparisons of the Poisson and Gaussian distributions showed that the Poisson model was appropriate for SB, while the Gaussian was appropriate for NT. The fitted GWAS models indicated 18 and 65 significant SNPs with one and nine quantitative trait locus (QTL) regions within which 18 and 57 related genes were identified for SB and NT, respectively. Based on the related TF, we selected the most representative TF for each trait and constructed a gene-TF network of gene-gene interactions and identified new candidate genes. Our comparative analyses showed that the Poisson model presented the best fit for SB. Thus, to increase the accuracy of GWAS, counting models should be considered for this kind of trait. We identified multiple candidate genes (e.g. PTP4A2, NPHP1, and CYP24A1 for SB and YLPM1, SYNDIG1L, TGFB3, and VRTN for NT) and TF (e.g. NF-κB and KLF4 for SB and SOX9 and ELF5 for NT), which were consistent with known newborn survival traits (e.g. congenital heart disease in fetuses and kidney diseases and diabetes in the mother) and mammary gland biology (e.g. mammary gland development and body length).

Malagasy tenrecs belong to the Afrotherian clade of placental mammals and comprise three subfamilies divided in eight genera (Tenrecinae: Tenrec, Echinops, Setifer and Hemicentetes; Oryzorictinae: Oryzorictes, Limnogale and Microgale; Geogalinae:Geogale). The diversity of their morphology and incomplete taxon sampling made it difficult until now to resolve phylogenies based on either morphology or molecular data for this group. Therefore, in order to delineate the evolutionary history of this family, phylogenetic and dating analyses were performed on a four nuclear genes dataset (ADRA2B, AR, GHR and vWF) including all Malagasy tenrec genera. Moreover, the influence of both taxon sampling and data partitioning on the accuracy of the estimated ages were assessed.