science.sciencemag.org/cgi/content/full/science.aay5433/DC1

Supplementary Materials for

Evolution of carnivorous traps from planar leaves through simple shifts in gene expression

Christopher D. Whitewoods*, Beatriz Gonçalves*, Jie Cheng*, Minlong Cui,

Richard Kennaway, Karen Lee, Claire Bushell, Man Yu, Chunlan Piao, Enrico Coen†

*These authors contributed equally to this work. †Corresponding author. Email: enrico.coen@jic.ac.uk

Published 21 November 2019 on Science First Release

DOI: 10.1126/science.aay5433

This PDF file includes:

Materials and Methods Figs. S1 to S10 Tables S1 to S6 Captions for Movies S1 to S10 Caption for Data S1 References

Other Supporting Online Material for this manuscript includes the following: (available at science.sciencemag.org/cgi/content/full/science.aay5433/DC1)

Movies S1 to S10 Data S1 (Excel)

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Materials and Methods

Plant material

U. gibba was obtained from The Fly Trap Plants (Cookes Road, Berghapton, NR15

1BA, UK). Plants were grown clonally in liquid medium (2.2 g/l MS, 2.5 g/l sucrose, pH 5.8) 30

in a controlled environment room at 25 °C, with 16-hour light and 8-hour dark cycles.

To identify candidate genes in U. gibba the Langebio genome assembly (9,10) was queried

by BLAST using AtPHV and AtFIL. The top hits for each search were used for further

analysis.

Phylogenetic analysis 35

Phylogenetic relationships were inferred using the Maximum Likelihood method based

on the JTT matrix-based model implemented on MEGA6 after alignment using MUSCLE

(27). A complete list of sequences used to generate YABBY, HDZIP-III and KANADI

phylogenies is available in Table S1.

RNAseq 40

For RNAseq analysis, traps were divided into six developmental stages based on length

(approximately 180µm, 270µm, 410µm, 620µm, 930µm and 1400µm) and collected in

triplicates of 5 to 20 traps each. A triplicate of leaflet tissue with no traps was also collected.

Total mRNA was extracted and converted to double stranded cDNA for Illumina Hiseq

sequencing (Earlham Institute). The 50bp single-end reads were processed and mapped 45

against the Langebio genome assembly1.

Sequencing was carried out by the Earlham Institute (then TGAC), Norwich. A total of

21 libraries were sequenced in one pool on two lanes of the Illumina HiSeq (Rapid Run mode

with on board clustering (duel indexing)) to generate 50 bp single-end reads. Reads were

filtered using fastq-mcf (28). Reads were trimmed after a position with a quality score below 50

20. After trimming, only reads with a minimum length of 30 bp and minimum mean quality

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score of 20 were kept. Reads with ambiguous bases were discarded. All sequencing cycles

were maintained and the adapter trimmer was turned off.

The filtered FASTQ reads (50 bp single end) from each sample were mapped to the U.

gibba published genome (9) using the software TopHat v. 2.0.10 (29). Minimum intron 55

length was set 40 bp and the maximum intron length was set to 100,000 bp. Mapped reads

were assembled into transcripts using cufflinks v. 2.1.1 (30). The min-isoform-fraction option

was used and set to 0.05, and intron size was set to a maximum of 100 kb. Option –u was

used to correct for reads mapping to multiple locations. Transcript expression was normalised

by the upper quartile of the number of fragments mapping to each gene. The new bias 60

detection and correction algorithm was run to improve accuracy of transcript abundance

estimates. The transcript assemblies were merged into a single global transcript list using the

cuffmerge tool from cufflinks, following default settings.

The global transcript file generated in the previous step was used to calculate each

transcript’s expression value. Normalised expression values (FPKM) were calculated for each 65

transcript using the cuffdiff tool in the cufflinks package (31).

