q 2017 by The University of Chicago. All rights reserved. DOI: /690105

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1 q 2017 by The University of Chicago. All rights reserved. DOI: / Appendix from R. M. Cox et al., Hormonally Mediated Increases in Sex-Biased Gene Expression Accompany the Breakdown of Between- Sex Genetic (Am. Nat., vol. 189, no. 3, p. 315) Supplemental Tables A1 A7 and Figure A1 1

2 Table A1: Quantitative genetics of sexual dimorphism in growth rate Trait and age Sample size (N ) Residual variance (V E ) Additive genetic variance (V A ) Heritability (h 2 ) Male Female Male Female Male Female Male Female Between-sex covariance Between-sex r mf SVL growth: # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # Mass growth: # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # Note: Quantitative-genetic parameter estimates for growth in SVL (in millimeters per day) or body mass (in grams per day) over five age intervals. Parameter estimates (5SE) are derived from animal model analysis using a paternal half-sib breeding design. Boldface indicates values of V A, h 2, and rmf significantly (P! :05) greater than 0. 2

3 Table A2: Gene ontology terms significantly enriched for sex-biased genes Gene ontology category Sex-biased genes Adjusted P Metabolic process Single-organism metabolic process Organic substance metabolic process Primary metabolic process Regulation of metabolic process Response to wounding Hemostasis Negative regulation of multicellular organismal process Steroid metabolic process Digestion 18!.0001 Protein activation cascade 16!.0001 Protein maturation Acute inflammatory response 15!.0001 Neutral lipid metabolic process Acylglycerol metabolic process Triglyceride metabolic process Protein processing Platelet degranulation Humoral immune response Complement activation 11!.0001 Regulation of coagulation Regulation of hemostasis Regulation of wound healing Regulation of blood coagulation Negative regulation of coagulation Negative regulation of hemostasis Acute-phase response Negative regulation of blood coagulation Regulation of ARF protein signal transduction Regulation of ARF GTPase activity Fibrolysis 8!.0001 Complement activation, alternative pathway 7!.0001 Regulation of protein processing Lipid digestion 7!.0001 Complement activation, classical pathway Regulation of fibrolysis Positive regulation of hemostasis Intestinal cholesterol absorption Negative regulation of fibrolysis Multicellular organismal lipid catabolic process Note: Significance was assessed following correction for multiple comparisons (Benjamini and Hochberg 1995). Gene ontology categories are sorted by the number of sex-biased genes and often represent subsets of larger categories. 3

