Supporting Information, including Supporting Tables and Supporting Figures

Adaptive sequence evolution is driven by biotic stress in a pair of orchid species

(Dactylorhiza) with distinct ecological optima

Francisco Balao, Emiliano Trucchi, Thomas Wolfe, Bao-Hai Hao, Maria Teresa Lorenzo,

Juliane Baar, Laura Sedman, Carolin Kosiol, Fabian Amman, Mark W. Chase, Mikael

Hedrén, Ovidiu Paun

METHODS

RNAseq library construction and sequencing

The leaf material was fixed in RNAlater in the same morning, and was left at 4 °C overnight,

before transferring it to -80 °C for storage until required. Total RNA was extracted using the

RNeasy Plant Mini Kit (Qiagen) following the manufacturer’s instructions. The purified RNA

was stored at -80 °C. The concentration of RNA extracts was first measured with a NanoDrop

ND-1000 Spectrophotometer (Thermo Scientific) and its purity estimated according to the

wavelength ratio of A260/280. The quantification and quality of the RNA was then confirmed

with an RNA 6000 Nano-kit on a 2100 BioAnalyzer (Agilent Technologies). The ribosomal

RNA was depleted with the RiboMinusTM

Plant Kit for RNA-Seq (Invitrogen) following the

manufacturer’s protocol. RNA fragmentation was done by hydrolysis for 2 min at 94 °C and

used 2 µl 10x buffer RT (SuperScript III First-Strand Synthesis System for RT-PCR,

Invitrogen) plus 75mM MgCl2 in 12 µl concentrated RNA. After a cleaning step with an

RNeasy spin column (from an RNeasy Plant Mini Kit, Qiagen), the first cDNA strand has

been synthesized with the Superscript III First-Strand Synthesis System for RT-PCR

(Invitrogen) and random hexamers. Surplus dNTPs have been eliminated with a Mini Quick

Spin Column for DNA (Roche). The second strand cDNA synthesis has been performed with

dUTPs, to allow for strand distinction upon Illumina sequencing. After a final clean up step

with a MiniElute Reaction Cleanup kit (Qiagen) and quantification using a Quant-iT

Picogreen dsDNA assay in a NanoDrop Fluorospectrometer ND-3300 (Thermo Scientific),

the final RNAseq library preparation (using NEBnext Ultra RNA kit and an UGdase

Balao et al. SI - Transcriptomic divergence in Dactylorhiza

2

treatment) and directional Illumina sequencing as 100bp paired-end reads was performed at

the CSF Vienna (www.csf.ac.at/ngs). Samples fB4, iB0 and iS4 (Table 1) were sequenced as

half lanes, whereas sample fP7 has been sequenced as two half lanes. Six other samples were

sequenced as full lanes.

After controlling read quality with FastQC v0.11.2 (available from http://www.bioinformatics

.babraham.ac.uk/projects/fastqc/), filtering of the raw reads was done with Trimmomatic

v0.32 (Bolger et al., 2014) with default settings, except for increasing the threshold for

average quality per base to 20 when scanning the reads over four-base sliding windows, and

only retaining adaptor-free, high-quality reads that are at least 50 bases long. After quality

filtering 78.6% of the read pairs were retained (Table S1). We further used only the paired-

end filtered reads, as the single-end files produced by Trimmomatic contained only a

negligible amount of orphan reads. The reads were finally filtered against a database of all

whole genome Virus and Bacteria from NCBI (ftp://ftp.ncbi.nlm.nih.gov/genomes/Bacteria/

and ftp://ftp.ncbi.nlm.nih.gov/genomes/Viruses/ version 2014-07-03) by using Bowtie2 v2.2.3

(Langmead and Salzberg, 2012) with default setting. Less than 1% of reads were found to be

potential contamination from viruses or bacteria.

