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Transcriptomics of post-anthesis colour change in Lotus Boehm, Mannfred M. A. 2017

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  TRANSCRIPTOMICS OF POST-ANTHESIS COLOUR CHANGE IN LOTUS by   Mannfred M.A. Boehm  B.Sc, Nova Scotia Agricultural College (Dalhousie University), 2014  A THESIS SUBMITTED IN PARTIAL FULLFILLMENT OF THE REQUIREMENTS OF FOR THE DEGREE OF   MASTER OF SCIENCE  in  THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Botany)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  January 2017   © Mannfred M.A. Boehm, 2017ii   Abstract    Post-anthesis colour change (PACC) is widely thought to be an adaptation to signal floral viability to pollinators, and may be a developmental pre-pattern for bird pollination in Lotus. Lotus filicaulis and Lotus sessilifolius are insect-pollinated herbaceous legumes with flowers that open yellow, shift to orange and finally, red. This thesis examines the ecological and molecular basis for floral colour change in these Lotus species.   L. filicaulis was cultivated in a glasshouse from which pollinating insects (bees) were excluded, and the rate of colour change was recorded in both unpollinated and manually pollinated flowers. Unpollinated flowers from both the yellow stage and red stage were sampled for sequencing. The transcriptomes of L. filicaulis and L. sessilifolius of both colour stages were analyzed for differentially expressed genes and enriched ontologies.   Pollination significantly accelerates PACC in L. filicaulis, consistent with the hypothesis that PACC increases pollination efficiency by directing pollinators to unpollinated flowers. RNA-seq results show the synchronized upregulation of the entire cyanidin biosynthesis pathway in the red stage of PACC of both Lotus species – possibly at the expense of adjacent pathways competing for the same substrate. This thesis is the foundation for understanding the molecular evolution of PACC in Lotus and will be useful in testing the hypothesis that PACC is a preadaptation to bird pollination in Lotus.                   iii  Preface    This thesis is a draft manuscript that will be submitted for publication: Boehm,  M.M.A., Ojeda D.I., and Cronk Q.C.B. 2016. Dissecting the ‘bacon-and-eggs’ phenotype: transcriptomics of floral colour change in Lotus. The idea came from discussions with Q.C.B Cronk and D.I. Ojeda. M.M.A. Boehm designed the analytical pipeline, performed all the analyses, conducted the hand pollination experiment and wrote the manuscript. D.I. Ojeda designed the transcriptomics experiment, collected plant material and processed tissue for RNA sequencing. Q.C.B. Cronk contributed to experimental and analytical design, contributed to the analysis of data, and provided feedback on this manuscript.                        iv  Table of Contents    Abstract………………………………………………………………………………………….. ii Preface…………………………………………………………………………………………... iii Table of Contents……………………………………………………………………………….iv List of Tables……………………………………………………………………………………vii List of Figures…………………………………………………………………………………. viii Acknowledgements……………………………………………………………………………... ix  1 Introduction........................................................................................................................1 1.1 What is post-anthesis colour change (PACC)? ...................................................... 1 1.2 Historical record of PACC ..................................................................................... 1 1.3 Evolutionary ecology of PACC  ............................................................................ 2 1.4 Insect vision and colours of PACC ........................................................................ 2 1.5 Molecular basis of PACC ...................................................................................... 3 1.6 The genus Lotus ..................................................................................................... 4 1.7 This study ............................................................................................................... 4   2 Materials and Methods..................................................................................................... 8 2.1 Plant material and cultivation ................................................................................ 8 2.2 Hand pollination experiment ................................................................................. 8 2.3 Transcriptome sequencing ..................................................................................... 9 2.4 Sequence analysis .................................................................................................. 9 2.5 TopHat Alignment ................................................................................................. 9 2.6 STAR Alignment ................................................................................................. 10 2.7 De novo assembly by Trinity ............................................................................... 10 2.8 Nucleotide alignments ......................................................................................... 11 2.9 Gene family composition ..................................................................................... 