1 Supplementary Figures and Tables 2 3 for manuscript: 4 Time course analysis of the brain transcriptome during transitions between brood care and 5 reproduction in the clonal raider ant 6 Romain Libbrecht*1,2, Peter R. Oxley*1,3, Daniel J. C. Kronauer1 7 *co-first authors 8 1 Laboratory of Social Evolution and Behavior, The Rockefeller University, 1230 York Avenue, New York, 9 NY 10065, USA 10 2 Institute of Organismic and Molecular Evolution, Johannes Gutenberg University, Johannes-von-Müller- 11 Weg 6, 55128 Mainz, Germany 12 3 Samuel J. Wood Library, Weill Cornell Medicine, 1300 York Avenue, New York, NY 10065, USA 13 Correspondence: [email protected] 14 [email protected] 15 A B C 1.5 RC12 Number of genes with more 0.0 than 2-fold change in expression RC06 RC12 RB12 BR96 RB06 BC06 RB24 BR06 RC06 12- through 96-hour time points RB48 RB06 BR96 BC06 RB96 BR12 BR48 BC12 564 BC12 BR06 BR24 BR12 BR48 RB96 BR24 RB12 RB24 18 RB48 16 34 6-hour BR 0 1 4 6-hour RB 16 RC12 BR96 BC06 BR06 RC06 RB06 RB96 BC12 BR12 BR48 BR24 RB12 RB24 RB48 17 Supplementary Figure 1 18 Outlier analysis. PCA and distance map of genes showing greater than 2-fold change in 19 expression. A) PCA plot of brood-swap and control samples. Clustering was based on the mean 20 gene expression of each group, for 967 genes with more than 2-fold change in expression 21 between samples. Percentages on each axis indicate the proportion of variance explained by 22 the indicated principal component. The color of each sample indicates the expected similarity to 23 the control samples; dark blue indicates reproductive phase, dark green indicates brood care 24 phase. Sample names are as per Figure 1. B) Heatmap showing Euclidean distance between all 25 samples, based on all genes with more than 2-fold change in expression, and clustered 26 according to average distances between samples. Blue and green color bar above heatmap 27 indicates similarity to control samples, as in A. Sample names are as per Figure 1. C) Venn 28 diagram showing outcome of eliminating the 6-hour time points. Numbers in the small circles 29 indicate genes with > 2-fold change in expression between 6-hour control and treatment 30 samples in the reproduction to brood care transition (blue) and brood care to reproduction 31 transition (green) (a priori true positives). Red numbers indicate genes that show > 2-fold 32 change in expression after removal of the 6-hour time points. Thus, elimination of the 6-hour 33 samples does not substantially reduce the number of DEGs identified with large expression 34 changes. 35 Brood care to reproduction Reproduction to brood care ● ● 100000 ● − ● ● 100000 − ● ● ● ●● ● ● ●●● ● ● ● ●●●● ● ● ● ● ● ●●●● ● ● ● ● ● ● ● ● ● ●●●●●● ● ● ● ● ● ● ● ● ●●● ●● ● ● ● ● ● ● ● ● ● ● ● ● 150000 ● − 150000 − 200000 − 200000 − BIC BIC 250000 − 250000 BIC score BIC score − ● 300000 − 300000 − 350000 − 350000 ● ● ●●●●●●●●●●●●●●●●●●●●●●●● − ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 0 20 40 60 80 100 120 0 20 40 60 80 100 Number Gof clusters Number ofG clusters 36 37 Supplementary Figure 2 38 Evaluation of BIC scores for selection of optimal number of clusters. Genes were clustered into 39 all even numbered cluster sizes between 2-120 (brood care to reproduction) or 2-110 40 (reproduction to brood care). The optimal cluster size was determined to be the cluster with the 41 lowest BIC score after stabilization to the plateau seen on the right of each graph. Arrows show 42 the cluster selected for each transition. 43 100% 80% Edges linking clusters 60% of genes regulated in: Same direction 40% Opposite direction 20% 0% Proportion of edges linking clusters between transitions Complete Conserved network network 44 45 Supplementary Figure 3 46 If the same genes regulated both transitions, we would expect network edges that link clusters 47 of genes regulated in opposite direction between transitions to represent a higher proportion of 48 edges in the conserved network (with only non-random connections) compared to the complete 49 network (which includes random connections). Contrary to this expectation, we found a lower 50 proportion in the complete network (χ2 = 22.4, p < 0.00001). 