De novo transcriptome assemblies and annotation

The transcriptome of each sequenced accession was individually assembled from its cleaned

reads using Trinity version r20140413 (Grabherr et al., 2011; Haas et al., 2013) with a kmer

setting of 25, strand-specificity (i.e., FR orientation) and retaining only contigs with a

minimum length of 200 bp. One individual reference per species has been then retained based

on the total length, N50 and the percentage of reads that uniquely mapped back to each

reference by using the CLC Genomics Workbench v7.5 (Qiagen) with strand-specificity, a

minimum similarity fraction of 0.95 over at least 0.95 of the length and

mismatch/insertion/deletion costs of 2/3/3. A multi-individual assembly was also attempted

for each species, however this failed to extend the N50 length significantly or increase the

back-mapping rate (results not shown), and was discarded to avoid the risk of chimeric

contigs.

Ecologically-relevant genes are expected to be generally expressed at low rates, and hence

may be missed in individual assemblies. To account for this, we have retained for each

accession the un-mapping reads when aligning them to the selected species-specific assembly,

and these reads were further combined per species and were re-assembled with Trinity with

Balao et al. SI - Transcriptomic divergence in Dactylorhiza

3

the same settings as above. The D. fuchsii, and, respectively, D. incarnata “lowly expressed”

contigs were then added to their specific reference. A common reference for the two species

has been constructed by pooling together the two individual references. We finally removed

redundancy from each of the three constructed references by using the clustering algorithm of

CD-HIT-EST (Fu et al., 2012) with a global identity of 80% over at least 70% of the length of

the shorter sequence (i.e., -aS 0.7) and by comparing only the 5’-3’ strands (i.e., -r No). The

final references are hereafter referred to as “f_reference”, “i_reference” and the Dactylorhiza

(i.e., “f_i”) reference.

The individual Trinity assemblies contained between 7,686 (fA6, Table S1) and 70,193

contigs (iS8), with N50 ranging from 303 (for fA6) to 472 bp (for fB4). Based on the richness

(i.e., the total length, Table S1), contiguity (i.e., N50 estimates) and completeness (i.e.,

mapping rate) we retained the assemblies of fB5 and iS8 as the best representative

transcriptomes for D. fuchsii and respectively D. incarnata. In order to get better

representation of the lowly expressed genes, species-specific Trinity assembly of all D. fuchsii

and, respectively, D. incarnata reads that did not map to these reference transcriptomes were

performed and resulted in 3,113 and 2,064 transcripts for D. fuchsii and, respectively, for D.

incarnata. After combining the different assemblies and removing redundancy as explained

above, the final combined fi_reference contains 33.8 Mbp within 101,010 transcripts (52.3%

originated from D. incarnata and 47.7% from D. fuchsii; Table S2). This transcriptome will

be of high value for further studies of gene expression alterations following whole genome

duplication events in Dactylorhiza.

Functional annotation analyses of the fi_reference were performed in Blast2Go v.3.2.7

(Conesa and Götz, 2008) using cloud-based NCBI BLAST+ searches against the

Viridiplantae database v. 17.01.2016 under the BLASTX algorithm and a minimum e-value

of 10-6

. Blast2Go was further used to assign gene ontology (GO) terms to the contigs, to

identify signatures of protein domains using InterProScan (Quevillon et al., 2005), and to

annotate non-coding RNAs and cis-regulatory elements by performing Rfam scans against

Xfam servers. Of the 101,010 transcripts in the fi_reference, 42.2% had significant BLASTX

hits in Blast2Go. After mapping these contigs and integrating InterProScan results, 30,046

contigs were successfully annotated with GO terms (Fig. S1). In addition, 121 contigs

received annotations from the Rfam scan, with 80 of them representing an rRNA type and 16

intronic regions.

