Supplementary Materials Table S1: Sample list and Reduced Representation Bisulfite Sequencing (RRBS) results of livers of fathers before and after heat treatment (F0LC and F0LH) No. Animal Treatment Total ID group reads Mapped reads Mapping ratios Unique CpGs 1 2 3 4 5 6 7 8 9 10 9,510,592 15,391,210 8,764,587 12,159,704 14,951,128 8,165,391 14,642,985 12,991,491 12,303,243 15,392,933 37.52% 38.19% 34.06% 35.95% 38.75% 38.58% 38.01% 37.89% 38.29% 37.14% 2,246,347 2,596,622 2,039,169 2,233,706 2,514,712 2,385,717 2,645,863 2,808,114 2,246,672 2,098,495 F0LC-F F0LC-G F0LC-H F0LC-I F0LC-J F0LH-F F0LH-G F0LH-H F0LH-I F0LH-J control control control control control heat heat heat heat heat 25,347,599 40,301,574 25,730,278 33,821,458 38,585,298 21,167,448 38,527,683 34,284,688 32,127,702 41,447,873 CpG Cov. (X) 13 18 13 16 17 10 17 14 16 20 BS Conv. rate 97.00% 98.23% 98.10% 97.79% 97.56% 98.28% 99.13% 98.78% 98.88% 95.26% Sequencing and mapping results from male F0 Wild guinea pig liver samples (F0LC and F0LH). Treatment groups are the five F0 fathers: either untreated (control: F0LC-F to F0LC-J) or exposed for 2 months to 30°C ambient temperature (heat: F0LH-F to F0LH-J). Table S2: Sample list and Reduced Representation Bisulfite Sequencing (RRBS) results of livers of sons sired before and after heat treatment of their fathers (F1LC and F1LH) No. Animal ID Treatmen Total reads Mapped t groups reads Mappin g ratios Unique CpGs CpG Cov. () 1 F1LC-F1 control 18,029,577 6,680,432 37.05% 3,309,580 7 BS Conv. rate 98.88% 2 F1LC-F2 control 16,059,059 6,066,076 37.77% 3,035,843 7 98.85% 3 F1LC-F3 control 22,521,636 8,711,906 38.68% 3,641,346 8 98.80% 4 F1LC-F4 control 24,850,183 9,780,109 39.36% 3,519,830 9 98.75% 5 F1LC-F5 control 21,141,617 7,974,523 37.72% 3,492,059 7 98.80% 6 F1LC-G1 control 29,401,669 11,042,394 37.56% 3,719,956 10 98.79% 7 F1LC-G2 control 22,183,595 8,798,513 39.66% 3,468,454 8 98.90% 8 F1LC-H1 control 20,205,777 7,907,817 39.14% 3,326,548 8 98.82% 9 F1LC-H2 control 24,959,451 9,423,385 37.75% 3,736,674 8 98.79% 10 F1LC-H3 control 15,427,493 6,206,456 40.23% 3,406,705 6 98.78% 11 F1LC-H4 control 20,939,405 8,425,873 40.24% 3,426,396 8 98.78% 12 F1LC-I1 control 22,053,277 8,336,943 37.80% 3,279,487 8 98.84% 13 F1LC-I2 control 16,424,032 6,771,022 41.23% 2,859,973 7 98.96% 14 F1LC-J1 control 17,776,958 7,315,599 41.15% 3,395,498 7 99.05% 15 F1LC-J2 control 19,264,973 7,702,700 39.98% 3,590,438 7 98.82% 16 F1LC-J3 control 24,919,184 9,606,720 38.55% 3,797,379 8 98.81% 17 F1LH-F1 heat 18,532,689 7,066,493 38.13% 3,516,644 6 98.82% 18 F1LH-F2 heat 28,644,120 10,804,520 37.72% 3,690,266 9 98.87% 19 F1LH-G1 heat 20,150,827 7,569,801 37.57% 3,389,117 7 98.84% 20 F1LH-G2 heat 19,321,403 6,701,990 34.69% 3,083,698 7 98.90% 21 F1LH-G3 heat 17,132,234 6,246,304 36.46% 3,300,790 6 98.93% 22 F1LH-G4 heat 19,276,868 6,637,384 34.43% 3,263,209 7 98.81% 23 F1LH-H1 heat 21,540,466 7,881,769 36.59% 3,425,142 7 98.98% 24 F1LH-H2 heat 18,390,731 7,099,149 38.60% 3,337,873 7 98.97% 25 F1LH-H3 heat 27,543,256 9,748,162 35.39% 3,632,603 9 98.99% 26 F1LH-H4 heat 33,786,783 11,335,437 33.55% 3,767,440 10 98.89% 27 F1LH-H5 heat 36,834,048 13,105,295 35.58% 