Changes in brain architecture are consistent with altered fear processing in domestic rabbits.

The most characteristic feature of domestic animals is their change in behavior associated with selection for tameness. Here we show, using high-resolution brain magnetic resonance imaging in wild and domestic rabbits, that domestication reduced amygdala volume and enlarged medial prefrontal cortex volume, supporting that areas driving fear have lost volume while areas modulating negative affect have gained volume during domestication. In contrast to the localized gray matter alterations, white matter anisotropy was reduced in the corona radiata, corpus callosum, and the subcortical white matter. This suggests a compromised white matter structural integrity in projection and association fibers affecting both afferent and efferent neural flow, consistent with reduced neural processing. We propose that compared with their wild ancestors, domestic rabbits are less fearful and have an attenuated flight response because of these changes in brain architecture.

Endemic plant species are more palatable to introduced herbivores than non-endemics.

Islands harbour a spectacular diversity and unique species composition. This uniqueness is mainly a result of endemic species that have evolved in situ in the absence of mammal herbivores. However, island endemism is under severe threat by introduced herbivores. We test the assumption that endemic species are particularly vulnerable to generalist introduced herbivores (European rabbit) using an unprecedented dataset covering an entire island with enormous topographic, climatic and biological diversity (Tenerife, Canary Islands). With increasing endemism, plant species are more heavily browsed by rabbits than non-endemic species with up to 67% of endemics being negatively impacted by browsing, indicating a dramatic lack of adaptation to mammal herbivory in endemics. Ecosystems with high per cent endemism are most heavily browsed, suggesting ecosystem-specific vulnerability to introduced herbivores, even within islands. Protection of global biodiversity caused by disproportionally high endemism on oceanic islands via ecosystem-specific herbivore control and eradication measures is of utmost importance.

A genomic map of clinal variation across the European rabbit hybrid zone.

Speciation is a process proceeding from weak to complete reproductive isolation. In this continuum, naturally hybridizing taxa provide a promising avenue for revealing the genetic changes associated with the incipient stages of speciation. To identify such changes between two subspecies of rabbits that display partial reproductive isolation, we studied patterns of allele frequency change across their hybrid zone using whole-genome sequencing. To connect levels and patterns of genetic differentiation with phenotypic manifestations of subfertility in hybrid rabbits, we further investigated patterns of gene expression in testis. Geographic cline analysis revealed 253 regions characterized by steep changes in allele frequency across their natural region of contact. This catalog of regions is likely to be enriched for loci implicated in reproductive barriers and yielded several insights into the evolution of hybrid dysfunction in rabbits: (i) incomplete reproductive isolation is likely governed by the effects of many loci, (ii) protein-protein interaction analysis suggest that genes within these loci interact more than expected by chance, (iii) regulatory variation is likely the primary driver of incompatibilities, and (iv) large chromosomal rearrangements appear not to be a major mechanism underlying incompatibilities or promoting isolation in the face of gene flow. We detected extensive misregulation of gene expression in testis of hybrid males, but not a statistical overrepresentation of differentially expressed genes in candidate regions. Our results also did not support an X chromosome-wide disruption of expression as observed in mice and cats, suggesting variation in the mechanistic basis of hybrid male reduced fertility among mammals.

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.

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.

Selection against domestication alleles in introduced rabbit populations.

Humans have moved domestic animals around the globe for thousands of years. These have occasionally established feral populations in nature, often with devastating ecological consequences. To understand how natural selection shapes re-adaptation into the wild, we investigated one of the most successful colonizers in history, the European rabbit. By sequencing the genomes of 297 rabbits across three continents, we show that introduced populations exhibit a mixed wild-domestic ancestry. We show that alleles that increased in frequency during domestication were preferentially selected against in novel natural environments. Interestingly, causative mutations for common domestication traits sometimes segregate at considerable frequencies if associated with less drastic phenotypes (for example, coat colour dilution), whereas mutations that are probably strongly maladaptive in nature are absent. Whereas natural selection largely targeted different genomic regions in each introduced population, some of the strongest signals of parallelism overlap genes associated with neuronal or brain function. This limited parallelism is probably explained by extensive standing genetic variation resulting from domestication together with the complex mixed ancestry of introduced populations. Our findings shed light on the selective and molecular mechanisms that enable domestic animals to re-adapt to the wild and provide important insights for the mitigation and management of invasive populations.