In situ hybridisation

In situ hybridisation was performed as previously described (33) with minor

modifications: The pronase digestion step was performed at 37ºC, the two probes were

hydrolysed for 60 minutes, hybridisation was overnight at a range of temperature from 48.5ºC 70

to 50ºC and staining and detection was over 60h. To produce digoxigenin labelled antisense

RNA probes, coding sequences for UgPHV1, UgFIL1, UgKAN1, UgKAN2 and UgKAN6

were synthesized (Lifetech) and cloned between T7 promoters using Golden Gate

technology. The cloned fragments were amplified using forward-specific primers (UgPHV1

5’- AGTCTGGAGCTTTTCCGAGC-3’, UgFIL1 5’-CTTCCTTCCCATTTGCAGCG-3’, 75

UgKAN1 5’-CTTCAGTGGCGGAAACCAAC-3’, UgKAN2 5’-

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ATCACGGGCTTCACAGATCG-3’, UgKAN6 5’- GGCCGCTCTCGTTTCTATCT-3’) and

a vector-specific primer. These yielded a 657bp fragment for UgPHV1, 1038bp for UgFIL1,

968bp for UgKAN1, 791bp for UgKAN2 and 821bp for UgKAN6. PCR products were

purified and processed for hybridisation as previously described (32). Results are shown for 80

UgPHV1, UgFIL1 and UgKAN1. The probes for UgKAN2 and UgKAN6 generated similar

results to UgKAN1.

Plant transformation

The p35S::KANR_pHS18.2::CRE_p35S:LoxP-eGFP-LoxP-UgPHV1-CFP construct was

assembled by Golden Gate cloning (33), using the complete coding sequence of UgPHV1 85

with domesticated sites to remove BsaI, BpiI and DraIII restriction sites and render the

putative microRNA binding site resistant to cleavage. U.gibba tissue was grown on solid ½

MS (4.4g/L MS, 25g/L sucrose) before transfer to solid callus-inducing media (½ MS, 1

mg/L 6-Benzylaminopurine (BA, Sigma-Aldrich, B3408) and 0.5 mg/L 1-Naphthaleneacetic

acid (NAA, N0640)) for 3 to 6 weeks. Agrobacterium tumefaciens (GV3101) was 90

transformed with a binary plasmid containing the construct described above and used as a

vector for transformation of callus tissue. The strain was grown, pelleted and resuspended at

0.1-0.2 OD in liquid callus media with 20 mg/L Acetosyringone (AC, Sigma-Aldrich,

D134406). Callus tissue was infected by vacuum infiltration for 1 minute followed by co-

culture in solid callus media with 20 mg/L AC for 3 days at 23ºC in the dark. Co-culture 95

tissue was transferred to solid callus media with 250mg/L CEF (Cefotaxime, Sigma Aldrich,

C7039) for 2 weeks to kill the Agrobacterium then transferred to same media with 150mg/L

G418 (Sigma Aldrich, A1720) for selection of transformed tissues. Transformation of 16

independent plants was confirmed after 4 weeks by presence of GFP fluorescence. All 16

lines had wild-type phenotype and 4 of these showed consistent phenotypes when heat-100

shocked. Single transgene insertion in these lines was checked by copy number analysis using

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qPCR (iDNA Genetics) with primers for the nptII resistance gene and a single copy control

gene, and two lines were used in subsequent experiments.

Induction experiments

For induction experiments, HS-UgPHV1 plant tissue was grown under standard 105

conditions (25°C, with 16-hour light and 8-hour dark cycles as described above), and 1cm-

long sections of including the apical spiral were transferred to liquid media in 6-well plates

and heat-shocked for 12 minutes at 48ºC in a water-bath, before being transferred back to

standard growth conditions. Plants in were imaged at daily intervals as described below.

Control plants were grown and imaged in the same except for the heat-shock treatment. 110

The sizes of leaf primordia within the spiral at the time of induction were inferred as

follows: Node 0 was defined as the node bearing the smallest visible leaf outside of the spiral

(mean leaf length = 3mm, n=10, Table S2). The number of leaves initiating per day was

counted and used to calculate the average plastochron (12 hrs, n=18, Table S3). The

plastochron and leaf size at node 0 were then used in combination with the growth rate of 115

leaves, calculated from tracking the growth over 24 hours (Data S1), to infer the size of

leaves on younger nodes (-1 to -4; Table S4). The growth equation for leaves was

y=1.647e0.0353t where y is leaf length in millimeters and t is time in hours (Data S1).

The sizes of trap primordia within the spiral at the time of induction were inferred as

follows: The size of traps on node 0 at the time of induction (time 0) were measured (mean= 120

201m, n=9; Table S5), and the plastochron (12 hours; Table S2), and trap growth equation

(y=9.86e0.018x; taken from 7) used to infer the size of traps on younger nodes (-1 to -4; Table

S6).