Balao et al. SI - Transcriptomic divergence in Dactylorhiza

4

Small RNA library preparation and sequencing

Isolation of RNA with smRNA enrichment was performed with the mirVana miRNA

Isolation Kit (Life Technologies) following the manufacturer’s instructions. Up to 90 mg of

the same tissue fixations as for the RNAseq experiment were used. The concentration of the

raw smRNA isolates was measured with a Quant-iT Picogreen dsDNA assay (Invitrogen) and

a NanoDrop Fluorospectrometer ND-3300 (Thermo Scientific). The quantification and quality

of the RNA isolate was then confirmed with a 2100 Bioanalyzer (Agilent Technologies) using

a small RNA analysis kit. The RNA extracts were denatured with loading buffer at 95°C for 3

min and further purified by gel size selection in a XCell SureLock Mini-Cell (Life

Technologies) using a microRNA Marker (NEB) and 15% TBE-Urea pre-casted gels (Life

Technologies) stained with 2x SYBR Green II (Life Technologies) for 1 h at RT. The smRNA

samples were re-isolated from gel slices by overnight incubation in 0.3M NaCl at 4 °C,

followed by filtration through Ultrafee-MC Durapore Filter Units 0.22µm (Merck Millipore)

and precipitation for 2 h at -20 °C in 2.5x volume 100% ethanol, together with 1 µl

GlycoBlue (Ambion). After resuspension in RNase-free water the smRNA isolates were again

checked on a Bioanalyzer small RNA chip. For individual iB6 the isolate has yielded

insufficient smRNA quantity and as no tissue was further available, the sample has not been

processed further. The smRNA libraries were prepared with the NEBNext Multiplex Small

RNA Library Prep Set for Illumina (Set 1 and 2, NEB) following the manufacturer’s protocol,

except for diluting the adapters 1:2 prior to use due to low sample concentration (130<mg)

and using only 1.5 µl instead of 2.5µl of the primers. The samples were purified on a 1.5 %

agarose gel with 1x TBE buffer. The DNA was extracted from the gel slices with a MinElute

Gel Extraction clean-up Kit (Qiagen) and eluted in water. Illumina sequencing as a half-lane

of 50 bp single-end reads was performed at the CSF Vienna (www.csf.ac.at/ngs). The

individual samples were demultiplexed with the BamIndexDecoder tool of the Illumina2Bam

software collection (available from http://gq1.github.io/illumina2bam/) and adapter sequences

have been removed using Trimmomatic v0.30 (Bolger et al., 2014). After demultiplexing and

adapters removal, the total number of reads longer than 15 nucleotides averaged ca. 9.9

million (std. 5.3 million) across all samples of the two species (D. fuchsii mean = 13.2, std. =

4.2; D. incarnata mean = 5.8, std. = 3.4).

Balao et al. SI - Transcriptomic divergence in Dactylorhiza

5

REFERENCES Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer for Illumina

sequence data. Bioinformatics 30: 2114-2120.

Conesa A, Götz S. 2008. Blast2GO: a comprehensive suite for functional analysis in plant

genomics. Int. J. Plant Genomics 2008: 619832.

Fu L, Niu B, Zhu Z, Wu S, Li W. 2012. CD-HIT: accelerated for clustering the next-

generation sequencing data. Bioinformatics 28: 3150-3152.

Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, et al. 2011. Full-length

transcriptome assembly from RNA-Seq data without a reference genome. Nat.

Biotechnol. 29: 644-652.

Haas BJ, Papanicolaou A, Yassour M, Grabherr M, Blood PD, et al. 2013. De novo

transcript sequence reconstruction from RNA-seq using the Trinity platform for

reference generation and analysis. Nat. Protoc 8: 1494-1512.

Langmead B, Salzberg SL. (2012). Fast gapped-read alignment with Bowtie 2. Nature

Methods 9: 357-359.

Quevillon E, Silventoinen V, Pillai S, Harte N, Mulder N, Apweiler R, Lopez R. 2005.

InterProScan: protein domains identifier. Nucleic Acids Res. 33: W116-W120.