51 BR RB GO:0003824 [MF] 'catalytic activity' GO:0004252 [MF] 'serine-type endopeptidase activity' GO:0005216 [MF] 'ion channel activity' GO:0005615 [CL] 'extracellular space' GO:0005319 [MF] 'lipid transporter activity' GO:0005789 [CL] 'endoplasmic reticulum membrane' GO:0005506 [MF] 'iron ion binding' GO:0005834 [CL] 'heterotrimeric G-protein complex' GO:0006030 [BP] 'chitin metabolic process' GO:0008237 [MF] 'metallopeptidase activity' GO:0005576 [CL] GO:0006508 [BP] 'proteolysis' GO:0015986 [BP] 'ATP synthesis coupled proton 'extracellular region' GO:0006811 [BP] 'ion transport' transport' GO:0052689 [MF] GO:0006869 [BP] 'lipid transport' GO:0035176 [BP] 'social behavior' 'carboxylic ester hydro- GO:0007160 [BP] 'cell-matrix adhesion' GO:0035556 [BP] 'intracellular signal transduction' lase activity' GO:0008061 [MF] 'chitin binding' GO:0043565 [MF] 'sequence-specific DNA binding' GO:0009166 [BP] 'nucleotide catabolic process' GO:0046034 [BP] 'ATP metabolic process' GO:0016705 [MF] 'oxidoreductase activity, acting on paired donors, with incorporation GO:0046933 [MF] 'proton-transporting ATP synthase or reduction of molecular oxygen' activity, rotational mechanism' GO:0016787 [MF] 'hydrolase activity' GO:0055114 [BP] 'oxidation-reduction process' GO:0020037 [MF] 'heme binding' GO:0043169 [MF] 'cation binding' 52 53 Supplementary Figure 4 54 GO terms significantly enriched in clusters enriched for DEGs. Only two of these GO terms were 55 common to both transitions (RB: transition from reproduction to brood care; BR: transition from 56 brood care to reproduction). The diameter of the circles is proportional to the number of 57 enriched GO terms. 58 1.5 0.0 RB48 BR12 BR48 BR24 RB96 BC12 RB24 RB12 RC12 BR96 59 RB48 BR12 BR48 BR24 RB96 BC12 RB24 RB12 RC12 BR96 60 Supplementary Figure 5 61 Genes associated with forkhead also segregate with position in the colony cycle. Heatmap 62 showing Euclidean distances between all samples for the 438 genes that contained at least one 63 TFBS for forkhead with a minimum score of 95%. The dendrogram was constructed using the 64 average distances between samples. The blue and green color bar above the heatmap 65 indicates average ovary activation score, as in Figure 2A. Sample names are as per Figure 1. 66 67 68 Supplementary Table 1 69 All 596 DEGs ranked according to p-value (smaller to larger). 70 Rank Gene ID Adjusted p-value Annotation 1 LOC105277456 2.86E-27 protein-L-isoaspartate(D-aspartate) O-methyltransferase 2 LOC105277457 2.09E-24 equilibrative nucleoside transporter 1 3 LOC105278524 5.91E-18 ILP2 4 LOC105281428_Q 1.25E-16 queen vitellogenin 5 LOC105275236 1.75E-16 carboxypeptidase M 6 LOC105284539 2.37E-12 sodium- and chloride-dependent glycine transporter 1 7 LOC105279766 2.72E-12 uncharacterized LOC105279766 8 LOC105279851 2.05E-11 yrdC domain-containing protein 9 LOC105275986 8.19E-11 uncharacterized LOC105275985 10 LOC105276495 1.17E-10 general odorant-binding protein 69a 11 LOC105285597 3.44E-10 uncharacterized LOC105285597 12 LOC105281011 1.12E-09 transferrin 13 LOC105285466 2.11E-09 uncharacterized LOC105285466 14 LOC105283669 7.35E-09 spermine synthase 15 LOC105276905 8.06E-09 neural/ectodermal development factor IMP-L2 16 LOC105275762 1.75E-08 protein aubergine 17 LOC105280170 4.95E-08 PI-PLC X domain-containing protein 2 18 LOC105281012 4.95E-08 uncharacterized LOC105281012 19 LOC105285380 6.73E-08 chitooligosaccharidolytic beta-N-acetylglucosaminidase 20 LOC105277400 2.52E-07 aldose 1-epimerase 21 LOC105279907 4.37E-07 uncharacterized LOC105279907 22 LOC105285376 4.71E-07 uncharacterized LOC105285376 23 LOC105280731 5.52E-07 inositol monophosphatase 1 24 LOC105275113 8.88E-07 pancreatic lipase-related protein 2-like 25 LOC105276066 1.03E-06 uncharacterized LOC105276066 26 LOC105275269 1.09E-06 uncharacterized LOC105275269 27 LOC105276731 1.09E-06 uncharacterized LOC105276731 28 LOC105283821 1.09E-06 nucleolar protein 6 29 LOC105278211 1.54E-06 centromere protein I 30 LOC105276018 1.94E-06 peroxisomal hydratase-dehydrogenase-epimerase 31 LOC105277056 1.94E-06 neuroparsin-A 32 LOC105288064 3.80E-06 uncharacterized LOC105288064 33 LOC105281928 5.80E-06 L-asparaginase 1 34 LOC105285607 6.17E-06 probable ribosome production factor 1 35 LOC105286129 6.88E-06 homeobox protein TGIF2LX 36 LOC105280465 9.64E-06 uncharacterized LOC105280465 37 LOC105281340 9.64E-06 sphingomyelin phosphodiesterase 38 LOC105283223 9.64E-06 putative mediator of RNA polymerase II transcription subunit 26 39 LOC105279511 9.67E-06 uncharacterized LOC105279511 40 LOC105281817 1.08E-05 uncharacterized LOC105281817 41 LOC105286755 1.08E-05 peptidyl-prolyl cis-trans isomerase 5 42 LOC105282988 1.18E-05 sodium/potassium-transporting ATPase subunit alpha 43 LOC105277203 1.24E-05 RNA polymerase I-specific transcription initiation factor RRN3 44 LOC105281008 1.45E-05 glutamate receptor ionotropic 45 LOC105286732 1.65E-05 anillin 46 LOC105281609 1.97E-05 small subunit processome component 20 homolog 47 LOC105285940 2.17E-05 THAP domain-containing protein 3 48 LOC105281708 2.20E-05 uncharacterized LOC105281708 49 LOC105281726 2.43E-05 ferrochelatase 50 LOC105280087 2.72E-05 protein regulator of cytokinesis 1 51 LOC105279867 2.74E-05 growth factor receptor-bound protein 14 52 LOC105283177 4.99E-05 glutathione S-transferase 53 LOC105276918 5.07E-05 14-3-3 protein epsilon 54 LOC105285920 5.07E-05