11    v  3  Results...............................................................................................................................13 3.1 Pollination accelerates colour change in L. filicaulis ........................................... 13 3.2 Illumina HiSeq Library ........................................................................................ 13 3.3 De novo assembly of non-model organism RNA reads outperforms alignment of reads to a reference genome of a closely related species...................................... 14 3.4 The cyanidin pathway is upregulated in the red stage of PACC in L. filicaulis and L. sessilifolius ................................................................................. 14            3.5 The carotenoid pathway is upregulated in the yellow stage of PACC in L. filicaulis and L. sessilifolius ............................................................................ 14            3.6        Differences between L. filicaulis and L. sessilifolius .......................................... 15   4 Discussion.........................................................................................................................27                        4.1 The significance of pollination as a trigger for PACC ........................................ 27  4.2 De novo assembly is preferred for RNA-seq data without a reference  genome ................................................................................................................. 27  4.3 PACC is associated with the wholesale upregulation of the cyanidin  pathway ................................................................................................................ 28  4.4 PACC is associated with highly specific upregulation of members of  gene families ........................................................................................................ 28  4.5 Upregulation of flavonoid biosynthesis may be antagonistic to other  phenylpropanoid pathways .................................................................................. 30  4.6 Carotenoid biosynthesis is downregulated during PACC ................................... 30  4.7 Is MAX1 a master regulator of PACC?  ..............................................................30  4.8 The role of FLS in PACC in Lotus sessilifolius .................................................. 31 4.9 The role of red in PACC ...................................................................................... 32    5 Conclusion......................................................................................................................34                                      Bibliography.....................................................................................................................35                                                                                     vi  Appendix 1 Sequences for Trinity contigs listed in Table 5....................................................44                                                                                                                           Appendix 2 Sequences for Trinity contigs listed in Table 6….................................................50                                                                                                                                               Appendix 3 Supplemementary Tables S1-42...........................................................................54                                                                                                                                                Appendix 4 Supplementary Figures S1 – S10.........................................................................162             Appendix 5 Supplementary Figures S11 – S17.......................................................................176                 Appendix 6 Supplementary Figures S18 – S19.......................................................................187                                                                                                                                              Appendix 7 Supplementary Figures S20 – S28.......................................................................189                                                                                                                                              Appendix 8 Supplementary Figures S29 – S30.......................................................................201                                                                                                                                           Appendix 9 Supplementary Figures S31 – S34.......................................................................203            Appendix 10 Supplementary Figure S35................................................................................208            Appendix 11 Supplementary Figures S36 – S40....................................................................209            Appendix 12 Supplementary Figures S41 – S47....................................................................215    Appendix 13 Supplementary Figures S48 – S54....................................................................224            Appendix 14 Supplementary Figures S55..............................................................................232            Appendix 15 Supplementary Figures S56 – S57...................................................................233            Appendix 16 Supplementary Figures S58 – S60...................................................................235            Appendix 17 Supplementary Figure 61.................................................................................238 vii  List of Tables    Table 1  Next-generation library construction for L. filicaulis ............................................. 