Balao et al. SI - Transcriptomic divergence in Dactylorhiza

6

Table S1. Summary of individual RNAseq Trinity assemblies. The uniquely and,

respectively, total mapping rates refer to mapping with CLC Genomics Workbench of the

reads of one sample to the assembly produced by those respective reads with Trinity.

Accession Filtered pairs

of reads (M)

Transcripts Contigs Total

Megabases

N50

(bp)

GC

(%)

Unique/Total

mapping (%)

fA6 183.9 7,686 6,163 2.5 303 50.3 -

fP1 40.5 15,373 13,348 5.9 391 51.0 85.4/85.7

fP7 112.5* 48,895 35,771 19.0 385 52.9 26.4/47.9

fB4 91.4 61,109 42,999 26.4 472 49.7 18.1/79.9

fB5 190.3 62,338 46,340 23.0 361 50.4 86.8/87.3

iA6 168.1 24,785 19,818 8.3 311 51.2 95.0/95.2

iS4 73.6 31,404 24,208 12.2 391 50.8 35.9/81.2

iS8 148.7 70,193 53,126 23.7 322 51.2 86.2/86.6

iB6 74.4 17,125 14,411 5.9 328 52.7 86.6/86.8

iB0 66.8 29,697 22,604 12.2 431 50.7 16.3/79.2

*summed over two half lanes

Table S2. Summary of the final reference transcriptomes. Species Assembly Transcripts Contigs Total base pairs N50

D. fuchsii f_reference 54,596 43,834 19,420,738 356

D. incarnata i_reference 61,841 49,960 20,394,752 314

Combined fi_reference 101,010 88,461 33,794,033 319

Supporting Figure S1. Map of the native localities of the Dactylorhiza samples analysed here. The samples were transplanted in a common garden at least one growing season before the material was fixed for analyses.

(e)0 1,000 2,000 3,000 4,000

#Seqs

Oxidoreductases

Transferases

Hydrolases

Lyases

Isomerases

Ligases

(a)0 20,000 40,000 60,000 80,000 100,000

Total Sequences

With InterProScan

With Blast Hits

With Mapping

With Annotation

(d)

(c)

(b)0 2,000 4,000 6,000 8,000 10,000

Elaeis guineensisPhoenix dactylifera

Musa acuminata mal.Vitis vinifera

Nelumbo nuciferaOryza sativa Japonica

Jatropha curcasZea mays

Vigna angularisGlycine max

Erythranthe guttataBeta vulgaris vulgaris

Citrus sinensisGossypium raimondii

Brassica napusTheobroma cacao

Prunus persicaMalus domestica

Nicotiana sylvestrisSesamum indicum

Amborella trichopodaEucalyptus grandis

Morus notabilisPrunus mumeSetaria italica

Ricinus communisMedicago truncatulaOryza sativa Indica

Cicer arietinumothers

(f)

Supporting Figure S2. Results of annotation analyses of the combined Dactylorhiza fi_reference with Blast2Go. (a) Data distribution. (b) Blast top-hit species distribution. (c) Sequences with length(x) annotated. (d) GO distribution by level (2) - top 20. (e) Enzyme code distribution. (f) Rfam biotypes seqeunce distribution.

1000 2000 3000 4000 5000 6000 7000 8000Length (bp)

20%

30%

40%

50%

60%

70%

80%

90%

100%

10%0 4,000 8,000 12,000 16,000 20,000

#Seqs

metabolic processcellular process

single-organism processbiological regulation

regulation of biological processresponse to stimulus

CC organization/biogenesislocalization

developmental processmulticellular organismal process

signalingreproduction

reproductive process+ regulation of biological process

multi-organism process- regulation of biological process

growthimmune system process

detoxificationrhythmic process

cellcell part

organellemembrane

membrane partmacromolecular complex

organelle partmembrane-enclosed lumen

cell junctionsymplast

extracellular regionsupramolecular fiber

virionvirion part

nucleoidextracellular region part

other organismother organism partextracellular matrix

extracellular matrix component

bindingcatalytic activity

transporter activitystructural molecule activity

nucleic acid binding TF activitymolecular function regulator

molecular transducer activityelectron carrier activity

antioxidant activityTF activity, protein binding

nutrient reservoir activitymetallochaperone activity

protein tagtranslation regulator activity

Biological Process:

