bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 2 Co-expression clustering across flower development

3 identifies modules for diverse floral forms in 4 Achimenes (Gesneriaceae) 5 6 Wade R Roberts1,2, Eric H Roalson1 7 8 1 School of Biological Sciences, Washington State University, Pullman, WA, United States 9 2 Current address: Biological Sciences, University of Arkansas, Fayetteville, AR, United States 10 11 Corresponding Author: 12 Wade Roberts1 13 Biological Sciences, University of Arkansas, SCEN 601, Fayetteville, AR, 72701, United States 14 Email address: [email protected] 15 16 Abstract 17 Background. Genetic pathways involved with flower color and shape are thought to play an 18 important role in the development of flowers associated with different pollination syndromes, 19 such as those associated with bee, butterfly, or hummingbird pollination. Because pollination 20 syndromes are complex traits that are orchestrated by multiple genes and pathways, the gene 21 networks have not been explored. Gene co-expression networks provide a systems level 22 approach to identify important contributors to floral diversification. 23 Methods. RNA-sequencing was used to assay gene expression across two stages of flower 24 development (an early bud and an intermediate stage) in 10 species of Achimenes 25 (Gesneriaceae). Two stage-specific co-expression networks were created from 9503 orthologs 26 and analyzed to identify module hubs and the network periphery. Module association with bee, 27 butterfly, and hummingbird pollination syndromes was tested using phylogenetic mixed models.

28 The relationship between network connectivity and evolutionary rates (dN/dS) was tested using 29 linear models. 30 Results. Networks contained 65 and 62 modules that were largely preserved between 31 developmental stages and contained few stage-specific modules. Over a third of the modules in 32 both networks were associated with flower color, shape, and pollination syndrome. Within these 33 modules, several hub nodes were identified that related to the production of anthocyanin and 34 carotenoid pigments and the development of flower shape. Evolutionary rates were decreased in 35 highly connected genes and elevated in peripheral genes. 36 Discussion. This study aids in the understanding of the genetic architecture and network 37 properties underlying the development of floral form and provides valuable candidate modules 38 and genes for future studies. bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

39 40 Introduction 41 42 Flowers display a diverse range of colors, shapes, and sizes, and understanding the 43 ecological and genetic factors contributing to their diversity across angiosperms has long been a 44 major goal in biology. Often this diversity has been attributed to pollinator-mediated selection 45 (Stebbins 1970; O’Meara et al. 2016; Gervasi and Schiestle 2017) and the relationship between a 46 plant and its mode of pollination is considered to be one of the key innovations contributing to 47 angiosperm diversification (Faegri and van der Pijl 1979; Fenster et al. 2004; van der Niet and 48 Johnson 2012; Barrett 2013; Sauquet and Magallón 2018). Most plants evolved to rely on biotic 49 or abiotic means in order to move pollen and ensure reproductive success (Faegri and van der 50 Pijl 1979; Fenster et al. 2004). These biotic pollinators are often attracted to flowers that contain 51 specific traits, such as red flowers that provide high nectar rewards for bird visitors (Cronk and 52 Ojeda 2008) or the ultraviolet markings on some flowers that attract bee visitors (Papiorek et al 53 2016). A wide range of floral traits are thought to contribute to successful pollination, such as 54 color (Sletvold et al. 2016), odor (Piechowski et al. 2010), nectar composition (Amorim et al. 55 2013), and time of flowering (Cortés-Flores et al. 2017). The genetic basis for these and other 56 traits and their role in floral and pollination syndrome divergence has been examined extensively 57 in model systems, particularly in Mimulus (Bradshaw et al. 1998; Yuan et al. 2016) and Petunia 58 (Hoballah et al. 2007; Hermann et al. 2015), and more recently in many non-model systems 59 (Wessinger et al. 2014; Alexandre et al. 2015). 60 Among the most widely used large-scale experimental approaches to investigate genome 61 function are transcriptome analyses (Pickrell et al. 2010; Raherison et al. 2015), particularly in 62 non-model organisms where no reference genome or functional genomics data exists. Changes in 63 expression often result from the combinatorial action of genetic regulatory pathways 64 orchestrating development and responses to environmental stimuli. Therefore, the transcriptome 65 can be viewed as a link between the genotype and phenotype and may be acted upon through 66 selection (Romero et al. 2012; Prasad et al. 2013). As changes in gene expression may underlie 67 many of the phenotypic changes between species (Brawand et al. 2011; Romero et al. 2012; 68 Uebbing et al. 2016), studying the transcriptome may shed light on important pathways and 69 targets of selection. Phenotypic changes can frequently arise through functional changes in 70 conserved developmental pathways among closely related species. For example, changes in 71 enzyme function and transcriptional regulation of the anthocyanin pathway has been implicated 72 frequently across angiosperms in the evolution of red flowers (Des Marais and Rausher 2010; 73 Smith and Rausher 2011). It is now recognized that most genes act as members of biological 74 pathways or of co-regulated modules (Hollender et al. 2014; Ma et al. 2018). 75 Here, we undertake a comparative study of gene expression during flower development 76 in the genus Achimenes. This group is a member of the diverse African violet family 77 (Gesneriaceae) and distributed throughout Mexico and Central America. Achimenes is a young 78 lineage (c. 7-12 Mya; Roalson and Roberts 2016) known for its floral diversity (Figure 1), a bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

79 feature thought to be associated with speciation (Ramírez Roa 1987; Roalson et al. 2003). Four 80 pollination syndromes are found in Achimenes, including melittophily (bees), psychophily 81 (butterflies), euglossophily (female euglossine bees), and ornithophily (hummingbirds) (Figure 82 2). These syndromes have traditionally been defined on the basis of flower color and flower 83 shape (Figure 2; Ramírez Roa 1987) and recently through pollinator observations (Martén- 84 Rodríguez et al. 2015; Ramírez-Aguirre et al. 2019). Currently there have been pollinator 85 observations made for four Achimenes species: A. antirrhina (hummingbirds, Amazilia 86 beryllina), A. flava (bees, Anthophoridae), A. obscura (bees, Trigona fulviventris), and A. patens 87 (unidentified butterflies) (Martén-Rodriguez et al. 2015; Ramírez-Aguirre et al. 2019). Based on 88 these observations and the floral traits of these species (Figure 2), we can hypothesize the likely 89 pollinators for the other Achimenes species (Figure 2; Table 1). Repeated origins and transitions 90 between these pollination syndromes have been hypothesized in Achimenes (Roalson et al. 91 2003), making this small lineage an attractive system to understand the genetic and ecological 92 factors underlying floral diversification. 93 In this study, we aimed to characterize and compare patterns of gene co-expression 94 during flower development across 10 Achimenes species. So far, few studies have investigated 95 the genetic basis of floral development in the Gesneriaceae family (Alexandre et al. 2015; 96 Roberts and Roalson 2017). Given that bee, butterfly, and hummingbird pollination syndromes 97 evolved in parallel across Achimenes, we hypothesized that: (1) distinct sets of co-expressed 98 genes would correlate to each syndrome during flower development, and (2) genes in pathways 99 involved with flower color and shape may be central in each network during flower 100 development. Using a comparative transcriptome approach, we clustered a shared set of 9503 101 orthologs into two different co-expression networks, each corresponding to two stages of flower 102 development: an early bud stage and an intermediate stage prior to anthesis. This strategy 103 allowed us to compare gene co-expression patterns temporally. We compared these networks and 104 tested whether gene co-expression clusters correlated with different floral traits (pollination 105 syndrome, flower color, flower shape, corolla spurs). We also tested whether network properties, 106 such as connectivity and expression, influence evolutionary constraints. Our results allowed us to 107 quantify the extent of shared gene co-expression among closely related species and to identify 108 pathways and genes that may be associated with the repeated evolution of flower types 109 underlying pollinator specificity. 110 111 Materials & Methods 112 113 Plant materials 114 Ten of the 26 currently recognized species in Achimenes were sampled: five from Clade 1 115 and five from Clade 2 (Figure 2). These species were chosen based on their hypothesized close 116 relationships and their diversity in floral form (Figure 1; Roalson et al. 2003). On the basis of 117 previous molecular studies (Roalson and Roberts 2016), two species were chosen as outgroups to 118 represent related lineages: Eucodonia verticillata and Gesneria cuneifolia (Figures 1-2). All bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

119 selected species were grown in standard greenhouse conditions at Washington State University, 120 under 16-hour days, 24-27°C, and 80-85% humidity. 121 Whole flower buds from two stages of flower development were sampled for each 122 species: an immature bud stage (Bud stage; Figure 1) and an intermediate stage before anthesis 123 (D stage; Figure 1). Our definitions of the floral developmental stages in Achimenes were 124 adapted from the stages determined for Antirrhinum majus, which uses a morphological and 125 temporal framework (Vincent and Enrico 2004). The Bud stage represents the smallest 126 reproductive bud distinguishable from vegetative buds, having lengths between 1-2 mm (Figure 127 1). The appearance is marked by the emergence of the floral primordia, no accumulation of 128 anthocyanin pigments, and no elongation or growth of corolla tissue cells. The D stage represents 129 a halfway point between the Bud and a pre-anthesis flower, where the length is about half the 130 length of a fully developed flower (Figure 1). Its appearance is marked by the accumulation of 131 anthocyanins in the corolla tissue, the cells have elongated in the corolla tube, and trichomes 132 have emerged. Corolla spurs are found only in A. grandiflora and A. patens, and the spur begins 133 development during the D stage as an outward growth of the corolla in the opposite direction 134 from the tube opening (Figure 1). 135 Each stage was sampled with two or three biological replicates, contributing four or six 136 samples for each species. The bud stage was chosen to represent a baseline level of gene 137 expression when floral phenotypes are largely similar between species that could then be 138 compared to the intermediate stage where floral phenotypes diverge and many important 139 pathways involved in flower development and pigmentation show increased expression levels 140 (Roberts and Roalson 2017). All tissues were immediately frozen into liquid nitrogen and stored 141 at -80°C. 142 143 RNA extraction and RNA-seq library construction 144 Total RNA was extracted from frozen tissue using the RNEasy Plant Kit (Qiagen) and 145 ribosomal depleted RNA samples were prepared using the RiboMinus Plant Kit (Thermo Fisher 146 Scientific). Stranded libraries were prepared using the NEBNext Ultra Directional RNA Library 147 Kit (New England Biolabs), barcoded, and pooled based on nanomolar concentrations. Library 148 quality and quantity were assessed using the Qubit (dsDNA HS assay; Thermo Fisher Scientific) 149 and BioAnalyzer 2100 (Agilent). The library pool was sequenced across 4 lanes of an Illumina 150 HiSeq2500 for paired-end 101 bp reads at the Genomics Core Lab at Washington State 151 University, Spokane. All new sequence data were deposited in the NCBI Sequence Read Archive 152 (BioProject: PRJNA435759). 153 Single replicate libraries for four of the species in the current study (A. cettoana, A. 154 erecta, A. misera, and A. patens) were previously sequenced for the same two timepoints (Bud 155 and D stages) (Roberts and Roalson 2017). These samples were combined with the newly 156 generated data to assembly de novo transcriptomes and perform co-expression clustering. This 157 data was deposited in the NCBI Sequence Read Archive (BioProject: PRJNA340450). 158 bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

159 Transcriptome assembly 160 Per base quality, adapter contamination, and per base sequence content of raw reads was 161 assessed with FastQC tools (http://bioinformatics.babraham.ac.uk/projects/fastqc). One library 162 (AT6) returned poor yields and low-quality reads and was excluded from further analyses. 163 Trimmomatic v.0.36 (Bolger et al. 2014) was then used to remove contaminating adapter 164 sequences and dynamically trim low-quality bases (ILLUMINACLIP:TruSeq3-PE-2.fa:2:30:10 165 HEADCROP:13 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:50). Trimmed 166 reads derived from an individual species were combined and de novo assembled into contigs 167 using Trinity (--SS_lib_type RF; Grabherr et al. 2011; Haas et al. 2013), generating a reference 168 transcriptome for each species. Sequence redundancy was reduced with CDHIT-EST (-c 0.99 -n 169 5; Li and Godzik 2006). Open reading frames were then predicted for each transcriptome 170 assembly with TransDecoder (Haas et al. 2013) using a BLASTp search against the SwissProt 171 database (www.uniprot.org) to maximize sensitivity. Finally, transcriptome assembly 172 completeness was assessed using BUSCO (Simão et al. 2015) against a set of single-copy plant 173 orthologs (embryophyte_odb10 dataset). 174 175 Orthogroup inference 176 To help facilitate orthogroup inference, transcriptome assemblies from flowers in 177 Sinningia eumorpha and S. magnifica were downloaded and combined with our assemblies 178 (https://doi.org/10.5061/dryad.4r5p1; Serrano-Serrano et al. 2017a). Sinningia is closely related 179 to our focal lineage in the Neotropical Gesneriaceae (Roalson and Roberts 2016). To identify 180 orthogroup sequences across all 14 species, we used a modified version of the approach 181 described in Yang and Smith (2014). All peptide sequences were clustered using an all-by-all 182 BLASTp search performed using DIAMOND (--evalue 1e-6; Buchfink et al. 2014) and results 183 were clustered using MCL v14-137 (-I 1.4; Enright et al. 2002). Homolog clusters were aligned 184 using MAFFT v7.271 (--genafpair --maxiterate 1000; Katoh and Standley 2013) and alignments 185 were trimmed using Phyutility v2.7.1 (-clean 0.1; Smith and Dunn 2008). Codon alignments 186 were then produced for each cluster using PAL2NAL v14 (Suyama et al. 2006) and maximum- 187 likelihood trees were constructed using FastTree v2.1.8 (Price et al. 2010) using the GTR model. 188 Trees were then trimmed to exclude branches >10 times longer than its sister or longer than 0.2 189 substitutions per site. Final homolog group alignments were created from the trimmed trees using 190 MAFFT and used for final homolog tree inference using RAxML v8.2.9 (Stamatakis 2014) and 191 the GTRGAMMA model. Orthogroups were inferred from the final homolog trees using the 192 rooted ingroup (RT) method of Yang and Smith (2014), using S. eumorpha and S. magnifica as 193 outgroups. Homolog trees were trimmed to include at least 2 ingroup species, resulting in 83595 194 orthogroups. 195 196 Quantifying gene expression 197 Trimmed reads for each sample were aligned to the corresponding transcriptome 198 assembly to quantify expression using default parameters in Kallisto v0.43.0 (Bray et al. 2016). bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

199 The resulting counts for each contig were matched with the inferred orthogroups derived above, 200 creating two separate matrices for the Bud and D stage samples. In the resulting gene expression 201 matrices, rows corresponded to orthogroups and columns corresponded to individual samples 202 (Supplemental Data). Counts for the Bud and D stage samples were separately transformed using 203 variance-stabilizing transformation implemented in the Bioconductor package DESeq2 (Love et 204 al. 2014) and quantile normalized using the Bioconductor package preprocessCore (Bolstad 205 2018). Lastly, counts for both stages were separately corrected for confounding effects, such as 206 batch or species effects, using principal component regression with the ‘sva_network’ function in 207 the Bioconductor package sva (Parsana et al. 2019). 208 209 Co-expression networks and identification of modules 210 The Bud and D stage RNAseq data was clustered into gene co-expression networks using 211 the R package WGCNA (Langfelder and Horvath 2008). Prior to network construction, 212 orthogroups were filtered to remove any with >50% missing values or <0.3 expression variance. 213 In total, 9503 orthogroups common to both Bud and D stage data were used to construct separate 214 networks for each stage. Parameters for the Bud stage network were as follows: power = 18, 215 networkType = “signed”, corType = “bicor”, maxPOutliers = 0.05, TOMType = “signed”, 216 deepSplit = 2, mergeCutHeight = 0.25. For the D stage network, the following parameters were 217 used: power = 16, networkType = “signed”, corType = “bicor”, maxPOutliers = 0.05, TOMType 218 = “signed”, deepSplit = 2, mergeCutHeight = 0.25. Soft-thresholding powers for each network 219 (“power” parameter) were chosen as the lowest value such that the scale free topology model fit 220 (R^2) was ≥ 0.9 (Supplemental Data). Signed networks consider positively correlated nodes 221 connected, while unsigned networks consider both positively and negatively correlated nodes 222 connected (van Dam et al. 2018). We used signed networks here (“networkType” and 223 “TOMType” parameters) because this type is considered more robust to biological functions 224 with more specific expression patterns (Mason et al. 2009; Song et al. 2012). Unsigned networks 225 can capture only strong correlations and many negative regulatory relationships in biological 226 systems are weak or moderate (Ritchie et al. 2009). Biweight midcorrelation (“corType” 227 parameter) was used as this method is often more robust than Pearson correlation and more 228 powerful than Spearman correlation (Song et al. 2012). All other WGCNA parameters were kept 229 at their default values. 230 Module eigengenes and orthogroup connectivity were calculated in each network 231 separately using the ‘moduleEigengenes’ and ‘intramodularConnectivity’ functions in WGCNA, 232 respectively. The module eigengene is defined as the first principal component of a module and 233 represents the gene expression profile of a sample within a module (Langfelder and Horvath 234 2008). Connectivity refers to the sum of connection strengths a node has to other network nodes. 235 Module hub genes (defined as the most highly connected nodes within a module) were 236 identified in both networks based on module membership (kME) scores > 0.9, calculated using 237 the ‘signedKME’ function of WGCNA. kME is defined as the correlation between the 238 expression levels of a gene and the module eigengene (Horvath and Dong 2008). Additionally, bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

239 using connectivity measures, we defined peripheral genes in each network as the lowest 10% of 240 connected nodes. 241 We tested whether Bud stage modules were preserved in the D stage and vice versa using 242 module preservation statistics in WGCNA (Langfelder et al. 2011). Median Rank and Zsummary 243 statistics were computed using the ‘modulePreservation’ function of WGCNA, using 200 244 permutations (Langfelder et al. 2011). Median rank statistics were compared to the “gold” 245 module, which consists of 1000 randomly selected orthogroups. Modules with a lower median 246 rank exhibit stronger preservation than modules with a higher median rank. Zsummary scores > 247 10 indicate strong evidence of preservation and scores < 2 indicate no evidence of preservation. 248 249 Phylogeny 250 We previously inferred a phylogeny for the 12 sampled species using 1306 single-copy 251 orthologs identified from the same transcriptome dataset used here (Roberts and Roalson 2018). 252 For comparative analyses of module-trait relationships, we randomly sampled 50 single-copy 253 ortholog gene trees and rescaled branch lengths to be proportional to time (ultrametric) using the 254 ‘chronos’ function in the R package ape (Paradis et al. 2004). Bootstrap support was 100 for 255 nearly every branch in the Roberts and Roalson (2018) phylogeny, therefore we chose to use 256 randomly sampled ortholog trees to account for phylogenetic uncertainty. 257 258 Module and trait relationship 259 We correlated external traits (e.g., red flower color) with the module eigengenes using a 260 phylogenetic Markov Chain Monte Carlo method, implemented in the R packages MCMCglmm 261 (Hadfield 2010) and mulTree (Guillerme and Healy 2014). These analyses were performed 262 individually for each module in both the Bud and D stage networks. Three floral traits and 263 pollination syndrome were coded: primary flower color (purple, red, white, yellow), flower 264 shape (funnelform, salverform, tubular), corolla spur (absent, present), and syndrome (bee, 265 butterfly, hummingbird). We fit a multivariate mixed model with a non-informative prior, where 266 the shape and scale parameters equal 0.001, and residual variance was fixed. Analyses were 267 performed over the 50 randomly selected ortholog trees to account for phylogenetic uncertainty. 268 A phylogenetic model was used to account for any interspecific non-dependency in the dataset 269 by using a random effects structure that incorporated a phylogenetic tree model. Floral traits 270 were set as categorical response variables and the module eigengenes were set as the predictor 271 variable. For each tree, two MCMC chains were run for 250k generations and discarding the first 272 50k as burn-in. This process was run individually over each module in both the Bud and D 273 stages. We checked for convergence between model chains using the Gelman-Rubin statistic 274 (Gelman and Rubin 1992). Potential scale reduction values were all less than 1.1 and effective 275 sample sizes for all fixed effects were greater than 400. We considered fixed effects to be 276 statistically significant when the probabilities in the 95% credible region did not include zero 277 (Supplemental Figures 3-6). 278 bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

279 Evolutionary rate analysis 280 Codon alignments of the filtered orthogroups (n=9503) were produced using PAL2NAL 281 v14 (Suyama et al. 2006). Subsequent alignments and corresponding gene trees were used as

282 input to the codeml package in PAML v4.9 (Yang 2007) to estimate dN/dS (omega, ω). dN/dS for 283 each orthogroup was estimated using a one-ratio model (model = 0, NSsites = 0), providing a 284 single estimate for each orthogroup to match other single orthogroup metrics, such as

285 connectivity. If dN/dS values were exceptionally large (= 999) because of zero synonymous 286 differences, those values were removed from the dataset. 287 First, we performed linear regression (LM) to test the effect of orthogroup connectivity,

288 average expression levels, and their interactions (explanatory variables) on the estimated dN/dS

289 values (main effect), LM = dN/dS ~ connectivity * expression. This was performed using the lm 290 function in R with 10000 bootstrap pseudoreplicates. Second, we ran two-sample t-tests with 291 10000 permutations to test whether hub nodes and periphery nodes in each network had lower

292 (for hubs) or higher (for periphery) dN/dS values than all background nodes. Third, we fit a LM 293 with 10000 bootstrap pseudoreplicates to test whether modules associated with a pollination

294 syndrome (bee, butterfly, or hummingbird; see Results below) had increased dN/dS values, LM =

295 dN/dS ~ syndrome. For all LM and permutation analyses, dN/dS, connectivity, and expression values 296 were log transformed to reach a normal distribution before being processed. Analyses were 297 performed separately for each network using the separately estimated connectivity and 298 expression levels. 299 300 Functional annotation and GO enrichment analysis 301 Filtered orthogroups (n=9503) were annotated based on a search against all green plant 302 proteins in the SwissProt database (release 2018_07; The UniProt Consortium 2017) using 303 Diamond BLASTp (--evalue 1e-6). Results were used to assign annotations and gene ontology 304 (GO) terms. GO enrichment analyses were performed using the R package TopGO with the 305 default “weight01” algorithm with the recommended cutoff of p < 0.05 (Alexa et al. 2006). 306 Enriched GO term redundancy was removed and summarized using the semantic similarity 307 measures implemented in the REVIGO web server (Supek et al. 2011). 308 309 Data and code availability 310 Raw sequencing reads are available from the NCBI Sequence Read Archive under 311 BioProjects: PRJNA435759 and PRJNA340450. Data and code for all analyses can found from 312 https://drive.google.com/open?id=1pvzw1t1pObY_kAEYmxQ484PCTuEWrsrb. 313 314 Results 315 316 Transcriptome assembly 317 Twelve transcriptomes were sequenced (72 libraries) and assembled in 12 species after 318 sampling across two floral developmental stages (Supplemental Table 1). The transcriptomes had bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

319 an average assembly length of 244.9 Mb (± 33.9 Mb), an average of 217,510 (± 47k) transcripts, 320 and an average N50 length of 1988 bp (± 306 bp). Between 22,942 and 33,501 putative genes 321 were detected with 2.34 (± 0.35) putative isoforms. BUSCO identified an average of 87% of the 322 complete, conserved single-copy plant orthologs (Supplemental Table 1). Our focus in our 323 comparative study was on the presence of conserved orthologs, and not on the presence of 324 taxonomically restricted genes, which are not likely preserved across species. 325 326 Constructing the co-expression network for 12 species 327 We identified 83595 orthologous clusters (orthogroups), each containing at least 2 328 species. After removing 74092 orthogroups with too many missing values (> 50% missing) or 329 low expression variance (< 0.3 variance) and keeping common orthogroups shared between the 330 Bud and D stages, 9503 orthogroups were used to construct two co-expression networks using 331 the weighted gene correlation network analysis (WGCNA) approach. Modules of co-expressed 332 orthogroups are inferred using the expression profiles of each sample regardless of species. 333 Clustering identified 65 and 62 co-expression modules in the Bud and D stage networks, 334 respectively (Figure 3; Supplemental Table 2). Module size in the Bud stage ranged from 22 335 (module ME63) to 592 (ME1) orthogroups (mean = 146) and ranged from 21 (ME61) to 704 336 (ME1) orthogroups (mean = 153) in the D stage. Out of the 9503 orthogroups, 7845 (83%) and 337 8735 (92%) were assigned to modules in each network. 338 The remaining unassigned genes from each network that were not placed into a well- 339 defined module were assigned to the ME0 module by WGCNA. Despite being unplaced, these 340 orthogroups may still be functionally relevant within each network. Unassigned genes in the 341 ME0 module were enriched for many processes, including transcription (Bud, n = 173; D, n = 342 84) and organ development (D, n = 55), among other functions (Supplemental Tables 3-4). 343 We tested whether modules in the Bud stage were preserved in the D stage, and vice 344 versa. Overall, preservation of modules between the Bud and D stage networks was high. Eight 345 of the 65 Bud stage modules (ME46, ME62, ME44, ME57, ME42, ME41, ME55, ME40) were 346 not strongly preserved in the D stage, while only 1 of the 62 D stage modules (ME59) was not 347 strongly preserved in the Bud stage (Supplemental Table 5). These patterns suggest that these 348 non-preserved modules may play unique roles within their network. Among the 8 Bud stage- 349 specific modules were many orthogroups related to primary cell growth, meristem identity and 350 differentiation, stamen development, and flowering (Supplemental Table 3). In the single D 351 stage-specific module were many orthogroups related to methylation, flowering, and carotenoid 352 biosynthesis (Supplemental Table 4). Despite the relatively high preservation, correspondence 353 between module membership in the Bud and D stages was moderate (Figure 4). Orthogroup 354 module membership tended to shift between each network. For example, most members of 355 module ME15 in the Bud stage are transferred to modules ME3 and ME7 in the D stage (Figure 356 4). 357 We defined network hubs (the most highly connected nodes within a module) with 358 module membership (kME) scores > 0.9. Using this definition, all modules in each network bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