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 gene's 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.

Steep clines within a highly permeable genome across a hybrid zone between two subspecies of the European rabbit.

Maintenance of genetic distinction in the face of gene flow is an important aspect of the speciation process. Here, we provide a detailed spatial and genetic characterization of a hybrid zone between two subspecies of the European rabbit. We examined patterns of allele frequency change for 22 markers located on the autosomes, X-chromosome, Y-chromosome and mtDNA in 1078 individuals sampled across the hybrid zone. While some loci revealed extremely wide clines (w ≥ 300 km) relative to an estimated dispersal of 1.95-4.22 km/generation, others showed abrupt transitions (w ≈ 10 km), indicating localized genomic regions of strong selection against introgression. The subset of loci showing steep clines had largely coincident centers and stepped changes in allele frequency that did not co-localize with any physical barrier or ecotone, suggesting that the rabbit hybrid zone is a tension zone. The steepest clines were for X- and Y-chromosome markers. Our results are consistent with previous inference based on DNA sequence variation of individuals sampled in allopatry in suggesting that a large proportion of each genome has escaped the overall barrier to gene flow in the middle of the hybrid zone. These results imply an old history of hybridization and high effective gene flow and anticipate that isolation factors should often localize to small genomic regions.

Responses of a top and a meso predator and their prey to moon phases.

We compared movement patterns and rhythms of activity of a top predator, the Iberian lynx Lynx pardinus, a mesopredator, the red fox Vulpes vulpes, and their shared principal prey, the rabbit Oryctolagus cuniculus, in relation to moon phases. Because the three species are mostly nocturnal and crepuscular, we hypothesized that the shared prey would reduce its activity at most risky moon phases (i.e. during the brightest nights), but that fox, an intraguild prey of lynx, would avoid lynx activity peaks at the same time. Rabbits generally moved further from their core areas on darkest nights (i.e. new moon), using direct movements which minimize predation risk. Though rabbits responded to the increased predation risk by reducing their activity during the full moon, this response may require several days, and the moon effect we observed on the rabbits had, therefore, a temporal gap. Lynx activity patterns may be at least partially mirroring rabbit activity: around new moons, when rabbits moved furthest and were more active, lynxes reduced their travelling distances and their movements were concentrated in the core areas of their home ranges, which generally correspond to areas of high density of rabbits. Red foxes were more active during the darkest nights, when both the conditions for rabbit hunting were the best and lynxes moved less. On the one hand, foxes increased their activity when rabbits were further from their core areas and moved with more discrete displacements; on the other hand, fox activity in relation to the moon seemed to reduce dangerous encounters with its intraguild predator.

Phylogeography and genetic structure of the red-legged partridge (Alectoris rufa): more evidence for refugia within the Iberian glacial refugium.

The Pleistocene climatic oscillations promoted the diversification in avian species during the last glacial period. The red-legged partridge (Alectoris rufa, Family Phasianidae) has a large natural distribution extending from the Mediterranean to humid temperate zones. However, the genetic structure for this species is unknown. The present study investigates the phylogeography, genetic structure and demographic history of A. rufa across its distribution, employing both mitochondrial DNA control region sequences and nuclear microsatellite loci. Our results propose that this species was greatly affected by Pleistocene glaciations. The mismatch analyses suggest that the current populations resulted from post-glacial expansion and subsequent differentiation resulting in five diagnosable genetic clusters: Southwestern, Central-eastern, Northwestern, Balearic and French and Italian. Further, we found evidence of three glacial refugia within the currently recognized Iberian glacial refugium. The intraspecific structure revealed by both maternal and biparental phylogeographic analyses was not resolved in the phylogenetic analyses. Based on all considerations, we recommended that five management units be recognized.

