(b)

(f)

P70

0+ le

vels

(d)

P70

0+ le

vels

P70

0+ le

vels

(a)

F. brownii (C4-like) F. palmeri (C4-like)

F. anomala (C3-C4) F. ramosissima (C3-C4)

F. pringlei (C3) F. robusta (C3) 0

0.2

0.4

0.6

0.8

1.0

(c)

time, s 0 5 10 15 20 25 30

(e)

time, s 0 5 10 15 20 25 30

(f)

0

0.2

0.4

0.6

0.8

1.0

0

0.2

0.4

0.6

0.8

1.0

Figure S1. 　 P700 oxidation kinetics in leaf discs infiltrated with distilled water (dH2O) or methyl viologen (MV) under far-red light illumination in C3, C3-C4, and C4-like Flaveria species. P700 oxidation kinetics in leaf discs of F. pringlei (a), F. robusta (b), F. anomala (c), F. ramosissima (d), F. brownii (e), and F. palmeri (f) infiltrated with dH2O (grey line) or 50 μmol MV (black line). Plants were dark-adapted for 2 h. Kinetics shown are the average of three individual plants.

116 88

62

47

35

28

(kDa)

PEPCPPDK

NADP-ME

rbcL (RuBisCO large subunit)

F. bid

entis

(C4)

F. an

omal

a (C

3-C

4)

F. pa

lmer

i (C 4

-like)

F. br

ownii

(C4-lik

e)

F. ro

busta

(C3)

F. pr

inglei

(C3)

F. r

amos

issim

a (C 3

-C 4)

Figure S2. 　 SDS-PAGE of total leaf soluble proteins from C3, C3-C4, C4-like, and C4 Flaveria species. Total soluble protein (20 μg) extracted from leaves of C3 F. pringlei, C3 F. robusta, C3-C4 F. anomala, C3-C4 F. ramosissima, C4-like F. brownii, C4-like F. palmeri, C4 F. bidentis, and C4 F. trinervia were separated by electrophoresis on 12.5% acrylamide gels and stained with Coomassie brilliant blue R-250.

(a)C

hlo

rop

hy

ll f

luo

res

ce

nc

e

time

(b)

MLOFF

ALON

SP

FR ON

MLON

Ch

loro

ph

yll

flu

ore

sc

en

ce

ALOFF

1min 30s4min

1 min 30 s

ML OFFFR ONAL OFF

B

A

Se-ries1

00.010.020.030.040.050.060.070.080.090.1

ND

H a

ctiv

ity (

a. u

.)

(c)

F. bidentis

F. brownii

F. anomala

F. pringlei

F. palmeri

F. robusta

F. ramosissim

a

F. pringlei (C3)

F. anomala (C3-C4) F. ramosissima (C3-C4)

F. robusta (C3)

F. bidentis (C4)

F. brownii (C4-like) F. palmeri (C4-like)

Figure S3. 　 NDH activity in C3, C3-C4, C4-like, and C4 Flaveria species.

(a) Left curve shows typical trace of chlorophyll fluorescence of C4 F. bidentis. Plants

were dark-adapted for 2 h and then exposed to actinic light (AL; 53 μmol photon m -2 s-1) for 4 min. Transient increase in chlorophyll fluorescence after actinic light illumination is ascribed to NDH activity. Right box shows magnified trace of transient increase in chlorophyll fluorescence from left curve. A and B indicate maximum values and minimum values, respectively, for amplitude of transient increase in chlorophyll fluorescence. (b) Magnified trace of transient increase in chlorophyll fluorescence after actinic light illumination in leaves of C3 F. pringlei, C3 F. robusta C3-C4 F. anomala, C3-C4 F.

ramosissima, C4-like F. brownii, C4-like F. palmeri, C4 F. bidentis, and C4 F. trinervia.

Chlorophyll fluorescence levels were normalised to Fm (maximum fluorescence at closed

PSII centre in the dark). (c) Quantification of NDH activity estimated from peak amplitude of transient increase in chlorophyll fluorescence in each Flaveria species. Values for NDH activity were calculated as follows: NDH activity = (A-B)/maximum fluorescence. Values are means ± SD (n=3).

(a) (b) (c)

(d) (e) (f)

Figure S4. 　 Background labelling with rabbit serum in in situ immunolocalisation. Transverse sections were labelled with rabbit serum and then labelled with secondary antibodies conjugated to FITC. FITC fluorescence in green (a-c) and overlaid images of FITC fluorescence in green and bright field (d-f) were visualised by confocal microscopy. Scale bar = 50 μm.

Bundle-sheath chloroplasts

Mesophyll chloroplasts

(a) (b)

(c) (d)

(e) (f)

Figure S5. 　Measurement of total length of grana thylakoid and stroma thylakoid membranes of BS chloroplasts or M chloroplasts in C4 F. bidentis. Electron micrograph shows part of BS chloroplast (a, c, e) and M chloroplast (b, d, f) used for measurements. Pink lines in c and d show trace of grana thylakoid and those in e and f show stroma thylakoid.