359 contained at least one hub, with the percentage of hubs ranging from 1% to 24% of orthogroups 360 in a module (Figures 5-6; Supplemental Table 6). Of the 632 and 691 hubs identified in the Bud 361 and D stages, a significant portion (110) were shared between networks (Fisher’s Exact Test, 362 odds ratio = 3.02, P < 0.001). Functional enrichment of hubs in the Bud stage revealed that many 363 were involved in the regulation of development and metabolic processes (n = 40), transport and 364 protein localization (n = 39), biosynthetic processes (n = 19), and DNA methylation (n = 7), 365 consistent with the notion that hubs usually play important roles in integrating other genes during 366 network evolution (Ravasz et al. 2002) (Table 2). In the D stage, hubs were enriched for 367 functions related to translation (n = 33), transport (n = 9), ethylene and abscisic acid signaling 368 pathways (n = 17), carotenoid and anthocyanin biosynthetic processes (n = 11), and the 369 regulation of gene expression (n = 20), among others (Table 2). 370 Nodes in the periphery of each network were loosely defined by considering them to be

371 the lowest 10% connected (connectivityBud < 2.824, connectivityD < 4.651). These definitions 372 identified 951 periphery nodes (Supplemental Table 7), of which 301 were peripheral in both 373 networks (Fisher’s Exact Test, odds ratio = 5.67, P < 0.001). These periphery nodes in the Bud 374 stage were enriched for functions related to reproductive structure development (n = 75), 375 transport and localization (n = 107), and signaling (n = 389), among others (Table 3). In the D 376 stage, the periphery was enriched for functions related to the regulation of flower development (n 377 = 64), the regulation of flowering time (n = 18), the regulation of flavonoid biosynthesis (n = 7), 378 and cell fate specification (n = 12), among others (Table 3). Greater than expected numbers of 379 peripheral nodes in the D stage were related to regulation (Fisher’s exact test, odds ratio = 1.191, 380 p = 0.02752) and transcription (Fisher’s exact test, odds ratio = 1.370, p = 0.001549). In contrast, 381 there were no differences in the expected number of peripheral nodes related to regulation 382 (Fisher’s exact test, odds ratio = 0.955, p = 0.7044) or transcription (Fisher’s exact test, odds 383 ratio = 1.047, p = 0.3549) in the Bud stage. 384 385 Modules associated with floral traits 386 We calculated module eigengenes, single values which represent the gene expression 387 profiles of a sample within a module (Supplemental Figures 1-2). The extent of module 388 involvement in various biological processes can be tested by correlating eigengenes with external 389 traits, such as flower color. We tested the relationship between the module eigengenes and the 390 four floral traits (pollination syndrome [bee, butterfly, hummingbird], flower color [purple, red, 391 yellow, white], flower shape [funnelform, salverform, tubular], and corolla spurs [absence, 392 presence]), while controlling for any phylogenetic bias in the dataset using a phylogenetic mixed 393 model (Figures 5-6; Supplemental Figures 3-6). We also examined whether eigengene values (> 394 0.1 or < -0.1) in single species contributed to a strong correlation or whether eigengene values (> 395 0.1 or < -0.1) in multiple species contributed to a strong correlation (Figures 5-6; Supplemental 396 Figures 3-6). We considered there to be strong evidence for trait association when a significant 397 correlation from MCMCglmm was coupled with elevated eigengene (> 0.1) or decreased 398 eigengene (< -1.0) expression in multiple species that shared any of the significant traits. bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

399 Likewise, weak evidence for trait association was considered when the MCMCglmm results 400 were significant, but only a single species or no species sharing the significant traits had elevated 401 (> 0.1) or decreased (< -1.0) eigengene expression. WGCNA does not use trait information when 402 inferring modules, so modules that correlated strongly with any floral trait were inferred without 403 a priori knowledge. 404 Pollination syndrome—We included 3 species with the bee pollination syndrome (A. 405 candida, A. misera, and E. verticillata), 4 species with the butterfly pollination syndrome (A. 406 cettoana, A. grandiflora, A. longiflora, and A. patens ‘Major’), and 5 species with the 407 hummingbird pollination syndrome (A. admirabilis, A. antirrhina, A. erecta, A. pedunculata, and 408 G. cuneifolia) (Figure 2; Table 1). These pollination syndromes are traditionally defined in 409 Achimenes and other gesneriads based on primary flower color, flower shape, and the presence 410 of corolla spurs (Wiehler 1983; Ramírez Roa 1987; Roalson et al. 2003). Expression of 23 (35%) 411 and 21 (34%) modules were positively associated with the pollination syndromes in the Bud and 412 D stage networks, consisting of 2967 and 2880 orthogroups, respectively. (Figures 5-6). There 413 were 3 (4.6%), 7 (10.8%), and 13 (20%) modules associated with bee, butterfly, and 414 hummingbird pollination syndrome in the Bud stage network, while 1 (1.5%), 8 (12.9%), and 12 415 (19.4%) modules were associated with the same syndromes in the D stage. Modules were never 416 associated with more than one syndrome. Butterfly and hummingbird syndromes were never 417 correlated to the same modules and were often correlated in opposite directions (i.e., butterfly 418 was positive correlated and hummingbird was negative correlated). 419 Among the 3 modules that were correlated to the bee pollination syndrome in the Bud 420 stage were many orthogroups involved in hormone signaling pathways (particularly ethylene and 421 abscisic acid, n = 14), cell wall and lipid biosynthesis (n = 7), photomorphogenesis (n = 2), 422 nitrogen compound metabolism (n = 4), and the transport of potassium and monocarboxylic 423 acids (n = 5) (Table 4). In contrast, the 1 module correlated to the bee pollination syndrome in 424 the D stage was enriched for different functions from the Bud stage, including the positive 425 regulation of molecular functions and response to biotic stimuli (n = 7), phospholipid catalysis (n 426 = 2), xyloglucan biosynthesis (n = 2), mRNA stability and catalysis (n = 5), and chromatin 427 modification (n = 4) (Table 5). 428 The 7 modules correlated to the butterfly pollination syndrome in the Bud stage 429 contained orthogroups involved in RNA splicing (n = 16), fertilization (n = 3), DNA methylation 430 (n = 2), cell growth and shoot formation (n = 8), the development of organ boundaries (n = 3), 431 and stress responses (n = 83) (Table 4). Within the 8 modules correlated with butterfly 432 pollination in the D stage were orthogroups enriched for functions in photorespiration (n = 7), 433 microtubule organization (n = 5), chromatin silencing (n = 3), red and far-red light responses (n 434 = 3), modified amino acid biosynthesis and metabolism (n = 10), and mRNA transport (n = 9) 435 (Table 5). 436 Thirteen modules were correlated to the hummingbird pollination syndrome in the Bud 437 stage and the orthogroups were enriched for numerous functions, including many developmental 438 processes (Table 4). These modules were enriched for floral whorl development (n = 12), RNA bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

439 and mRNA modifications (n = 19), cell growth and division (n = 54), organelle assembly (n = 3), 440 transportation of carbohydrates and potassium (n = 22), and responses to external stimuli (n = 441 17) (Table 4). Many of the same enrichments were seen in the 12 modules correlated to the 442 hummingbird pollination syndrome in the D stage. These modules had enrichments for floral 443 whorl development (n = 14), cell polarity and proliferation (n = 8), adaxial/abaxial specification 444 (n = 8), organ formation (n = 3), the regulation of growth (n = 26), and endosperm development 445 (n = 6). Additionally, these modules had enrichment for RNA and mRNA modification (n = 79), 446 transcription (n = 3), protein-DNA complex formation (n = 7), signaling (n = 25), and UV 447 response (n = 9) (Table 5). 448 Flower color—Primary flower color is closely associated to pollination syndrome in 449 Achimenes (Ramírez Roa 1987; Roalson et al. 2003), and we identified 26 (40%) and 17 (27%) 450 modules associated to any flower color in the Bud and D stage networks, consisting of 2935 and 451 1500 orthogroups respectively (Figures 5-6). Red and yellow are associated with hummingbird 452 pollination, purple with butterfly pollination, and white with bee pollination. In the Bud stage, 453 there were 14 (21.5%), 11 (16.9%), and 1 (1.5%) module associated with red, purple, or yellow 454 flower color. In the D stage, no modules were associated with yellow, but 8 (12.9%) and 9 455 (14.5%) modules were associated with red or purple flower color. In both networks, no modules 456 were corelated to white flower color. Similar to syndrome, purple and red flower color were 457 never associated with the same modules and were always correlated in opposite directions. 458 The 14 modules associated with red flowers in the Bud stage overlapped significantly 459 with the modules associated with the hummingbird pollination syndrome in the Bud stage and 460 contained the same functional enrichments (Table 4; Supplemental Table 8). However, in the 8 D 461 stage modules associated with red flowers, the orthogroups were enriched for functions involved 462 in flower morphogenesis (n = 8), cell wall modification (n = 3), positive regulation of gene 463 expression (n = 14), and histone modification (n = 7), among others (Supplemental Table 8). 464 Within the 11 modules associated with purple flowers in the Bud stage were orthogroups 465 enriched for transcription (n = 14), meristem initiation (n = 4), DNA methylation (n = 3), 466 fertilization (n = 5), ethylene signaling (n = 5), response to biotic stimuli (n = 55), and others 467 (Supplemental Table 8). In contrast, the 9 modules associated with purple flowers in the D stage 468 were enriched for orthogroups involved in anther and endosperm development (n = 9), 469 translation (n = 4), the regulation of developmental processes (n = 9), lipid modification (n = 8), 470 cell division (n = 3), and hormone signaling (gibberellic acid and jasmonic acid, n = 6), among 471 many other processes (Supplemental Table 8). 472 A single module (ME36) was associated with yellow flower color, only in the Bud stage, 473 and contained few orthogroups involved in floral meristem determinacy (n = 1), floral organ 474 identity (n = 1), gene silencing (n = 1), sulfur compound biosynthesis (n = 2), and vegetative 475 phase change (n = 1) (Supplemental Table 8). 476 Flower shape—Flower shape is also important but not as closely associated to pollination 477 syndromes as primary flower color in Achimenes (Ramírez Roa 1987; Roalson et al. 2003). 478 Flowers with bee pollination tend to have funnelform flowers, butterfly pollinated flowers all bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

479 have salverform flowers, and hummingbird pollinated flowers have either salverform or tubular 480 flowers (Table 2). Overall, there were 14 (22%) and 13 (21%) modules associated with any of 481 the flower shapes in the Bud and D stages, consisting of 2940 and 2504 orthogroups, 482 respectively (Figures 5-6). Here, there were 1 (1.5%), 8 (12.3%), and 5 (7.7%) modules whose 483 expression correlated to funnelform, salverform, or tubular flowers in the Bud stage (Figure 5). 484 Fewer modules were correlated in the D stage network, but expression in 1 (1.6%), 7 (12.3%), 485 and 5 (8.1%) modules were associated with funnelform, salverform, and tubular shapes (Figure 486 4). No modules were positively associated with more than one flower shape. 487 Only two modules were associated with funnelform flowers, one in the Bud stage and the 488 other in the D stage, and these were enriched for functions related to ethylene signaling (n = 3), 489 lipid biosynthesis (n = 3), chromatin modification (n = 4), cell wall organization (n = 2), and 490 many metabolic processes (Supplemental Table 8). Within the 8 modules associated with 491 salverform flowers in the Bud stage, there were many orthogroups involved in mRNA processing 492 and modification (n = 50), ovule development (n = 7), chlorophyll biosynthesis (n = 4), and the 493 regulation of flower development (n = 20) (Supplemental Table 8). In the D stage, the 7 modules 494 associated with salverform flowers contained orthogroups involved in chromatin silencing (n = 495 7), anthocyanin biosynthesis (n = 10), cell cycle and cell division (n = 95), stamen development 496 (n = 12), auxin biosynthesis (n = 7), and transport (n = 17) (Supplemental Table 8). 497 Among the 5 modules associated with tubular flowers in the Bud stage were orthogroups 498 enriched for functions in the positive regulation of transcription and organelle assembly (n = 13), 499 RNA processing (n = 5), xylem development (n = 4), metabolism (n = 12), and many 500 biosynthetic processes (Supplemental Table 8). In the 5 modules associated with tubular flowers 501 in the D stage were enrichments for floral whorl development (n = 6), positive regulation of gene 502 expression (n = 15), mRNA modification (n = 5), and signaling (n = 12) (Supplemental Table 8). 503 Corolla spurs—The presence of corolla spurs is found only on butterfly pollinated 504 species, two of which were included: A. grandiflora and A. patens (Figure 1; Table 1). There was 505 expression in 5 (7.7%) modules associated with spurs in the Bud stage network, while only 1 506 (1.6%) module was associated in the D stage network (Figures 3-4). Within the 5 modules 507 associated with corolla spurs in the Bud stage were orthogroups related to miRNA processing (n 508 = 4), mRNA splicing (n = 9), the regulation of signal transduction pathways (n = 16), and pollen 509 tube growth (n = 6) (Supplemental Table 8). In the single module associated with corolla spurs in 510 the D stage (ME53) were very few orthogroups involved in microtubule organization (n = 2), 511 stamen development (n = 1), the positive regulation of cell division (n = 1), flavonoid 512 biosynthesis (n = 1), and indole acetic acid metabolism (n = 1) (Supplemental Table 8). 513 Non-associated—There were 13 and 9 modules not associated with any traits in the Bud 514 and D stage networks, respectively (Figures 5-6). Three of these modules in the Bud stage were 515 not-preserved in the D stage (modules ME46, ME44, and ME41; Figures 5-6; Supplemental 516 Table 5). The 9 non-associated modules in the D stage were also not associated with any traits in 517 the Bud stage (Figures 5-6) and the orthogroups had overlapping enrichments for many essential 518 processes (Supplemental Table 8). These modules were enriched for orthogroups involved in the bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

519 development of the inflorescence, stamens, and floral organs, biosynthesis of vitamins and 520 nucleotides, photosynthesis and photosynthetic electron transport, metabolism of phosphate 521 compounds, and cell surface receptor signaling (Supplemental Table 8). Numerous orthogroups 522 were also involved in the positive regulation of reproduction, the negative regulation of growth, 523 and the regulation of heterochronic development, histone methylation, and gibberellic acid 524 mediated signaling (Supplemental Table 8). 525 526 Evolutionary rates correlate to network location 527 We ran several analyses to explore how several network variables may affect

528 evolutionary rates of the orthogroups within each network (as measured by dN/dS). First, we tested

529 the effects of orthogroup connectivity, expression levels, and their interaction on the dN/dS values 530 (Supplemental Table 7). Results indicated a collective effect between connectivity and 531 expression on dN/dS in both the Bud stage (LM, F(3,9548) = 92.58, p = 0, � = 0.03) and the D 532 stage networks (LM, F(3,9548) = 78.36, p = 0, �= 0.02). We found that orthogroup connectivity

533 was negatively correlated with dN/dS in the Bud stage network (LM, t = -16.31, p = 0) and the D 534 stage network (LM, t = -15.13, p = 0) (Figure 7), while expression was positively correlated with

535 dN/dS in the Bud stage (LM, t = 2.12, p = 0.0344) and not in the D stage (LM, t = 1.51, p = 536 0.1324) (Supplemental Figure 7). The interaction between connectivity and expression also

537 indicated a marginal dependency of both predictors on dN/dS in the Bud stage (LM, t = -2.022, p = 538 0.0433) but not in the D stage network (LM, t = -1.823, p = 0.0683). These patterns have been 539 observed in model species and may be universal to all eukaryotes.

540 Second, we tested whether the nodes we defined as hubs in each network had lower dN/dS 541 values, suggestive of increased evolutionary constraint (Supplemental Table 9). Hubs are 542 considered to functionally significant and may control large portions of the network. We found

543 that the hubs in the Bud stage network had lower dN/dS than all background genes (Two-sample t- 544 test, t(679.31) = 7.0934, p = 1.65e-12, Cohen’s d = 0.383; Permutation t-test. mean diff. = 0.266,

545 p = 0.0001) (Supplemental Figure 8). Similarly, hubs in the D stage network also had lower dN/dS 546 than all background genes (Two-sample t-test, t(755.91) = 4.1918, p = 1.55e-05, Cohen’s d = 547 0.206; Permutation t-test, mean diff. = 0.143, p = 0.0001) (Supplemental Figure 8). 548 Third, we tested whether the most peripheral nodes (the lowest 10% connected nodes)

549 had higher dN/dS values, which may suggest relaxed evolutionary constraint (Supplemental Table 550 9). These nodes are loosely connected within the network and their roles may be more apt to

551 fluctuate during evolution and development. These peripheral genes had higher dN/dS than all 552 background genes in the Bud stage network (Two-sample t-test, t(1487.70) = -10.272, p = 0, 553 Cohen’s d = -0.253; Permutation t-test, mean diff. = -0.176, p = 0.0001) and the D stage network 554 (Two-sample, t(1512.30) = -10.387, p = 0, Cohen’s d = -0.252; Permutation t-test, mean diff. = - 555 0.175, p = 0.0001) (Supplemental Figure 8). 556 Lastly, we tested whether modules correlated with bee, butterfly, or hummingbird 557 pollination syndromes showed increased evolutionary rates over the non-associated modules.

558 Results indicated a very weak effect of syndrome association on dN/dS in the Bud network (LM, bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

559 F(3,9548) = 5.186, p = 0.001406, � = 0.0016) and the D network (LM, F(3,9548) = 4.277, p = 560 0.005038, �= 0.0013) (Supplemental Table 9). Only bee syndrome associated modules had

561 marginally lower dN/dS values in both networks (Bud stage, LM, t = -3.58, p = 0.000346; D stage,

562 LM, t = -2.508, p = 0.0122). Butterfly associated modules had marginally lower dN/dS only in the 563 D stage (LM, t = -2.548, p = 0.0108), while hummingbird associated modules did not have any 564 effect in either network. 565 566 Discussion 567 Here we report on patterns of gene co-expression across two stages of flower 568 development in 12 species of Achimenes, Eucodonia, and Gesneria. Our comparative and 569 phylogenetic context allowed us to reveal gene co-expression patterns across a group of closely 570 related plant species that correlated to diverse pollination syndromes and floral traits, including 571 flower color, flower shape, and corolla spurs. Our results suggest floral forms that are associated 572 with bee, butterfly, and hummingbird pollinator groups are correlated with many co-expression 573 modules. We found that nearly a third of modules in each network (23 and 21 modules in the 574 Bud and D stages, respectively) had evidence for association with the three pollination 575 syndromes (Figures 5-6). In our analysis, large fractions of our 9503 orthogroups set could be 576 partitioned into modules (Figure 3), network hubs and peripheral nodes identified (Supplemental 577 Tables 6-7), and several orthogroups were identified for their potential importance in floral 578 phenotypic differentiation. Lastly, orthogroup position in each network (measured by

579 connectivity) had an association with evolutionary rate (dN/dS) (Figure 7). These analyses provide 580 a first overview of floral transcriptional architecture across members of the same genus that 581 display diverse floral forms. 582 583 Hub nodes contain important candidates for floral form 584 To date, a large number of studies conducted in diverse angiosperm systems, have found 585 that many genes are associated with flower development (Quattrocchio et al. 1999; Howarth et 586 al. 2011). Despite evidence for extensive gene/genome duplications that could expand the 587 genetic repertoire (Tang et al. 2008), many recent studies have suggested that the generation of 588 novel genes is rare (Patterson et al. 2009; De Smet et al. 2013). Thus, the evolution and 589 development of diverse floral forms may predominantly occur through the co-option of existing 590 pathways (Preston et al. 2011). To our knowledge, gene co-expression network analyses have not 591 been previously used across multiple species in a non-model lineage to understand flower 592 development and evolution. 593 Hub nodes (genes) are considered to be conserved, highly connected nodes that are 594 central to network architecture and may directly orchestrate numerous biological processes 595 (Ravasz et al. 2002; Hu et al. 2016; Mähler et al. 2017). Our analyses identified over 600 gene 596 nodes as hubs within each network, the majority of which were only hubs within a specific floral

597 stage. These hub nodes tended to have lower dN/dS estimates, suggesting that they not prone to 598 rapid evolutionary change (Supplemental Table 9). Unsurprisingly, hub nodes in the Bud stage bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

599 were enriched for many processes related to early flower development, including cell growth, 600 leaf formation, and photosystem assembly (Table 2). Similarly, hub nodes in the D stage were 601 enriched for genes that may be related the development of distinct floral forms, including 602 carotenoid biosynthesis, anthocyanin biosynthesis, and cell morphogenesis (Table 2). Within this 603 context of flower development, we identified a number of hubs that we consider important 604 candidates for involvement in the production of flower color and the development of floral form. 605 Many of these candidates were considered previously (Roberts and Roalson 2017) as potentially 606 important for the diversification of floral form in Achimenes. Undoubtedly, this list of hubs 607 contains many genes that may important for other biological traits that we do not consider here, 608 such as scent, nectar, and pollen production. 609 Anthocyanin biosynthesis—Anthocyanins are common floral pigments that produce red, 610 purple, and blue colors and are produced via the highly conserved flavonoid biosynthetic 611 pathway (Holton and Cornish 1995). With a few exceptions, all anthocyanins are produced from 612 three precursor molecules: pelargonidin, cyanidin, and delphinidin (Grotewold 2006). There are 613 six enzymatic reactions required for the production of anthocyanins and many also serve as 614 branching points for the production of flavonoids, lignins, flavonols, and other phenolics that 615 have roles in stress response and physiology (Winkel-Shirley 2002; Tahara 2007). We identified 616 four homologs of anthocyanin biosynthetic pathway enzymes as network hubs: flavonoid 3’- 617 hydroxylase (F3’H; cluster8703_1rr.inclade1.ortho3; module ME12), dihydroflavonol 4- 618 reductase (DFR; cluster5082_1rr.inclade1.ortho6; module ME48), flavonoid 3’,5’- 619 methyltransferase (FAOMT; cluster11783_1rr.inclade1.ortho1.tre; module ME32), and flavonol 620 3-O-glucosyltransferase (UFOG; cluster8241_2rr.inclade1.ortho2.tre; module ME1). 621 These 4 enzymes are involved in distinct steps during anthocyanin production and 622 chemical modification in floral tissue. Activity of F3’H directs the pathway toward the 623 production of cyanidins and quercetin (Grotewold 2006). Cyanidin can accumulate in floral 624 tissue as a purple-hued pigment, while quercetin plays a large role in various stress-related 625 physiological functions (Pollastri and Tattini 2011), including response to high light and UVB 626 radiation (Gerhardt et al. 2008) and antioxidant properties (Rice-Evans et al. 1996). Therefore, 627 F3’H as an important hub in the networks makes sense given its enormous pleiotropic role in 628 various physiological responses. Just prior to the production of anthocyanins, DFR catalyzes the 629 reaction to produce pelargonidins, cyanidins, and delphinidins from the precursor molecules 630 dihydrokaempferol, dihydroquercetin, and dihyrdomyricetin, respectively (Grotewold 2006). 631 Shifts in DFR expression, function, and substrate specificity have been implicated in many 632 evolutionary transitions in flower color (Smith et al. 2013; Wu et al. 2013). 633 Further modifications to anthocyanins, such as methylation, glycosylation, and acylation, 634 produce a wide variety of compounds and hues (Wessinger and Rausher 2012). We identified 635 homologs to both a methyltransferase (FAOMT) and glucosyltransferase (UFOG) as hubs in the 636 networks. Methylation of anthocyanins have been widely studied in petunia (Provenzano et al. 637 2014), grapes (Hugueney et al. 2009) and tree peony (Du et al. 2015), demonstrating that 638 methylation can impact the hue, either shifting it more toward purple (Sakata et al. 1995) or bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