4,015,123 10 98.92% 28 F1LH-I1 heat 35,310,760 11,730,786 33.22% 3,834,311 10 98.97% 29 F1LH-I2 heat 28,842,334 9,439,364 32.73% 3,778,893 8 98.80% 30 F1LH-I3 heat 35,306,161 12,372,854 35.04% 3,918,431 10 98.91% 31 F1LH-I4 heat 39,554,259 15,022,978 37.98% 3,814,662 13 98.84% 32 F1LH-I5 heat 16,821,180 6,590,335 39.18% 2,988,359 7 99.04% 33 F1LH-I6 heat 57,468,348 20,094,726 34.97% 3,101,848 21 99.02% 34 F1LH-J1 heat 37,827,572 12,759,894 33.73% 3,824,993 11 98.96% Sequencing and mapping results from F1 Wild guinea pig liver samples (F1LC and F1LH). Treatment groups are the sons (Fn-Jn) of fathers either untreated (control: F1LC-F1 - F1LH-J3) or exposed for 2 months to 30°C ambient temperature (heat: F1LH-F1 - F1LH-J1). Table S3: Sample list and Reduced Representation Bisulfite Sequencing (RRBS) results of testis of sons sired before and after heat treatment of their fathers (F0TC and F0TH) No. Label 1 2 3 4 5 6 7 8 9 10 F0TC-F F0TC-G F0TC-H F0TC-I F0TC-J F0TH-F F0TH-G F0TH-H F0TH-I F0TH-J Treatment group Total reads Mapped reads Mapping ratio Unique CpGs control control control control control heat heat heat heat heat 34,941,270 32,377,904 30,188,067 30,078,031 31,940,052 35,502,770 40,249,815 33,981,470 40,151,093 30,637,704 12,661,449 12,016,680 11,317,550 11,325,352 12,143,485 13,115,294 14,883,650 12,863,185 14,686,904 11,240,908 36.24% 37.11% 37.49% 37.65% 38.02% 36.94% 36.98% 37.85% 36.58% 36.69% 4,252,743 3,885,977 3,994,599 4,223,878 4,266,646 4,427,390 4,378,046 4,177,212 4,367,847 4,128,242 CpG Cov. () 9 9 9 8 9 9 10 10 11 8 BS Conv. rate 99.29% 98.84% 99.33% 99.26% 99.28% 99.21% 98.95% 99.25% 99.22% 99.23% Sequencing and mapping results from F1 Wild guinea pig testis samples (F1TC and F1TH). Treatment groups are the sons of fathers that were either untreated (control: F0TC-F to F0TC-J) or exposed for 2 months to 30°C ambient temperature (heat: F0TH-F to F0TH-J). Testis DNA was pooled of sons from the same father resulting in 10 RRBS libraries. Table S4: Number of annotated regions with DMRs per father group 4/5 fathers promoter 11 5/5 fathers 0 CDS 33 12 CGI 45 15 The number of annotated regions including DMRs (annotated DMRs), which occurred in at least four fathers or in offspring of at least four out of five fathers (F-J), as well as in all comparison groups (F0LC vs. F0LH, F1LC vs. F1LH and F1TC vs. F1TH; Fig. 2) were listed according their annotation (promoter, CDS and CGI). Table S5: Annotated regions with DMRs per father group after comparing F0LC vs. F0LH, F1LC vs. F1LH and F1TC vs. F1TH ID Gene name F0LC vs. F0LH F1LC vs. F1LH F1TC vs. F1TH ENSCPOG00000001921_promoter ENSCPOG00000006486_promoter ENSCPOG00000012928_promoter ENSCPOG00000020508_promoter ENSCPOG00000020731_promoter ENSCPOG00000021486_promoter ENSCPOG00000023463_promoter ENSCPOG00000024113_promoter ENSCPOG00000024501_promoter ENSCPOG00000025237_promoter ENSCPOG00000025665_promoter ENSCPOG00000000591_CDS ENSCPOG00000000999_CDS ENSCPOG00000001618_CDS ENSCPOG00000002223_CDS ENSCPOG00000003802_CDS ENSCPOG00000007730_CDS ENSCPOG00000009376_CDS ENSCPOG00000010080_CDS ENSCPOG00000012359_CDS ENSCPOG00000013220_CDS ENSCPOG00000021078_CDS ENSCPOG00000026620_CDS