Keeping an eye on the use of eye-lens weight as a universal indicator of age for European wild rabbits.

Accurate methods for age determination are critical to the knowledge of wildlife populations' age structure and, therefore, to their successful management. The reliability of age estimation may have profound economic and ecological consequences on the management of the European wild rabbits, Oryctolagus cuniculus, in its native and introduced range, where it is a keystone species and a major pest, respectively. As in other mammal species, European rabbits' age is often estimated using the Gompertz relationship between age and lens' weight. The growth rate formula has been developed based on data collected from European rabbits introduced in Australia, where a single subspecies (O. cuniculus cuniculus, Occ) is present. However, this curve has never been validated in the species native range, the Iberian Peninsula, where two subspecies (Occ, and O. c. algirus, Oca) coexist naturally. In this study, we tested the relationship between age and lens' weight using 173 Occ and 112 Oca wild rabbits that were surveyed in two experimental facilities in Spain. Our findings show that, in the native range, the published growth curve formula fits well Occ but not Oca data. Therefore, we recommend using the formula reported in this study to estimate the age of Oca (Lens dry weight = 240 × 10(-64.9/(Age+32))). This study supports Oca rabbits' distinctiveness revealed by previous studies, which suggests that management interventions should be applied to protect this subspecies whose distribution range is very narrow and whose populations seem to be declining. More broadly, our findings point to the importance of testing the suitability of growth curves defined for other species with different genetic forms as occurs in the European wild rabbit case.

Individual fate and gut microbiome composition in the European wild rabbit (Oryctolagus cuniculus).

Studies connecting microbiome composition and functional performance in wildlife have received little attention and understanding their connections with wildlife physical condition are sorely needed. We studied the variation in gut microbiota (hard fecal pellets) between allopatric subspecies of the European wild rabbit in wild populations and in captured individuals studied under captivity. We evaluated the influence of environmental and host-specific factors. The microbiome of wild rabbit populations reduced its heterogeneity under controlled conditions. None of the host-specific factors tested correlated with the microbiota composition. We only observed significant intra-group dispersion for the age factor. The most diverse microbiomes were rich in Ruminococcaceae potentially holding an enriched functional profile with dominance of cellulases and xylanases, and suggesting higher efficiency in the digestion of fiber-rich food. Conversely, low diversity gut microbiomes showed dominance of Enterobacteriaceae potentially rich in amylases. We preliminary noticed geographical variations in field populations with higher dominance of Ruminococcaceae in south-western than in north-eastern Spain. Spatial differences appeared not to be subspecies driven, since they were lost in captivity, but environmentally driven, although differences in social structure and behavior may also play a role that deserve further investigations. A marginally significant relationship between the Ruminococcaceae/Enterobacteriaceae ratio and potential life expectancy was observed in captive rabbits. We hypothesize that the gut microbiome may determine the efficiency of feeding resource exploitation, and can also be a potential proxy for life expectancy, with potential applications for the management of declining wild herbivorous populations. Such hypotheses remain to be explored in the future.

Brain Transcriptomics of Wild and Domestic Rabbits Suggests That Changes in Dopamine Signaling and Ciliary Function Contributed to Evolution of Tameness.

