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  1. Supplementary figures (SF) and supplementary tables (ST) Genetic architecture of divergent circadian phenotypes in morning and evening emerging fruit flies Drosophila melanogaster Koustubh M Vaze, KL Nikhil and Vijay Kumar Sharma* Chronobiology Laboratory, Evolutionary and Organismal Biology Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, PO Box. 6436, Bangalore-56 0064, Karnataka, India Running Title: Genetic analyses of early and late flies. *Correspondence to: Vijay Kumar Sharma E-mail: vsharma@jncasr.ac.in/ vksharmas@gmail.com; Phone: +91-080-22082843 (office)/ 2844/45 (lab); Fax: +91-080-22081766; Url: http://www.jncasr.ac.in/vsharma.

  2. SF1. Circadian phenotypes of early, control and late stocks assayed at generation 165. (A)Morning and evening adult emergence, (B) Circadian period of activity/rest rhythm, (C) Adult emergence profiles of early, control and late stocks under LD12:12, and (D) Representative actograms showing differences in circadian period of early, control and late flies. White bar- early, black bar- control and gray bar- late. Error bars are 95% confidence intervals and therefore non-overlapping error bars indicate that means are significantly different from each other. 40 30 (A) (B) 20 10 % of flies emerged circadian period (hr) 00 24 hr 24 hr 24 hr morning evening control late early (C) early control % flies 18 00 06 12 18 late Zeitgeber Time (hr) (D) early control late Day1 Day2 Day3 . . . . Day7 Vaze et al. Supp. Figure 1

  3. SF 2. Variance estimates of 16 progeny generations plotted against coefficients for additive model used in the analysis of generation variances. The coefficients for parental and F1 generations are 0.00, and for backcross generations 0.5, and 1 for F2 generation. (A) morning emergence, (B) evening emergence, and (C) τ of activity/rest rhythm. (A) Blk1 ♂ 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.00 1.00 1.00 1.00 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 1.00 1.00 1.00 1.00 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 variance Blk2 ♀ Blk2 ♂ variance variance Blk3 ♂ Blk3 ♀ Blk1 ♀ Blk4 ♀ Blk4 ♂ variance Coefficients from additive model Vaze et al. Supp. Figure 2

  4. (B) Blk1 ♂ Blk1 ♀ variance 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.00 1.00 1.00 1.00 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 1.00 1.00 1.00 1.00 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 Blk2 ♂ Blk2 ♀ variance variance Blk3 ♂ Blk3 ♀ Blk4 ♂ Blk4 ♀ variance Coefficients from additive model Vaze et al. Supp. Figure 2

  5. (C) Blk1 ♂ Blk1 ♀ variance 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.00 1.00 1.00 1.00 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 1.00 1.00 1.00 1.00 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 Blk2 ♂ Blk2 ♀ variance variance Blk3 ♂ Blk3 ♀ Blk4 ♂ Blk4 ♀ variance Coefficients from additive model Vaze et al. Supp. Figure 2

  6. ST 1 (A).ANOVA on the percentage of adult emergence during morning and evening hours in early, control and late stocks. ST 1 (B).ANOVA on circadian period of activity/rest rhythms in early, control and late stocks. Vaze et al. Supp. Table 1

  7. ST 2 (A). Scheme of crosses between early (E) and late (L) stocks and inheritance of four hereditary factors in the progeny flies. Permanent Cytoplasmic factors Transient Maternal factors Female Male Cross No. ♀ ♂ XX X Y Autosome Cross number in brackets indicate equivalent cross number in de Belle and Sokolowski, 1987. Notations used for different forms of a hereditary factor arising from crosses between parental stocks F1 = F1 hybrid BE = Back-cross to early parent BL = Back-cross to late parent F2 = F2 hybrid Table modified from de Belle and Sokolowski, 1987 Vaze et al. Supp. Table 2A

  8. ST2 (B). Comparisons/contrasts used to test contribution of various hereditary factors to the difference between early and late circadian phenotypes. Test Comparison ♂ ♂ ♂ ♂ ♂ ♂ ♂ ♂ ♀ ♀ ♀ ♀ ♀ ♀ ♀ Vaze et al. Supp. Table 2B

  9. ST 3. Replicate wise ANOVA on the circadian phenotypes of early and late parental stocks and in their progeny. (A) morning emergence, (B) evening emergence, and (c) circadian period. (A) ♂ ♀ (B) ♂ ♀ (C) ♂ ♀ Vaze et al. Supp. Table 3

  10. ST 4. Coefficients used to estimate the model parameters. Parameter notations are according to Kearsey and Pooni (1996). Crosses ♀ ♂ d c dm m a a.d am a.a d.d Please note: order of crosses is different from the one in Table 1A . E – early parent, L – late parent m – mean, a – additive effect, d – dominance am – additive maternal effect, dm – dominance maternal effect c – cytoplasmic factors, a.a – additive-additive interaction a.d – additive-dominance interaction, d.d – dominance-dominance interaction Vaze et al. Supp. Table 4

  11. Keys for supplementary Tables ST5-ST10 Upper case alphabet(s) (A, D, M, E) in upper most row of each tables represent type of genetic models tested. Each of those alphabets represent presence of genetic effect(s) in the model being tested. A - m, a D - m, d M - m, am,dm, C E- m, a.a, a.d, d.d Small letter alphabets in the middle rows of each table show model parameters which were significant and their estimates were used in the calculation of expected generation means . m – mean, a – additive effect, d – dominance am – additive maternal effect, dm – dominance maternal effect c – cytoplasmic factors, a.a – additive-additive interaction a.d – additive-dominance interaction, d.d – dominance-dominance interaction K = number of significant parameters df = degrees of freedom used for testing goodness of fit (df= 16-k) AIC = Akaike information criteria value ( calculated as described in Bieri and Kawecki, 2003). Non significant values are shown in bold letters. Smallest AIC values are shown in bold letters. c2

  12. ST 5. Results of all the models tested on generation means of males for morning emergence in four replicates. Parameters Parameters Parameters Parameters Vaze et al. Supp. Table 5

  13. ST 6. Results of all the models tested on generation means of females for morning emergence in four replicates. Parameters Parameters Parameters Parameters Vaze et al. Supp. Table 6

  14. ST 7. Results of all the models tested on generation means of males for evening emergence in four replicates. Parameters Parameters Parameters Parameters Vaze et al. Supp. Table 7

  15. ST 8. Results of all the models tested on generation means of females for evening emergence in four replicates. Parameters Parameters Parameters Parameters Vaze et al. Supp. Table 8

  16. ST 9. Results of all the models tested on generation means of males for circadian period in four replicates. Parameters Parameters Parameters Parameters Vaze et al. Supp. Table 9

  17. ST 10. Results of all the models tested on generation means of females for circadian period in four replicates. Parameters Parameters Parameters Parameters Vaze et al. Supp. Table 10

  18. ST 11. Broad sense (h2b) and narrow sense (h2n) heritability estimates in all the replicate populations based on τ values of activity/rest rhythm, and morning and evening emergence. Heritability estimates were calculated by carrying out analysis of generational variances according to the protocol described in Kearsey and Pooni (1996) and Lynch and Walsh (1998). OR indicates ‘out of range’ heritability values (i.e., less than 0, or greater than 1). Out of range of heritability estimates appear to be due to lower values of F2 and backcross generational variance estimates rather than a result of parental and F1 generational variance, which is quite evident in supplementary figures SF 2A, SF 2B and SF 2C. morning emergence evening emergence τ of activity/rest rhythm Vaze et al. Supp. Table 11