ENSCPOG00000000387_CDS ENSCPOG00000000682_CDS ENSCPOG00000000777_CDS ENSCPOG00000001125_CDS ENSCPOG00000001215_CDS ENSCPOG00000002024_CDS ENSCPOG00000003039_CDS ENSCPOG00000003239_CDS ENSCPOG00000003371_CDS ENSCPOG00000006604_CDS Topaz1 Ticam1 A9lpa2 C5orf45 Kiaa1609 Zc3h18 Sncg C19orf52 Ak4 Svp Krtap17-1 Notch4 Icam5 Kcnh6 Myh14 Eps8l2 Rara Pclo Plekhh3 Map3k6 Hmcn2 Ntf4 Dock6 Phkg1 Pde11a Mast1 Palm3 Fgd2 Cmbl Rasal3 Apba1 Stab2 Sox13 4 5 5 4 4 5 4 4 5 5 4 5 5 5 5 5 5 5 5 5 5 5 5 4 5 5 5 5 5 5 5 5 4 5 4 4 5 4 4 5 4 4 5 4 5 5 5 5 5 5 5 5 5 5 5 5 5 4 4 4 4 4 4 4 4 5 5 4 4 4 4 5 5 5 5 4 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 4 5 5 5 5 5 4 4 No. of fathe rs 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 5 5 4 4 4 4 4 4 4 4 4 4 ENSCPOG00000006927_CDS ENSCPOG00000007786_CDS ENSCPOG00000009248_CDS ENSCPOG00000009593_CDS ENSCPOG00000009735_CDS ENSCPOG00000009742_CDS ENSCPOG00000009747_CDS ENSCPOG00000010411_CDS ENSCPOG00000011364_CDS ENSCPOG00000011963_CDS ENSCPOG00000012253_CDS ENSCPOG00000012929_CDS ENSCPOG00000012950_CDS ENSCPOG00000013646_CDS ENSCPOG00000013669_CDS ENSCPOG00000015562_CDS ENSCPOG00000019861_CDS ENSCPOG00000021309_CDS ENSCPOG00000021486_CDS ENSCPOG00000023599_CDS ENSCPOG00000024030_CDS ENSCPOG00000024356_CDS ENSCPOG00000025737_CDS CGI_scaffold137_2593957_2594458 CGI_scaffold24_26911431_26912365 CGI_scaffold25_8916300_8917298 CpG_Island_scaffold27_7015314_7016000 CpG_Island_scaffold347_182392_182891 CGI_scaffold4_14096129_14096696 CGI_scaffold53_10797058_10798420 CGI_scaffold53_12338161_12339640 CGI_scaffold65_4665560_4666457 CGI_scaffold70_8308344_8309287 CGI_scaffold8_8119090_8119649 CGI_scaffold912_7518_8043 CGI_scaffold927_16_3441 CGI_scaffold927_3618_16028 CGI_scaffold96_4454758_4455442 CGI_scaffold10_22854406_22855263 CGI_scaffold10_49324092_49324882 CGI_scaffold115_4041100_4041661 Bambam1 Ush2a Gnas Lama5 Wiz Pcdha5 Slc27a1 Fcho1 Inpp5d Cacna1i Ptpn21 Hspb2 Pik3cd Kcns1 Ehbp1l1 Gpt C9orf142 Tmem44 Zc3h18 Hgfac Atcay Ppp1r18 Fbll1 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 4 4 5 5 5 5 5 4 4 4 4 4 4 5 4 4 5 4 4 4 5 4 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 4 4 4 4 4 4 4 5 4 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 4 4 4 5 5 5 5 4 5 4 4 4 5 5 4 5 4 4 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 4 5 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 4 4 4 CGI_scaffold117_1774019_1774529 CGI_scaffold15_34244619_34245449 CGI_scaffold156_1121544_1122341 CGI_scaffold16_36817085_36817599 CGI_scaffold17_14716398_14717393 CGI_scaffold18_38301297_38301912 CGI_scaffold19_27074656_27075589 CGI_scaffold2_67477859_67478928 CGI_scaffold20_31185302_31186764 CGI_scaffold226_426882_427711 CGI_scaffold226_632579_633632 CGI_scaffold25_13488570_13489078 CGI_scaffold27_2619059_2619665 CGI_scaffold30_10384183_10384700 CGI_scaffold3088_1_2831 CGI_scaffold314_110299_111344 CGI_scaffold32_299187_300192 CGI_scaffold322_240862_241395 CGI_scaffold33_18212468_18213158 CGI_scaffold347_333299_334244 CGI_scaffold368_180343_181204 CGI_scaffold4_33132022_33132631 CGI_scaffold41_873326_873825 CGI_scaffold42_13041239_13041738 CGI_scaffold42_13324813_13325557 CGI_scaffold429_95158_95955 