639 sometimes red (Tanaka et al. 2008). Glycosylation stabilizes anthocyanins molecules for 640 subsequent transfer to acidic vacuoles (Saito and Yamazaki 2002), increasing their solubility, 641 and influencing their color variability by inter- and intra-molecular stacking (Springob et al. 642 2003). Finding homologs of FAOMT and UFOG as network hubs reflect the importance of 643 anthocyanin modification for different flower color hues among Achimenes species. 644 Carotenoid biosynthesis—Carotenoids are red, orange, and yellow pigments that are 645 produced in plastids and are essential for photosynthesis, but also accumulate as secondary 646 metabolites in flowers to attract pollinators (Kevan and Baker 1983). Floral and fruit carotenoids 647 can also be used to produce volatile apocarotenoids, which may further enhance plant-animal 648 interactions during pollination and seed dispersal (Dudareva et al. 2006). They may also serve as 649 precursors to the production of the hormone abscisic acid and strigalactones that are involved in 650 many plant developmental processes (Rosas-Saavedra et al. 2016). We identified 4 homologs of 651 carotenoid biosynthetic pathway enzymes as network hubs: geranylgeranyl pyrophosphate 652 synthase (GGPPS; cluster10945_2rr.inclade1.ortho1.tre; module ME35), phytoene 653 dehydrogenase (PDS; cluster13048_1rr.inclade1.ortho1.tre; module ME4), lycopene beta cyclase 654 (LCYB; cluster13774_1rr.inclade1.ortho1.tre; module ME7), and capsanthin-capsorubin synthase 655 (CCS; cluster13583_1rr.inclade1.ortho2.tre; module ME6). 656 Each of these 4 enzymes catalyzes reactions during different steps of carotenoid 657 production. GGPPS produces the main precursor molecular geranylgeranyl pyrophosphate 658 (GGPP) to carotenoid biosynthesis that is produced as the end-product of the plastid 659 methylerythritol 4-phosphate (MEP) pathway. Two GGPP molecules are then condensed into 660 phytoene and undergoes four successive dehydrogenations and isomerizations to produce 661 lycopene. PDS catalyzes the first two desaturation steps that transform the colorless phytoene 662 into the red-colored lycopene. PDS is a rate-limiting enzyme in carotenoid biosynthesis 663 (Chamovitz et al. 1993) and down-regulation has been shown to lead to large accumulations of 664 phytoene (Busch et al. 2002) and decreases in total carotenoid, chlorophyll, and photosynthetic 665 efficiency (Wang et al. 2009). 666 Lycopene is the main starting compound for a large variety of carotenoids. LCYB takes 667 lycopene as a substrate and subsequently produces beta-carotene. Many plants use tissue-specific 668 isoforms of LCYB genes that are expressed in fruits or flowers that often correlate with the 669 accumulation of beta-carotene or downstream xanthophylls (Ronen et al. 2000; Ahrazem et al. 670 2010; Devitt et al. 2010). Beta-carotenes are hypothesized to be the primary carotenoids in 671 Achimenes flowers (Roberts and Roalson 2017). Another functionally related carotenoid cyclase 672 enzyme is CCS (or neoxanthin synthase, NSY) which catalyzes the final step in the carotenoid 673 pathway and converts the yellow-colored violaxanthin into neoxanthin (Parry and Horgan 1991). 674 Both violaxanthin and neoxanthin can be further used for producing the plant hormone abscisic 675 acid (Neuman et al. 2014). Both enzymes are likely functionally important in the developing 676 flowers for the biosynthesis of beta-carotene and xanthophylls. 677 Flower development—Very few studies have so far investigated the molecular 678 mechanisms involved in the development of flower shape in the Gesneriaceae (Gao et al. 2008; bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

679 Zhou et al. 2008; Alexandre et al. 2015). However, extensive work has been done in model 680 organisms, such as Arabidopsis thaliana and Antirrhinum majus, which can provide insight into 681 the potential roles that many transcription factors might play in determining floral organ identity 682 and shape in Achimenes. Among the network hubs, we identified several homologs that are likely 683 involved in floral organ development, including mixta (MIXTA; 684 cluster8699_1rr.inclade1.ortho3.tre; modules ME5 and ME8), agamous (AG; 685 cluster7311_1rr.inclade1.ortho3.tre; module ME23), globosa (GLO; 686 cluster2750_1rr.inclade1.ortho3.tre; module ME37), and tcp4 (TCP4; 687 cluster12380_1rr.inclade1.ortho2.tre; module ME22). 688 Petal identity and petal surface morphology are both thought to be important components 689 for pollinator attraction (Noda et al. 1994; Whitney et al. 2011). MIXTA was first characterized 690 from Antirrhinum and controls the development of the conical cell shape in the petal epidermis 691 (Noda et al. 1994). Consequently, MIXTA and MIXTA-like genes have been characterized in 692 other model systems, such as Arabidopsis and Mimulus for their similar role in epidermal cell 693 morphogenesis (Baumann et al. 2007) as well as trichome development (Gilding and Marks 694 2010; Scoville et al. 2011). The homolog of MIXTA in our study was a hub node in both 695 networks, suggesting it might play an important role throughout flower development in 696 determining petal surface morphology. 697 Early floral organ determination requires the involvement of several MADS-box 698 transcription factors, such as AG and GLO. We identified homologs of AG as a hub in the Bud 699 stage and GLO as a hub in the D stage (Supplemental Table 7). AG has been shown in 700 Arabidopsis to be expressed in early flower development in order to terminate meristem activity 701 and promote the development of stamens and carpels, while working in combination with 702 APETALA3, PISTILLATA, and SEPALLATA (Gómez-Mena et al. 2005). More recently homologs 703 of AG were demonstrated to be involved in nectary development in both Arabidopsis and 704 Petunia (Morel et al. 2018), another floral trait important for plant-pollinator interaction. GLO 705 was first characterized in Antirrhinum and Arabidopsis and is also involved with stamen identity, 706 as well as petal identity (Tröbner et al. 1992; Goto and Meyerowitz 1994). Outside of these 707 model systems, its expression patterns and role in the evolution of complex floral forms have 708 been examined in numerous plant lineages, from monocots (Bartlett and Specht 2010) to 709 Solanaceae (Zhang et al. 2014). AG and GLO will be interesting candidates to begin exploring 710 further how petals, stamens, carpels, and nectary development in Achimenes. 711 Lastly, the homolog of TCP4 was identified as a hub during the Bud stage, and may be 712 important for its potential role in petal development and cell proliferation. Looking into the 713 genetic factors regulating the elaboration of petal growth will be important for understanding 714 how corolla spurs develop in Achimenes (Figure 1). The role of TCP4 been characterized in 715 Arabidopsis for its role in both petal growth, showing restricted expression to the developing 716 petal tissue (Nag et al. 2009). More recent work using gene regulatory networks in Arabidopsis 717 has shown that petal size is controlled by a SEPALLATA3-regulated miR319/TCP4 module 718 (Chen et al. 2018). Outside of Arabidopsis, TCP4 was also implicated for a having a role in the bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

719 development of the petal spur in Aquilegia (Yant et al. 2015). Therefore, considering both the 720 role for TCP4 in petal and spur development demonstrated in Arabidopsis and Aquilegia and its 721 identification as a hub gene in our networks, we think this gene requires further interrogation. 722 723 Peripheral genes are enriched for transcriptional regulators in later stages of development 724 Peripheral nodes have low connectivity within co-expression networks (Supplemental 725 Table 7), tend to have higher rates of molecular evolution (Masalia et al. 2017), and have been 726 hypothesized to contribute more to evolutionary innovations than network hubs (Ichihashi et al. 727 2014). Of the 951 nodes we defined as peripheral in both Bud and D stage networks, roughly a 728 third were consistently peripheral under our definitions (lowest 10% connected; Supplemental 729 Table 7). After identifying the nodes with homologies to transcriptional regulators, we found 730 there was no difference in the expected number in the early Bud stage, while there were greater 731 than expected numbers of transcriptional regulators in the periphery of the D stage (see Results). 732 No such patterns existed when we tested these transcriptional regulators as hub genes. While 733 Mähler et al. (2017) found an enrichment for transcription factors among hub genes in a single 734 species network of Populus tremula, our results in a multi-species network find the opposite 735 pattern. Shifting the expression patterns of various genes and pathways can lead to phenotypic 736 divergence among closely related species (West-Eberhard 1989). This can be seen during later 737 stages of flower development when distinct traits, such as red or purple flower color, can appear 738 in different species due to shifting expression patterns within the anthocyanin biosynthetic 739 pathway (Smith and Rausher 2011). The connection between the apparent overabundance of 740 transcriptional regulators in later stages of flower development and the rapid phenotypic 741 divergence in floral form will need to be explored further. 742 Several notable peripheral nodes were identified for their potential involvement in 743 important floral processes (Supplemental Table 7). Three homologs were identified in the 744 periphery of the Bud stage with potential involvement in the determination of zygmorphic 745 (bilateral) floral symmetry: two homologs of divaricata (DIV; 746 cluster13684_1rr.inclade1.ortho1.tre, cluster8585_1rr.inclade1.ortho1.tre; ME26 and ME0) and 747 one homolog of dichotoma (DICH; cluster13245_1rr.inclade1.ortho1.tre; ME0). Bilateral 748 symmetry has been proposed as a mechanism that facilitates pollen transfer by insects (Galliot et 749 al. 2006). Both DIV and DICH are TCP transcription factors that were first characterized in 750 Antirrhinum (Almeida et al. 1997; Luo et al. 1996), and show localized expression to the ventral 751 and dorsal portions of the petal, respectively. Achimenes flowers at the Bud stage are lacking or 752 only just beginning to establish floral asymmetry and the relegation of both DIV and DICH to the 753 network periphery may reflect this small role. The role of these genes has not been studied in the 754 Gesneriaceae, but have been examined more recently in other non-model systems for their role in 755 the evolution of pollination syndromes (Zhang et al. 2010; Preston et al. 2011). Additionally, a 756 homolog of the homeobox wuschel gene (WUS; cluster9273_1rr.inclade1.ortho1.tre; module 757 ME0) was identified from the periphery of the D stage network. WUS was first characterized in 758 Arabidopsis for its role in meristem cell maintenance (Laux et al. 1996) and its expression is bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

759 localized only to the meristem (Mayer et al. 1998). Once the floral meristem has been initiated, 760 WUS starts to be repressed by AG (hub gene, described above) (Sun et al. 2009). The relegation 761 of WUS to the periphery in our networks at the D stage might then be expected given that it plays 762 such a prominent role during early floral development and the vegetative to reproductive 763 transition. 764 765 Evolutionary rates are correlated to network connectivity 766 Increased protein evolutionary rates during rapid diversification has been suggested in 767 other radiations of animals and plants (Kapralov et al. 2013; Brawand et al. 2014; Pease et al. 768 2016). Our study is among the first to examine patterns of network evolution across multiple 769 closely-related species of plants with multiple transitions in flower type and pollination 770 syndrome. Hummingbirds are hypothesized to have had an important influence on diversification 771 in Neotropical Gesneriaceae (Roalson and Roberts 2016; Serrano-Serrano et al. 2017b), while 772 the role of butterflies has not been explored. Modules correlated with hummingbird or butterfly 773 pollination syndromes did not exhibit clear increases or decreases in evolutionary rates compared 774 to other modules (Supplemental Table 9). Detecting selection is subject to many factors, such as 775 a gene’s transcriptional abundance and its importance within the protein interaction network 776 (Lemos et al. 2005). For instance, we found that higher network connectivity was strongly 777 associated with lower evolutionary rates across both networks in our study (Figure 7; 778 Supplemental Table 9). This corroborates the recent results found in other eukaryotic systems 779 (Morandin et al. 2016; Masalia et al. 2017; Josephs et al. 2017; Mähler et al. 2017) while also 780 showing the apparent strength of correlation between connectivity and evolutionary rate differs 781 between studies and systems. 782 Network effects (such as the type of network analysis employed) and the expression 783 levels of highly abundant genes are known to affect evolutionary rates, potentially confounding

784 analysis of selection pressure (Krylov et al. 2003). We found that dN/dS in early development may 785 be dependent on both connectivity and expression levels, while this interactive effect was gone 786 during later development (Supplemental Table 9). As there are few highly connected genes 787 (hubs, which are important determinants of the observed network structure), a random mutation 788 would be less likely to affect such a gene. Changing the amino acid sequence of a hub gene 789 could have multiple associated effects, some deleterious (Hahn and Kern 2004; Luisi et al. 790 2015). In comparison, a random mutation would be more likely to affect a gene with lower 791 connectivity, while also resulting in variants with fewer negative consequences. The hub genes 792 have more direct interactions and are more likely to be essential and involved in more biological 793 processes, placing them under greater constraint. 794 795 Numerous network modules correlated to floral form 796 One primary use for gene co-expression network analyses is to identify modules of co- 797 expressed genes whose overall expression (as measured by the eigengene) may correlate with 798 different traits. These traits can be both qualitative or quantitative, such as social caste (Morandin bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

799 et al. 2016), seed oil production (Hu et al. 2016), or anther development (Hollender et al. 2014). 800 While we tested only qualitative traits here, future studies could also correlate eigengene 801 expression to quantitative variables such as nectary size or sucrose concentration, since these 802 appear to be another important component of pollination syndrome delimitation (Perret et al. 803 2001; Katzer et al. 2019; Vandelook et al. 2019). Since our dataset was composed of samples 804 from 12 species, we performed correlation analyses using a phylogenetic model to test whether 805 module expression correlated to different flower colors, flower shapes, corolla spurs, and 806 pollination syndromes. Nearly half of our modules in both networks had evidence for an 807 association with these floral traits (Figures 5-6). There were no modules whose correlation to a 808 pollination syndrome had increased eigengene expression in all species sharing that trait (Figures 809 5-6). Instead many modules with correlation to a trait showed a phylogenetic pattern with a 810 shared increased eigengene expression among the most closely related species. For example, 811 module ME23 in the Bud stage network had a correlation to butterfly pollination and increased 812 eigengene expression in A. cettoana and A. longiflora, both members of Clade 1 (Figure 2). Our 813 aim was to identify sets of co-expressed genes that would provide candidates for involvement in 814 the development of these traits. 815 For instance, many of the candidate hubs we identified (see above) were found in 816 modules that were correlated to one or multiple traits to which they might contribute, such as 817 PHAN (module ME4, correlated to corolla spurs) and DFR (module ME48, correlated to 818 hummingbird pollination and purple flowers) (Supplemental Table 2). Additionally, modules 819 correlated to various floral traits were enriched for multiple genes that might be involved in the 820 development of those traits (Supplemental Table 8). Module ME53 was correlated to the corolla 821 spurs found in A. grandiflora and A. patens in the D stage network and was enriched for many 822 genes involved in cell division and cell growth (Figure 6; Supplemental Table 4). Other times, 823 modules were correlated to a specific trait but the enriched GO terms did not provide a clear idea 824 of how these co-expressed orthogroups may contribute (Supplemental Tables 3-4). In our 825 analyses, we considered relatively few traits and only focused on those that have traditionally 826 been considered the most important for the differentiation of floral form in Achimenes, namely 827 flower color, flower shape, and corolla spurs. Undoubtedly there are a myriad number of 828 molecular and biochemical pathways that we have not considered here that underlie the 829 development of these complex floral phenotypes. With the idea that networks represent complex 830 systems of gene and protein interactions, it may be that these seemingly simple and generalized 831 floral phenotypes are produced through much more complex means than many have previously 832 considered. 833 834 Strengths and limitations of network analyses in non-model organisms 835 Our analysis covers genes with orthologs found in most species, representing a set of 836 evolutionarily conserved genes. While no reference genome currently exists for Neotropical 837 Gesneriaceae, we constructed, annotated, and presented de novo transcriptomes for 10 838 Achimenes species as well as 2 outgroup species, E. verticillata and G. cuneifolia. Our approach bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

839 enables functional genomic studies in this non-model system, but the potential for misassemblies 840 may bias our downstream results (Hornett and Wheat 2012). Using a gene tree-based approach to 841 carefully identify orthogroups, we focused our attention on conserved gene regulatory machinery 842 and not on taxonomically restricted genes. The latter may be involved in species-specific 843 functions. Nevertheless, it is worth noting that many recent studies have indicated that transitions 844 in floral form may be due to the shifting expression of genes found in conserved genetic 845 pathways (Wessinger and Rausher 2014). Taxonomically restricted genes are likely interacting 846 with existing regulatory pathways, and the manner in which they integrate into the co-expression 847 modules identified here will be a fascinating topic for future research. With extended sampling 848 within Achimenes we could infer species-specific co-expression networks and perform more 849 detailed cross-species comparisons. We expect that these taxonomically restricted genes may be 850 poorly connected with the network, which may allow for increased diversification. 851 We characterized gene expression patterns by sequencing RNA across two timepoints 852 during flower development. Previous gene expression experiments across three timepoints (early 853 Bud, intermediate D, and late Pre-anthesis stages) in four Achimenes species suggested that the 854 majority of differential gene expression occurred between the early Bud stage and the 855 intermediate D stage (Roberts and Roalson 2017). We believe our approach allows us to capture 856 dynamic transcriptional patterns during flower development for comparison across multiple 857 species. We found that the co-expression networks for the Bud and D stages were largely 858 preserved, despite there being a few stage specific modules (Supplemental Table 5). However, 859 this approach may greatly over-simplify the nature of transcriptional regulation in these species. 860 Organ- and structure-specific expression studies may have greater power to detect floral form- 861 specific differences, both in terms of the number of differentially expressed genes, and co- 862 expression network structure (Hollender et al. 2014; Suzuki et al. 2017; Shahan et al. 2018). As a 863 result, our data likely represents an underestimate of the true number of modules. Future studies 864 could focus on the comparative analysis of expression patterns in specific organs, such as the 865 petals, stigma, and stamens, and additional timepoints, which would provide greater functional 866 insight into the evolution, development, and diversification of these organs. 867 Network analysis represents a more complex approach than differential expression 868 analyses because it can capture system-level properties (Langfelder and Horvath 2008). Most 869 phenotypes involve interactions of proteins from diverse biochemical pathways. Although 870 inferred modules do not necessarily correspond to entire biochemical pathways, or other 871 components of cellular organization, the approach performs well in reconstructing the 872 complexity of protein-protein interaction networks (Allen et al. 2012). In many systems this 873 approach has succeeded in identifying candidate genes for various biological traits, for example 874 floral organ development in strawberry (Hollender et al. 2014; Shahan et al. 2018) or carotenoid 875 accumulation in carrots (Iorizzo et al. 2016). Our results demonstrate the difficulty in identifying 876 gene clusters that underlie highly correlated floral traits, especially in non-model systems, as 877 seen by the overlapping correlation of modules associated with flower shape and color (Figures 878 5-6). It has been shown in many plant systems that floral traits tend to be selected together by bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

879 pollinators (Cuartas-Domínguez and Medel 2010; Sletvold et al. 2010), making it challenging to 880 separate correlated modules from causal modules. Co-expression networks are foundationally 881 about correlations between genes (van Dam et al. 2018), making them useful in identifying novel 882 sets of genes that may be functionally relevant for trait of interest. Other approaches, such as 883 quantitative trait locus (QTL) analyses may provide more promise in identifying distinct genes 884 that are involved in the divergence of floral traits. QTL studies have already shown promise in 885 identifying genetic loci that underlie pollination syndrome divergence in Mimulus (Yuan et al. 886 2013), Penstemon (Wessinger et al. 2014), and Rhytidophyllum (Alexandre et al. 2015). 887 We were able to identify numerous candidate orthogroups involved in the development of 888 floral form in our non-model system. While no functional genomic data exists yet for Achimenes, 889 our annotations are based on the manually curated SwissProt database. De novo assemblies 890 provide important data for non-model organisms, but there can be biases introduced during 891 assembly and annotation due to potentially missing genes (Hornett and Wheat 2012). We 892 attempted to limit this bias by focusing only on shared orthogroups found in the majority (at least 893 6) of our sampled Achimenes. Applying this network-based approach to the diversification of 894 flowers across multiple species in Achimenes, we recovered distinct modules of co-expressed 895 genes that may underlie a range of floral phenotypic traits tied to different pollination 896 syndromes. Although our data come from whole flowers during development, and do not allow 897 us to test specific hypotheses regarding whether particular genes have shifted expression location 898 in the developing flower, they do suggest that there are conserved regulatory modules that may 899 have been co-opted in convergent flower types multiple times. It will be interesting to apply 900 network analyses to study floral diversification in additional lineages and examine how 901 conserved or divergent the patterns are across angiosperms. 902 903 Conclusions 904 Floral forms corresponding to bee, butterfly, and hummingbird pollination have evolved 905 multiple times across the Neotropical plant genus Achimenes. Genome-wide gene expression 906 estimates were taken from flowers in 12 species across two development stages in order to 907 construct, analyze, and compare stage-specific gene co-expression networks. We hypothesized 908 that numerous modules in each network would correlate to these pollination syndromes and that 909 central genes in the network may be candidates for involvement in the development of important 910 floral traits, such as flower color and shape. We found that nearly a third of modules were 911 correlated to the pollination syndromes and many more were correlated to different flower colors 912 and shapes. The outgroup species, E. verticillata (bee pollinated) and G. cuneifolia 913 (hummingbird pollinated), displayed correlation patterns similar to the ingroup species that 914 shared floral traits, suggesting that some co-expression patterns might be shared across 915 evolutionary distances. Several of the hub genes in the networks were homologs of the 916 anthocyanin and carotenoid biosynthetic pathways, important for the production of floral 917 pigments that attract different pollinators. A negative relationship between network connectivity

918 and dN/dS corroborates the findings in model systems that more centrally located nodes (likely bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

919 hubs) are under increased evolutionary constraint. We found that the less connected genes 920 (peripheral) are under more relaxed constraints and contained numerous transcriptional 921 regulators. Our results demonstrate the utility of applying co-expression network analyses in 922 non-model plant lineages to begin identifying important modules and pathways that will be 923 useful starting points for additional analyses into the evolution and development of floral form. 924 925 Acknowledgements 926 We thank Joanna Kelley, Amit Dhingra, and Andrew McCubbin for helpful comments that 927 improved this work. Sequencing was performed at the Genomics Core Lab at Washington State 928 University, Spokane, and the Genomics Sequencing and Analysis Facility at the University of 929 Texas, Austin. 930 931 References 932 Ahrazem O, Rubio-Moraga A, López RC, Gómez-Gómez L. 2010. The expression of a 933 chromoplast-specific lycopene beta cyclase gene is involved in high production of 934 saffron’s apocarotenoid precursors. J Exp Bot 61:105-119. 935 Alexa A, Rahnenführer J, Lengauer T. 2006. Improved scoring of functional groups from gene 936 expression data by decorrelating GO graph structure. Bioinformatics 22:1600-1607. 937 Alexandre H, Vrignaud J, Mangin B, Joly S. 2015. Genetic architecture of pollination syndrome 938 transition between hummingbird-specialist and generalist species in the genus 939 Rhytidophyllum (Gesneriaceae). PeerJ 3:e1028. 940 Allen JD, Xie Y, Chen M, Girard L, Xiao G. 2012. Computing statistical methods for 941 constructing large scale gene networks. PLOS One 7:e29348. 942 Almeida J, Rocheta M, Galego L. 1997. Genetic control of flower shape in Antirrhinum majus. 943 Development 124:1387-1392. 944 Amorim FW, Galetto L, Sazima M. 2013. Beyond the pollination syndrome: nectar ecology and 945 the role of diurnal and nocturnal pollinators in the reproductive success of Inga sessilis. 946 Plant Biol 15:317-327. 947 Baumann K, Perez-Rodriguez M, Bradley D, Venail J, Bailey P, Jin H, Koes R, Roberts K, 948 Martin C. 2007. Control of cell and petal morphogenesis by R2R3 MYB transcription 949 factors. Development 134:1691-1701. 950 Barrett SC. 2013. The evolution of plant reproductive systems: how often are transitions 951 irreversible? P Roy Soc B-Biol Sci 280: 20130913. 952 Bartlett ME, Specht CD. 2010. Evidence for the involvement of GLOBOSA-like gene 953 duplications and expression divergence in the evolution of floral morphology in the 954 Zingiberales. New Phytol 187:521-541. 955 Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer for Illumina sequence 956 data. Bioinformatics 30:2114-2120. 957 Bolstad B. 2018. preprocessCore: A collection of pre-processing functions. R package version 958 1.46.0, https://github.com/bmbolstad/preprocessCore. bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

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Tables

Table 1. Definition of pollination syndromes in Achimenes, Eucodonia, and Gesneria.

Table 2. Gene Ontology enrichment for the network hubs.

Table 3. Gene Ontology enrichment for the network periphery.

Table 4. Gene Ontology enrichment in Bud stage modules correlated to pollination syndrome.

Table 5. Gene Ontology enrichment in D stage modules correlated to pollination syndrome.

Figures

Figure 1. Achimenes flowers and the sampled developmental stages. Flowers of the twelve sampled species: (A) A. admirabilis, (B) A. antirrhina, (C) A. candida, (D) A. cettoana, (E) A. erecta, (F) A. grandiflora, (G) A. longiflora, (H) A. misera, (I) A. patens, (J) A. pedunculate, (K) E. verticillate, and (L) G. cuneifolia. Sampled Bud and D stage flowers for (M-N) A. cettoana, (O-P) A. erecta, (Q-R) A. misera, and (S-T) A. patens. Corolla spurs found in (U) A. grandiflora and (V) A. patens are indicated with arrows. Scale bars equal 1 cm. Abbreviations: A, Achimenes; E, Eucodonia; G, Gesneria.

Figure 2. Phylogenetic relationships and floral traits in Achimenes. Phylogeny of Achimenes adapted from Roberts and Roalson (2018). Clade 1 and Clade 2 (sensu Roalson et al. 2003) are indicated on the phylogeny with a circle and box, respectively. Flower traits for each species are shown at the tips of the phylogeny, including flower color, flower shape, presence of corolla spurs, pollination syndrome, and pollinator observations.

Figure 3. Co-expression clustering of the Bud and D stages. Hierarchical clustering by WGCNA showing co-expressed modules for (A) the Bud Stage and (B) the D stage. Each of the 9503 orthogroups were assigned to 65 and 62 modules, respectively, and the color rows underneath show the module assignment.

Figure 4. Correspondence of the Bud and D stage networks. Each row corresponds to one Bud stage module (labeled by module name and module size), and each column corresponds to one D stage module (labeled by module name and module size). Numbers in the table indicate gene counts in the intersection of the corresponding modules. Coloring of the table encodes - log(p), with p being the Fisher’s exact test p-value for the overlap of the two modules. The stronger the red color, the more significant the overlap.