Imaging

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Live plant tissues were imaged in water using a Leica M205C stereomicroscope with 125

Leica DFC495 camera. In situ hybridised material was imaged using a Leica DM6000 and a

Zeiss Axio Imager. For confocal imaging, samples were fixed in 10% acetic acid and 50%

methanol and stained using modified pseudo-Schiff propidium iodide (mPS-PI) protocol (35).

Imaging was performed on a Leica SP5-II confocal microscope and mPS-PI stained tissues

were excited with a 488-nm helium–neon laser and fluorescence was collected between 485–130

565nm.

In situ and optical images from live tissue were analysed using ImageJ software

(http://imagej.nih.gov/ij/). Confocal image Z-stacks were saved as .png format image

sequences using ImageJ software (http://imagej.nih.gov/ij/), for visualization, measurement,

virtual dissection and volume rendering with VolViewer software 135

(http://cmpdartsvr3.cmp.uea.ac.uk/wiki/BanghamLab/index.php/VolViewer). Measurements

were taken as shown in Fig. 1 and Fig. S1.

Computational Modelling

All 3D and 2D models used GFtbox (14), a toolkit implemented in Matlab. It is

available for downloading from (http://sourceforge.net/projects/gftbox/). 140

We used the growing polarised tissue (GPT) modelling framework, in which tissue is

treated as a continuous volume of material (14). We assume that for each region of tissue,

there is a specified growth rate that defines how much, and in which orientations, that region

would grow in mechanical isolation from neighbouring tissue. Because of the constraints of

mechanical connectivity, in general, there will be no deformation field in which every region 145

of the tissue achieves its specified growth. Thus, resultant growth (i.e. how each region grows

in the context of mechanical constraints) will therefore, typically differ from specified

growth. The difference between the specified and resultant growth is the residual strain, and

produces a proportionate residual stress. The actual deformation resulting from the field of

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specified growth is taken to be whatever shape minimizes the residual strain energy. 150

Residual strain is assumed to dissipate after each growth step, reflecting the irreversible

plastic flow involved in plant growth (35).

The models have two coordinated networks: the Polarity Regulatory Network (PRN)

which sets up the polarity fields, and the Growth Regulatory Network (KRN) which defines

specified growth rates. The resultant growth depends on specified growth rates and 155

mechanical constraints.

Description of 3D models

The initial state is a hemispherical mesh of unit radius consisted of 92,096 finite

elements, which were not subdivided during the simulation. A latitudinal and longitudinal

grid was superimposed on the initial hemisphere to show the deformation of mesh during 160

growth. All models were run for 12 time units.

The adaxial-abaxial system was implemented by identity factors ADAXIAL (AD, blue

in Fig. 4D), ABAXIAL (AB, brown in Fig. 4D). AD and AB domains each generated a

diffusible signal. MIDPLANE (MIP, green in Fig. 4D), was activated where both the AD

and AB signals fell below a threshold value. An alternative implementation would be to have 165

MIP activated when the AD signal is above a threshold value, but in absence of AD identity.

To specify the growth orientations in three-dimensional space, we defined two polarity

fields – orthoplanar polarity field and proximodistal polarity field. The orthoplanar polarity

field was established by a diffusible morphogen POL generated by a source (plus organiser)

at the surfaces of mesh, and removed at a sink (minus organiser) activated by MIP. Other 170

implementations for an orthoplanar polarity field are also possible, such as the plus organiser

being on the adaxial surface and the minus organiser on the abaxial surface or vice versa.

The proximodistal polarity field was established by another diffusible morphogen POL2

generated by a source at the base of the primordium (magenta, Fig. 4A), and removed at a

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sink at the tip of the primordium (yellow, Fig. 4A). Another possibility is a proximo-marginal 175

polarity field, with the sink at the intersection between MIP and the surface of the

primordium. Specified growth rates parallel to the orthoplanar polarity field were named Kop

(kpar in GFtbox), growth rates parallel to the proximodistal polarity field named Kpd (kpar2

in GFtbox), and growth rates perpendicular to both Kop and Kpd named Kper (kper in GFtbox).