CGI_scaffold45_11118285_11118918 CGI_scaffold509_141021_141523 CGI_scaffold53_11638212_11638734 CGI_scaffold6_3164487_3165970 CGI_scaffold6_598782_599924 CGI_scaffold61_9054899_9055727 CGI_scaffold687_5368_6432 CGI_scaffold7_45860487_45861026 CGI_scaffold70_8735848_8736348 CGI_scaffold72_7980612_7981332 CGI_scaffold72_8633449_8633948 CGI_scaffold72_8651524_8652515 CGI_scaffold81_1145048_1146070 CGI_scaffold86_6254900_6255830 CGI_scaffold86_6260228_6261562 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 4 4 5 5 5 5 5 5 4 4 5 4 4 5 4 5 5 4 5 4 4 4 4 4 4 4 5 5 4 5 4 4 5 4 4 5 4 4 5 4 5 5 4 4 4 4 4 5 4 4 4 4 5 4 4 5 4 4 4 4 4 4 4 4 4 4 4 5 4 4 4 5 5 4 5 5 4 4 4 4 5 4 5 5 5 4 5 5 4 5 5 5 5 4 5 5 5 5 4 5 5 4 5 4 5 5 5 4 4 4 5 5 4 5 5 5 5 5 4 5 5 4 5 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 CGI_scaffold90_5370669_5371337 NA 4 5 5 4 All annotated regions with DMRs among at least four father groups are listed. The ID includes the Ensembl-ID as well as regulatory regions followed by gene name. To localize CGIs also the scaffold and position were included. The table was sorted by regulatory regions, starting with promoters, followed by CDS and CGIs and by their occurrence in five and four father groups, for F0LC vs. F0LH, F1LC vs. F1LH and F1TC vs. F1TH separately, as well as for all three combined (No. of fathers) as shown in Fig. 2. Table S6: Number of genes in CpGs and DMRs F0LC vs. F0LH F1LC vs. F1LH F1TC vs. F1TH Genes incl. mCpGs Genes incl. mCpGs sign. Genes incl. mCpGs insign. Genes incl. DMRs 10,860 13,900 12,522 8,235 7,311 6,795 10,589 13,845 12,461 304 103 353 The number of genes are listed including the methylated CpGs (mCpGs) in the fathers` livers before and after heat exposure (F0LC vs. F0LH) and sons sired before and after the fathers` exposure in liver (F1LC vs. F1LH) and testis (F1TC vs. F1TH). Most of the genes had several CpGs, which were partly significantly (mCpGs sign.) and partly insignificantly (mCpGs insign.) changed in their methylation levels. In addition, the table gives the number of genes that were located in DMRs. Fig. S1: DMRs located in the Seminal vesicle polypeptide (Svp) gene Hypermethylated (red bars) and hypomethylated (blue bars) DMRs detected in the Seminal vesicle polypeptide (Svp) gene in the heat-treated father groups. This male-specific gene indicated 1) a tissue-specific pattern and 2) a composition of DMRs for regulatory control. Whereas Svp was hypomethylated in livers samples of fathers (F0LH), it showed hypermethylation in the son’s testis (F1TH) and an additional DMR located further upstream of the DMRs detected in livers. Fig. S2: Thermoregulation pathway genes showing single mCpG changes detected in heat group compare to control Thermoregulation genes with significantly changed methylation ratio (CpGn) in the heat exposed father livers (F0LC vs. F0LH) and their sons` livers and testes (F1LC vs. F1LH and F1TC vs. F1TH F0LH). The genomic location, such as coding sequence (CDS) illustrated as circles and promoter illustrated as triangle, as well as gene methylation states (blue hypomethylated, red hypermethylated) are given. GO terms describe the thermoregulatory pathway the genes are involved in.