Figure 5. Summary of Bud stage network module association to floral traits and pollination syndrome. Each row represents an individual module in the Bud stage network and shows the total size (column All), the number of hubs (column Hubs), and whether the module is preserved in the D stage. Each column in the central table represents the floral traits (Pollination Syndrome, bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Flower Color, Flower Shape, and Spurs) that were tested for an association to module eigengenes using MCMCglmm. The eigengene expression is indicated for each module in which all three replicates from an individual species had > 1.0 or < -1.0 expression. An association was considered strong when both MCMCglmm results were significant and the eigengene expression was > 1.0 or < -1.0 in multiple species that shared the significant trait. An association was considered weak when MCMCglmm results were significant but the eigengene was > 1.0 or < - 1.0 in a single species or no species that shared the significant trait. Eigengene expression for species that share traits to those found significant by MCMCglmm are in bold.

Figure 6. Summary of D stage network module association to floral traits and pollination syndrome. Each row represents an individual module in the D stage network and shows the total size (column All), the number of hubs (column Hubs), and whether the module is preserved in the Bud stage. Each column in the central table represents the floral traits (Pollination Syndrome, Flower Color, Flower Shape, and Spurs) that were tested for an association to module eigengenes using MCMCglmm. The eigengene expression is indicated for each module in which all three replicates from an individual species had > 1.0 or < -1.0 expression. An association was considered strong when both MCMCglmm results were significant and the eigengene expression was > 1.0 or < -1.0 in multiple species that shared the significant trait. An association was considered weak when MCMCglmm results were significant but the eigengene was > 1.0 or < - 1.0 in a single species or no species that shared the significant trait. Eigengene expression for species that share traits to those found significant by MCMCglmm are in bold.

Figure 7. Relationship between orthogroup dN/dS (omega) and orthogroup connectivity. (A) Bud stage network. (B) D stage network. Each point represents an individual orthogroup. The red line indicates the linear regression line for the relationship between dN/dS and connectivity. The dotted line indicates dN/dS = 1 with orthogroups above this line considered to be under relaxed or positive selection. bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Table 1. Definition of pollination syndromes in Achimenes, Eucodonia, and Gesneria.

Pollination syndrome Primary flower color Flower shape Presence of Species corolla spurs Melittophily (bees) White or Purple with Funnelform No Achimenes candida yellow or purple Achimenes misera spotted throat Eucodonia verticillata Ornithophily Red and yellow Tubular, No Achimenes admirabilis (hummingbirds) Salverform Achimenes antirrhina Achimenes erecta Achimenes pedunculata Gesneria cuneifolia Psychophily Purple Salverform Yes Achimenes cettoana (butterflies) Achimenes grandiflora Achimenes longiflora Achimenes patens

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Table 2. Gene Ontology enrichment for the network hubs. GO ID Term Annotated Significant Expected P A. Bud stage Hubs GO:0031325 positive regulation of cellular metabolic process 224 16 14.52 0.000 GO:0006607 NLS-bearing protein import into nucleus 7 4 0.45 0.001 GO:0015991 ATP hydrolysis coupled proton transport 9 4 0.58 0.002 GO:0046470 phosphatidylcholine metabolic process 9 4 0.58 0.002 GO:0018298 protein-chromophore linkage 32 7 2.07 0.004 GO:0070507 regulation of microtubule cytoskeleton organization 6 3 0.39 0.005 GO:1990778 protein localization to cell periphery 6 3 0.39 0.005 GO:0051592 response to calcium ion 7 3 0.45 0.008 GO:0006412 translation 233 24 15.10 0.009 GO:0042594 response to starvation 60 10 3.89 0.012 GO:0007035 vacuolar acidification 8 3 0.52 0.012 GO:0006534 cysteine metabolic process 9 4 0.58 0.012 GO:0009833 plant-type primary cell wall biogenesis 15 4 0.97 0.013 GO:0048831 regulation of shoot system development 123 12 7.97 0.017 GO:0032509 endosome transport via multivesicular body sorting pathway 9 3 0.58 0.017 GO:0051568 histone H3-K4 methylation 9 3 0.58 0.017 GO:0000271 polysaccharide biosynthetic process 123 11 7.97 0.023 GO:0006732 coenzyme metabolic process 116 8 7.52 0.023 GO:0010338 leaf formation 5 2 0.32 0.037 GO:0048564 photosystem I assembly 5 2 0.32 0.037 GO:0017001 antibiotic catabolic process 26 3 1.69 0.037 GO:0044282 small molecule catabolic process 75 10 4.86 0.038 GO:0006833 water transport 13 3 0.84 0.048 GO ID Term Annotated Significant Expected P B. D stage Hubs GO:0006412 translation 233 30 16.03 0.001 GO:0042939 tripeptide transport 8 4 0.55 0.001 GO:0016117 carotenoid biosynthetic process 20 6 1.38 0.002 GO:0006744 ubiquinone biosynthetic process 5 3 0.34 0.003 GO:0032259 methylation 88 15 6.05 0.004 GO:0045814 negative regulation of gene expression, epigenetic 37 5 2.55 0.005 GO:0009206 purine ribonucleoside triphosphate biosynthetic process 44 6 3.03 0.013 GO:0000271 polysaccharide biosynthetic process 123 11 8.46 0.013 GO:0009873 ethylene-activated signaling pathway 78 12 5.37 0.014 GO:0015991 ATP hydrolysis coupled proton transport 9 3 0.62 0.020 GO:0009718 anthocyanin-containing compound biosynthetic process 26 5 1.79 0.030 GO:0000018 regulation of DNA recombination 5 2 0.34 0.041 GO:0015976 carbon utilization 5 2 0.34 0.041 GO:0034314 Arp2/3 complex-mediated actin nucleation 5 2 0.34 0.041 GO:0080144 amino acid homeostasis 5 2 0.34 0.041 GO:1901615 organic hydroxy compound metabolic process 121 9 8.32 0.041 GO:0000902 cell morphogenesis 174 9 11.97 0.042

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Table 3. Gene Ontology enrichment for the network periphery. GO.ID Term Annotated Significant Expected P A. Bud stage periphery GO:0016579 protein deubiquitination 24 16 8.70 0.002 GO:0048825 cotyledon development 21 12 7.61 0.005 GO:0015846 polyamine transport 5 5 1.81 0.006 GO:0034314 Arp2/3 complex-mediated actin nucleation 5 5 1.81 0.006 GO:0006020 inositol metabolic process 12 7 4.35 0.011 GO:0010229 inflorescence development 9 7 3.26 0.014 GO:0009744 response to sucrose 31 16 11.24 0.021 GO:1903338 regulation of cell wall organization or biogenesis 11 7 3.99 0.026 GO:0006809 nitric oxide biosynthetic process 6 5 2.18 0.026 GO:0009773 photosynthetic electron transport in photosystem I 8 6 2.90 0.030 GO:0034613 cellular protein localization 183 75 66.34 0.031 GO:0006004 fucose metabolic process 12 8 4.35 0.032 GO:0009926 auxin polar transport 42 22 15.23 0.037 GO:0010090 trichome morphogenesis 38 19 13.78 0.041 GO:0009414 response to water deprivation 182 69 65.98 0.045 GO:0009873 ethylene-activated signaling pathway 78 38 28.28 0.046 GO:0007166 cell surface receptor signaling pathway 93 42 33.71 0.046 GO:0006163 purine nucleotide metabolic process 96 38 34.80 0.047 GO:0035195 gene silencing by miRNA 15 7 5.44 0.048 GO:0016236 macroautophagy 21 9 7.61 0.048 GO:0055083 monovalent inorganic anion homeostasis 13 5 4.71 0.048 GO:0051338 regulation of transferase activity 38 13 13.78 0.048 GO:0006473 protein acetylation 23 7 8.34 0.048 GO.ID Term Annotated Significant Expected P B. D stage periphery GO:0045490 pectin catabolic process 20 19 12.88 0.002 GO:0007166 cell surface receptor signaling pathway 93 71 59.90 0.006 GO:0000398 mRNA splicing, via spliceosome 46 38 29.63 0.007 GO:0009740 gibberellic acid mediated signaling pathway 31 24 19.97 0.015 GO:0006310 DNA recombination 54 41 34.78 0.015 GO:0009909 regulation of flower development 80 57 51.53 0.017 GO:0009058 biosynthetic process 2121 1360 1366.19 0.019 GO:0042545 cell wall modification 34 26 21.90 0.021 GO:0001708 cell fate specification 13 12 8.37 0.027 GO:0042538 hyperosmotic salinity response 25 21 16.10 0.027 GO:0002181 cytoplasmic translation 21 18 13.53 0.029 GO:0006897 endocytosis 40 27 25.76 0.029 GO:0010197 polar nucleus fusion 20 17 12.88 0.039 GO:0071702 organic substance transport 492 319 316.91 0.046 GO:0006949 syncytium formation 7 7 4.51 0.046 GO:0008295 spermidine biosynthetic process 7 7 4.51 0.046 GO:0009963 positive regulation of flavonoid biosynthetic process 7 7 4.51 0.046

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Table 4. Gene Ontology enrichment in Bud stage modules correlated to pollination syndrome. A. Bee pollination GO.ID Term Annotated Significant Expected P GO:0046686 response to cadmium ion 99 16 5.69 0.000 GO:0010104 regulation of ethylene-activated signaling pathway 12 3 0.69 0.003 GO:0010189 vitamin E biosynthetic process 8 3 0.46 0.008 GO:0070085 glycosylation 72 7 4.13 0.009 GO:0009226 nucleotide-sugar biosynthetic process 21 5 1.21 0.011 GO:0042136 neurotransmitter biosynthetic process 9 4 0.52 0.018 GO:0070592 cell wall polysaccharide biosynthetic process 32 4 1.84 0.018 GO:0051173 positive regulation of nitrogen compound metabolic process 192 4 11.03 0.019 GO:0046777 protein autophosphorylation 71 9 4.08 0.019 GO:0009651 response to salt stress 215 19 12.35 0.022 GO:0009738 abscisic acid-activated signaling pathway 135 11 7.75 0.028 GO:0015718 monocarboxylic acid transport 12 3 0.69 0.028 GO:0046889 positive regulation of lipid biosynthetic process 12 3 0.69 0.029 GO:0006809 nitric oxide biosynthetic process 5 2 0.29 0.029 GO:0006974 cellular response to DNA damage stimulus 99 9 5.69 0.035 GO:0010099 regulation of photomorphogenesis 6 2 0.34 0.042 GO:0071805 potassium ion transmembrane transport 6 2 0.34 0.042 B. Butterfly pollination GO.ID Term Annotated Significant Expected P GO:0009415 response to water 155 13 11.47 0.016 GO:0008380 RNA splicing 122 16 9.03 0.016 GO:0010223 secondary shoot formation 14 4 1.04 0.016 GO:0009624 response to nematode 15 4 1.11 0.021 GO:0009695 jasmonic acid biosynthetic process 9 3 0.67 0.024 GO:0090691 formation of plant organ boundary 9 3 0.67 0.024 GO:0006950 response to stress 1173 83 86.79 0.028 GO:0006353 DNA-templated transcription, termination 17 4 1.26 0.032 GO:0009567 double fertilization forming a zygote and endosperm 11 3 0.81 0.042 GO:0009835 fruit ripening 11 3 0.81 0.042 GO:0009607 response to biotic stimulus 374 35 27.67 0.045 bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

GO:0010216 maintenance of DNA methylation 5 2 0.37 0.047 GO:0010363 regulation of plant-type hypersensitive response 5 2 0.37 0.047 GO:0071577 zinc ion transmembrane transport 5 2 0.37 0.047 GO:0009825 multidimensional cell growth 19 4 1.41 0.047 C. Hummingbird pollination GO.ID Term Annotated Significant Expected P GO:0009451 RNA modification 353 89 62.34 0.000 GO:1900618 regulation of shoot system morphogenesis 9 5 1.59 0.001 GO:0009826 unidimensional cell growth 95 24 16.78 0.002 GO:0016556 mRNA modification 35 11 6.18 0.002 GO:0006012 galactose metabolic process 5 4 0.88 0.004 GO:0031425 chloroplast RNA processing 18 8 3.18 0.008 GO:0008643 carbohydrate transport 44 15 7.77 0.010 GO:1905428 regulation of plant organ formation 6 4 1.06 0.011 GO:0006888 ER to Golgi vesicle-mediated transport 19 8 3.36 0.011 GO:0044272 sulfur compound biosynthetic process 58 12 10.24 0.011 GO:0045944 positive regulation of transcription by RNA polymerase II 37 13 6.53 0.013 GO:0002239 response to oomycetes 18 6 3.18 0.019 GO:0006813 potassium ion transport 16 7 2.83 0.020 GO:0043650 dicarboxylic acid biosynthetic process 10 5 1.77 0.020 GO:0051053 negative regulation of DNA metabolic process 7 4 1.24 0.022 GO:0051301 cell division 147 30 25.96 0.027 GO:0000079 regulation of cyclin-dependent protein serine/threonine kinase activity 11 5 1.94 0.031 GO:0046131 pyrimidine ribonucleoside metabolic process 12 3 2.12 0.031 GO:0032509 endosome transport via multivesicular body sorting pathway 8 4 1.41 0.037 GO:0071496 cellular response to external stimulus 71 17 12.54 0.041 GO:1902117 positive regulation of organelle assembly 5 3 0.88 0.041 GO:0048438 floral whorl development 69 12 12.19 0.042 GO:0009651 response to salt stress 215 49 37.97 0.043

bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Table 5. Gene Ontology enrichment in D stage modules correlated to pollination syndrome. A. Bee pollination GO.ID Term Annotated Significant Expected P GO:0061014 positive regulation of mRNA catabolic process 6 3 0.26 0.002 GO:0010189 vitamin E biosynthetic process 8 3 0.35 0.004 GO:0044093 positive regulation of molecular function 46 4 2.01 0.006 GO:0046686 response to cadmium ion 99 10 4.33 0.011 GO:0006974 cellular response to DNA damage stimulus 99 8 4.33 0.017 GO:0002833 positive regulation of response to biotic stimulus 19 3 0.83 0.017 GO:0009395 phospholipid catabolic process 5 2 0.22 0.017 GO:0009969 xyloglucan biosynthetic process 5 2 0.22 0.017 GO:0006544 glycine metabolic process 7 2 0.31 0.035 GO:0043488 regulation of mRNA stability 7 2 0.31 0.035 GO:0016569 covalent chromatin modification 58 4 2.54 0.044 GO:0043902 positive regulation of multi-organism process 22 3 0.96 0.045 GO:0009631 cold acclimation 8 2 0.35 0.045 B. Butterfly pollination GO.ID Term Annotated Significant Expected P GO:0009853 photorespiration 19 7 2.00 0.002 GO:0043622 cortical microtubule organization 13 5 1.37 0.008 GO:0031349 positive regulation of defense response 51 8 5.37 0.010 GO:0006575 cellular modified amino acid metabolic process 35 7 3.69 0.018 GO:0070919 production of siRNA involved in chromatin silencing by small RNA 6 3 0.63 0.018 GO:0042398 cellular modified amino acid biosynthetic process 7 3 0.74 0.029 GO:0009808 lignin metabolic process 22 5 2.32 0.031 GO:2000030 regulation of response to red or far red light 9 3 0.95 0.031 GO:0019220 regulation of phosphate metabolic process 39 6 4.11 0.031 GO:0010197 polar nucleus fusion 13 4 1.37 0.040 GO:0051028 mRNA transport 44 9 4.63 0.041 GO:0006833 water transport 13 4 1.37 0.043 GO:0043902 positive regulation of multi-organism process 22 4 2.32 0.044 C. Hummingbird pollination GO.ID Term Annotated Significant Expected P bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

GO:0009960 endosperm development 10 6 1.53 0.002 GO:0007163 establishment or maintenance of cell polarity 5 4 0.77 0.002 GO:0000272 polysaccharide catabolic process 50 14 7.65 0.005 GO:0010224 response to UV-B 24 9 3.67 0.007 GO:0009089 lysine biosynthetic process via diaminopimelate 7 4 1.07 0.013 GO:0009451 RNA modification 353 70 54.01 0.015 GO:0007166 cell surface receptor signaling pathway 86 21 13.16 0.018 GO:0008285 negative regulation of cell proliferation 8 4 1.22 0.023 GO:0042939 tripeptide transport 8 4 1.22 0.023 GO:0009955 adaxial/abaxial pattern specification 17 8 2.60 0.023 GO:0051656 establishment of organelle localization 36 9 5.51 0.023 GO:0006213 pyrimidine nucleoside metabolic process 14 3 2.14 0.023 GO:0033014 tetrapyrrole biosynthetic process 39 6 5.97 0.024 GO:0009267 cellular response to starvation 47 14 7.19 0.025 GO:0006635 fatty acid beta-oxidation 16 6 2.45 0.026 GO:0016556 mRNA modification 35 9 5.36 0.026 GO:0048438 floral whorl development 69 14 10.56 0.028 GO:0010103 stomatal complex morphogenesis 14 5 2.14 0.028 GO:0006383 transcription by RNA polymerase III 5 3 0.77 0.028 GO:0040008 regulation of growth 126 26 19.28 0.029 GO:0010019 chloroplast-nucleus signaling pathway 9 4 1.38 0.036 GO:0010506 regulation of autophagy 9 4 1.38 0.036 GO:0043547 positive regulation of GTPase activity 9 4 1.38 0.036 GO:0018298 protein-chromophore linkage 27 8 4.13 0.044 GO:0031425 chloroplast RNA processing 18 6 2.75 0.045 GO:0051187 cofactor catabolic process 34 9 5.20 0.050 GO:0002238 response to molecule of fungal origin 6 3 0.92 0.050 GO:0032958 inositol phosphate biosynthetic process 6 3 0.92 0.050 GO:0042219 cellular modified amino acid catabolic process 6 3 0.92 0.050 GO:1905428 regulation of plant organ formation 6 3 0.92 0.050 GO:0065004 protein-DNA complex assembly 45 8 6.89 0.050 bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Figure 1. Achimenes flowers and the sampled developmental stages. bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Flower Flower Corolla Pollination Pollinator Color Shape Spurs Syndrome Observation

G. cuneifolia Tubular Absent ornithophily

E. verticillata Funnelform Absent melittophily

A. misera Funnelform Absent melittophily

A. admirabilis Salverform Absent ornithophily Clade 1

A. erecta Salverform Absent ornithophily

A. longiflora Salverform Absent psychophily

A. cettoana Salverform Absent psychophily

A. candida Funnelform Absent melittophily

Clade Martén-Rodriguez et al. 2015; Pollination Syndromes 2 A. antirrhina Absent ornithophily Tubular Ramírez-Aguirre et al. 2019 Melittophilly (Bees) A. pedunculata Tubular Absent ornithophily Ornithophily (Hummingbirds) A. grandiflora Salverform Present psychophily Psychophily (Butterflies) Martén-Rodriguez et al. 2015; 0.006 A. patens Salverform Present psychophily Ramírez-Aguirre et al. 2019 Figure 2. Phylogenetic relationships and floral traits in Achimenes. bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

A Cluster Dendrogram 1.0 0.9 0.8 Height 0.7 0.6

Module colors

as.dist(dissTom) fastcluster::hclust (*, "average")

B Cluster Dendrogram 1.0 0.9 0.8 Height 0.7 0.6

Module colors

as.dist(dissTom) fastcluster::hclust (*, "average")

Figure 3. Co-expression clustering of the Bud and D stages. bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Correspondence of Bud and D stage modules