Specified growth rates per time unit for the different models were: 180

Fig. 4, A to C

Kpd = 0.25

Kop = 0.001

Kper = 0.001

Fig. 4, D to G, Fig. 4, J to P, Fig. S7, A to B, D to E and G to H, and Fig. S8, A to B 185

Kpd = 0.25

Kop = 0.001

Kper = 0.2

Fig. 4, H to I, and Fig. S7, C and F

Kpd = 0.25 190

Kop = 0.001

Kper = 0.05

Fig. S8C

Kpd = 0.3

Kop = 0.001 195

Kper = 0.15

Fig. S9, A and B

Kpd = 0.25

Kop = 0.001

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Kper = 0.2 * inh (10, ipetiole), 200

where ipetiole denotes the level of factor PETIOLE and inh (z, ipetiole) denotes multiply by 1/ (1

+ zipetiole). The petiole domain is defined by a new morphogen named PETIOLE expressed at

the base of the primordium (red, Fig. S9A).

Fig. S9C

Left to right, 205

1. Kpd : Kper : Kop = 1 : 0.8 (0.08 in the petiole domain) : 0.004

2. Kpd : Kper : Kop = 1 : 0.36 (0.036 in the petiole domain) : 0.003

3. Kpd : Kper : Kop = 1 : 0.2 (0.02 in the petiole domain) : 0.002

4. Kpd : Kper : Kop = 1 : 0.1 (0.01 in the petiole domain) : 0.001

The petiole domain is defined by a new morphogen named PETIOLE expressed at the base of 210

the primordium (red, Fig. S9A).

Fig. S9, D to E

The stalk domain is defined by a new morphogen named STALK expressed at the base of the

primordium (red, Fig. S9D).

Kpd = 0.25 215

Kop = 0.001

Kper = 0.2 * inh (10, istalk),

where istalk denotes the level of factor STALK and inh (z, istalk) denotes multiply by 1/ (1 +

zistalk).

Description of 2D models 220

The initial state was a circular mesh with unit radius (r=1) consisting of 2,400 finite

elements, which were not subdivided during the simulation. AD, AB and MIP domains were

set up as described for the 3D models. A single polarity field (orthoplanar) was established

by a diffusible morphogen POL generated by a source (plus organiser) at the outer rim of the

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mesh and removed at a sink (minus organiser) activated by MIP. Two growth rates were 225

specified: growth rate parallel to the polarity field named Kop (kapar/kbpar in GFtbox);

growth rate perpendicular to the polarity field named Kper (kaper/kbper in GFtbox).

To simulate cell division, we superimposed polygons (number=100) on the initial state to

represent cells. The vertices of polygons were anchored and moved during the tissue growth.

New vertices were introduced as cells according to the rule that the new wall follows the 230

shortest path passing through the cell centre (6). For visualisation purposes, three cell factors

were defined corresponding to the AD, AB and MIP regional identities.

Fig. 4 Q to S

Model was run for 7 time units with specified growth rates per time unit of:

Kop = 0.001 235

Kper = 0.2

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Fig. S1.

Trap Height, Width and Thickness plotted against trap Length. Measurements taken as shown 240

in inset. Color reference matched to Figure 1.

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Fig. S2. 245

HDZIP-III, YABBY and KANADI family members in Utricularia gibba. A-C, Molecular

Phylogenetic analysis by Maximum Likelihood method of HDZIP-III (A), YABBY (B) and

KANADI (C) family members. Circles represent character states for expression pattern of the

genes and presence absence of lamina as indicated in the box. D-F, Heatmap of transcript

RNAseq levels for the candidate genes found in samples of a range of traps sizes and one 250

sample of leaves, displayed in fragments per kilobase of transcript per million mapped reads.

Species used in phylogenetic analysis: Am, Antirrhinum majus; At, Arabidopsis thaliana;

Cci, Cabomba caroliniana; Cco, Coffea canephora; Cfol, Cephalotus follicularis; Ds,

Dawsonia superba; Ed, Equisetum diffusum; Ep, Elaphoglossum peltatum; Gb, Ginkgo

biloba; GSVIV, Vitis vinifera; Or, Osmunda regalis; Os, Oryza sativa; Pg, Pilularia 255

globulifera; Pm, Pseudotsuga menziesii; Pn, Psilotum nudum; Potri, Populus trichocarpa; Pp

or PP, Physcomytrella patens; Ps, Papaver somniferum; Sb, Sorghum bicolor; Sk, Selaginella

kraussiana; Solyc, Solanum lycopersicum; Sp, Sarracenia purpurea; Sr, Streptocarpus rexii;

Ta, Triticum aestivum; Tm, Tropeaoleum majus; Ug, Utricularia gibba; Zm, Zea mays.