4 Table A3: Mean expression of genes in the GH/IGF network by sex and age Juveniles (cpm) Subadults (cpm) Ensembl ID Gene Ensembl description Male Female Male Female ENSACAT GHR Growth hormone receptor ENSACAT GHRRH Growth hormone releasing hormone receptor ENSACAT IGF1 Insulin-like growth factor 1 (somatomedin C) ENSACAT IGF1BP1 Insulin-like growth factor binding protein ENSACAT IGF1BP2 Insulin-like growth factor binding protein ENSACAT IGF1BP3 Insulin-like growth factor binding protein ENSACAT IGF1BP4 Insulin-like growth factor binding protein ENSACAT IGF1BP5 Insulin-like growth factor binding protein ENSACAT IGF1BP6 Insulin-like growth factor binding protein ENSACAT IGF1BP7 Insulin-like growth factor binding protein ENSACAT IGF1R Insulin-like growth factor 1 receptor ENSACAT IGF2 Insulin-like growth factor 2 (somatomedin A) ENSACAT IGF2BP1 Insulin-like growth factor 2 mrna binding protein ENSACAT IGF2BP2 Insulin-like growth factor 2 mrna binding protein ENSACAT IGF2BP3 Insulin-like growth factor 2 mrna binding protein ENSACAT SHBG Sex hormone binding globulin Note: This table shows 16 genes whose transcripts were quantified by RNA-seq analysis of Anolis sagrei livers and assigned to the growth hormone/insulin-like growth factor (GH/IGF) network based on the annotated cdna set of the congener Anolis carolinensis (AnoCar 2.0; Ensembl release 75; Alfioldi et al. 2011), the KEGG database (Kanehisa et al. 2004), and WikiPathways (Kelder et al. 2011). Table A4: Mean expression of genes in the mtor network by sex and age Juveniles (cpm) Subadults (cpm) Ensembl ID Gene Ensembl description Male Female Male Female ENSACAT AKT1 V-AKT murine thymoma viral oncogene homolog ENSACAT BRAF B-RAF protooncogene, serine/threonine kinase ENSACAT EIF4EBP1 Eukaryotic translation initiation factor 4E binding protein ENSACAT H1F1AN Hypoxia inducible factor 1, a subunit inhibitor ENSACAT IRS1 Insulin receptor substrate ENSACAT MTOR Mechanistic target of rapamycin ENSACAT PIK3CA Phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit a ENSACAT PIK3R5 Phosphoinositide-3-kinase, regulatory subunit ENSACAT PRKAA1 Protein kinase, AMP-activated, a 1 catalytic subunit ENSACAT PRKAA2 Protein kinase, AMP-activated, a 2 catalytic subunit ENSACAT PRKAB2 Protein kinase, AMP-activated, b 2 noncatalytic subunit ENSACAT PRKAG1 Protein kinase, AMP-activated, g 1 noncatalytic subunit ENSACAT PRKAG2 Protein kinase, AMP-activated, g 2 noncatalytic subunit ENSACAT PRKAG3 Protein kinase, AMP-activated, g 3 noncatalytic subunit ENSACAT PRKCH Protein kinase C ENSACAT PTEN Phosphatase and tensin homolog ENSACAT RICTOR RPTOR independent companion of MTOR, complex ENSACAT RPTOR Regulatory associated protein of MTOR, complex ENSACAT RPS6KA2 Ribosomal protein S6 kinase ENSACAT RPS6KC1 Ribosomal protein S6 kinase ENSACAT STRADA STE20-related kinase adaptor a ENSACAT STRADB STE20-related kinase adaptor b ENSACAT TSC1 Tuberous sclerosis ENSACAT TSC2 Tuberous sclerosis ENSACAT VEGFA Vascular endothelial growth factor A Note: This table shows 25 genes whose transcripts were quantified by RNA-seq analysis of Anolis sagrei livers and assigned to the mechanistic target of rapamycin (mtor) network based on the annotated cdna set of the congener Anolis carolinensis (AnoCar 2.0; Ensembl release 75; Alfioldi et al. 2011), the KEGG database (Kanehisa et al. 2004), and WikiPathways (Kelder et al. 2011). 4