Bud ME8: 276 0 2 5 7 0 2 0 1 0 0 0 0 5 2 0 2 0 1 1 0 3 0 2 0 8 26 4 5 61 6 12 61 29 4 0 1 2 0 0 0 0 1 0 0 0 0 2 3 0 1 1 0 0 2 0 4 1 0 0 0 0 9 Bud ME35: 58 0 0 3 9 0 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 6 2 24 1 0 0 2 0 0 0 0 0 0 0 0 1 0 0 1 0 1 1 0 0 0 0 0 0 0 0 0 0 3 Bud ME59: 29 0 0 0 0 0 0 1 0 2 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 5 6 4 0 3 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 4 Bud ME11: 237 0 0 0 1 0 0 1 0 1 1 0 0 9 8 0 1 0 2 0 0 0 0 1 1 25 10 10 3 82 8 14 12 17 2 1 0 0 0 0 0 0 0 0 0 0 0 1 2 0 3 0 7 0 1 0 0 0 3 1 1 0 8 50 Bud ME9: 266 0 0 0 0 0 2 2 1 0 2 0 0 1 6 2 18 2 0 3 0 1 2 2 0 1 0 3 11 109 14 22 10 16 2 1 0 0 0 0 0 0 2 0 0 0 0 0 5 0 0 2 4 3 2 0 1 0 0 0 0 0 14 Bud ME14: 211 1 0 0 4 1 4 1 0 1 5 0 0 1 0 0 11 0 2 0 0 12 1 1 0 0 2 2 6 20 26 12 67 11 1 0 0 0 0 0 0 0 0 0 0 0 0 0 4 0 0 1 0 0 0 0 0 1 0 0 0 0 13 Bud ME15: 177 0 0 0 0 0 1 7 0 1 0 0 0 67 51 6 1 1 3 3 1 0 0 0 0 0 0 0 0 3 0 0 0 1 0 10 0 0 0 0 0 0 3 0 0 0 0 0 2 1 1 0 3 0 1 0 0 0 4 2 1 0 3 Bud ME51: 35 0 0 0 0 0 0 0 0 0 0 0 0 2 9 1 1 0 9 0 2 0 0 0 0 0 0 0 0 3 0 1 0 0 1 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 0 0 0 1 Bud ME32: 70 0 0 0 0 0 1 4 0 0 0 0 1 3 9 25 4 1 1 4 2 0 0 1 0 0 0 0 0 1 0 1 0 0 0 0 1 0 0 0 0 0 0 2 1 1 0 0 0 0 0 0 2 0 0 1 1 0 0 0 0 0 3 Bud ME41: 48 0 0 0 1 0 0 0 0 0 0 0 0 13 6 8 7 1 1 0 0 0 1 0 0 0 0 0 0 4 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 5 Bud ME29: 84 0 0 4 0 0 0 1 1 0 1 0 0 2 2 0 2 0 0 1 2 1 2 4 5 0 0 1 0 2 0 0 0 1 2 2 0 0 0 0 0 1 1 0 0 0 2 1 24 3 0 0 0 1 1 0 0 2 0 0 0 0 12 Bud ME13: 233 3 0 0 1 3 3 3 0 0 4 0 2 2 11 2 8 1 56 8 6 42 1 0 15 1 0 0 0 6 1 0 5 1 1 0 1 0 0 0 0 2 3 1 0 1 0 1 0 0 0 0 3 0 0 0 1 0 8 0 4 0 22 Bud ME42: 48 0 0 0 3 0 5 3 1 1 1 0 1 5 0 0 0 0 4 1 0 2 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 4 1 0 0 1 1 0 3 0 1 0 1 0 0 0 0 0 5 Bud ME44: 47 4 2 1 1 0 0 2 0 1 0 0 0 2 2 1 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 3 0 0 1 4 4 5 0 0 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 8 Bud ME57: 30 7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 1 1 8 0 0 0 1 0 0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 2 0 4 Bud ME30: 74 0 0 2 1 0 1 2 0 0 0 0 0 5 3 0 0 0 1 0 1 2 0 0 0 2 0 0 0 1 0 2 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 18 1 18 0 0 0 0 0 1 6 0 0 6 40 Bud ME56: 31 1 0 0 0 0 0 0 0 0 0 0 0 7 4 0 0 0 2 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 7 0 0 0 0 0 1 3 0 0 2 Bud ME7: 291 1 0 2 4 0 2 11 0 3 0 3 2 37 60 13 5 6 4 3 3 3 1 0 0 0 1 0 0 17 0 1 7 3 3 17 4 2 0 0 3 0 11 0 0 1 1 0 4 1 0 1 6 1 6 3 1 0 5 1 1 1 26 Bud ME34: 59 0 0 0 0 0 0 4 0 0 1 0 0 11 21 1 1 0 3 2 0 0 0 0 0 0 0 0 1 8 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 1 0 1 1 0 0 0 1 0 0 0 0 0 0 0 0 0 1 Bud ME4: 344 0 1 3 11 1 0 3 1 0 1 0 0 51 12 0 1 0 3 0 1 0 0 1 0 10 2 0 0 13 0 1 3 2 3 17 7 3 4 2 2 0 5 0 0 3 0 10 5 0 2 6 1 0 3 1 2 4 80 10 5 12 36 Bud ME10: 249 0 0 0 2 0 0 3 0 0 0 0 0 123 21 1 1 0 1 1 0 0 0 0 0 2 0 0 0 9 0 0 0 1 2 12 1 0 1 0 0 0 6 0 0 1 0 0 1 0 1 2 2 0 2 1 2 0 30 6 1 0 13 Bud ME28: 108 0 0 3 2 1 2 2 0 0 0 0 0 3 1 0 0 0 0 0 0 0 0 0 0 2 0 1 0 4 1 0 12 22 0 0 0 0 0 0 0 0 0 0 0 0 0 5 22 11 0 0 0 0 0 0 5 2 0 1 2 0 4 Bud ME45: 46 0 0 7 9 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 1 0 6 1 0 1 12 0 0 0 0 0 0 0 0 0 0 0 2 3 Bud ME58: 30 0 0 0 7 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 0 0 0 0 17 0 0 0 0 0 0 0 0 0 0 0 1 Bud ME31: 70 0 0 4 50 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 4 0 0 0 3 1 5 0 0 0 0 0 0 0 0 0 0 2 Bud ME1: 591 2 0 68 259 11 1 0 16 2 8 3 10 4 0 0 1 0 1 0 0 9 0 0 0 2 2 0 0 3 1 3 18 1 7 0 2 1 0 2 0 0 0 0 0 52 0 1 3 1 13 1 2 1 3 0 0 2 2 0 8 32 33 Bud ME24: 121 0 0 6 47 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 2 0 0 0 1 0 0 0 1 0 4 5 1 0 0 0 0 0 1 0 0 0 0 0 6 0 0 0 0 27 9 0 1 1 0 0 0 0 0 0 1 7 30 Bud ME20: 133 2 0 5 24 14 0 0 3 0 21 1 14 0 0 0 1 0 0 0 0 11 0 0 0 0 0 0 0 1 2 1 17 0 0 0 0 0 0 0 0 0 0 0 0 7 0 0 1 1 1 0 0 0 0 0 0 0 0 0 0 0 6 Bud ME26: 108 3 0 0 3 17 2 0 5 0 5 0 0 0 0 0 0 0 1 1 0 55 0 0 0 0 0 0 0 0 0 0 9 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 3 Bud ME49: 37 0 0 3 25 0 0 0 0 0 1 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 Bud ME63: 22 0 0 0 2 0 0 0 1 0 0 4 10 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 Bud ME25: 111 0 0 1 4 0 0 0 0 0 0 0 0 8 1 0 3 0 0 0 0 0 1 0 0 0 1 0 0 1 0 0 1 0 0 1 0 0 0 3 0 0 2 0 0 1 0 4 7 1 1 5 2 0 48 1 1 1 1 1 1 1 8 Bud ME53: 33 0 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 18 0 0 3 0 0 4 0 1 1 0 0 0 0 0 2 0 0 0 0 0 0 0 0 Bud ME50: 36 0 0 3 10 0 0 0 0 0 0 2 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 0 1 6 0 1 2 0 0 0 2 0 0 0 0 0 1 2 Bud ME62: 26 0 0 2 6 1 0 0 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 3 0 0 0 0 0 0 2 0 0 0 0 1 1 0 0 0 0 1 0 0 0 3 0 0 0 0 0 0 0 3 Bud ME38: 49 0 0 2 3 0 0 1 0 1 0 21 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 1 2 0 0 1 0 0 0 0 0 0 0 0 0 0 0 10 0 0 0 0 1 0 0 0 0 0 0 0 0 2 Bud ME12: 234 0 0 1 3 0 0 3 0 1 29 2 5 33 5 10 28 9 0 0 0 4 6 0 0 0 1 0 0 4 0 0 3 1 0 2 2 0 0 0 0 0 4 1 0 5 0 1 8 1 3 1 1 0 9 0 0 0 0 0 0 0 48 Bud ME17: 153 0 0 7 22 0 0 2 2 1 52 7 2 0 0 1 0 1 0 0 0 2 10 0 2 0 0 0 0 1 1 2 4 0 0 0 1 0 0 1 0 0 0 0 0 4 0 0 5 1 1 0 0 1 0 0 0 0 0 0 0 0 20 Bud ME39: 49 1 0 0 0 0 0 0 0 0 1 0 0 3 0 1 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 2 1 15 11 1 4 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 3 20 Bud ME55: 31 0 0 0 1 1 0 0 0 0 9 1 0 1 1 1 6 4 0 0 0 3 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 1 Bud ME5: 320 6 0 0 0 0 15 109 27 45 0 1 0 6 23 4 0 0 6 8 0 4 1 0 0 0 0 0 11 6 0 1 2 0 0 0 1 1 0 0 0 0 0 0 1 2 0 0 13 0 0 0 0 0 0 2 7 0 1 0 0 0 17 Bud ME19: 145 0 2 2 2 0 0 9 1 4 0 0 0 1 4 0 0 0 0 0 0 0 0 0 0 3 0 0 1 4 0 2 6 1 0 2 1 14 1 0 0 0 2 0 2 2 0 2 1 0 3 3 0 0 0 1 29 21 7 7 1 0 4 Bud ME47: 42 0 1 0 1 0 0 0 5 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 1 1 0 2 2 0 0 0 0 0 0 2 23 0 0 0 1 1 Bud ME22: 123 2 1 1 1 2 4 4 70 13 0 2 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1 1 0 0 7 0 0 0 0 0 0 0 0 0 0 0 1 2 0 0 2 0 1 0 0 0 0 0 0 1 0 0 0 0 6 Bud ME36: 52 1 0 0 3 0 16 8 3 2 0 0 0 0 1 0 0 0 3 4 0 2 0 0 0 0 0 0 0 1 0 0 1 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 3 Bud ME60: 27 0 0 0 0 0 6 7 3 1 0 0 0 0 0 0 0 0 0 5 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Bud ME2: 514 1 4 13 101 0 1 3 3 2 0 2 5 77 8 7 1 1 2 2 0 1 0 1 0 4 1 0 0 7 0 1 9 2 3 6 7 3 0 2 0 0 6 1 1 9 1 1 11 4 33 31 9 6 8 3 1 1 9 16 7 8 68 Bud ME46: 43 0 0 0 2 0 1 4 0 0 0 0 0 1 3 17 1 0 1 0 0 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 0 0 0 0 1 0 1 0 0 1 0 0 2 0 0 0 1 2 0 0 0 0 1 0 0 0 2 Bud ME64: 22 0 0 0 0 0 0 14 3 2 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 Bud ME33: 60 0 0 0 0 0 0 2 1 2 0 0 0 0 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 6 0 33 10 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 10 Bud ME6: 317 1 0 0 0 0 0 2 0 0 1 0 0 13 5 1 0 0 3 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 6 0 1 9 3 11 7 196 9 11 16 2 0 3 0 0 0 1 0 0 0 1 0 2 1 0 0 8 Bud ME54: 31 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 2 1 3 2 3 16 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 Bud ME48: 38 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 20 1 0 0 3 1 0 6 1 1 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 Bud ME27: 108 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 3 3 1 24 3 1 59 2 1 1 0 2 0 0 1 0 1 0 0 0 0 0 0 4 Bud ME3: 349 2 0 1 1 1 1 0 3 1 2 2 1 0 0 1 0 0 3 1 1 4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 1 1 2 6 16 9 19 13 234 10 0 0 0 1 0 0 0 0 0 0 0 0 0 1 0 9 Bud ME21: 126 0 0 0 3 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 4 2 0 1 13 0 1 83 7 0 0 0 1 1 0 0 0 0 0 1 0 0 0 0 4 Bud ME43: 47 0 0 0 0 0 1 1 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 1 0 3 10 20 0 1 0 0 0 0 0 0 0 2 0 0 0 0 0 3 Bud ME61: 27 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 6 0 0 3 10 0 1 0 1 0 0 0 0 0 0 0 1 0 0 0 1 Bud ME18: 152 0 0 69 17 2 0 0 2 1 1 0 1 0 0 0 0 0 0 0 0 0 0 1 0 0 4 0 0 3 0 0 0 0 6 3 20 3 0 0 0 0 0 0 0 3 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 1 13 Bud ME40: 48 0 0 2 0 0 0 1 0 0 0 0 0 2 0 3 1 1 0 0 1 0 0 0 0 0 1 0 0 2 0 0 1 0 1 11 8 5 0 0 1 0 1 0 0 1 0 0 0 0 0 0 1 0 1 0 0 0 2 0 0 0 1 Bud ME37: 50 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 4 2 1 0 1 20 0 13 0 0 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 Bud ME52: 33 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 17 3 3 0 0 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 0 Bud ME16: 163 1 0 4 1 0 0 4 0 4 0 0 0 4 2 2 1 2 1 4 3 0 0 0 0 0 4 0 1 6 0 0 0 0 4 35 48 13 0 0 2 0 2 0 0 1 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 0 12 Bud ME23: 123 1 0 1 0 0 0 7 0 1 0 0 0 14 7 0 0 2 2 1 1 0 0 0 0 1 0 0 1 5 0 0 0 0 2 41 7 11 0 0 1 0 1 0 1 0 0 0 1 0 0 0 0 0 0 1 0 0 6 1 1 0 5 Bud ME0: 1658 32 13 40 46 4 30 38 18 24 19 15 6 56 37 17 23 36 28 11 28 41 17 42 19 9 17 6 9 62 17 13 31 10 48 22 32 14 11 4 11 6 31 6 9 55 7 35 60 6 22 7 27 21 15 4 11 14 29 18 58 5 256

D ME37:D ME59:D 73 ME9:D 27 ME1: 266D ME48: 703 D61 ME8: 269 D ME45:D ME46:D 68 ME3:D 65 ME7: 579 326 D ME40:D 70 ME43:D ME47: 69 D 63 ME56:D ME51:D 44 ME55:D 56 ME39:D 46 ME38:D 71 ME60:D 73 ME50:D 27 ME4:D 58 ME29: 462D ME26:D 87 ME6: 99 332D ME27: 98 D ME32:D ME52:D 82 ME53:D 53 ME31:D 52 ME57:D 85 ME5:D 42 ME49: 383D ME30:D 60 ME2:D 87 ME42: 635D ME41: 69 D 69 ME54:D 50 ME33:D 80 ME58:D 39 ME61:D ME35:D 21 ME36: 74 D 74 ME34:D ME28:D 75 ME44:D 94 ME0: 68 768 D ME24: D103 ME14:D ME22:D 172 ME15: 120 165 D ME20:D ME19: 129D 130 ME18: 146D ME11: 210 D ME21:D 127 ME12:D ME17: 201 157 D ME10:D 221 ME16:D 159 ME25:D 102 ME23: 111 D ME13: 198

Figure 4. Correspondence of the Bud and D stage networks. bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Module Membership Preserved Pollination syndrome Flower color Flower shape Spurs Eigengene expression All Hubs in D stage Bee Butterfly Hummingbird Red Purple White Yellow Funnelform Salverform Tubular Present > 1.0 < -1.0 ME61 27 4 AA, AL, EV AE, GC ME47 42 9 AD, AP, AT AG, AM, AN ME7 291 24 AE, AM AD, AL ME2 515 49 AA, AC, AD AE, AM, AN ME3 349 49 AN, EV AM, GC ME46 43 3 No AD, AL, AN AE, AG ME60 27 2 AL, AN, AT AA, AG, AP ME62 26 3 No AE, AG, AP AC, AD, AL, AT ME14 211 3 AN, GC AP, EV ME22 123 20 AD, AE, AT AG, AM ME24 121 14 AC, AD AL, AM, AN, EV ME34 59 6 AA, AM, GC AD, AL ME33 60 9 AT, EV AG, GC ME26 108 3 AD, AE, AT AM, AP ME45 46 6 AA, AD, AE, AL AC, AM ME21 126 13 AD, EV AM, AN, GC ME59 29 7 AE, GC AC, AD, EV ME48 38 7 AG, AT, EV AN, GC ME23 123 6 AC, AL, AM AA, AD, AE ME44 47 4 No AC, AE AA, AD, AL ME5 320 21 AM, AN, AP, AT AD, AG ME11 237 14 AM, GC AD, EV ME0 1659 0 AG, AL, AT AC, AN, AP ME17 153 23 AD, AG, AN AM, AT ME58 30 3 AA, AC, AD, AE AL, AM ME43 47 3 AL, AT, EV AC, AG, GC ME57 30 2 No AC, AE, AT AA, AD, AL, AP ME16 163 17 AC, AE, AL AA, AD ME42 48 8 No AA, AE, AM AC, AD, AL ME18 152 9 AC, AD, AE, AL AA, AM, AN ME56 31 5 AA, AC, AM AD, AE, AL ME41 48 1 No AG, AM AD, AE, AT ME19 145 6 AP, AT AG, AN ME9 266 13 AM, AN, GC AD, EV ME55 31 4 No AD, AG, AN AE, AP, AT ME63 22 5 AD, AN AG, AM, AP, AT ME40 48 8 No AC, AD, AL AA, AE ME15 177 7 AC, AM AD, AE ME54 31 3 AE, AN, EV AD, GC ME25 111 5 AG, AP AN, AT ME39 49 4 AG, EV AE, AT, GC ME31 70 6 AA, AD AM, AN ME53 33 4 AG, AP, EV AN, AT, GC ME8 276 20 AD, GC AE, EV ME38 49 3 AD, AL, AN, AP AG, AT ME49 37 8 AD AM, AT ME10 249 14 AM, AP AD, AE, AN ME6 317 36 AM, EV AD, GC ME20 133 5 AD, AE, AN, GC AL, AM, AP, AT ME29 84 13 AE AC, AD ME13 233 5 AE, AG, AM, AT AC, AD, AL, AP ME52 33 2 AE, AL, EV AA, AD, GC ME35 58 11 AD, GC AM, EV ME28 108 1 AA, AL, AT, GC AC, EV ME64 22 5 AT AE, AG ME37 50 2 AC, AL, EV AA, AE, GC ME30 74 7 AA, AC AD, AL ME12 236 7 AG, AN, AP AD, AT ME51 35 4 AA, AG, AM, AT AE, AP ME50 36 4 AE, AP AC, AG, AN, AT ME1 592 22 AD, AE AL, AM, AN ME32 70 4 AM, AN AD ME27 108 26 AD, EV AE, AM, GC ME4 346 16 AE, AG, AM, AP AD, AL, AN ME36 52 3 AN, AT AG, AP Summary Number modules correlated 3 (4.6%) 7 (10.8%) 13 (20%) 14 (21.5%) 11 (16.9%) 0 1 (1.5%) 1 (1.5%) 8 (12.3%) 5 (7.7%) 5 (7.7%) Number orthogroups correlated 551 (5.8%) 680 (7.2%) 1736 (18.3%) 1766 (18.6%) 1117 (11.8%) 0 52 (0.06%) 317 (3.3%) 1747 (18.4%) 876 (9.2%) 752 (7.9%)

Strongly positive Weakly positive Strongly negative Weakly negative Abbreviations: AA, A. admirabilis; AC, A. cettoana; AD, A. candida; AE, A. erecta; AG, A. grandiflora; AL, A. longiflora; AM, A. misera; AN, A. pedunculata; AP, A. patens; AT, A. antirrhina; EV, E. verticillata; GC, G. cuneifolia.

Figure 5. Summary of Bud stage network module association to floral traits and pollination syndrome. bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Module Membership Preserved Pollination syndrome Flower color Flower shape Spurs Eigengene expression All Hubs in Bud stage Bee Butterfly Hummingbird Red Purple White Yellow Funnelform Salverform Tubular Present > 1.0 < -1.0 ME61 21 4 AP AE, AG, AN, AT ME47 63 1 AC, AG, AN, AT AA, AD, AP ME7 326 35 AM, AT AD, AE ME2 635 63 AE, EV AM, GC ME3 580 41 AM, AP AD, AE, AT ME46 65 9 AE, AN AG, AM, AP, AT ME60 27 2 AL, GC AC, EV ME14 172 25 AD, AE, AN, AT AG, AM ME22 121 12 AN, AP, AT AG ME24 103 3 AN, AT AG, AP ME34 75 2 AA, AC, AM, AP, AT AE, AL, AN ME33 80 3 AA, AP AL, AN ME26 99 5 AC, GC AL, EV ME45 68 6 AN, AP AG, AT ME21 127 7 AA, GC AC, EV ME59 27 3 No AC, AD, AE, AT AA, AL, AM, AN ME48 61 15 AD, AE, AT AM, AP ME23 111 11 AG, AP AE, AN, AT ME44 68 13 AD, AP, AT AM, AN ME5 383 46 AM, EV AD, AE, GC ME11 210 8 AE, AG, AN, AT AL, AM, AP ME0 768 0 AA, AN AL, AT ME17 157 11 AC, AE, AL AA ME58 39 3 AA, AC, AN, AP AG, AL, AT ME43 69 2 AM, AN, AT AA, AD, AG, AP ME57 42 4 AG, AL, EV AA, AC, AP, GC ME16 160 5 AA, AC, AD AL, AM, AN ME42 69 6 AA, AL, AT, EV AC, GC ME18 146 6 AG, AM, AN, AT AD, AE, AP ME56 45 2 AG, AL, AN AC, AP, AT ME41 69 4 AA, AG, AL, AT AC, AN ME19 131 1 AG, AM, AN, GC AP, AT, EV ME9 266 22 AD, AE AA, AM, AN, AT ME55 46 2 AA, AG, AL, AN, AT AC, AP ME40 70 3 AC, AG, AL, AM, AN AA, AD, AT ME15 165 8 AD, AE, AG, AN AM, AP, AT ME54 50 4 AA, AD AC, AM ME25 102 6 AA, AC, AM AE, AL ME39 71 2 AT, GC AN, EV ME31 85 8 AC, AL, EV AA, GC ME53 52 3 AG, AP, EV AN, AT, GC ME8 270 23 AM, AN, AT AD, AE, AG ME38 73 3 AP, GC AA, AN, AT, EV ME49 60 3 AG, AN, EV AP, GC ME10 221 4 AA, AL AC ME6 332 25 AD, AE, GC AM, EV ME20 129 5 AM, AN AD, AE, AG, AT ME29 87 2 AN, GC AA, AP, EV ME13 198 19 AG, AM, AP, AT AE, AN ME52 53 10 AG, AT, EV AN, GC ME35 74 8 AL, AP, AT AE, AG, AN ME28 94 6 AG, AT AN, AP ME37 73 3 AC, AE, AN AA, AL, AP ME30 87 7 AN, AT, EV AG, GC ME12 201 14 AC, AL, AM AA, AD, AE ME51 56 2 AG, AL, AT AA, AC, AP ME50 58 8 AN, AT, GC AG, EV ME1 704 76 AD, AE AL, AM, AN, AT ME32 82 4 AC, AL, AP, AT AA, AN ME27 98 2 AC, AG AA, AN, AP ME4 462 43 AM, GC AD, AE, EV ME36 74 9 AD, AP, AT AG, AN Summary Number modules correlated 1 (1.5%) 8 (12.9%) 12 (19.4%) 8 (12.9%) 9 (14.5%) 0 0 1 (1.6%) 7 (12.3%) 5 (8.1%) 1 (1.6%) Number orthogroups correlated 383 (4.0%) 969 (10.2%) 1528 (16.1%) 680 (7.2%) 820 (8.6%) 0 0 61 (0.06%) 1838 (19.3%) 605 (6.4%) 52 (0.06%)

Strongly positive Weakly positive Strongly negative Weakly negative Abbreviations: AA, A. admirabilis; AC, A. cettoana; AD, A. candida; AE, A. erecta; AG, A. grandiflora; AL, A. longiflora; AM, A. misera; AN, A. pedunculata; AP, A. patens; AT, A. antirrhina; EV, E. verticillata; GC, G. cuneifolia. Figure 6. Summary of D stage network module association to floral traits and pollination syndrome. bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

A B Bud stage D stage

1.50 1.50

1.25 1.25

1.00 1.00

0.75 0.75

0.50 0.50 Omega (dN/dS)

0.25 0.25

0.00 0.00

0 20 40 60 80 0 50 100 150 Connectivity Connectivity

Figure 7. Relationship between orthogroup dN/dS (omega) and orthogroup connectivity. bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

antiquewhite4ME61 darkturquoiseME23 mediumpurple3ME40 ME64skyblue2 0.2 0.3 0.1 0.1 0.0 0.1 −0.1 −0.1 −0.1 −0.2 eigengene expression eigengene expression eigengene expression eigengene expression −0.3 −0.3 −0.4 −0.3

AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3

ME47bisque4 floralwhiteME44 midnightblueME15 ME37skyblue3 0.2 0.2 0.1 0.1 0.0 0.0 −0.1 −0.1 −0.2 eigengene expression eigengene expression eigengene expression eigengene expression −0.2 −0.3 −0.3 −0.4

AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3

ME7black ME5green navajowhite2ME54 ME30steelblue 0.3 0.2 0.1 0.0 0.1 0.0 −0.1 −0.2 −0.1 −0.2 eigengene expression eigengene expression eigengene expression eigengene expression −0.4 −0.3 −0.3 −0.4

AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3

ME2blue greenyellowME11 ME25orange ME12tan 0.3 0.3 0.2 0.3 0.1 0.1 0.0 0.1 −0.1 −0.1 −0.2 −0.1 eigengene expression eigengene expression eigengene expression eigengene expression −0.3 −0.3 −0.4 −0.3

AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3

ME3brown ME0grey orangered4ME39 ME51thistle1 0.3 0.1 0.2 0.1 0.1 0.0 −0.1 −0.1 −0.1 eigengene expression eigengene expression eigengene expression eigengene expression −0.2 −0.3 −0.3 −0.3

AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3

ME46brown4 ME17grey60 paleturquoiseME31 ME50thistle2 0.3 0.2 0.4 0.3 0.1 0.0 0.2 0.1 −0.1 −0.2 0.0 −0.1 eigengene expression eigengene expression eigengene expression eigengene expression −0.3 −0.4 −0.3 −0.2

AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3

ME60coral1 honeydew1ME58 palevioletred3ME53 ME1turquoise 0.2 0.3 0.2 0.1 0.0 0.1 0.0 −0.1 −0.2 −0.1 eigengene expression eigengene expression eigengene expression eigengene expression −0.2 −0.3 −0.4 −0.3

AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3

ME62coral2 ME43ivory ME8pink ME32violet 0.3 0.4 0.3 0.1 0.2 0.1 0.1 0.0 −0.1 −0.1 −0.1 eigengene expression eigengene expression eigengene expression eigengene expression −0.2 −0.3 −0.3 −0.3

AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3

ME14cyan lavenderblush3ME57 ME38plum1 ME27white 0.3 0.2 0.3 0.2 0.1 0.1 0.1 0.0 0.0 −0.1 −0.1 eigengene expression eigengene expression eigengene expression eigengene expression −0.2 −0.2 −0.3 −0.3

AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3

ME22darkgreen lightcyanME16 ME49plum2 ME4yellow 0.2 0.4 0.1 0.0 0.0 0.2 −0.1 0.0 −0.2 −0.2 eigengene expression eigengene expression eigengene expression eigengene expression −0.3 −0.2 −0.4 −0.4

AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3

ME24darkgrey lightcyan1ME42 ME10purple yellowgreenME36 0.3 0.3 0.2 0.3 0.1 0.1 0.1 0.1 0.0 −0.1 −0.1 −0.1 −0.2 eigengene expression eigengene expression eigengene expression eigengene expression −0.3 −0.3 −0.3

AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3

darkmagentaME34 ME18lightgreen ME6red 0.3 0.3 0.1 0.1 0.1 −0.1 −0.1 −0.1 eigengene expression eigengene expression eigengene expression −0.3 −0.3 −0.3

AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3

darkolivegreenME33 lightpink4ME56 ME20royalblue 0.2 0.1 0.1 0.0 −0.1 −0.1 eigengene expression eigengene expression eigengene expression −0.2 −0.3 −0.3

AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3

darkorangeME26 lightsteelblue1ME41 saddlebrownME29 0.1 0.0 0.0 −0.2 −0.1 −0.2 eigengene expression eigengene expression eigengene expression −0.4 −0.4 −0.3

AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3

darkorange2ME45 lightyellowME19 ME13salmon 0.2 0.2 0.2 0.1 0.0 0.0 −0.1 −0.2 eigengene expression eigengene expression eigengene expression −0.2 −0.4 −0.3

AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3

ME21darkred ME9magenta ME52salmon4 0.3 0.3 0.1 0.1 0.1 −0.1 −0.1 −0.1 eigengene expression eigengene expression eigengene expression −0.3 −0.3 −0.3

AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3

darkseagreen4ME59 ME55maroon ME35sienna3 0.3 0.2 0.1 0.1 0.0 −0.1 −0.1 eigengene expression eigengene expression eigengene expression −0.3 −0.2 −0.3

AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3

darkslateblueME48 mediumorchidME63 ME28skyblue 0.3 0.1 0.1 0.1 −0.1 −0.1 eigengene expression eigengene expression eigengene expression −0.1 −0.3 −0.3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3 AA1 AA2 AA3 AC1 AC2 AC3 AD1 AD2 AD3 AE1 AE2 AE3 AG1 AG2 AG3 AL1 AL2 AL3 AM1 AM2 AM3 AN1 AN2 AN3 AP1 AP2 AP3 AT1 AT2 AT3 EV1 EV2 EV3 GC1 GC2 GC3

Supplemental Figure 1. Module eigengenes in the Bud stage network. bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

antiquewhite4ME61 floralwhiteME44 navajowhite2ME54 ME12tan 0.2 0.1 0.3 0.0 0.0 0.1 −0.2 −0.1 −0.2 eigengene expression eigengene expression eigengene expression eigengene expression −0.1 −0.4 −0.3 −0.4

AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6

ME47bisque4 ME5green ME25orange ME51thistle1 0.3 0.3 0.1 0.1 0.1 0.1 −0.1 −0.1 −0.1 −0.1 eigengene expression eigengene expression eigengene expression eigengene expression −0.3 −0.3 −0.3 −0.3

AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6

ME7black greenyellowME11 orangered4ME39 ME50thistle2 0.3 0.3 0.0 0.1 0.1 0.1 −0.2 −0.1 −0.1 −0.1 eigengene expression eigengene expression eigengene expression eigengene expression −0.3 −0.3 −0.4 −0.3

AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6

ME2blue ME0grey paleturquoiseME31 ME1turquoise 0.3 0.3 0.2 0.1 0.1 0.1 0.0 −0.1 −0.1 −0.1 −0.2 eigengene expression eigengene expression eigengene expression eigengene expression −0.3 −0.3 −0.4 −0.3

AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6

ME3brown ME17grey60 palevioletred3ME53 ME32violet 0.2 0.3 0.2 0.1 0.0 0.1 0.0 −0.1 −0.2 −0.1 eigengene expression eigengene expression eigengene expression eigengene expression −0.2 −0.4 −0.3 −0.3

AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6

ME46brown4 honeydew1ME58 ME8pink ME27white 0.3 0.4 0.1 0.1 0.1 0.2 −0.1 −0.1 −0.1 0.0 eigengene expression eigengene expression eigengene expression eigengene expression −0.3 −0.3 −0.3 −0.2

AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6

ME60coral1 ME43ivory ME38plum1 ME4yellow 0.3 0.3 0.3 0.2 0.1 0.1 0.1 0.0 −0.1 −0.1 −0.1 eigengene expression eigengene expression eigengene expression eigengene expression −0.3 −0.2 −0.3 −0.3

AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6

ME14cyan lavenderblush3ME57 ME49plum2 yellowgreenME36 0.3 0.0 0.1 0.1 0.1 −0.2 −0.1 −0.1 −0.1 eigengene expression eigengene expression eigengene expression eigengene expression −0.4 −0.3 −0.3 −0.3

AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6

ME22darkgreen ME16lightcyan ME10purple 0.2 0.2 0.2 0.0 0.0 0.0 −0.2 −0.2 −0.2 eigengene expression eigengene expression eigengene expression −0.4 −0.4 −0.4

AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6

ME24darkgrey lightcyan1ME42 ME6red 0.3 0.2 0.1 0.1 0.0 −0.1 −0.1 eigengene expression eigengene expression eigengene expression −0.2 −0.3 −0.3

AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6

darkmagentaME34 ME18lightgreen royalblueME20 0.2 0.4 0.1 0.0 0.2 0.0 −0.1 −0.2 eigengene expression eigengene expression eigengene expression −0.2 −0.4 −0.3

AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6

darkolivegreenME33 ME56lightpink4 saddlebrownME29 0.3 0.4 0.1 0.1 0.2 −0.1 0.0 −0.1 eigengene expression eigengene expression eigengene expression −0.3 −0.2 −0.3

AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6

ME26darkorange lightsteelblue1ME41 ME13salmon 0.3 0.2 0.1 0.1 0.0 −0.1 −0.1 −0.2 eigengene expression eigengene expression eigengene expression −0.3 −0.4 −0.3

AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6

darkorange2ME45 ME19lightyellow ME52salmon4 0.1 0.1 0.1 −0.1 −0.1 −0.1 eigengene expression eigengene expression eigengene expression −0.3 −0.3 −0.3

AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6

ME21darkred ME9magenta ME35sienna3 0.3 0.3 0.2 0.1 0.1 0.0 −0.1 −0.1 eigengene expression eigengene expression eigengene expression −0.2 −0.3 −0.3

AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6

darkseagreen4ME59 ME55maroon ME28skyblue 0.3 0.1 0.2 0.1 −0.1 0.0 −0.1 eigengene expression eigengene expression eigengene expression −0.3 −0.3 −0.2

AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6

darkslateblueME48 mediumpurple3ME40 ME37skyblue3 0.1 0.2 0.1 −0.1 0.0 −0.1 eigengene expression eigengene expression eigengene expression −0.3 −0.2 −0.3

AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6

darkturquoiseME23 midnightblueME15 ME30steelblue 0.2 0.0 0.1 0.1 −0.2 −0.1 −0.1 eigengene expression eigengene expression eigengene expression −0.4 −0.3 −0.3

AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6 AA4 AA5 AA6 AC4 AC5 AC6 AD4 AD5 AD6 AE4 AE5 AE6 AG4 AG5 AG6 AL4 AL5 AL6 AM4 AM5 AM6 AN4 AN5 AN6 AP4 AP5 AP6 AT4 AT5 EV4 EV5 EV6 GC4 GC5 GC6

Supplemental Figure 2. Module eigengenes in the D stage network. bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

ME61 ME24 ME5 ME56 ME39 ME13 ME1 30 15 20 30 10 20 20 20 10 ● ● ● ● ● 20 ● ●

● 5

5 ● 10 10 10 ● ● ● 10 ● ● ● 10 ● ● ● ● ● ● ●

0 ● ● ● ● 1 1 1 1 1 1 1

0 ● ● ● ● ● ● ● ● ● 0 ● ● ● ● ● ● ● 0 ● ● ● ● ● 0 ● ●

0 ●

● 0 ● ● ● ● ● ● ● ● −5 ● ● ● ● ● ● ● ● ● ● −5

50 ●

● ● ● ● ● 0 ● ● ● ● −10 −50 ● 1

● −100 −150 −200

1 ● (Intercept) residual.variance phylogenetic.variance traitred.1:MEskyblue3 traitspur.1:MEskyblue3 traitwhite.1:MEskyblue3 traityellow.1:MEskyblue3 traitpurple.1:MEskyblue3 traittubular.1:MEskyblue3 traitsalverform.1:MEskyblue3 traitfunnelform.1:MEskyblue3 ● −10 ● ● ● −10 ● ● −10 −10 −10 −20 −10 −20 −15

1 1 1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) residual.variance residual.variance residual.variance residual.variance residual.variance residual.variance residual.variance traitred.1:MEgreen traitspur.1:MEgreen traitred.1:MEsalmon traitwhite.1:MEgreen traitspur.1:MEsalmon traityellow.1:MEgreen traitred.1:MEdarkgrey traitpurple.1:MEgreen phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitwhite.1:MEsalmon traittubular.1:MEgreen traitred.1:MEturquoise traitred.1:MElightpink4 traitspur.1:MEdarkgrey traityellow.1:MEsalmon traitspur.1:MEturquoise traitpurple.1:MEsalmon traitspur.1:MElightpink4 traitwhite.1:MEdarkgrey traittubular.1:MEsalmon traitwhite.1:MEturquoise traitwhite.1:MElightpink4 traitred.1:MEorangered4 traityellow.1:MEdarkgrey traitpurple.1:MEdarkgrey traityellow.1:MEturquoise traittubular.1:MEdarkgrey traitpurple.1:MEturquoise traityellow.1:MElightpink4 traitspur.1:MEorangered4 traitpurple.1:MElightpink4 traittubular.1:MEturquoise traitsalverform.1:MEgreen traittubular.1:MElightpink4 traitfunnelform.1:MEgreen traitwhite.1:MEorangered4 traitred.1:MEantiquewhite4 traityellow.1:MEorangered4 traitsalverform.1:MEsalmon traitpurple.1:MEorangered4 traitfunnelform.1:MEsalmon traitspur.1:MEantiquewhite4 traittubular.1:MEorangered4 traitwhite.1:MEantiquewhite4 traitsalverform.1:MEdarkgrey traitfunnelform.1:MEdarkgrey traitsalverform.1:MEturquoise traitfunnelform.1:MEturquoise traitsalverform.1:MElightpink4 traityellow.1:MEantiquewhite4 traitpurple.1:MEantiquewhite4 traitfunnelform.1:MElightpink4 traittubular.1:MEantiquewhite4 traitsalverform.1:MEorangered4 traitfunnelform.1:MEorangered4 traitsalverform.1:MEantiquewhite4 traitfunnelform.1:MEantiquewhite4

ME47 ME34 ME11 ME41 ME31 ME52 ME32 15 30 15 10

10 ● ● ● 10 10 ● 20 ● 0 ● 20 ● 10 ● ● ● ● ● ● ● ● 5 ● ● ● ● ● ● ● ● 5

5 ● −10

0 ● ● 10 ● ● ● 10 ● ● ● ● ● ● 1 1 1 1 1 1 1 0 ● −20 ● ● 0 ● ● ● ● ● ● ●

0 ● ● ● ● ● ● ● ● ● ● ● 0 ● −10

● 0 ● ● −30 ● ● ● ● ● −5 ● ●

● −5 ● ● ● ● ● ● −5

● ● −40 ● −10 −20 −10 −10 −10 −50 −10 −15

1 1 1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) residual.variance residual.variance residual.variance residual.variance residual.variance residual.variance residual.variance traitred.1:MEviolet traitspur.1:MEviolet traitwhite.1:MEviolet traitred.1:MEbisque4 traityellow.1:MEviolet traitpurple.1:MEviolet traitred.1:MEsalmon4 traittubular.1:MEviolet phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitspur.1:MEbisque4 traitspur.1:MEsalmon4 traitwhite.1:MEbisque4 traitwhite.1:MEsalmon4 traityellow.1:MEbisque4 traitpurple.1:MEbisque4 traittubular.1:MEbisque4 traityellow.1:MEsalmon4 traitpurple.1:MEsalmon4 traitred.1:MEgreenyellow traittubular.1:MEsalmon4 traitsalverform.1:MEviolet traitfunnelform.1:MEviolet traitspur.1:MEgreenyellow traitred.1:MEdarkmagenta traitred.1:MEpaleturquoise traitwhite.1:MEgreenyellow traitspur.1:MEdarkmagenta traitred.1:MElightsteelblue1 traitspur.1:MEpaleturquoise traityellow.1:MEgreenyellow traitpurple.1:MEgreenyellow traitsalverform.1:MEbisque4 traitwhite.1:MEdarkmagenta traitfunnelform.1:MEbisque4 traitspur.1:MElightsteelblue1 traittubular.1:MEgreenyellow traitwhite.1:MEpaleturquoise traitsalverform.1:MEsalmon4 traitfunnelform.1:MEsalmon4 traityellow.1:MEdarkmagenta traitpurple.1:MEdarkmagenta traitwhite.1:MElightsteelblue1 traityellow.1:MEpaleturquoise traitpurple.1:MEpaleturquoise traittubular.1:MEdarkmagenta traittubular.1:MEpaleturquoise traityellow.1:MElightsteelblue1 traitpurple.1:MElightsteelblue1 traittubular.1:MElightsteelblue1 traitsalverform.1:MEgreenyellow traitfunnelform.1:MEgreenyellow traitsalverform.1:MEdarkmagenta traitfunnelform.1:MEdarkmagenta traitsalverform.1:MEpaleturquoise traitfunnelform.1:MEpaleturquoise traitsalverform.1:MElightsteelblue1 traitfunnelform.1:MElightsteelblue1

ME7 ME33 ME0 ME19 ME53 ME35 ME27 30 20 15 40 15 15 20 10 ● ● ● ● 10 20 10

● 10 ●

● 10 ● ● 10 ● ● ● ● ● ● ● 0 ● ● ● ● ● ● ● 0 ● ● ● 5 ● ● ●

5 ● 0 ● ● ● ●

● 5 ● ●

0 ● 1 1 1 1 1 ● ● 1 1 ● ● ● ●

0 ● ● ● ● ● ● ● ● ● ● ● −10 ● ● −20 ● −10

0 ● ● ●

0 ● ● ● ● ● ● ● ● ● ● −5 ● ● ● ● ● −40 ● −20 −10 −20 −5 −5 −10 −30 −60 −15 −30 −10 −10 −20

1 1 1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) residual.variance residual.variance residual.variance residual.variance residual.variance residual.variance residual.variance traitred.1:MEgrey traitred.1:MEblack traitred.1:MEwhite traitspur.1:MEgrey traitspur.1:MEblack traitspur.1:MEwhite traitwhite.1:MEgrey traitwhite.1:MEblack traitwhite.1:MEwhite traityellow.1:MEgrey traitpurple.1:MEgrey traittubular.1:MEgrey traitred.1:MEsienna3 traityellow.1:MEblack traityellow.1:MEwhite traitpurple.1:MEblack traitpurple.1:MEwhite traittubular.1:MEblack traittubular.1:MEwhite phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitspur.1:MEsienna3 traitwhite.1:MEsienna3 traitred.1:MElightyellow traityellow.1:MEsienna3 traitpurple.1:MEsienna3 traitspur.1:MElightyellow traittubular.1:MEsienna3 traitsalverform.1:MEgrey traitfunnelform.1:MEgrey traitwhite.1:MElightyellow traitsalverform.1:MEblack traitsalverform.1:MEwhite traitfunnelform.1:MEblack traitfunnelform.1:MEwhite traityellow.1:MElightyellow traitpurple.1:MElightyellow traittubular.1:MElightyellow traitred.1:MEpalevioletred3 traitred.1:MEdarkolivegreen traitspur.1:MEpalevioletred3 traitsalverform.1:MEsienna3 traitfunnelform.1:MEsienna3 traitspur.1:MEdarkolivegreen traitwhite.1:MEpalevioletred3 traitwhite.1:MEdarkolivegreen traityellow.1:MEpalevioletred3 traitpurple.1:MEpalevioletred3 traittubular.1:MEpalevioletred3 traitsalverform.1:MElightyellow traityellow.1:MEdarkolivegreen traitfunnelform.1:MElightyellow traitpurple.1:MEdarkolivegreen traittubular.1:MEdarkolivegreen traitsalverform.1:MEpalevioletred3 traitfunnelform.1:MEpalevioletred3 traitsalverform.1:MEdarkolivegreen traitfunnelform.1:MEdarkolivegreen

ME2 ME26 ME17 ME9 ME8 ME28 ME4 30 15 15 30 30 15 10 10 20 ● ● ● 10 ● 20 ●

20 ● ● 5 ● ●

● ● ● 10 ● ● 5 ●

10 ●

0 ● ● ● ● 10 ●

0 ● ● ● ● 10 ● ● ● ● ●

● 5 ● 0 ● ● 1 1 1 1 1 1 ● 1 ●

● 0 ● ● ● 0 ● ● ● ● ● ● ● ● ● −5 ● ● ● ● 0 ● ● ● ● ● ● ● ● ● −10 ●

−5 ●

0 ● ● ● ● ● ● ● −10 −10 ● ● −10 ● ● ● −10

● −10 −5 −15 −20 −20 −20 −15 −20 −20 −30 −10

1 1 1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) residual.variance residual.variance residual.variance residual.variance residual.variance traitred.1:MEpink residual.variance residual.variance traitred.1:MEblue traitspur.1:MEpink traitspur.1:MEblue traitwhite.1:MEpink traitred.1:MEyellow traitwhite.1:MEblue traitred.1:MEgrey60 traityellow.1:MEpink traityellow.1:MEblue traitpurple.1:MEpink traitspur.1:MEyellow traitpurple.1:MEblue traitred.1:MEskyblue traittubular.1:MEpink traittubular.1:MEblue traitspur.1:MEgrey60 traitwhite.1:MEyellow traitspur.1:MEskyblue traitred.1:MEmagenta traitwhite.1:MEgrey60 phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traityellow.1:MEyellow traitpurple.1:MEyellow traitwhite.1:MEskyblue traittubular.1:MEyellow traityellow.1:MEgrey60 traitspur.1:MEmagenta traitpurple.1:MEgrey60 traittubular.1:MEgrey60 traityellow.1:MEskyblue traitpurple.1:MEskyblue traitwhite.1:MEmagenta traittubular.1:MEskyblue traitsalverform.1:MEpink traitred.1:MEdarkorange traitsalverform.1:MEblue traitfunnelform.1:MEpink traitfunnelform.1:MEblue traityellow.1:MEmagenta traitpurple.1:MEmagenta traittubular.1:MEmagenta traitspur.1:MEdarkorange traitwhite.1:MEdarkorange traitsalverform.1:MEyellow traitfunnelform.1:MEyellow traitsalverform.1:MEgrey60 traitfunnelform.1:MEgrey60 traityellow.1:MEdarkorange traitpurple.1:MEdarkorange traitsalverform.1:MEskyblue traittubular.1:MEdarkorange traitfunnelform.1:MEskyblue traitsalverform.1:MEmagenta traitfunnelform.1:MEmagenta traitsalverform.1:MEdarkorange traitfunnelform.1:MEdarkorange

ME3 ME45 ME58 ME55 ME38 ME64 ME36 20 15 15 20 40 10 15 5 10 10 ● ● ● 20 10 ● ● 5 ● ● ● ● ●

10 ● 5 ● ● ●

5 ● ● ● 0 ● ● ● ● ● 5 ● ●

● 0 ●

● 0 ● ● ● ● ● ●

0 ● 1 1 1 1 1 ● ● 1 1 ● ● ● ● ● ● ●

0 ● ● ● ● ● ● 0 ● ● ● ● ● 0 ● ● ● ● ● ● ● ● ● ● −5 ● −5 ● ● −20 −5 ● ● ● ● ● ●

● −5 ● ● −5 ● −40 −10 −10 −10 −10 −10 −10 −15 −15 −60 −15

1 1 1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) residual.variance residual.variance residual.variance residual.variance residual.variance residual.variance residual.variance traitred.1:MEbrown traitred.1:MEplum1 traitspur.1:MEbrown traitspur.1:MEplum1 traitred.1:MEmaroon traitwhite.1:MEbrown traitwhite.1:MEplum1 traitspur.1:MEmaroon phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitred.1:MEskyblue2 traityellow.1:MEbrown traityellow.1:MEplum1 traitpurple.1:MEbrown traitpurple.1:MEplum1 traittubular.1:MEbrown traitwhite.1:MEmaroon traittubular.1:MEplum1 traitspur.1:MEskyblue2 traityellow.1:MEmaroon traitpurple.1:MEmaroon traitwhite.1:MEskyblue2 traittubular.1:MEmaroon traitred.1:MEhoneydew1 traityellow.1:MEskyblue2 traitpurple.1:MEskyblue2 traitred.1:MEyellowgreen traittubular.1:MEskyblue2 traitspur.1:MEhoneydew1 traitred.1:MEdarkorange2 traitspur.1:MEyellowgreen traitsalverform.1:MEbrown traitsalverform.1:MEplum1 traitwhite.1:MEhoneydew1 traitfunnelform.1:MEbrown traitfunnelform.1:MEplum1 traitspur.1:MEdarkorange2 traitwhite.1:MEyellowgreen traityellow.1:MEhoneydew1 traitpurple.1:MEhoneydew1 traitwhite.1:MEdarkorange2 traitsalverform.1:MEmaroon traityellow.1:MEyellowgreen traittubular.1:MEhoneydew1 traitpurple.1:MEyellowgreen traitfunnelform.1:MEmaroon traittubular.1:MEyellowgreen traityellow.1:MEdarkorange2 traitpurple.1:MEdarkorange2 traitsalverform.1:MEskyblue2 traittubular.1:MEdarkorange2 traitfunnelform.1:MEskyblue2 traitsalverform.1:MEhoneydew1 traitfunnelform.1:MEhoneydew1 traitsalverform.1:MEyellowgreen traitfunnelform.1:MEyellowgreen traitsalverform.1:MEdarkorange2 traitfunnelform.1:MEdarkorange2

ME46 ME21 ME43 ME63 ME49 ME37 20 15 10 10 10 50 ● 10

● 5 ● 10 ● ● 5 ● ● 5 ● ● ● ● ● 0 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 0 ●

0 ●

0 ● ● ● 5 ● ● ● ● ● ●

0 ● ● −50 1 1 1 1 ● ● 1 1 ● ● ● ● ● ● −5 ● ● ● −5 ● ● 0 ● ● ● ● ● ● −10 ● ●

● −100 −5 ● ● ● −10 ● −10 −5 −15 −20 −150 −15 −10 −10 −20 −200 −30 −20

1 1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) residual.variance residual.variance residual.variance residual.variance residual.variance residual.variance traitred.1:MEivory traitspur.1:MEivory traitred.1:MEplum2 traitwhite.1:MEivory traitspur.1:MEplum2 traitred.1:MEbrown4 traitred.1:MEdarkred traityellow.1:MEivory traitpurple.1:MEivory traitwhite.1:MEplum2 traittubular.1:MEivory traitspur.1:MEbrown4 traitspur.1:MEdarkred phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitred.1:MEskyblue3 traityellow.1:MEplum2 traitpurple.1:MEplum2 traitwhite.1:MEbrown4 traittubular.1:MEplum2 traitwhite.1:MEdarkred traitspur.1:MEskyblue3 traityellow.1:MEbrown4 traitpurple.1:MEbrown4 traityellow.1:MEdarkred traitpurple.1:MEdarkred traitwhite.1:MEskyblue3 traittubular.1:MEbrown4 traittubular.1:MEdarkred traityellow.1:MEskyblue3 traitpurple.1:MEskyblue3 traitsalverform.1:MEivory traitfunnelform.1:MEivory traittubular.1:MEskyblue3 traitsalverform.1:MEplum2 traitfunnelform.1:MEplum2 traitred.1:MEmediumorchid traitsalverform.1:MEbrown4 traitfunnelform.1:MEbrown4 traitsalverform.1:MEdarkred traitfunnelform.1:MEdarkred traitspur.1:MEmediumorchid traitsalverform.1:MEskyblue3 traitwhite.1:MEmediumorchid traitfunnelform.1:MEskyblue3 traityellow.1:MEmediumorchid traitpurple.1:MEmediumorchid traittubular.1:MEmediumorchid traitsalverform.1:MEmediumorchid traitfunnelform.1:MEmediumorchid

ME60 ME59 ME57 ME40 ME10 ME30 30 15 20 15 15

● 20 10 10 20 10 ● ● 10

● ● ● ● ● ● ● ● ●

0 ● 5 ● ● 5 ● ● ●

● 5 10 ● ● 10 ● ● ● 1 1 1 1 1 1 0 ● ● ● ● ● ● −10 0 ● ● ● ● ●

0 ●

0 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● −5 ●

● 0 ● ● −20 ● ● ● ● −5 ● ● ● ● −5

−10 ● −10 ● −30 −10 −15 −10 −10 −20 −40

1 1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) residual.variance residual.variance residual.variance residual.variance residual.variance residual.variance traitred.1:MEcoral1 traitred.1:MEpurple traitspur.1:MEcoral1 traitspur.1:MEpurple traitwhite.1:MEcoral1 traitwhite.1:MEpurple phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitred.1:MEsteelblue traityellow.1:MEcoral1 traitpurple.1:MEcoral1 traityellow.1:MEpurple traitpurple.1:MEpurple traittubular.1:MEcoral1 traittubular.1:MEpurple traitspur.1:MEsteelblue traitwhite.1:MEsteelblue traityellow.1:MEsteelblue traitpurple.1:MEsteelblue traittubular.1:MEsteelblue traitsalverform.1:MEcoral1 traitfunnelform.1:MEcoral1 traitsalverform.1:MEpurple traitfunnelform.1:MEpurple traitred.1:MEdarkseagreen4 traitred.1:MElavenderblush3 traitred.1:MEmediumpurple3 traitspur.1:MEdarkseagreen4 traitspur.1:MElavenderblush3 traitsalverform.1:MEsteelblue traitfunnelform.1:MEsteelblue traitspur.1:MEmediumpurple3 traitwhite.1:MEdarkseagreen4 traitwhite.1:MElavenderblush3 traitwhite.1:MEmediumpurple3 traityellow.1:MEdarkseagreen4 traityellow.1:MElavenderblush3 traitpurple.1:MEdarkseagreen4 traitpurple.1:MElavenderblush3 traityellow.1:MEmediumpurple3 traittubular.1:MEdarkseagreen4 traittubular.1:MElavenderblush3 traitpurple.1:MEmediumpurple3 traittubular.1:MEmediumpurple3 traitsalverform.1:MEdarkseagreen4 traitsalverform.1:MElavenderblush3 traitfunnelform.1:MEdarkseagreen4 traitfunnelform.1:MElavenderblush3 traitsalverform.1:MEmediumpurple3 traitfunnelform.1:MEmediumpurple3

ME62 ME48 ME16 ME15 ME6 ME12 50 15 15 20 20 40 20 10 10 ● ● ● 10 30 ● ● ● 5 ● 10 5 ● ● ●

20 ● 10 ● ● ● ● ● ● ● ● ● ● ● ● ● 0 ● 1 1 1 1 1 1 0 ● ● ● ● ● 0 ● ● ●

● 10

0 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●

● 0 ● 0 ● −5 ● ● −5 ● ● ● ● ● ● −10 ● ● ● ●

−10 ●

−10 ● −10 −10 −20 −10 −20 −15 −20

1 1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) traitred.1:MEtan traitred.1:MEred residual.variance residual.variance residual.variance residual.variance residual.variance residual.variance traitspur.1:MEtan traitspur.1:MEred traitwhite.1:MEtan traitwhite.1:MEred traityellow.1:MEtan traitred.1:MEcoral2 traitpurple.1:MEtan traityellow.1:MEred traitpurple.1:MEred traittubular.1:MEtan traittubular.1:MEred traitspur.1:MEcoral2 traitwhite.1:MEcoral2 phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitred.1:MElightcyan traityellow.1:MEcoral2 traitpurple.1:MEcoral2 traittubular.1:MEcoral2 traitspur.1:MElightcyan traitsalverform.1:MEtan traitsalverform.1:MEred traitfunnelform.1:MEtan traitfunnelform.1:MEred traitwhite.1:MElightcyan traityellow.1:MElightcyan traitpurple.1:MElightcyan traittubular.1:MElightcyan traitred.1:MEmidnightblue traitsalverform.1:MEcoral2 traitred.1:MEdarkslateblue traitfunnelform.1:MEcoral2 traitspur.1:MEmidnightblue traitspur.1:MEdarkslateblue traitwhite.1:MEmidnightblue traitwhite.1:MEdarkslateblue traityellow.1:MEmidnightblue traitpurple.1:MEmidnightblue traitsalverform.1:MElightcyan traittubular.1:MEmidnightblue traitfunnelform.1:MElightcyan traityellow.1:MEdarkslateblue traitpurple.1:MEdarkslateblue traittubular.1:MEdarkslateblue traitsalverform.1:MEmidnightblue traitfunnelform.1:MEmidnightblue traitsalverform.1:MEdarkslateblue traitfunnelform.1:MEdarkslateblue