260

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Fig. S3.

UgPHV1 and UgFIL1 expression patterns in Utricularia gibba leaf and trap

development. A-D, UgPHV1 expression in leaflet primordia (A,B) and young leaflet (C,D)

in longitudinal (A,C) and cross section (B,D). E-H, UgFIL1 expression in leaflet primordia 265

(E,F) and young leaflet (G,H) in longitudinal (E,G) and cross section (F,H). I-J, Volume

rendering (see ‘Imaging’ in Materials and Methods) of two leaflet developmental stages

imaged by confocal microscopy, with regions colored in according to expression identity

(UgPHV1 in blue and UgFIL1 in yellow). K-P, UgPHV1 expression in traps at representative

developmental stages, in longitudinal (K-O) and cross section (L-P). Q-V, UgFIL1 270

expression in traps at representative developmental stages, in longitudinal (Q-U) and cross

section (R-V). W-Y, Volume rendering (see ‘Imaging’ in Materials and Methods) of three

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trap developmental stages imaged by confocal microscopy, with regions colored in according

to expression identity (UgPHV1 in blue and UgFIL1 in yellow). Lt leaflet in transverse

(cross) section, Ll leaflet in longitudinal section, Lo leaflet in oblique section, Tt trap in 275

transverse (cross) section, Tl trap in longitudinal section. Scale bars, 50 m. Images are

duplicated from main text figures as follows: A, B, E, F, K, L, Q, R, M, O, S and U from Fig.

2 A, B, C, D, E, F, G, H, I, J, K and L respectively and I, J, W and X from Fig. 1 G, H, J and

L respectively.

280

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Fig. S4.

Heat-shock induction of UgPHV1. A-D, Control (uninduced) plants show uniform GFP

expression over 3 days of growth. E-H, Plants that undergo a 10 minute heat-shock induction

of UgPHV1 lose GFP in newly developed tissue after 3 days. I-J, In situ hybridization of 285

UgPHV1 in transverse sections of control (I) and HS-UgPHV1 induced (J) tissues. Induced

developing primordia show a broader domain of UgPHV1 expression. A, C, E and G are

transmitted light images and B, D, F and H are GFP fluorescence. Scale bars: 5 mm (A-H);

100μm (I,J). Large traps often maintain GFP expression in glandular cells, suggesting that the

induction of PHV may not be efficient in older tissues. 290

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Fig. S5.

Induction of UgPHV1 induces radial leaflets. A-F, Confocal micrographs of six different

leaflet primordia in induced plants (10 days after heat-shock) have circular transverse 295

sections. Sections of non-induced leaves are oval at similar sizes (See Fig. 1). Scale bars:

10μm.

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Fig. S6. 300

UgPHV1 induction disrupts trap development. A-B, Plant expressing 35S::UgPHV1 on

day 0 (A) and 7 days after heat-shock induction (B). Nodes are numbered according to their

position at induction (day 0), and color coded for each phenotypic region. Red for nodes with

no traps visible on the stereomicroscope (upper region), blue for nodes with traps visible but

stunted (middle region), and black for nodes with traps unaffected by induction (lower 305

region). The regions of the stolon at day 0 that give rise to these regions are marked in (B) in

the same colours. C-E, Confocal micrographs of small malformed structures that are

observed in the position of traps in the upper region. F-H, Small traps located in the middle

region, visible by stereomicroscope (F,G) and confocal imaging (H). Scale bars: 5mm (A,B);

50μm (C-E, H); 100μm (F,G). Panels A and B are reproduced from Fig. 3 C and D. 310

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Fig. S7.