Molecular Function:

Cellular Component:

0 10 20 70 80Sequences

IntronGene

Gene; snRNA; snoRNA; HACA-boxGene; rRNA

Gene; snRNA; snoRNA; CD-boxCis-reg; frameshift_element

Cis-reg; riboswitchCis-reg

Gene; snRNA; splicingGene; miRNA

0.1D. incarnata B0

D. fuchsii B5

D. incarnata S4

Orchis italica

D. incarnata A6

D. incarnata B6

D. fuchsii B4

D. fuchsii P1

D. incarnata S8

D. fuchsii P7

8 8

100

89

90

100

100

100

Supporting Figure S3. Maximum-Likelihood RAxML phylogenetic tree based on 449,518 high-quality cSNPs, illustrating relationships between the Dactylorhiza accessionsanalysed. Bootstrap percentages are indicated.

−0.4 −0.2 0 0.2 0.4

−0.6

−0.2

0.2

0.6

V1 (66.4%)

V2 (5

.9%

)

iB6

iB0

iS4

iS8

iA6

fP1

fP7

fB5

fB4

-0.2

-0.4

0

V1 (53.9%)

V2 (1

2.0%

) fP1

fP7

fB5

fB4

fA6

iB0

iS4

iS8

iA6

0 0.2

0.4

V1 (55.8%)

V2 (1

2.5%

)

(a) (b)

(c)-0

.6-0

.40.

4

0-0.2 0.40.2

fP1

fP7fB5

fB4

fA6 iB0

iS4

iS8

iA6

(d)-0

.20

0.2

0.4

0.2

-0.2

Supporting Figure S4. Transcriptome variation within and between D. fuchsii and D. incarnata. (a) SNPRelate PCA on 129,511 filtered biallelic cSNP variants. (b) PCA showing the largest components of variance in gene expression as uncoveredby edgeR. (c) PCA drawn with EDAseq on the patterns of expressed miRNA and tasiRNA (i.e., analysis of 20-22nt small RNAs). (d) EDAseq PCA on patterns of siRNA expression (i.e., analysis of 24nt small RNAs).

−6 −3 0 3 6

V1 (70.5%)

V2 (1

3.3%

)−4

−20

24

iB6

iB0iS4

iS8

iA6

fP1

fP7

fB5

fB4

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

00.

10.

20.

3

D. fuchsii vs. O. italica

Ks

Ka

D. incarnata vs. D. fuchsii

00.

10.

20.

3K

a

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

Ka/Ks > 1Ka/Ks ≤ 1

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

00.

10.

20.

3 D. incarnata vs. O. italicaK

a

Ks

D. fuchsiivs.

D. incarnata

D. fuchsiivs.

O. italica

D. incarnatavs.

O. italica

Ka

00.

050.

100.

15

D. fuchsiivs.

D. incarnata

D. fuchsiivs.

O. italica

D. incarnatavs.

O. italica

00.

20.

40.

6

Supporting Figure S5. Representation of the synonymous and non-synonimous sustitution rates. (a) KaKs plots of pairwise analyses. The red dots represent the putative CDS showing signals of positive selction; the grey dots indicate putative CDS that show signals of purifying selction. (b) Boxplots of the synonymous substitution rates (Ks) and the non-synonymous substitution rate (Ka) in intergeneric (blue) and intrageneric (green) comparisons. Note the different scales on the Y-axes.