ME14 ME23 ME42 ME54 ME20 ME51 20 50 15 100 20 20 40

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1 1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) residual.variance residual.variance residual.variance residual.variance residual.variance residual.variance traitred.1:MEcyan traitspur.1:MEcyan traitwhite.1:MEcyan traitred.1:MEthistle1 traityellow.1:MEcyan traitpurple.1:MEcyan traitspur.1:MEthistle1 traittubular.1:MEcyan phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitred.1:MEroyalblue traitwhite.1:MEthistle1 traitspur.1:MEroyalblue traityellow.1:MEthistle1 traitred.1:MElightcyan1 traitpurple.1:MEthistle1 traittubular.1:MEthistle1 traitwhite.1:MEroyalblue traitspur.1:MElightcyan1 traityellow.1:MEroyalblue traitsalverform.1:MEcyan traitpurple.1:MEroyalblue traitwhite.1:MElightcyan1 traitfunnelform.1:MEcyan traittubular.1:MEroyalblue traitred.1:MEnavajowhite2 traityellow.1:MElightcyan1 traitpurple.1:MElightcyan1 traittubular.1:MElightcyan1 traitred.1:MEdarkturquoise traitspur.1:MEnavajowhite2 traitsalverform.1:MEthistle1 traitfunnelform.1:MEthistle1 traitspur.1:MEdarkturquoise traitwhite.1:MEnavajowhite2 traitwhite.1:MEdarkturquoise traityellow.1:MEnavajowhite2 traitpurple.1:MEnavajowhite2 traitsalverform.1:MEroyalblue traitfunnelform.1:MEroyalblue traittubular.1:MEnavajowhite2 traityellow.1:MEdarkturquoise traitpurple.1:MEdarkturquoise traittubular.1:MEdarkturquoise traitsalverform.1:MElightcyan1 traitfunnelform.1:MElightcyan1 traitsalverform.1:MEnavajowhite2 traitfunnelform.1:MEnavajowhite2 traitsalverform.1:MEdarkturquoise traitfunnelform.1:MEdarkturquoise

ME22 ME44 ME18 ME25 ME29 ME50 15 30 30 10 30 10

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1 1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) residual.variance residual.variance residual.variance residual.variance residual.variance residual.variance traitred.1:MEorange traitred.1:MEthistle2 traitspur.1:MEorange traitspur.1:MEthistle2 phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitwhite.1:MEorange traitwhite.1:MEthistle2 traitred.1:MElightgreen traityellow.1:MEorange traitpurple.1:MEorange traityellow.1:MEthistle2 traitred.1:MEfloralwhite traitpurple.1:MEthistle2 traitred.1:MEdarkgreen traittubular.1:MEorange traittubular.1:MEthistle2 traitspur.1:MElightgreen traitspur.1:MEfloralwhite traitspur.1:MEdarkgreen traitwhite.1:MElightgreen traitwhite.1:MEfloralwhite traitwhite.1:MEdarkgreen traitred.1:MEsaddlebrown traityellow.1:MElightgreen traitpurple.1:MElightgreen traityellow.1:MEfloralwhite traityellow.1:MEdarkgreen traitpurple.1:MEfloralwhite traitpurple.1:MEdarkgreen traittubular.1:MElightgreen traittubular.1:MEfloralwhite traittubular.1:MEdarkgreen traitspur.1:MEsaddlebrown traitsalverform.1:MEorange traitsalverform.1:MEthistle2 traitfunnelform.1:MEorange traitfunnelform.1:MEthistle2 traitwhite.1:MEsaddlebrown traityellow.1:MEsaddlebrown traitpurple.1:MEsaddlebrown traittubular.1:MEsaddlebrown traitsalverform.1:MElightgreen traitfunnelform.1:MElightgreen traitsalverform.1:MEfloralwhite traitsalverform.1:MEdarkgreen traitfunnelform.1:MEfloralwhite traitfunnelform.1:MEdarkgreen traitsalverform.1:MEsaddlebrown traitfunnelform.1:MEsaddlebrown

Supplemental Figure 3. Effects of floral traits on the eigengene expression in the Bud stage modules summarized over 50 phylogenetic trees.

bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

ME35 ME61 ME26 ME57 ME25 15 30 15 20 10 10

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1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) residual.variance residual.variance residual.variance residual.variance residual.variance traitbee.1:MEorange traitbee.1:MEsienna3 phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitbutterfly.1:MEorange traitbee.1:MEdarkorange traitbutterfly.1:MEsienna3 traitbee.1:MEantiquewhite4 traitbee.1:MElavenderblush3 traitbutterfly.1:MEdarkorange traithummingbird.1:MEorange traithummingbird.1:MEsienna3 traitbutterfly.1:MEantiquewhite4 traitbutterfly.1:MElavenderblush3 traithummingbird.1:MEdarkorange traithummingbird.1:MEantiquewhite4 traithummingbird.1:MElavenderblush3 ME47 ME45 ME16 ME39 ME28 10 15 20 10 20 ● ● 10 5 ● ● 10 ● ● ● 5

0 ● ● ● 10 1 1 1 1 1

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1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) residual.variance residual.variance residual.variance residual.variance residual.variance traitbee.1:MEskyblue traitbee.1:MEbisque4 phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitbee.1:MElightcyan traitbee.1:MEorangered4 traitbutterfly.1:MEskyblue traitbutterfly.1:MEbisque4 traitbee.1:MEdarkorange2 traitbutterfly.1:MElightcyan traitbutterfly.1:MEorangered4 traitbutterfly.1:MEdarkorange2 traithummingbird.1:MEskyblue traithummingbird.1:MEbisque4 traithummingbird.1:MElightcyan traithummingbird.1:MEorangered4 traithummingbird.1:MEdarkorange2

ME7 ME21 ME42 ME31 ME64 20 50 10 15 40 10 5 10 5 ● 30 ●

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0 ● ● ● ● ● −5 −5 −5 −10 −10 ● −15 −20 −10 −10

1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) residual.variance residual.variance residual.variance residual.variance residual.variance traitbee.1:MEblack traitbee.1:MEdarkred phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitbee.1:MEskyblue2 traitbutterfly.1:MEblack traitbee.1:MElightcyan1 traitbutterfly.1:MEdarkred traitbutterfly.1:MEskyblue2 traitbee.1:MEpaleturquoise traitbutterfly.1:MElightcyan1 traithummingbird.1:MEblack traithummingbird.1:MEdarkred traitbutterfly.1:MEpaleturquoise traithummingbird.1:MEskyblue2 traithummingbird.1:MElightcyan1 traithummingbird.1:MEpaleturquoise ME2 ME59 ME18 ME53 ME37 30 ● ● 15 10 ● 0 ● ●

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0 ●

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1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) residual.variance residual.variance residual.variance residual.variance residual.variance traitbee.1:MEblue phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitbutterfly.1:MEblue traitbee.1:MEskyblue3 traitbee.1:MElightgreen traitbutterfly.1:MEskyblue3 traithummingbird.1:MEblue traitbee.1:MEpalevioletred3 traitbutterfly.1:MElightgreen traitbee.1:MEdarkseagreen4 traithummingbird.1:MEskyblue3 traitbutterfly.1:MEpalevioletred3 traithummingbird.1:MElightgreen traitbutterfly.1:MEdarkseagreen4 traithummingbird.1:MEpalevioletred3 traithummingbird.1:MEdarkseagreen4 ME3 ME48 ME56 ME8 ME30 25 20 15 10 20 15 10 5 5

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1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) residual.variance residual.variance residual.variance residual.variance residual.variance traitbee.1:MEpink traitbee.1:MEbrown phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitbutterfly.1:MEpink traitbee.1:MEsteelblue traitbee.1:MElightpink4 traitbutterfly.1:MEbrown traitbutterfly.1:MEsteelblue traitbee.1:MEdarkslateblue traithummingbird.1:MEpink traitbutterfly.1:MElightpink4 traithummingbird.1:MEbrown traitbutterfly.1:MEdarkslateblue traithummingbird.1:MEsteelblue traithummingbird.1:MElightpink4 traithummingbird.1:MEdarkslateblue ME46 ME23 ME41 ME38 ME12 10 30 10 10 20

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1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) traitbee.1:MEtan residual.variance residual.variance residual.variance residual.variance residual.variance traitbee.1:MEplum1 traitbee.1:MEbrown4 traitbutterfly.1:MEtan phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitbutterfly.1:MEplum1 traitbutterfly.1:MEbrown4 traithummingbird.1:MEtan traitbee.1:MEdarkturquoise traitbee.1:MElightsteelblue1 traithummingbird.1:MEplum1 traithummingbird.1:MEbrown4 traitbutterfly.1:MEdarkturquoise traitbutterfly.1:MElightsteelblue1 traithummingbird.1:MEdarkturquoise traithummingbird.1:MElightsteelblue1 ME60 ME44 ME19 ME49 ME51 6 5 5 5 5 4

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1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) residual.variance residual.variance residual.variance residual.variance residual.variance traitbee.1:MEcoral1 traitbee.1:MEplum2 traitbee.1:MEthistle1 phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitbee.1:MEfloralwhite traitbee.1:MElightyellow traitbutterfly.1:MEcoral1 traitbutterfly.1:MEplum2 traitbutterfly.1:MEthistle1 traitbutterfly.1:MEfloralwhite traitbutterfly.1:MElightyellow traithummingbird.1:MEcoral1 traithummingbird.1:MEplum2 traithummingbird.1:MEthistle1 traithummingbird.1:MEfloralwhite traithummingbird.1:MElightyellow

ME62 ME5 ME9 ME10 ME50 20 10 5 20 5

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1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) residual.variance residual.variance residual.variance residual.variance residual.variance traitbee.1:MEgreen traitbee.1:MEcoral2 traitbee.1:MEpurple traitbee.1:MEthistle2 phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitbee.1:MEmagenta traitbutterfly.1:MEgreen traitbutterfly.1:MEcoral2 traitbutterfly.1:MEpurple traitbutterfly.1:MEthistle2 traitbutterfly.1:MEmagenta traithummingbird.1:MEgreen traithummingbird.1:MEcoral2 traithummingbird.1:MEpurple traithummingbird.1:MEthistle2 traithummingbird.1:MEmagenta ME14 ME11 ME55 ME6 ME1 10 15 10 80 10 10 ● 5 ● 5

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1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) traitbee.1:MEred residual.variance residual.variance residual.variance residual.variance residual.variance traitbee.1:MEcyan traitbutterfly.1:MEred traitbee.1:MEmaroon phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitbutterfly.1:MEcyan traitbee.1:MEturquoise traitbutterfly.1:MEmaroon traitbee.1:MEgreenyellow traithummingbird.1:MEred traitbutterfly.1:MEturquoise traithummingbird.1:MEcyan traitbutterfly.1:MEgreenyellow traithummingbird.1:MEmaroon traithummingbird.1:MEturquoise traithummingbird.1:MEgreenyellow ME22 ME0 ME63 ME20 ME32 10 10 10 30 15

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1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) residual.variance residual.variance residual.variance residual.variance residual.variance traitbee.1:MEgrey traitbee.1:MEviolet phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitbutterfly.1:MEgrey traitbee.1:MEroyalblue traitbutterfly.1:MEviolet traitbee.1:MEdarkgreen traitbutterfly.1:MEroyalblue traithummingbird.1:MEgrey traitbee.1:MEmediumorchid traitbutterfly.1:MEdarkgreen traithummingbird.1:MEviolet traithummingbird.1:MEroyalblue traitbutterfly.1:MEmediumorchid traithummingbird.1:MEdarkgreen traithummingbird.1:MEmediumorchid ME24 ME17 ME40 ME29 ME27 20 30 30 10

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1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) residual.variance residual.variance residual.variance residual.variance residual.variance traitbee.1:MEwhite traitbee.1:MEgrey60 phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitbee.1:MEdarkgrey traitbutterfly.1:MEwhite traitbutterfly.1:MEgrey60 traitbee.1:MEsaddlebrown traitbutterfly.1:MEdarkgrey traithummingbird.1:MEwhite traitbee.1:MEmediumpurple3 traithummingbird.1:MEgrey60 traitbutterfly.1:MEsaddlebrown traithummingbird.1:MEdarkgrey traitbutterfly.1:MEmediumpurple3 traithummingbird.1:MEsaddlebrown traithummingbird.1:MEmediumpurple3 ME34 ME58 ME15 ME13 ME4 15 15 20 10 10 5

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1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) residual.variance residual.variance residual.variance residual.variance residual.variance traitbee.1:MEyellow traitbee.1:MEsalmon phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitbutterfly.1:MEyellow traitbee.1:MEhoneydew1 traitbutterfly.1:MEsalmon traitbee.1:MEmidnightblue traitbee.1:MEdarkmagenta traithummingbird.1:MEyellow traitbutterfly.1:MEhoneydew1 traithummingbird.1:MEsalmon traitbutterfly.1:MEmidnightblue traitbutterfly.1:MEdarkmagenta traithummingbird.1:MEhoneydew1 traithummingbird.1:MEmidnightblue traithummingbird.1:MEdarkmagenta ME33 ME43 ME54 ME52 ME36 15 15 40 10 10 10 10

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1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) residual.variance residual.variance residual.variance residual.variance residual.variance traitbee.1:MEivory phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitbee.1:MEsalmon4 traitbutterfly.1:MEivory traitbee.1:MEyellowgreen traitbutterfly.1:MEsalmon4 traitbee.1:MEnavajowhite2 traithummingbird.1:MEivory traitbee.1:MEdarkolivegreen traitbutterfly.1:MEyellowgreen traitbutterfly.1:MEnavajowhite2 traithummingbird.1:MEsalmon4 traitbutterfly.1:MEdarkolivegreen traithummingbird.1:MEyellowgreen traithummingbird.1:MEnavajowhite2 traithummingbird.1:MEdarkolivegreen

Supplemental Figure 4. Effects of pollination syndrome on the eigengene expression in the Bud stage modules summarized over 50 phylogenetic trees. bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

ME61 ME34 ME0 ME41 ME53 ME35 ME4 15 40 15 15 10 10

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traitspur.1:MEskyblue3 ● traitwhite.1:MEskyblue3 traityellow.1:MEskyblue3 traitpurple.1:MEskyblue3 traittubular.1:MEskyblue3 traitsalverform.1:MEskyblue3 ● ● traitfunnelform.1:MEskyblue3 ● ● ● ● ●

−5 ● ● ● ● ● ● ● ● −40 −10 −20 −5 −5 −20 −10 −60 −15 −30 −10 −10

1 1 1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) residual.variance residual.variance residual.variance residual.variance residual.variance residual.variance residual.variance traitred.1:MEgrey traitspur.1:MEgrey traitred.1:MEyellow traitwhite.1:MEgrey traitspur.1:MEyellow traityellow.1:MEgrey traitpurple.1:MEgrey traittubular.1:MEgrey traitred.1:MEsienna3 traitwhite.1:MEyellow phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitspur.1:MEsienna3 traityellow.1:MEyellow traitpurple.1:MEyellow traittubular.1:MEyellow traitwhite.1:MEsienna3 traityellow.1:MEsienna3 traitpurple.1:MEsienna3 traittubular.1:MEsienna3 traitsalverform.1:MEgrey traitfunnelform.1:MEgrey traitred.1:MEdarkmagenta traitsalverform.1:MEyellow traitfunnelform.1:MEyellow traitred.1:MEantiquewhite4 traitred.1:MEpalevioletred3 traitspur.1:MEdarkmagenta traitred.1:MElightsteelblue1 traitspur.1:MEantiquewhite4 traitspur.1:MEpalevioletred3 traitsalverform.1:MEsienna3 traitwhite.1:MEdarkmagenta traitfunnelform.1:MEsienna3 traitspur.1:MElightsteelblue1 traitwhite.1:MEantiquewhite4 traitwhite.1:MEpalevioletred3 traityellow.1:MEdarkmagenta traitpurple.1:MEdarkmagenta traitwhite.1:MElightsteelblue1 traittubular.1:MEdarkmagenta traityellow.1:MEantiquewhite4 traityellow.1:MEpalevioletred3 traitpurple.1:MEantiquewhite4 traitpurple.1:MEpalevioletred3 traityellow.1:MElightsteelblue1 traittubular.1:MEantiquewhite4 traitpurple.1:MElightsteelblue1 traittubular.1:MEpalevioletred3 traittubular.1:MElightsteelblue1 traitsalverform.1:MEdarkmagenta traitfunnelform.1:MEdarkmagenta traitsalverform.1:MEantiquewhite4 traitsalverform.1:MEpalevioletred3 traitfunnelform.1:MEantiquewhite4 traitfunnelform.1:MEpalevioletred3 traitsalverform.1:MElightsteelblue1 traitfunnelform.1:MElightsteelblue1 ME47 ME26 ME11 ME19 ME8 ME28 ME36 15 15 15 30 15 40 10 10 10 ● ● ● ● 10 ● 10 20 ● ● 20 ● ● ●

● 5 ● ● ● ● ● 0 ● ● 5 5 ● ● ● ● 5 10 ● ●

0 ● ● ● ● ● ● ●

● ● 0 ● ● 1 1 1 1 1 1 1 ● ● ● ● 0 ● ● ● ● ● ● ● 0 ● ● ● ● ● ● 0 ● ● ● ● ● ● ● 0 ● ● −20

● −10

−5 ● ● ● ● ● ● ● ● ● −5 ● ● ● ● ● −5 −10 −10

● −40 −5 −10 −20 −15 −20 −10 −60 −15 −10

1 1 1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) residual.variance residual.variance residual.variance residual.variance residual.variance traitred.1:MEpink residual.variance residual.variance traitspur.1:MEpink traitwhite.1:MEpink traityellow.1:MEpink traitpurple.1:MEpink traitred.1:MEskyblue traittubular.1:MEpink traitred.1:MEbisque4 traitspur.1:MEskyblue phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitspur.1:MEbisque4 traitwhite.1:MEskyblue traitwhite.1:MEbisque4 traitred.1:MElightyellow traityellow.1:MEskyblue traitpurple.1:MEskyblue traityellow.1:MEbisque4 traitpurple.1:MEbisque4 traittubular.1:MEskyblue traitspur.1:MElightyellow traitsalverform.1:MEpink traitred.1:MEdarkorange traittubular.1:MEbisque4 traitfunnelform.1:MEpink traitred.1:MEgreenyellow traitred.1:MEyellowgreen traitwhite.1:MElightyellow traitspur.1:MEdarkorange traitspur.1:MEgreenyellow traitspur.1:MEyellowgreen traityellow.1:MElightyellow traitwhite.1:MEdarkorange traitpurple.1:MElightyellow traittubular.1:MElightyellow traitwhite.1:MEgreenyellow traitwhite.1:MEyellowgreen traityellow.1:MEdarkorange traitpurple.1:MEdarkorange traitsalverform.1:MEskyblue traityellow.1:MEgreenyellow traittubular.1:MEdarkorange traityellow.1:MEyellowgreen traitpurple.1:MEgreenyellow traitfunnelform.1:MEskyblue traitpurple.1:MEyellowgreen traitsalverform.1:MEbisque4 traitfunnelform.1:MEbisque4 traittubular.1:MEgreenyellow traittubular.1:MEyellowgreen traitsalverform.1:MElightyellow traitfunnelform.1:MElightyellow traitsalverform.1:MEdarkorange traitfunnelform.1:MEdarkorange traitsalverform.1:MEgreenyellow traitsalverform.1:MEyellowgreen traitfunnelform.1:MEgreenyellow traitfunnelform.1:MEyellowgreen

ME7 ME33 ME17 ME55 ME38 ME37 30 20 10 50 15

5 ● 20

10 ● ● ● ● 10 ● 5 ● ● ● ● 0 ● ● ● ● ● ● ● ● ● ● ●

● ● 10

● 0 ●

5 ● ● ● ● ● ●

● 0 ● ● ● ●

0 ● 1 1 1 1 1 1 ● −50 ● ● ● ● ●

● ● 0 ● ●

0 ● ● ● ● ● ● ● ● ●

● ● −5 −5 ● ● ● −100 −5 ● ● ● ● −10 ● −10 −10 −10 −10 −150 −20 −15 −15 −200 −20

1 1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) residual.variance residual.variance residual.variance residual.variance residual.variance residual.variance traitred.1:MEblack traitred.1:MEplum1 traitspur.1:MEblack traitred.1:MEgrey60 traitspur.1:MEplum1 traitwhite.1:MEblack traitred.1:MEmaroon traitspur.1:MEgrey60 traitwhite.1:MEplum1 traityellow.1:MEblack traitpurple.1:MEblack traitspur.1:MEmaroon traittubular.1:MEblack traitwhite.1:MEgrey60 phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitred.1:MEskyblue3 traityellow.1:MEplum1 traitpurple.1:MEplum1 traitwhite.1:MEmaroon traittubular.1:MEplum1 traityellow.1:MEgrey60 traitspur.1:MEskyblue3 traitpurple.1:MEgrey60 traittubular.1:MEgrey60 traityellow.1:MEmaroon traitpurple.1:MEmaroon traitwhite.1:MEskyblue3 traittubular.1:MEmaroon traityellow.1:MEskyblue3 traitpurple.1:MEskyblue3 traittubular.1:MEskyblue3 traitsalverform.1:MEblack traitfunnelform.1:MEblack traitsalverform.1:MEplum1 traitfunnelform.1:MEplum1 traitsalverform.1:MEgrey60 traitfunnelform.1:MEgrey60 traitred.1:MEdarkolivegreen traitsalverform.1:MEmaroon traitfunnelform.1:MEmaroon traitspur.1:MEdarkolivegreen traitsalverform.1:MEskyblue3 traitfunnelform.1:MEskyblue3 traitwhite.1:MEdarkolivegreen traityellow.1:MEdarkolivegreen traitpurple.1:MEdarkolivegreen traittubular.1:MEdarkolivegreen traitsalverform.1:MEdarkolivegreen traitfunnelform.1:MEdarkolivegreen

ME2 ME45 ME58 ME9 ME49 ME30 15 15 20 30 10 10 20

● 10 ● 20

● 5 5 ● ● ● ● ●

10 ● ● ● ● 5

● ● 10 0 ● ●

0 ● ● 10 ● ● ● ● 1 1 1 1 1 ● ● ● ● ● 1

0 ● ● ● ● ● −5

● 0 ● ● ● −5 ● ● ● 0 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 0 ● ● ● −10 ● −10 ● ● −10 ● ● −5 ● ● −15 −15 −20 −10 −10 −10 −20 −20 −30

1 1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) residual.variance residual.variance residual.variance residual.variance residual.variance residual.variance traitred.1:MEblue traitspur.1:MEblue traitred.1:MEplum2 traitwhite.1:MEblue traityellow.1:MEblue traitspur.1:MEplum2 traitpurple.1:MEblue traittubular.1:MEblue traitwhite.1:MEplum2 traitred.1:MEmagenta phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitred.1:MEsteelblue traityellow.1:MEplum2 traitpurple.1:MEplum2 traittubular.1:MEplum2 traitspur.1:MEmagenta traitspur.1:MEsteelblue traitwhite.1:MEmagenta traitwhite.1:MEsteelblue traitsalverform.1:MEblue traitred.1:MEhoneydew1 traitfunnelform.1:MEblue traityellow.1:MEmagenta traitpurple.1:MEmagenta traityellow.1:MEsteelblue traitpurple.1:MEsteelblue traittubular.1:MEmagenta traitspur.1:MEhoneydew1 traittubular.1:MEsteelblue traitred.1:MEdarkorange2 traitsalverform.1:MEplum2 traitwhite.1:MEhoneydew1 traitfunnelform.1:MEplum2 traitspur.1:MEdarkorange2 traityellow.1:MEhoneydew1 traitpurple.1:MEhoneydew1 traitwhite.1:MEdarkorange2 traittubular.1:MEhoneydew1 traityellow.1:MEdarkorange2 traitpurple.1:MEdarkorange2 traitsalverform.1:MEmagenta traittubular.1:MEdarkorange2 traitfunnelform.1:MEmagenta traitsalverform.1:MEsteelblue traitfunnelform.1:MEsteelblue traitsalverform.1:MEhoneydew1 traitfunnelform.1:MEhoneydew1 traitsalverform.1:MEdarkorange2 traitfunnelform.1:MEdarkorange2

ME3 ME59 ME43 ME15 ME10 ME12 15 30 15 20 15 15 10 10 20 10

● 10 ● 10 ● ● ● ● 5

5 ● ● ● ● ● 5 5 10 ● ● ● ● ● ● ● ● ● ●

● ● 0 ● 0 0 1 1 1 1 1 ● 1 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 0 0 ● ● ● 0 ● ● ● ● ● ● ● ● ● ● ● ● ●

−5 ● −5 ● ● ● ● ● −10 ● ● ● −5 −5 −10

−10 ● −10 −10 −20 −10 −15 −20 −15

1 1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) traitred.1:MEtan residual.variance residual.variance residual.variance residual.variance residual.variance residual.variance traitspur.1:MEtan traitred.1:MEivory traitwhite.1:MEtan traitspur.1:MEivory traityellow.1:MEtan traitred.1:MEbrown traitpurple.1:MEtan traitred.1:MEpurple traittubular.1:MEtan traitwhite.1:MEivory traitspur.1:MEbrown traitspur.1:MEpurple traityellow.1:MEivory traitpurple.1:MEivory traitwhite.1:MEbrown traittubular.1:MEivory traitwhite.1:MEpurple phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traityellow.1:MEbrown traitpurple.1:MEbrown traityellow.1:MEpurple traitpurple.1:MEpurple traittubular.1:MEbrown traittubular.1:MEpurple traitsalverform.1:MEtan traitfunnelform.1:MEtan traitsalverform.1:MEivory traitfunnelform.1:MEivory traitred.1:MEmidnightblue traitsalverform.1:MEbrown traitfunnelform.1:MEbrown traitsalverform.1:MEpurple traitfunnelform.1:MEpurple traitspur.1:MEmidnightblue traitwhite.1:MEmidnightblue traitred.1:MEdarkseagreen4 traityellow.1:MEmidnightblue traitpurple.1:MEmidnightblue traitspur.1:MEdarkseagreen4 traittubular.1:MEmidnightblue traitwhite.1:MEdarkseagreen4 traityellow.1:MEdarkseagreen4 traitpurple.1:MEdarkseagreen4 traittubular.1:MEdarkseagreen4 traitsalverform.1:MEmidnightblue traitfunnelform.1:MEmidnightblue traitsalverform.1:MEdarkseagreen4 traitfunnelform.1:MEdarkseagreen4 ME46 ME21 ME57 ME40 ME6 ME51 20 50 15 15 20 10 40 ● 10 10 10 ● 10 ● 30