12  Table 2  Next-generation library construction for L. sessilifolius ........................................ 12  Table 3  Comparison of protocols used to detect differential expression of RNA in L. filicaulis ................................................................................................................. 21  Table 4  Comparison of protocols used to detect differential expression of RNA  in L. sessilifolius ..................................................................................................... 22  Table 5  Selected genes for enzymes involved in flavonoid biosynthesis in L.  filicaulis ................................................................................................................. 23  Table 6  Selected genes for enzymes involved in flavonoid biosynthesis in L.                          sessilifolius ........................................................................................................... 25  viii  List of Figures    Figure 1  Lotus filicaulis, exhibiting post-anthesis colour change .......................................... 6  Figure 2  Simplified phylogeny of the Lotus spp. used in this study ...................................... 7  Figure 3  Timing of stages in the hand pollinated treatment .................................................. 16  Figure 4 Graphical representation of the differential expression of genes (Trinity contigs) involved in flavonoid and lignin biosynthesis throughout PACC in Lotus filicaulis ................................................................................................................................ 17  Figure 5  Graphical representation of the differential expression of genes (Trinity contigs) involved in flavonoid and lignin biosynthesis throughout PACC in Lotus sessilifolius ............................................................................................................ 18  Figure 6  Graphical representation of the differential expression of genes  (Trinity contigs) involved in carotenoid biosynthesis throughout  PACC in Lotus filicaulis ........................................................................................ 19  Figure 7  Graphical representation of the differential expression of genes  (Trinity contigs) involved in carotenoid biosynthesis throughout                           PACC in Lotus sessilifolius ................................................................................ 20                        ix    Acknowledgements     Scripting the RNA-seq pipeline would not have been possible without the patient assistance of Sebastian Bailey, Alistair Blachford, Dan Bock, Melissa Guzman, Andy LeBlanc, Geneviève Leduc-Robert, Andrew MacDonald, Remi MattheyDoret, Diana Rennison, Saemundur Sveinsson, David Tack, and Silu Wang.    Growing healthy Lotus filicaulis and Lotus sessilifolius was greatly helped by Pierre Fobert, Melina Biron, and Dario Ojeda.    Insightful and often crucial feedback was provided by Brian Ellis, FLORUM, Armando Geraldes, Anastasia Kuzmin, the Angert Lab, the Otto Lab, the Samuels Lab, Noriko Okamoto, the Schluter Lab, and Santokh Singh.     I would like to especially acknowledge Prof. Quentin Cronk and Kristen Hudson for continuing to encourage my interest in plants.   1    1  Introduction     1.1  What is post anthesis colour change (PACC)?  In angiosperms, flowers are typically left on display until pollination or senescence (i.e. until the pollen becomes inviable or the stigma non-receptive), at which time the flower will generally wilt and cease reward production. Often the corolla abscises at this point, thus effectively excluding any further visits from pollinators (Faegri & Vanderpijl, 1979). However, instead of abscission, some species retain the corolla past the cessation of reward production and modify petal colour, a trait I herein refer to as post-anthesis colour change (PACC) (Mohan Ram & Mathur, 1984; Farzad et al., 2003). I use “post-anthesis colour change” instead of the broader “floral colour change” as many flowers change colour between pre-anthesis (bud) and anthesis (flower opening). This early maturation colour change is unrelated to PACC. Several species of Lotus L. (Fabaceae) exhibit yellow flowers at anthesis followed by a transition to red (Ojeda et al., 2013) thus these species have sometimes acquired the English vernacular name ‘bacon and eggs’ (Grigson, 1975) (Figure 1). A well-studied example is the North American lotoid legume  Acmispon glaber (Vogel) Brouillet (Lotus scoparius (Nutt.) Ottley) (Jones & Cruzan, 1999).    