Domestication has resulted in immense phenotypic changes in animals despite their relatively short evolutionary history. The European rabbit is one of the most recently domesticated animals, but exhibits distinct morphological, physiological, and behavioral differences from their wild conspecifics. A previous study revealed that sequence variants with striking allele frequency differences between wild and domestic rabbits were enriched in conserved noncoding regions, in the vicinity of genes involved in nervous system development. This suggests that a large proportion of the genetic changes targeted by selection during domestication might affect gene regulation. Here, we generated RNA-sequencing data for four brain regions (amygdala, hypothalamus, hippocampus, and parietal/temporal cortex) sampled at birth and revealed hundreds of differentially expressed genes (DEGs) between wild and domestic rabbits. DEGs in amygdala were significantly enriched for genes associated with dopaminergic function and all 12 DEGs in this category showed higher expression in domestic rabbits. DEGs in hippocampus were enriched for genes associated with ciliary function, all 21 genes in this category showed lower expression in domestic rabbits. These results indicate an important role of dopamine signaling and ciliary function in the evolution of tameness during rabbit domestication. Our study shows that gene expression in specific pathways has been profoundly altered during domestication, but that the majority of genes showing differential expression in this study have not been the direct targets of selection.

Key role of European rabbits in the conservation of the Western Mediterranean basin hotspot.

The Mediterranean Basin is a global hotspot of biodiversity. Hotspots are said to be experiencing a major loss of habitat, but an added risk could be the decline of some species having a special role in ecological relationships of the system. We reviewed the role of European rabbits (Oryctolagus cuniculus) as a keystone species in the Iberian Peninsula portion of the Mediterranean hotspot. Rabbits conspicuously alter plant species composition and vegetation structure through grazing and seed dispersal, which creates open areas and preserves plant species diversity. Moreover, rabbit latrines have a demonstrable effect on soil fertility and plant growth and provide new feeding resources for many invertebrate species. Rabbit burrows provide nest sites and shelter for vertebrates and invertebrates. In addition, rabbits serve as prey for a number of predators, including the critically endangered Iberian lynx (Lynx pardinus) and Spanish Imperial Eagle (Aquila adalberti). Thus, the Mediterranean ecosystem of the Iberian Peninsula should be termed "the rabbit's ecosystem." To our knowledge, this is the first empirical support for existence of a multifunctional keystone species in a global hotspot of biodiversity. Rabbit populations have declined drastically on the Iberian Peninsula, with potential cascading effects and serious ecological and economic consequences. From this perspective, rabbit recovery is one of the biggest challenges for conservation of the Mediterranean Basin hotspot.

A large-scale assessment of European rabbit damage to agriculture in Spain.

BACKGROUND: Numerous small and medium-sized mammal pests cause widespread and economically significant damage to crops all over the globe. However, most research on pest species has focused on accounts of the level of damage. There are fewer studies concentrating on the description of crop damage caused by pests at large geographical scales, or on analysis of the ecological and anthropogenic factors correlated with these observed patterns. We investigated the relationship between agricultural damage by the European rabbit (Oryctolagus cuniculus) and environmental and anthropogenic variables throughout Spain. RESULTS: Rabbit damage was mainly concentrated within the central-southern regions of Spain. We found that rabbit damage increased significantly between the early 2000s and 2013. Greater losses were typical of those areas where farming dominated and natural vegetation was scarce, where main railways and highways were present, and where environmental conditions were generally favourable for rabbit populations to proliferate. CONCLUSION: From our analysis, we suggest that roads and railway lines act as potential corridors along which rabbits can spread. The recent increase in Spain of such infrastructure may explain the rise in rabbit damage reported in this study. Our approach is valuable as a method for assessing drivers of wildlife pest damage at large spatial scales, and can be used to propose methods to reduce human - wildlife conflict. © 2017 Society of Chemical Industry.

Large-scale assessment of myxomatosis prevalence in European wild rabbits (Oryctolagus cuniculus) 60years after first outbreak in Spain.