(a)

(b)

methionineadenosyltransferase Ac

molybdate ion transmembranetransporter Ac

small conjugating protein Bi

dihydrokaempferol 4−reductase Ac

ubiquitin Bi potassium iontransmembrane transporter Ac−2.0

−1.6

(a)

semantic space Y

sem

antic

spa

ce X

copper ionBi

secondaryactive

transmembranetransporter

delta 3−trans−hexadecenoicacid phosphatidylglycerol

desaturase

methionineadenosyltransferase Ac

ammonia−lyase Ac

primary amineoxidase Ac

catechol oxidaseAc

oxidoreductase activity,oxidizing metal ions

hexosetransmembrane transporter

−2.0−1.5

(b)

Supporting Figure S6. Enriched molecular functions (p < 0.01) with elements targeted by positive selection in D. fuchsii (a), and in D. incarnata (b). Bubble size is proportional tothe frequency of the respective term in the public GO database. The colour represents the log10value of the significance of the Fisher’s tests of enrichment, corresponding to the indicated scale. Ac - activity; Bi - binding.

136672

381

200

13157

272

1237

edgeR DESeq2

baySeq

Supporting Figure S7. Differential gene expression analysis of D. incarnata versus D. fuchsii. (a) edgeR drawn MA-plot of the relative expression levels. The x-axis shows the log2 of the counts per million of mapped reads (CPM) for each cluster. The y-axis shows the log2 of the expression fold change (FC) for each transcript. The red dots represent the clusters that were DE between the two Dactylorhiza species. (b) Intersection of the DE results of the three tests performed at a level of false discovery rate (FDR) of 0.05. (c) Histogram of the log2 of the expression fold change values. (d) Heat map of the top 50 most differentially expressed clusters between D. incarnata andD. fuchsii.

(a) (b)

−10 −5 0 5 10

05K

10K

15K

logFC

Freq

uenc

y

(c) (d)

structural constituentof ribosome

aspartic−typeendopeptidaseAc

structural moleculeAc

waterchannel Ac

chlorophyllBi

ribulose−1,5−bisphosphatecarboxylase/oxygenaseactivator Ac

alliin lyase Ac

methionine−tRNAligase Ac

intramoleculartransferase

Ac, phosphotransferases

RNA−directedDNA polymerase Ac

pigment Bi

tetrapyrroleBi

nucleicacid Bi

RNA−DNA hybridribonuclease Ac

glycerol transmembranetransporter Ac

watertransmembrane

transporter Ac

peptidaseAc

sem

antic

spa

ce Y

−6

−3

structural constituentof ribosome

RNA−DNA hybridribonuclease

Ac structural molecule Ac

tetrapyrrole Bi

allene−oxide cyclase Ac

inositol 3−alpha−galactosyltransferase Ac

retinal dehydrogenaseAc

magnesium chelatase Ac

oxidoreductase Ac

pigment Bi

chlorophyll Bi

naringenin−chalconesynthase Ac

pantetheine−phosphate

adenylyltransferase Ac

GTP Bi

minus−end−directedmicrotubule motor Ac

hexosaminidase Ac

proteindisulfide

oxidoreductase Ac

semantic space X

−5

−3

Supporting Figure S8. Enriched molecular functions (p < 0.01) that are affected by overexpression in D. fuchsii (a) and D. incarnata (b). The log10 of the p-value of the enrichment test is shown by the colour of the bubbles, according to the indicated scale. The size of thebubbles is proportional with the frequency of that particular GO term in the public GO database. Ac, activity; Bi, binding.

(a)

(b)

semantic space y semantic space y

Supporting Figure S9. Enriched (p < 0.01) biological processes (a-b) and molecular functions (c-d) with elements differentially targeted (FDR < 0.05) by miRNAs/tasiRNAs between D. fuchsii and D. incarnata. (a, c) Enriched GO terms of genes with increased mi/tasiRNA targeting in D. fuchsii. (b, d) Enriched GO terms of genes with over-regulation by mi/tasiRNAs in D. incarnata. Bubble size is proportional to the frequency of the respective term in the public GO database. The colour represents the log10 value of the significance of the Fisher’s test of enrichment, corresponding to the indicated scale. C, compound; Ac, activity; SAc, synthase activity; Bi, binding; Sy, synthesis; Pr, process; MPr, metabolic process.