1.2  Historical record of PACC  The widespread (Weiss, 1995) and striking phenomenon of PACC has long interested botanists. One of the first scientific observations of PACC dates to a letter written from Fritz Müller to Charles Darwin, discussing the pollination ecology of Lantana camara L. (Müller, 1877; see also Weiss, 1991; Jones & Cruzan 1999). Müller notes that L. camara flowers open as yellow and over a three-day period shift to orange and finally purple. Tellingly, he also writes that butterflies tend to visit yellow flowers most often, occasionally visiting the orange flowers, and never the purple flowers. More recently the phenomenon has been documented in a variety of studies and reviews (Mohan Ram & Mathur, 1984; Weiss, 1991; Ojeda et al., 2013; Brito et al. 2015).       2    1.3  Evolutionary ecology of PACC  As anticipated by Müller, PACC is likely an adaptation to redirect pollinators at close range while maintaining long-distance appeal of the plant floral display (Weiss, 1991; Jones & Cruzan, 1999), hence the retention of the corolla instead of wilting or abscission. Indeed, in the North American Acmispon glaber (formerly known as Lotus scoparius) PACC is accelerated by pollination and corresponds temporally with the termination of reward production (Jones & Cruzan 1999). By differentiating between viable and unviable flowers, it is thought that plants are able to signal to pollinators which flowers possess a food reward (Weiss, 1991). This is of potential benefit for both organisms as the pollinator forages with greater efficiency, while the plant may receive a higher rate of successful pollination events. Pollinators could, of course, be directed to young flowers by the wilting or abscission of old flowers, but it is hypothesized that PACC provides a selective advantage by aiding long-distance attractiveness of the overall floral display.    1.4  Insect vision and colours of PACC  The flower colours involved in effective signalling are not random; PACC tends to be a shift from shorter reflective wavelengths (e.g. yellow), to longer ones (e.g. red). Like many floral traits, it is plausible that PACC has evolved through interaction with pollinators, and thus the characteristics of insect vision may be implicated in the colour palette of PACC. Generally, bees (Hymenoptera: Apidae) have three kinds of light receptors with peak sensitivities at 340nm (ultraviolet, Sreceptor), 430nm (blue, M-receptor), and 540nm (green, L-receptor) (Chittka, 1996). However, the physiology of hymenopteran light receptors (Chittka, 1996) and colour-reward experiments (Weiss, 1991; Chittka & Waser, 1997; MartínezHarms et al., 2010) suggest that bees are not incapable of detecting red light (620750nm), and electroretinogram recordings of Bombus dahlbomii (Apidae) have demonstrated that red light can induce an L-receptor response (Martínez-Harms et al., 2010). Indeed, in the wild B. dahlbomii is known to visit a variety of plant species with flowers that look red to humans, and they can be trained to visit artificial red targets ex situ (Martínez-Harms et al., 2010). Results of colourreward experiments suggest that bees are able to detect red flowers by achromatic contrast (Chittka & Waser, 1997; Martínez-Harms et al., 2010). That is, red flowers likely appear as a dark, hue-less patches 3    against a background of green plant foliage, providing sufficient contrast to locate such flowers (Martínez-Harms et al., 2010).   Given that a pollinator spends a finite amount of time at any one plant, PACC may be an adaptation to attract insect pollinators at long range by deceptively by retaining flowers, regardless of viability, while honestly directing pollinators at short range to the flowers most likely to benefit from pollen transfer (discussed in Brito et al., 2015). In numerous plant taxa the visual physiology of pollinators has meditated the evolution of flower colour (Rausher, 2008); in the case of PACC, it is conceivable that natural selection would favour a post-anthesis colour that is relatively less attractive than at anthesis, but not so unattractive as to be indistinguishable from its surroundings.       1.5  Molecular basis of  PACC  The timing of PACC is potentially tied to a host of environmental and biochemical triggers that influence pigment pathways in conjunction with the cessation of food rewards. In Lantana camara, Gossypium hirsutum L., and Viola cornuta L. PACC has been found to a result from changes in flavonoid biosynthetic processes, specifically those branches that produce anthocyanins and their methylated derivatives (Mohan Ram & Mathur, 1984; Farzad et al., 2003; Tan et al., 2013). The anthocyanin biosynthetic pathway (ABP) is a highly conserved and versatile pathway known to produce the orange, red and purple colouration in floral tissues - in addition to a range of other functions not related to pigmentation (Kong, 2003).  The regulation of PACC at the genetic level remains poorly known, however, presuming the anthocyanin pathway is involved, it is possible that PACC is regulated by MYB transcription factors (e.g. PAP1 and PAP2, Feller et al. 2011) as well as WD40s and basic helix-loop-helix proteins (Ramsay & Glover, 2005). These three proteins are known to produce a MYB-bHLH-WD40 transcription complex capable of regulating a range of target genes in numerous plant species (Ramsay & Glover, 2005), including those involved in anthocyanin biosynthesis (Spelt et al., 2002). Additionally, many flavonoid biosynthetic pathway genes are represented as gene families with multiple duplicated copies (Ober, 2005) and whether all or some of these copies are involved in PACC remains to be determined.     