5 Table A5: Mean expression of genes in the insulin-signaling network by sex and age Juveniles (cpm) Subadults (cpm) Ensembl ID Gene Ensembl description Male Female Male Female ENSACAT AKT1 V-AKT murine thymoma viral oncogene homolog ENSACAT CDKN1B Cyclin-dependent kinase inhibitor 1B ENSACAT EIF4E Eukaryotic translation initiation factor 4E ENSACAT EIF4EBP1 Eukaryotic translation initiation factor 4E binding protein ENSACAT ELK1 ELK1, member of ETS oncogene family ENSACAT FBP1 Fructose-1,6-biphosphatase 1 1, , , , ENSACAT FLOT1 Flotillin ENSACAT FLOT2 Flotillin ENSACAT G6PC2 Glucose-6-phosphatase, catalytic ENSACAT GNL3 Guanine nucleotide binding protein-like ENSACAT GRB2 Growth factor receptor-bound protein ENSACAT GYS1 Glycogen synthase 1 (muscle) ENSACAT CBL CBL protooncogene, E3 ubiquitin protein ligase ENSACAT LIPE Lipase, hormone sensitive ENSACAT IRS1 Insulin receptor substrate ENSACAT IRS4 Insulin receptor substrate ENSACAT MAP2K2 Mitogen-activated protein kinase ENSACAT MAP2K3 Mitogen-activated protein kinase ENSACAT MAP2K4 Mitogen-activated protein kinase ENSACAT MAP2K5 Mitogen-activated protein kinase ENSACAT MAP2K6 Mitogen-activated protein kinase ENSACAT MAP2K7 Mitogen-activated protein kinase ENSACAT MLST8 MTOR associated protein, LST8 homolog ENSACAT MYC V-MYC avian myelocytomatosis viral oncogene homolog ENSACAT NR3C1 Nuclear receptor subfamily 3, group C, member ENSACAT PDE3A Phosphodiesterase 3A, cgmp-inhibited ENSACAT PDPK1 3-phosphoinositide dependent protein kinase ENSACAT PCK1 Phosphoenolpyruvate carboxykinase ENSACAT PCK2 Phosphoenolpyruvate carboxykinase 2 1, , , , ENSACAT PPARA Peroxisome proliferator-activated receptor a ENSACAT PHKA2 Phosphorylase kinase, a ENSACAT PIK3CA Phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit a ENSACAT PIK3R5 Phosphoinositide-3-kinase, regulatory subunit ENSACAT PRKAA1 Protein kinase, AMP-activated, a 1 catalytic subunit ENSACAT PRKAA2 Protein kinase, AMP-activated, a 2 catalytic subunit ENSACAT PRKAB2 Protein kinase, AMP-activated, b 2 noncatalytic subunit ENSACAT PRKAG1 Protein kinase, AMP-activated, g 1 noncatalytic subunit ENSACAT PRKAG2 Protein kinase, AMP-activated, g 2 noncatalytic subunit ENSACAT PRKAG3 Protein kinase, AMP-activated, g 3 noncatalytic subunit ENSACAT PRKCH Protein kinase C ENSACAT PTEN Phosphatase and tensin homolog ENSACAT RAC1 RAS-related C3 botulinum toxin substrate ENSACAT RAC2 RAS-related C3 botulinum toxin substrate ENSACAT RAF1 RAF-1 protooncogene, serine/threonine kinase ENSACAT RAPGEF1 RAP guanine nucleotide exchange factor (GEF) ENSACAT RASD1 RAS, dexamethasone-induced ENSACAT RHEB RAS homolog enriched in brain ENSACAT RHO Anolis carolinenesis rhodopsin (rho), mrna ENSACAT RICTOR RPTOR independent companion of MTOR, complex ENSACAT RPS6KB1 Ribosomal protein S6 kinase, 70kDa, polypeptide ENSACAT RPS6L1 Ribosomal protein S6 kinase-like ENSACAT RPTOR Regulatory associated protein of MTOR ENSACAT SHC1 SHC (SRC homology 2 domain containing) transforming protein 1 ENSACAT SHC2 SHC (SRC homology 2 domain containing) transforming protein