Robustness of the model to initial primordium shapes. (A to C) Models initiated with an

elliptical primordium shape (hemiellipsoid). Initial (A) and final (B, C) states shown in 315

oblique view. Relative growth rates for (B) (Kpd : Kper : Kop = 1 : 0.8 : 0.004), generate a

flattened shape while growth rates in (C) (Kpd : Kper : Kop = 1 : 0.2 : 0.004), generate an

elliptical cylinder. (D to F) Leaf models initiated with an elongated primordium shape. Initial

(D) and final (E and F) states shown in oblique view. Relative growth rates in (E) (Kpd : Kper :

Kop = 1 : 0.8 : 0.004), generate flattened shape while growth rates in (F) (Kpd : Kper : Kop = 1 : 320

0.2 : 0.004), generate an elliptical cylinder. (G and H) Cup model initiated with an elongated

primordium shape. Initial (G) and final (H) states shown in oblique view. Relative growth

rates in (H) are (Kpd : Kper : Kop = 1 : 0.8 : 0.004). Adaxial (blue), abaxial (brown) and

midplane (green) domains colored. Proximodistal polarity runs from organizers at base to tip

and orthoplanar polarity (black arrows) runs from surface to midplane organizer. Both 325

proximodistal and orthoplanar polarity fields run through the whole primordium but only

orthoplanar polarity field is shown on the cutaway surface, for simplicity. Transverse or

longitudinal sections indicated by color-coded rectangles. Scale bars in arbitrary units.

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330 Fig. S8.

Modified model for trap development in Sarracenia with lid. (A to B) Initial (A) and final

(B) states shown in oblique and cutaway views with adaxial (blue), abaxial (brown) and

midplane (green) domains colored as in Fig. 4M. The distal part of the hemisphere has the

same pattern as in Fig. 4D. Relative growth rates in (B) are (Kpd : Kper : Kop = 1 : 0.8 : 0.004). 335

(C) Final state shown in oblique and cutaway views with modified grow rates (Kpd : Kper : Kop

= 1 : 0.5 : 0.003). (D) Three transverse sections, corresponding to three dissection levels of

the initial stage shown in (A). (E) Three transverse sections, corresponding to three dissection

levels of the final stage shown in (C). Proximodistal polarity (red arrows) runs from

organizers at base to tip and orthoplanar polarity (black arrows) runs from surface to 340

midplane organizer. Both proximodistal and orthoplanar polarity fields run through the whole

primordium but are shown only on the outer surface and cutaway surface, respectively, for

simplicity. Levels of transverse sections indicated by color-coded rectangles. Scale bars in

arbitrary units.

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345 Fig. S9.

Modified leaf and trap models with petiole/stalk. (A to C) Generation of diverse flat leaves

with additional petiole domain (red). Initial state (A) and development stages (B) of a flat leaf

shown in oblique view. (C) Morpho-space of flat leaves by varying Kpd/Kper, see Description

of 3D models in Methods for relative growth rates. (D and E) Generation of a cup with stalk 350

domain. Initial state (D) and development stages (E) states shown in oblique view. Relative

growth rates in (E) (Kpd : Kper : Kop = 1 : 0.8 (0.08 in the petiole/stalk domain) : 0.004). Scale

bars in arbitrary units.

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355

Fig. S10.

Evolutionary scenarios for the generation of diverse leaf forms. (1) Beginning with a

tapering radial cylinder as ground state, an ancestral flat leaf can be generated by introducing

the adaxial-abaxial system and a petiole. This form is similar to what is now seen in many

leaves such as Arabidopsis thaliana illustrated in the top right. (2-4) This flat leaf 360

developmental program can be modified through shifts in gene expression and growth rate

modulation to generate diverse forms. Generation of a filiform leaf by reducing Kper (such as

Utricularia gibba leaflet, (2)). Generation of a cup by restricting the adaxial domain (such as

Utricularia gibba trap, (3)). Generation of a pitcher leaf by restricting the adaxial domain

while retaining a proximal strip of activity (such as Sarracenia flava, (4)). 365

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Species Gene Genbank Reference

