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Supplementary figures (SF) and supplementary tables (ST)

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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: [email protected]/ [email protected]; Phone: +91-080-22082843 (office)/ 2844/45 (lab); Fax: +91-080-22081766; Url: http://www.jncasr.ac.in/vsharma.

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

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

(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

(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

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

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

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

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

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

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

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

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

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

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

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

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

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