The domesticated brain: genetics of brain mass and brain structure in an avian species

As brain size usually increases with body size it has been assumed that the two are tightly constrained and evolutionary studies have therefore often been based on relative brain size (i.e. brain size proportional to body size) rather than absolute brain size. The process of domestication offers an excellent opportunity to disentangle the linkage between body and brain mass due to the extreme selection for increased body mass that has occurred. By breeding an intercross between domestic chicken and their wild progenitor, we address this relationship by simultaneously mapping the genes that control inter-population variation in brain mass and body mass. Loci controlling variation in brain mass and body mass have separate genetic architectures and are therefore not directly constrained. Genetic mapping of brain regions indicates that domestication has led to a larger body mass and to a lesser extent a larger absolute brain mass in chickens, mainly due to enlargement of the cerebellum. Domestication has traditionally been linked to brain mass regression, based on measurements of relative brain mass, which confounds the large body mass augmentation due to domestication. Our results refute this concept in the chicken.

birds, before being weighed in the same manner as stated previously. Brain volumes 15 were calculated using the method detailed in 6 using a principal based on the changing 16 mass measurements of the brains when suspended in water as compared to their 17 standard weight. This method is used due to the increased accuracy as compared to 18 more typical water displacement methods. A Pearson correlation was used to test the 19 correlation between total brain mass and total brain volume using the R-statistical 20 software package 7 . 21

Genotyping, QTL and mapping 22
DNA preparation was performed by Agowa GmbH (Berlin, Germany), using a 23 standard salt extraction technique 8 . A total of 652 SNP markers were used to generate 24 a map of length ~92675cM, with an average marker spacing of ~16cM. SNPS were 25 chosen based on a previously obtained panel of 10000 SNPs that had been run on the 26 parental birds. Additional details of marker generation, map generation and the like 27 can be found in 9 . QTL analysis was performed using R/Qtl 10 for both standard 28 interval mapping and epistatic analyses. Interval mapping was performed using 29 additive and additive+dominance models. Map generation and permutation threshold 30 measures were performed using the F 8 dataset, to account for the map expansion from 31 against the observed number of overlaps between the detected QTL and the selective 85 sweeps. This was repeated 1000 times, with the number of overlaps recorded each 86 time used to generate a significance value. 87

Fecundity Phenotypic Measures 88
One major behavioural change caused by domestication in chickens is reduced 89 brooding behaviour. In RJF brooding behaviour in females is associated with the 90 cessation of egg laying followed by nesting after a clutch of 6-10 eggs have been laid, 91 but selection for persistent egg production during domestication has resulted in a 92 reduction in the incidence of this behaviour 18 particularly in Mediterranean breeds 93 such as the White Leghorn in which brooding behaviour is rarely observed 19 . 94 Therefore one method for ascertaining if a chicken is brooding is to perform two 95 fecundity trials, one in which the eggs are removed daily, followed by another in 96 which the birds are allowed to retain the eggs laid. The number of eggs laid in the 97 second trial is then deducted from the number of eggs laid in the first trial to calculate 98 a 'brooding index'. The lower this number is the less broody the individual is (with 99 negative values indicating a female laid more eggs during the brooding trial than the 100 fecundity trial). Initially birds were housed individually and eggs were collected daily 101 over a two-week period for the first trial. The second trial was performed immediately 102 after the first and was identical except birds were given two dummy eggs to incubate 103 and were allowed to keep all eggs laid over a ten-day period. Because the brooding 104 trial was four days shorter than the fecundity trial (with the exception of one batch), 105 and to make the brooding indices between the two trials more interpretable, we 106 extrapolated the number of eggs in the second trial to 14 days. We excluded 11 females that laid no eggs in the first trial and 55 that laid no eggs in the second trial. 108 Chickens were reared and tested in five separate batches. In the case of the first two 109 batches, the number of females exceeded the number of individual cages available for 110 testing, resulting in assays being staggered in two sub-batches. This was then included 111 as a covariate in subsequent QTL analyses. 112

Correlations Between Brain Region Mass And Brooding Behaviour 113
Correlations were performed using the linear model function in R 7 . Total mass and 114 proportion of total brain mass (i.e. region mass divided by total brain mass) for the 115 cerebellum and cerebral hemispheres were modelled against brooding behaviour. 116 Body mass at slaughter was added as covariate, whilst rearing batch was included as a 117 fixed factor. A total of 123 birds were used in the analysis. 118

Domestic and Wild Birds 120
The ontogenetic comparison of wild Red Junglefowl and domestic White Leghorn 121 birds was performed at each age point (six age points used in total -from weeks one, 122 two, four, ten, fifteen and adult). Eight to seventeen birds from each population (RJF 123 and WL) were used for each time point comparison (1 st week: 10-RJF and 10-WL, 2 nd 124 week: 10-RJF and 10-WL, 4 th week: 10-RJF and 10-WL, 10 th week: 10-RJF and 8-125 WL, 15 th week: 10-RJF and 8-WL, Adulthood: 11-RJF and 17-WL), with both 126 absolute and relative mass calculated. A 2-sample t-test was used to compare 127 differences between RJF and WL individuals for absolute brain region mass, using the

Brain regions exhibit consistently different mass between domestic and wild 131 birds 132
By measuring brains from RJF and domestic chickens (WL) from 1-week of age until 133 sexual maturity we show that RJF brain regions weigh about ~85% of the total mass 134 of their domestic counterparts (cerebral hemispheres ~83%, optic tectum ~88%, 135 brainstem ~90%, cerebellum ~81%), with this mass difference being largely while the relative mass of the brainstem region is essentially fixed and the relative 147 mass of the optic tectum decreases by 3%. The relative mass of the cerebellum 148 increases by 2% during development. The differences between RJF and domesticated 149 (WL) chickens is also generalised to broilers (chickens produced for meat), with 150 broilers at two weeks of age showing similar changes in brain composition as WL brain evolution. Nature Reviews Neuroscience 6, 151--159 (2005 Affecting a Quantitative Character. Genetics 142, 285--294 (1996).