Arabidopsis thaliana

ATHB15/AtCNA AT1G52150

TAIR

ATHB-8 AT4G32880

ATML1 At4g21750

AtPHB AT2G34710

AtPHV AT1G30490

AtREV AT5G60690

Cabomba caroliniana CcC3HDZ1 BAJ83628.1 Yamada 2011

Dawsonia superba DsC3HDZ1 AMB49032.1 Yip 2016

Elaphoglossum peltatum EpC3HDZ2 ANC28169.1 Vasco 2016

Equisetum diffusum EdC3HDZ2 AMS37122.1 Vasco 2016

Ginkgo biloba GbC3HDZ1 ABD75306.1 Floyd 2006

Osmunda regalis OrC3HDZ2 AMS37129.1 Vasco 2016

Physcomytrella patens

PpC3HDZ1 ABD75297.1

Yip 2016 PpC3HDZ3 AMB49028.1

PpC3HDZ4 AMB49029.1

Pilularia globulifera PglC3HDZ2 AMS37133.1 Vasco 2016

Pseudotsuga menziesii PmC3HDZ1 ABD75309.1 Vasco 2016

Psilotum nudum PnC3HDZ2 ABD75303.1 Vasco 2016

Sarracenia purpurea SpPHB BAQ19376.1 Fukushima 2015

Selaginella krausiana

SkC3HDZ1 ABD75300.1 Floyd 2006

SkC3HDZ2 ABD75301.1 Floyd 2006

Utricularia gibba

UgPHV1 Scf00004.g913

LANGEBIO v4.1,

named in this study

UgPHV2 Scf00001.g276

UgPHV3 Scf00044.g5034

UgREV1 Scf00042.g4804

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UgREV3 Scf00184.g11794

UgREV2 Scf00450.g17884

UgCNA Scf00691.g20852

Table S1A

List of sequences used in HDZIP-III phylogenetic analysis

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Species Gene Genbank Reference