−5

−3

sem

antic

spa

ce x

−5

−3

(c) (d)

aspartic−typeendopeptidase Ac

Bi

zinc ion Bi

catechol oxidase Ac

porphobilinogen SAc

enone reductase Ac

nucleic acid Bi

heterocyclic C Bi ion Bi

nuclease Accation Bi

organic cyclic C Bi

peptidase Ac

RNA−directed DNA polymerase Ac

Bi

nucleic acidBi

chlorophyll Bi

phenylalanineammonia-lyase Ac

ion Biserine−tRNA ligase Ac

zinc ion Bi

pigment Biheterocyclic C Bi

tetrapyrrole Bi

cysteine-typeendopeptidase Ac

alcohol Bi

cationBi

sem

antic

spa

ce x

−5−3

(a)

DNAintegration

chloroplast-nucleussignaling

nitrogen C MProrganiccyclic C MPr

cellular aromaticC MPr

box H/ACAsnoRNA 3'−endprocessing

porphyrin-containingC MPr

snRNAmodification

heterocycle MPr

mRNA pseudouridineSy

DNA MPr

nucleic acid MPr

cellular nitrogenC MPr

actinfilament organization

tetrapyrrole MPr

−5−3

(b)

photosynthesisDNA integration

photosynthesis, lightharvesting in photosystem I

photosynthesis, lightreaction

protein−chromophorelinkage

proteintargeting

to chloroplastseryl−tRNA

aminoacylation

maltose MPr

starch biosynthetic Prcinnamic acidbiosynthetic Pr

L-phenylalanine catabolic Pr

tetrapyrroleMPr DNA MPr

porphyrin-containing C MPr

semantic space y semantic space y

Supporting Fig. S10 Enriched (p < 0.01) biological processes (a-b) and molecular functions (c-d) with elements differentially targeted (FDR < 0.05) by siRNAs between D. fuchsii and D. incarnata.(a, c) Enriched GO terms of genes with increased siRNA targeting in D. fuchsii. (b, d) Enriched GOterms of genes with over-regulation by siRNAs in D. incarnata. Bubble size is proportional to the frequency of the respective term in the public GO database. The colour represents the log10 valueof the significance of the Fisher’s tests of enrichment, corresponding to the indicated scale. C, compound; Ac, activity; SAc, synthase activity; Bi, binding; Sy, synthesis; Pr, process; MPr, metabolicprocess.

−20−10

sem

antic

spa

ce x

−8−4

(c) (d)

ADP Bi

Bi

zinc ion Bi

pseudouridineSAc

cysteine−typeendopeptidase Ac

nucleic acidBi

heterocyclicC Bi

ion Bi

cation Bi

organiccyclic C Bi

serine−tRNAligase Ac

Bi

zincion Bi

serine-glyoxylate transaminase Ac

chlorophyll Binucleic acid Bi

phosphateion Bi

heterocyclicC Bi

ion Bi

cationBi

organiccyclic C Bi

ADP Bi

tetrapyrole Bi

−20−10

sem

antic

spa

ce x

(a)DNA integration

photosynthesis

organic cyclicC MPr

cellulararomaticC MPr

box H/ACAsnoRNA3'−endprocessing

snRNAmodification

snoRNA MPr

heterocycle MPr

mRNApseudouridine Sy

snRNAMPr

DNA MPr

macromolecule MPr

pseudouridine Sy

nucleicacid MPr

−10−5

(b)

regulation of tetrapyrrole MPr

DNAintegration

organic cyclicC MPr

cellulararomatic C MPr

seryl−tRNAaminoacylation

heterocycleMPr

DNA MPr

nucleicacid MPr

nitrogen C MPr

cellularMPr