6 Table A5 (Continued) Juveniles (cpm) Subadults (cpm) Ensembl ID Gene Ensembl description Male Female Male Female ENSACAT SHC4 SHC (SRC homology 2 domain containing) family, member 4 ENSACAT SOCS1 Suppressor of cytokine signaling ENSACAT SOCS2 Suppressor of cytokine signaling ENSACAT SOCS4 Suppressor of cytokine signaling ENSACAT SOCS5 Suppressor of cytokine signaling ENSACAT SOCS6 Suppressor of cytokine signaling ENSACAT SOCS7 Suppressor of cytokine signaling ENSACAT SOS1 Son of sevenless homolog ENSACAT SOS2 Son of sevenless homolog ENSACAT STAT1 Signal transducer and activator of transcription ENSACAT STAT2 Signal transducer and activator of transcription ENSACAT STAT3 Signal transducer and activator of transcription ENSACAT STAT6 Signal transducer and activator of transcription ENSACAT STK11 Serine/threonine kinase ENSACAT TGFB1 Transforming growth factor, b ENSACAT TGFB2 Transforming growth factor, b ENSACAT TGFB3 Transforming growth factor, b ENSACAT TGFBR3L Transforming growth factor, b receptor 3-like ENSACAT TGFBR1 Transforming growth factor, b receptor ENSACAT TGFBR2 Transforming growth factor, b receptor ENSACAT TGFBR3 Transforming growth factor, b receptor ENSACAT TSC1 Tuberous sclerosis ENSACAT TSC2 Tuberous sclerosis Note: This table shows 76 genes whose transcripts were quantified by RNA-seq analysis of Anolis sagrei livers and assigned to the insulin-signaling network based on the annotated cdna set of the congener Anolis carolinensis (AnoCar 2.0; Ensembl release 75; Alfioldi et al. 2011), the KEGG database (Kanehisa et al. 2004), and WikiPathways (Kelder et al. 2011). Table A6: Sex-biased expression of GH/IGF genes in juveniles and subadults Juveniles Subadults Gene Male (cpm) Female (cpm) Sex bias Uncorrected P Male (cpm) Female (cpm) Sex bias Uncorrected P GHR GHRRH IGF !.0001 IGFBP IGFBP IGFBP IGFBP IGFBP IGFBP IGFBP IGF1R IGF IGF2BP IGF2BP IGF2BP SHBG !.0001 Note: Sex bias in gene expression (fold difference in counts per million between males and females; positive values are male biased, and negative values are female biased) is shown for 16 genes assigned to the growth hormone/insulin-like growth factor (GH/IGF) network. Uncorrected P values are from pairwise exact tests with a negative binomial distribution. Boldface indicates significant sex-biased expression (uncorrected P! :0031 after Bonferroni correction for 16 comparisons). In subadults, two genes (IGFBP6, IGF2BP1) had undetectable expression levels for one or both sexes and therefore could not be assigned a fold difference in expression. 6

7 Table A7: IPA pathways significantly enriched for genes responsive to testosterone Top canonical pathways P Overlap Testosterone-responsive genes in pathway Factors promoting cardiogenesis in vertebrates Pyridoxal 5 0 -phosphate salvage pathway /92 BMP4, CER1, PRKCD, TDGF1, ACVR1, PRKCE, DKK1, GSK3B, PRKD3, BMP5, CDK2, PRKCA /64 PAK1, SGK1, PRKCD, PRKCE, PAK7, G6PC, MAP2K1, CDK2, LIMK1 Growth hormone signaling /81 GHR, IGF1, PRKCD, IRS1, CEBPA, IGFALS, PRKCE, RPS6KA5, PRKD3, PRKCA LPS/IL-1-mediated inhibition of RXR function /221 CHST4, SLC10A1, GSTM5, HS3ST4, SOD3, IL18, NR0B2, HS3ST2, SULT1A3/SULT1A4, FABP1, CYP2A6, CHST10, FABP5, PLTP, SULT1C3, ALDH16A1, FMO4, ALDH5A1 NRF2-mediated oxidative stress response /193 AKR7A3, GSTM5, PRDX1, DNAJC15, SOD3, DNAJC5G, FTL, PRKCD, IRS1, PRKCE, GSK3B, SQSTM1, MAP2K1, ACTC1, PRKD3, PRKCA Note: This table shows the top five pathways enriched for genes responsive to testosterone, arranged by increasing P values and shown alongside overlap (number of significantly responsive genes/total number of genes in the pathway that were expressed in the liver) and ortholog names for genes responsive to testosterone in juvenile females (P! :05 without false discovery rate correction). Analyses were conducted using ingenuity pathway analysis (IPA; Qiagen, Valencia, CA). 7

8 Figure A1: Ingenuity pathway analysis pathways significantly enriched for sex-biased genes, arranged by decreasing P values. Pathways that are predicted to be activated are shown in blue. The orange line corresponds to the proportion of sex-biased genes out of the total number of reference genes in each pathway. 8