Antirrhinum majus

AmGRAM AAS10177.1

Golz 2004

AmCRC AAS10180.1

AmINO AAS10181.1

AmPROL AAS10178.1

AmYAB2 AAS10179.1

Arabidopsis thaliana

AtCRC AT1G69180

TAIR

AtYAB2 AT1G08465

AtFIL AT2G45190

AtYAB3 AT4G00180

AtYAB2 AT1G08465

AtINO AT1G23420

AtYAB5 AT2G26580

Cabomba caroliniana

CcCRC BAJ83627.1

Yamada 2011

CcFIL BAJ83625.1

CcINO BAJ83626.1

CcYAB5 BAJ83624.1

CcYAB2 BAJ83623.1

Ginkgo biloba

GbYAB1B CTQ35246.1

Finet 2016 GbYAB2B CTQ35248.1

GbYABC CTQ35250.1

Oryza sativa

OsYABBY1 BAF45802.1 Toribe 2007

OsYABBY2 BAF45803.1 Toribe 2007

OsYABBY4 BAF45805.1 Liu 2007

OsDL XP_015630075.1 Yamaguchi 2004

Papaver somniferum PsYAB3 XP_026389545.1 Vosnakis 2012

Pseudotsuga menziesii PmYABC CTQ35254.1 Finet 2016

Submitted Manuscript: Confidential

26

Sarracenia purpurea SpFIL BAQ19377.1 Fukushima 2015

Sorghum bicolor SbDL BAH83537.1 Ishikawa 2009

Streptocarpus rexii SrGRAM ACL68660.1 Tononi 2010

Triticum aestivum TaDL BAJ54068.1 Ishikawa 2009

Tropaeolum majus TmYABBY AAV74414.1 Gleissberg 2005

Utricularia gibba

UgFIL1 Scf00036.g4336

LANGEBIO v4.1,

named in this study

UgFIL2 Scf00038.g4513

UgFIL3 Scf00297.g14946

UgFIL4 Scf02045.g25594

UgYAB2 Scf00886.g22488

UgCRC Scf00096.g8262

UgINO Scf00100.g8452

Zea mays ZmDL NP_001148730.1 Ishikawa 2009

Table S1B 370

List of sequences used in YABBY phylogenetic analysis

Submitted Manuscript: Confidential

27

Species Gene Genbank Reference

Arabidopsis thaliana

AtATS AT5G42630

TAIR

AtKAN1 AT5G16560

AtKAN2 AT1G32240

AtKAN3 AT4G17695

Coffea canephora

CcKAN1 GSCOCG00024897001

Genoscope v1

CcCYP450 GSCOCG00024897001

Cephalotus follicularis

Cfol−v3−23798

Fukushima 2017 Cfol−v3−01995

Cfol−v3−19237

Physcomitrella patens

PP00114G00880

Phytozome PP00009G01280

PP00049G00800

Populus trichocarpa

Potri.014G037200.1

Phytozome

Potri.002G130200.1

Potri.017G137600.1

Potri.004G082400.1

Potri.012G042100.1

Potri.015G031600.1

Potri.001G137600.1

Potri.003G096300.1

Solanum lycopersicum Solyc11g011770.1.1

Phytozome

Solyc08g005260.1.1

Vitis vinifera

GSVIVT01026319001

Phytozome GSVIVT01024383001

GSVIVT01013085001

Utricularia gibba UgKAN1 Scf00203.g12422

Submitted Manuscript: Confidential

28

UgKAN2 Scf00280.g14523

LANGEBIO v4.1,

named in this study

UgKAN3 Scf00151.g10673

UgKAN4 Scf00082.g7466

UgKAN5 Scf00335.g15719

UgKAN6 Scf00022.g3054

UgKAN7 Scf02105.g25653

UgKAN8 Scf00665.g20623

Zea mays

ZmKANADI1 ACH68606.1

Candela 2008 ZmKANADI3 ACH56978.1

ZmMWP1 ACH56977.1

Table S1C

List of sequences used in KANADI phylogenetic analysis

Submitted Manuscript: Confidential

29

Length of leaf on node 0 measured in m at induction

2476

3563

2619

2863

1823

1929

1645

3937

4573

5116

Average

3054.4

Table S2.

Length (m) of leaves on node 0 at induction. 375

Submitted Manuscript: Confidential

30

Plant How many nodes on day 7… … since day Nodes per day Plastochron in hours

1a 9 1 1.50 16

1b 14 0 2.00 12

1d 14 0 2.00 12

1e 6 1 1.00 24

1f 11 1 1.83 13

2a 17 0 2.43 10

2b 12 1 2.00 12

2c 17 0 2.43 10

2d 10 1 1.67 14

2e 17 0 2.43 10

2f 14 0 2.00 12

3a 8 3 2.00 12

3b 20 0 2.86 8

3c 15 1 2.50 10

3d 16 0 2.29 11

3f 14 0 2.00 12

Average: 2.06 12.35

Table S3.

Calculation of plastochron. Number of nodes initiated over growth period, used to calculate

the number of nodes per day and then the period of one plastochron in hours. 380

Submitted Manuscript: Confidential

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Node Length of leaf (m)

-4 570.99

-3 863.92

-2 1315.48

-1 2004.46

Table S4.

Predicted sizes of leaves on nodes inside spiral on the day of induction (day 0). These were

calculated from Table S2, S3 and Data S1 as described in Methods.

Submitted Manuscript: Confidential

32

Length of trap on node 0 (m) at induction

280

184

162

300

129

210

245

98

Average

201

385

Table S5.

Size (m) of traps on node 0 at induction.

Submitted Manuscript: Confidential

33

Node Length of Trap (m)

-4 85.28

-3 105.67

-2 131.31

-1 160.03

Table S6.

Predicted sizes of traps on nodes inside spiral on the day of induction (day 0). These were

calculated from Table S2, S5 and Data S1 as described in Methods. 390

Submitted Manuscript: Confidential

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Movie S1

Movie of model shown in Fig. 4, A to C.

Movie S2

Movie of model shown in Fig. 4, D to G.

Movie S3 395

Movie of model shown in Fig. 4, H and I.

Movie S4

Movie of model shown in Fig. 4, J to L.

Movie S5

Movie of model shown in Fig. 4, M and N. 400

Movie S6

Movie of model shown in Fig. 4, Q to S (left panel).

Movie S7

Movie of model shown in Fig. 4, Q to S (right panel).

Movie S8 405

Movie for model shown in Fig S8 A, B

Movie S9

Movie for model shown in Fig S8 C

Movie S10

Movie for model shown in Fig S9 A, B 410

Data S1

Data used to calculate leaf growth rates. Length of leaves at node 0 was measured over a 48

hour period and used to calculate a growth rate. Growth rate reduced as leaves reached

maturity, so for calculations of primordia size the growth of leaves over the first 24 hours. 415

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