Myxomatosis is a viral disease that affects European rabbits (Oryctolagus cuniculus) worldwide. In Spain, populations of wild rabbits drastically decreased in the 1950s after the first outbreak of myxomatosis. Since that first appearance, it seems to be an annual epizootic in Spain with periodic outbreaks, predominantly in summer and autumn. Taking into account rabbit population structure, abundance, and genetic lineage, this paper attempts to make a large-scale characterization of myxomatosis seroprevalence based on the immune status of 29 rabbit populations distributed throughout Spain, where O. cuniculus cuniculus and O. c. algirus, the two known rabbit subspecies, naturally inhabit. A total of 654 samples were collected between 2003 and 2009, and seroprevalence of antibodies against Myxoma virus (MYXV) was determined. Overall, our results revealed that 53% of the rabbit samples were positive to antibodies against MYXV. Newborn and juvenile rabbits were the most susceptible animals to the virus, with 19% and 16% seropositivity for newborn and juveniles, respectively, while adult rabbits were the most protected, with 65% of seropositive samples. This suggests that prevalence is negatively related to the proportion of newborn and juvenile rabbits in a population. Our results also showed that seroprevalence against MYXV tended to be higher in high-abundance populations. In contrast, no differences were detected in seroprevalence between rabbit subspecies. This study confirms that >60years since first outbreak, myxomatosis is an endemic disease in Spain. Based on the results, the establishment of a myxomatosis surveillance protocol is proposed.

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

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.

Speciation in the European rabbit (Oryctolagus cuniculus): islands of differentiation on the X chromosome and autosomes.

Studies of gene flow between closely related taxa can provide insight into the genetic basis of speciation. To evaluate the importance of the X chromosome in reproductive isolation between subspecies of the European rabbit and to study the genomic scale over which islands of differentiation extend, we resequenced a total of 34 loci distributed along the X chromosome and chromosome 14. Previous studies based on few markers suggested that loci in centromeric regions were highly differentiated between rabbit subspecies, whereas loci in telomeric regions were less differentiated. Here, we confirmed this finding but also discovered remarkable variation in levels of differentiation among loci, with F(ST) values from nearly 0 to 1. Analyses using isolation-with-migration models suggest that this range appears to be largely explained by differential levels of gene flow among loci. The X chromosome was significantly more differentiated than the autosomes. On chromosome 14, differentiation decayed very rapidly at increasing distances from the centromere, but on the X chromosome distinct islands of differentiation encompassing several megabases were observed both at the centromeric region and along the chromosome arms. These findings support the idea that the X chromosome plays an important role in reproductive isolation between rabbit subspecies. These results also demonstrate the mosaic nature of the genome at species boundaries.

Field experimental vaccination campaigns against myxomatosis and their effectiveness in the wild.

We conducted a field experiment in SW Spain to test the efficacy of a myxomatosis vaccine, a viral disease strongly affecting wild rabbit populations, by assessing individual survival and antibody seroprevalence of monthly live-trapped, vaccinated (N=466) and unvaccinated (N=558) juvenile wild rabbits, between April and October 2007. Eight percent of all juveniles caught from April to June showed maternal antibodies against myxomatosis, whereas all animals were seropositive to the disease after the outbreak. Juveniles vaccinated before the outbreak showed 17% higher survival (31% vs. 14%) and an increased mortality probability of 8% after the outbreak. Results suggest that only a costly and systematic vaccination performed before the annual myxomatosis outbreak, would improve the survival of juvenile rabbits, a premise not always accomplished that compromises its efficacy in the field.

The effects of fasting and refeeding on biochemical parameters in the red-legged partridge (Alectoris rufa).

Every year, about three million farm-reared red-legged partridges are released in Spain for hunting purposes, most of them for restocking where the success is very low. Some factors such as the sudden shift from abundant commercial energy-rich diet to natural food and absence of experience at looking for natural food resources could cause a rapid physical deterioration, being more vulnerable to predation and disease. To determine fasting capacity in red-legged partridges, we fasted 32 birds during 4 days and then we refed them, collecting blood samples and body masses during the experiment. Red-legged partridges entered in the third critic phase of fasting, in which body proteins became the main energetic source, after 48 h of fasting. The fasting period caused an average loss of 17% of their initial body mass and only attained a 93.6% of their initial body mass after 24 days of refeeding. The limited capacity to resist fasting in the red-legged partridge could be one of the reasons for the low success in restocking, mainly in those where neither supplementary feeding nor acclimation period is allowed for birds before they are finally released.