5 ● ● ● ● ● ● ● ●

● ● 5

0 ● ● ● ● ● 5 ● ● ● ● 0 ● ● ● ● 20 ● ● ● ● ● ● 0 ● 1 1 1 1 1 1 ● ● ● ● 0 ● ● −10

● 0 ● ● ● 10 ● ● ● ● ● ●

−10 ● ● ● ● ● ● 0 ● ● −20

−5 ● ● ● ● −5 −5 ● ● ● ● −10

−20 ● −30 −10 −10 −10 −20 −40 −30

1 1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) traitred.1:MEred residual.variance residual.variance residual.variance residual.variance residual.variance residual.variance traitspur.1:MEred traitwhite.1:MEred traityellow.1:MEred traitpurple.1:MEred traittubular.1:MEred traitred.1:MEthistle1 traitred.1:MEbrown4 traitred.1:MEdarkred traitspur.1:MEthistle1 traitspur.1:MEbrown4 traitspur.1:MEdarkred phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitwhite.1:MEthistle1 traitwhite.1:MEbrown4 traitwhite.1:MEdarkred traityellow.1:MEthistle1 traitpurple.1:MEthistle1 traityellow.1:MEbrown4 traitpurple.1:MEbrown4 traitsalverform.1:MEred traityellow.1:MEdarkred traittubular.1:MEthistle1 traitfunnelform.1:MEred traitpurple.1:MEdarkred traittubular.1:MEbrown4 traittubular.1:MEdarkred traitsalverform.1:MEthistle1 traitfunnelform.1:MEthistle1 traitsalverform.1:MEbrown4 traitfunnelform.1:MEbrown4 traitsalverform.1:MEdarkred traitfunnelform.1:MEdarkred traitred.1:MElavenderblush3 traitred.1:MEmediumpurple3 traitspur.1:MElavenderblush3 traitspur.1:MEmediumpurple3 traitwhite.1:MElavenderblush3 traitwhite.1:MEmediumpurple3 traityellow.1:MElavenderblush3 traitpurple.1:MElavenderblush3 traityellow.1:MEmediumpurple3 traittubular.1:MElavenderblush3 traitpurple.1:MEmediumpurple3 traittubular.1:MEmediumpurple3 traitsalverform.1:MElavenderblush3 traitfunnelform.1:MElavenderblush3 traitsalverform.1:MEmediumpurple3 traitfunnelform.1:MEmediumpurple3 ME60 ME48 ME16 ME54 ME20 ME50 15 20 20 15 20 10 20

15 ● 10 ●

● 10 ● ●

● 5 ● ● ● 10 10 5 ●

10 ● ● ● ● ● ● ● ● 0 ● ● 5 ● ● 1 1 1 1 1 1 0 ● ● 0 ● ● ● ● ● ● ● ● ● ● 0 ● ● ● ● ● ● ● ● ● ● 0 ● ●

● ● −5 −5 ● ● ● 0 ● ● ● ● ● ● ● ● ● ● ● ● ● −5

● −10

−10 ● −10 −10 −10 −15 −10 −15 −20 −20

1 1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) residual.variance residual.variance residual.variance residual.variance residual.variance residual.variance traitred.1:MEcoral1 traitspur.1:MEcoral1 traitred.1:MEthistle2 traitwhite.1:MEcoral1 traitspur.1:MEthistle2 phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitred.1:MElightcyan traitred.1:MEroyalblue traityellow.1:MEcoral1 traitwhite.1:MEthistle2 traitpurple.1:MEcoral1 traittubular.1:MEcoral1 traitspur.1:MElightcyan traitspur.1:MEroyalblue traityellow.1:MEthistle2 traitpurple.1:MEthistle2 traittubular.1:MEthistle2 traitwhite.1:MElightcyan traitwhite.1:MEroyalblue traityellow.1:MElightcyan traitpurple.1:MElightcyan traityellow.1:MEroyalblue traitpurple.1:MEroyalblue traittubular.1:MElightcyan traittubular.1:MEroyalblue traitred.1:MEnavajowhite2 traitsalverform.1:MEcoral1 traitred.1:MEdarkslateblue traitfunnelform.1:MEcoral1 traitspur.1:MEnavajowhite2 traitsalverform.1:MEthistle2 traitspur.1:MEdarkslateblue traitfunnelform.1:MEthistle2 traitwhite.1:MEnavajowhite2 traitwhite.1:MEdarkslateblue traityellow.1:MEnavajowhite2 traitsalverform.1:MElightcyan traitpurple.1:MEnavajowhite2 traitsalverform.1:MEroyalblue traitfunnelform.1:MElightcyan traityellow.1:MEdarkslateblue traitfunnelform.1:MEroyalblue traitpurple.1:MEdarkslateblue traittubular.1:MEnavajowhite2 traittubular.1:MEdarkslateblue traitsalverform.1:MEnavajowhite2 traitfunnelform.1:MEnavajowhite2 traitsalverform.1:MEdarkslateblue traitfunnelform.1:MEdarkslateblue ME14 ME23 ME42 ME25 ME29 ME1 50 100 30 30 20 20 40 ●

● 20 ● 50 ● 30 ● 20 10 ● ● ● ● 10

● ● 10 20 ● ● ●

0 ● 1 1 1 1 1 ● ● ● ● 1 0 ● ● ●

● 10 ● ● ● ● ●

0 ● ● ● ● ● 10 ●

0 ● ● ● ● ● ● ● ● ● ● ●

−10 ● ● ● ● ● 0 ●

0 ● ●

● −10 ● ● ● ● ● ● ● −50 ● ● ● ●

−20 ● −10 −10 −20 −10 −20 −30 −30 −100

1 1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) residual.variance residual.variance residual.variance residual.variance residual.variance residual.variance traitred.1:MEcyan traitspur.1:MEcyan traitwhite.1:MEcyan traitred.1:MEorange traityellow.1:MEcyan traitpurple.1:MEcyan traitspur.1:MEorange traittubular.1:MEcyan phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitwhite.1:MEorange traitred.1:MEturquoise traityellow.1:MEorange traitpurple.1:MEorange traitred.1:MElightcyan1 traitspur.1:MEturquoise traittubular.1:MEorange traitspur.1:MElightcyan1 traitwhite.1:MEturquoise traitsalverform.1:MEcyan traitwhite.1:MElightcyan1 traitfunnelform.1:MEcyan traityellow.1:MEturquoise traitpurple.1:MEturquoise traitred.1:MEsaddlebrown traittubular.1:MEturquoise traityellow.1:MElightcyan1 traitpurple.1:MElightcyan1 traittubular.1:MElightcyan1 traitred.1:MEdarkturquoise traitspur.1:MEsaddlebrown traitsalverform.1:MEorange traitfunnelform.1:MEorange traitspur.1:MEdarkturquoise traitwhite.1:MEsaddlebrown traitwhite.1:MEdarkturquoise traityellow.1:MEsaddlebrown traitpurple.1:MEsaddlebrown traittubular.1:MEsaddlebrown traitsalverform.1:MEturquoise traityellow.1:MEdarkturquoise traitfunnelform.1:MEturquoise traitpurple.1:MEdarkturquoise traittubular.1:MEdarkturquoise traitsalverform.1:MElightcyan1 traitfunnelform.1:MElightcyan1 traitsalverform.1:MEsaddlebrown traitfunnelform.1:MEsaddlebrown traitsalverform.1:MEdarkturquoise traitfunnelform.1:MEdarkturquoise ME22 ME44 ME18 ME39 ME13 ME27 30 15 30 10 30 20 20 ● ●

10 ● ●

● ● 20 20 10 5 ● ● ● ● 5 ●

10 ● ● ● 10 10 ● ● 0 ● ● ● ● ● ● ● ● ● ● ● 1 1 1 1 1 ● ● 1 ●

0 ● ●

0 ● ● ● ● ● ● 0 ● ● ●

0 ● ● ● ●

● 0 ● ● −10 ● ● ● ● ● ● ●

● −5 ● ● ●

−10 ● ● ● −20 −10 −5 ● ● ● ● −10 −10 −20 −30 −20 −15 −10 −30

1 1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) residual.variance residual.variance residual.variance residual.variance residual.variance residual.variance traitred.1:MEwhite traitspur.1:MEwhite traitwhite.1:MEwhite traitred.1:MEsalmon traityellow.1:MEwhite traitspur.1:MEsalmon traitpurple.1:MEwhite traittubular.1:MEwhite phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitwhite.1:MEsalmon traitred.1:MElightgreen traitred.1:MEfloralwhite traitred.1:MEdarkgreen traityellow.1:MEsalmon traitpurple.1:MEsalmon traittubular.1:MEsalmon traitspur.1:MElightgreen traitspur.1:MEfloralwhite traitspur.1:MEdarkgreen traitred.1:MEorangered4 traitwhite.1:MElightgreen traitwhite.1:MEfloralwhite traitwhite.1:MEdarkgreen traitsalverform.1:MEwhite traitspur.1:MEorangered4 traitfunnelform.1:MEwhite traityellow.1:MElightgreen traitpurple.1:MElightgreen traityellow.1:MEfloralwhite traityellow.1:MEdarkgreen traitpurple.1:MEfloralwhite traitpurple.1:MEdarkgreen traittubular.1:MElightgreen traitwhite.1:MEorangered4 traittubular.1:MEfloralwhite traittubular.1:MEdarkgreen traityellow.1:MEorangered4 traitsalverform.1:MEsalmon traitpurple.1:MEorangered4 traitfunnelform.1:MEsalmon traittubular.1:MEorangered4 traitsalverform.1:MElightgreen traitfunnelform.1:MElightgreen traitsalverform.1:MEfloralwhite traitsalverform.1:MEdarkgreen traitfunnelform.1:MEfloralwhite traitfunnelform.1:MEdarkgreen traitsalverform.1:MEorangered4 traitfunnelform.1:MEorangered4

ME24 ME5 ME56 ME31 ME52 ME32 30 30 20 10 10 ● ● ● 20

● 20 ● 0

20 ● ● ● ● ● ● ● ● ● 5 ●

10 ● 10 −10 ● 10 ● 10 ● ● ● ● ● ● ● 1 1 1 1 1 1 ● 0 ● −20

● ● 0 ● ● ● ● ● 0 ● ● ● ● ● ● ● ● ● ● ● ● ● ● 0 ●

0 ●

● −30 ● ● ● ● ● ● ● ● ●

−5 ● −10

● ● ● ● −40 ● −10 −10 −10 −20 −50 −10

1 1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) residual.variance residual.variance residual.variance residual.variance residual.variance residual.variance traitred.1:MEviolet traitred.1:MEgreen traitspur.1:MEviolet traitspur.1:MEgreen traitwhite.1:MEviolet traitwhite.1:MEgreen traityellow.1:MEviolet traitpurple.1:MEviolet traitred.1:MEsalmon4 traityellow.1:MEgreen traittubular.1:MEviolet traitred.1:MEdarkgrey traitpurple.1:MEgreen phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traittubular.1:MEgreen traitred.1:MElightpink4 traitspur.1:MEsalmon4 traitspur.1:MEdarkgrey traitwhite.1:MEsalmon4 traitspur.1:MElightpink4 traitwhite.1:MEdarkgrey traityellow.1:MEsalmon4 traitwhite.1:MElightpink4 traitpurple.1:MEsalmon4 traityellow.1:MEdarkgrey traitpurple.1:MEdarkgrey traittubular.1:MEsalmon4 traittubular.1:MEdarkgrey traityellow.1:MElightpink4 traitsalverform.1:MEviolet traitpurple.1:MElightpink4 traitfunnelform.1:MEviolet traitsalverform.1:MEgreen traittubular.1:MElightpink4 traitfunnelform.1:MEgreen traitred.1:MEpaleturquoise traitspur.1:MEpaleturquoise traitwhite.1:MEpaleturquoise traitsalverform.1:MEsalmon4 traitfunnelform.1:MEsalmon4 traitsalverform.1:MEdarkgrey traitfunnelform.1:MEdarkgrey traityellow.1:MEpaleturquoise traitpurple.1:MEpaleturquoise traitsalverform.1:MElightpink4 traitfunnelform.1:MElightpink4 traittubular.1:MEpaleturquoise traitsalverform.1:MEpaleturquoise traitfunnelform.1:MEpaleturquoise

Supplemental Figure 5. Effects of floral traits on the eigengene expression in the D stage modules summarized over 50 phylogenetic trees.

bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

ME37 ME61 ME45 ME16 ME31 10 10 10 10 10 ● ● ● 5 5 5 ● ● ● ● ● ● 0 ● 0 ● ● ●

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0 ●

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1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) residual.variance residual.variance residual.variance residual.variance residual.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitbee.1:MElightcyan traitbee.1:MEskyblue3 traitbee.1:MEdarkorange2 traitbutterfly.1:MElightcyan traitbutterfly.1:MEskyblue3 traitbee.1:MEpaleturquoise traitbee.1:MEantiquewhite4 traitbutterfly.1:MEdarkorange2 traitbutterfly.1:MEpaleturquoise traitbutterfly.1:MEantiquewhite4 traithummingbird.1:MElightcyan traithummingbird.1:MEskyblue3 traithummingbird.1:MEdarkorange2 traithummingbird.1:MEpaleturquoise traithummingbird.1:MEantiquewhite4 ME47 ME21 ME42 ME53 ME30 15 15 20

● ● ● 0 ● ● 20 10 10 5

● ● −20 ● 5 ● 5 10 ●

● −40

0 ● 1 1 1 1 ● 1 ● ● 0 ● ● ●

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● −5 ● −80 ● −5 −5 −10 −100 −10 −10 −120

1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) residual.variance residual.variance residual.variance residual.variance residual.variance traitbee.1:MEdarkred traitbee.1:MEbisque4 phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitbee.1:MEsteelblue traitbee.1:MElightcyan1 traitbutterfly.1:MEdarkred traitbutterfly.1:MEbisque4 traitbutterfly.1:MEsteelblue traitbee.1:MEpalevioletred3 traitbutterfly.1:MElightcyan1 traithummingbird.1:MEdarkred traithummingbird.1:MEbisque4 traitbutterfly.1:MEpalevioletred3 traithummingbird.1:MEsteelblue traithummingbird.1:MElightcyan1 traithummingbird.1:MEpalevioletred3 ME59 ME18 ME8 ME12 30 10 ME7 15 15 20 10 ● 5 10 5 ● ● 10 5 ●

● 5

● ● 1 1 1 1 ● 1 ●

0 ●

0 ●

0 ● ● ● 0 ● ●

● ● 0 ● ● ● ● ● ● ● ● ●

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1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) traitbee.1:MEtan residual.variance residual.variance residual.variance residual.variance residual.variance traitbee.1:MEpink traitbee.1:MEblack traitbutterfly.1:MEtan phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitbutterfly.1:MEpink traitbutterfly.1:MEblack traitbee.1:MElightgreen traithummingbird.1:MEtan traithummingbird.1:MEpink traitbutterfly.1:MElightgreen traithummingbird.1:MEblack traitbee.1:MEdarkseagreen4 traithummingbird.1:MElightgreen traitbutterfly.1:MEdarkseagreen4 traithummingbird.1:MEdarkseagreen4 ME38 ME2 ME48 ME56 ME50 20 20 20 10 10 15 15 15

●

● 5 10 10 10 ● 5 ● ● 5 5 5 ● 1 1 1 1 ● ● 1 0 ●

0 ● ● ● ● ● ● 0 ● ● 0 ●

0 ● ● ● ● ● ● ● ● ● −5 ● −5

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1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) residual.variance residual.variance residual.variance residual.variance residual.variance traitbee.1:MEblue traitbee.1:MEplum1 traitbee.1:MEthistle1 phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitbutterfly.1:MEblue traitbee.1:MElightpink4 traitbutterfly.1:MEplum1 traitbutterfly.1:MEthistle1 traitbee.1:MEdarkslateblue traithummingbird.1:MEblue traitbutterfly.1:MElightpink4 traithummingbird.1:MEplum1 traithummingbird.1:MEthistle1 traitbutterfly.1:MEdarkslateblue traithummingbird.1:MElightpink4 traithummingbird.1:MEdarkslateblue ME3 ME23 ME41 ME49 ME50 40 15 10 10 20 5 ● ● 20 ● ● 5

● 5

● 10 ● ● ● 0 1 1 1 1 ● ● ● 1 0 ●

0 ● ● ● ● ●

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−5 ● −10 −10 −15 −40 −10

1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) residual.variance residual.variance residual.variance residual.variance residual.variance traitbee.1:MEbrown traitbee.1:MEplum2 traitbee.1:MEthistle2 phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitbutterfly.1:MEbrown traitbutterfly.1:MEplum2 traitbutterfly.1:MEthistle2 traitbee.1:MEdarkturquoise traitbee.1:MElightsteelblue1 traithummingbird.1:MEbrown traithummingbird.1:MEplum2 traithummingbird.1:MEthistle2 traitbutterfly.1:MEdarkturquoise traitbutterfly.1:MElightsteelblue1 traithummingbird.1:MEdarkturquoise traithummingbird.1:MElightsteelblue1 ME46 ME44 ME19 ME10 ME1 20 10 10 10 5

● 10 5 5 5 ● ● ● ● ● ● ● ●

0 ●

0 ● ● ● 0 ● ● 1 1 1 1 ● ● 1 ● ● ●

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● −5 −5 −5 −20 −10 −10 −10 −10

1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) residual.variance residual.variance residual.variance residual.variance residual.variance traitbee.1:MEpurple traitbee.1:MEbrown4 phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitbee.1:MEturquoise traitbee.1:MEfloralwhite traitbee.1:MElightyellow traitbutterfly.1:MEpurple traitbutterfly.1:MEbrown4 traitbutterfly.1:MEturquoise traitbutterfly.1:MEfloralwhite traitbutterfly.1:MElightyellow traithummingbird.1:MEpurple traithummingbird.1:MEbrown4 traithummingbird.1:MEturquoise traithummingbird.1:MEfloralwhite traithummingbird.1:MElightyellow ME60 ME5 ME9 ME6 ME32 20 30 10 10 20

10 ● ● ● 20 5 ●

● 5 ●

● 10

0 ● ● ● 10 0 ● ● 1 1 1 1 ● ● 1 ● 0 ● 0 ● ● ● ● ●

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● −10 ● −5 −20 −10 −10 −20 −20 −30

1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) traitbee.1:MEred residual.variance residual.variance residual.variance residual.variance residual.variance traitbee.1:MEviolet traitbee.1:MEgreen traitbee.1:MEcoral1 traitbutterfly.1:MEred phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitbee.1:MEmagenta traitbutterfly.1:MEviolet traitbutterfly.1:MEgreen traitbutterfly.1:MEcoral1 traithummingbird.1:MEred traitbutterfly.1:MEmagenta traithummingbird.1:MEviolet traithummingbird.1:MEgreen traithummingbird.1:MEcoral1 traithummingbird.1:MEmagenta ME14 ME11 ME55 ME20 ME27 15 30 20 30 5 ● ● 10 20 ● ● 20 ● 10 ● 5 10

0 ●

10 ● 1 1 1 1 1 ● ●

0 ● ● ● 0 ● ● ● ● ● 0 ● ● ● 0 ● ● ● ● ●

−5 ● −5 ● ● −10 ● −10 −10 −10 −20 −10 −20

1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) residual.variance residual.variance residual.variance residual.variance residual.variance traitbee.1:MEcyan traitbee.1:MEwhite traitbee.1:MEmaroon phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitbutterfly.1:MEcyan traitbee.1:MEroyalblue traitbutterfly.1:MEwhite traitbutterfly.1:MEmaroon traitbee.1:MEgreenyellow traitbutterfly.1:MEroyalblue traithummingbird.1:MEcyan traithummingbird.1:MEwhite traitbutterfly.1:MEgreenyellow traithummingbird.1:MEmaroon traithummingbird.1:MEroyalblue traithummingbird.1:MEgreenyellow ME22 ME0 ME40 ME29 ME4 10 15 20 20 20 10 5 ● ● ● 5

● 10 ● 10 10 ● ● ●

0 ● ● ● 0 1 1 1 1 ● 1

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0 ●

0 ● ● ● ●

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● −10 −10 −10 −10 −15 −20 −10

1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) residual.variance residual.variance residual.variance residual.variance residual.variance traitbee.1:MEgrey traitbee.1:MEyellow phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitbutterfly.1:MEgrey traitbee.1:MEdarkgreen traitbutterfly.1:MEyellow traitbee.1:MEsaddlebrown traithummingbird.1:MEgrey traitbutterfly.1:MEdarkgreen traithummingbird.1:MEyellow traitbee.1:MEmediumpurple3 traitbutterfly.1:MEsaddlebrown traithummingbird.1:MEdarkgreen traitbutterfly.1:MEmediumpurple3 traithummingbird.1:MEsaddlebrown traithummingbird.1:MEmediumpurple3 ME24 ME17 ME15 ME13 ME36 40 20 250 30 5 5 200 ● ● 10 20

150 ● ● ● ● ● 1 1 1 1 1 10

0 ●

0 ● ● 100 ●

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50 ● ●

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● −50 −20

1 1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) (Intercept) residual.variance residual.variance residual.variance residual.variance residual.variance traitbee.1:MEgrey60 traitbee.1:MEsalmon phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitbee.1:MEdarkgrey traitbutterfly.1:MEgrey60 traitbutterfly.1:MEsalmon traitbee.1:MEyellowgreen traitbee.1:MEmidnightblue traitbutterfly.1:MEdarkgrey traithummingbird.1:MEgrey60 traitbutterfly.1:MEyellowgreen traithummingbird.1:MEsalmon traitbutterfly.1:MEmidnightblue traithummingbird.1:MEdarkgrey traithummingbird.1:MEyellowgreen traithummingbird.1:MEmidnightblue ME34 ME58 ME54 ME52 10 15 15 10 5 10

5 ● ●

5 ● ● 5 ● ● ● ● 0 ● ● ● 1 1 1 1 ● 0 ●

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1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) residual.variance residual.variance residual.variance residual.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitbee.1:MEsalmon4 traitbee.1:MEhoneydew1 traitbutterfly.1:MEsalmon4 traitbee.1:MEnavajowhite2 traitbee.1:MEdarkmagenta traitbutterfly.1:MEhoneydew1 traitbutterfly.1:MEnavajowhite2 traitbutterfly.1:MEdarkmagenta traithummingbird.1:MEsalmon4 traithummingbird.1:MEhoneydew1 traithummingbird.1:MEnavajowhite2 traithummingbird.1:MEdarkmagenta ME33 ME43 ME25 ME35 10 10 10 5 5 5 ● 5 ● ● ● ● ●

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1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) residual.variance residual.variance residual.variance residual.variance traitbee.1:MEivory traitbee.1:MEorange traitbee.1:MEsienna3 phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitbutterfly.1:MEivory traitbutterfly.1:MEorange traitbutterfly.1:MEsienna3 traithummingbird.1:MEivory traitbee.1:MEdarkolivegreen traithummingbird.1:MEorange traithummingbird.1:MEsienna3 traitbutterfly.1:MEdarkolivegreen traithummingbird.1:MEdarkolivegreen

ME26 ME57 ME39 ME28 6 20 10 10 4

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1 1 1 1 (Intercept) (Intercept) (Intercept) (Intercept) residual.variance residual.variance residual.variance residual.variance traitbee.1:MEskyblue phylogenetic.variance phylogenetic.variance phylogenetic.variance phylogenetic.variance traitbee.1:MEdarkorange traitbee.1:MEorangered4 traitbutterfly.1:MEskyblue traitbee.1:MElavenderblush3 traitbutterfly.1:MEdarkorange traitbutterfly.1:MEorangered4 traithummingbird.1:MEskyblue traitbutterfly.1:MElavenderblush3 traithummingbird.1:MEdarkorange traithummingbird.1:MEorangered4 traithummingbird.1:MElavenderblush3

Supplemental Figure 6. Effects of pollination syndrome on the eigengene expression in the D stage modules summarized over 50 phylogenetic trees. bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Supplemental Figure 7. Relationship between orthogroup dN/dS (omega) and average expression.

bioRxiv preprint doi: https://doi.org/10.1101/817221; this version posted February 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

A B Bud stage D stage

1.50 1.50

1.25 1.25

1.00 1.00

0.75 0.75

0.50 0.50 Omega (dN/dS)

0.25 0.25

0.00 0.00 Non−hubs Hubs Non−hubs Hubs

C D Bud stage D stage

1.50 1.50

1.25 1.25

1.00 1.00

0.75 0.75

0.50 0.50 Omega (dN/dS)

0.25 0.25

0.00 0.00 Non−periphery Periphery Non−periphery Periphery

Supplemental Figure 8. Distribution of dN/dS (omega) among hubs and periphery nodes.