4      1.6  The genus Lotus  Lotus is a genus with a diverse range of floral traits to attract pollinators (Ojeda et al., 2013), including varying sugar compositions, corolla shapes, petal micromorpholgies, and petal pigmentations (Ojeda et al., 2012; Ojeda et al., 2013).  It is a promising genus for studying the evolution of pollination syndromes, and PACC is particularly interesting because it has been suggested that it is a preadaptation (developmental ‘pre-pattern’), for the evolution of bird pollination in Lotus section Rhyncholotus (Ojeda et al., 2013). Lotus includes the well-studied L. corniculatus L., which is of considerable economic importance as a forage crop (Duke, 1981) and exhibits PACC. However, L. corniculatus is a polyploid and therefore a complex organism for studying gene expression.   The anthocyanin pathway in Lotus japonicus (Regel) K. Larsen (L. corniculatus L. var. japonicus Regel) has been well studied, but it does not exhibit PACC. Chromatographic analysis of L. japonicus aerial tissue has revealed that cyanidin and peonidin make up the flavonoid profile of this species (Suzuki et al., 2008); both of these pigments are known to reflect red and purple light and it is possible these same branches of the anthocyanin pathway are involved in the Lotus spp. that exhibit PACC. For this reason I have chosen to leverage the genomic resources available for L. japonicus by studying PACC in the closely related L. filicaulis Durieu, a diploid perennial herbaceous legume (Ferreira & Pedrosa-Harand, 2014) notable for its post-anthesis transition from yellow to red flowers (Figure 1). For comparison I also investigate another species, Lotus sessilifolius DC, which has been the subject of previous studies (Ojeda et al., 2013) and may play a significant role in the evolution of pollination syndromes in Lotus (Ojeda et al. 2012).  Additionally, an analysis of the pigment composition of L. sessilifolius floral tissue suggests that FLAVONOL SYNTHASE (FLS) may play an important role in PACC of this species (Ojeda et al. 2013).     1.7  This Study  In this study I first investigate whether PACC is triggered by pollination in Lotus filicaulis. I then compare RNA expression at anthesis (yellow) and during PACC (red) in Lotus filicaulis and Lotus sessilifolius to describe the pigment pathways involved, and propose several candidate genes and pathways that I believe play a key role in post-anthesis colour change in these Lotus 5    species. This is the first transcriptome-wide analysis of PACC to date. PACC appears to have evolved independently in L. filicaulis and L. sessilifolius (Ojeda et al. 2013; Figure 2) as they belong to different clades of Lotus. L. sessilifolius is in the Canary Island clade of Lotus and apparently a tetraploid (Ojeda and Santos-Guerra, 2011, Ojeda et al. 2013). However, L. sessilifolius (insect-pollinated) is the sister taxon to the bird pollinated ‘rhyncholotus group’ in Lotus (Ojeda et al. 2013), and further studies on this species can provide additional information about the evolution of bird pollination in this group. I discuss the results in the context of observations regarding PACC in other plant species, the role of pollinators in driving the evolution of PACC, the role of gene duplication, and known molecular pathways of pigment biosynthesis.                    6      Figure 1 Lotus filicaulis, exhibiting post-anthesis colour change from yellow to red, a trait commonly referred to as the ‘bacon and eggs’ phenotype. Photo taken at the Horticulture Glasshouse, University of British Columbia.                   7      Figure 2 Simplified phylogeny of the Lotus spp. used in this study (modified from Ojeda et al. 2013, reproduced in Supplementary Figure S54). Red branches show clades where PACC has evolved, and dotted branches indicate the presence of adjacent clades.                                   8    2  Materials and Methods      2.1  Plant material and cultivation  The North African species, Lotus filicaulis Durieu (voucher: Ojeda 71, herb. UBC) was used for experiments on floral manipulation; L. filicaulis is sometimes regarded as synonymous with L. tenuis Willd.  A second species, Lotus sessilifolius DC. (voucher: Ojeda 225, herb. UBC) from the Canary Islands, Spain, was added as a comparison for RNAseq experiments. Both species exhibit strong PACC with a yellow to red colour change. The plants were propagated from seeds in the horticulture glasshouse, University of British Columbia, Vancouver, Canada. All plants were grown in pots of 10-20 cm in diameter at 20-25 °C and were more than 6 weeks old when flowers were collected for analyses.      2.2  Hand pollination experiment  In order to standardize the stages of PACC, five genetic individuals of L. filicaulis were tagged for both control and treatment (hand pollination), monitored daily for colour change, and photographed to capture distinct colour stages. 3 flowers were measured per plant for both the control and treatment. Hand pollination was conducted one day after anthesis (flower opening) to ensure the keel petals had separated.  Pollen was transferred between flowers using an implement of a Victorinox SwissToolTM (unpointed blade).   The rate of progression though these stages was modelled using the lme4 package (Bates et al. 2015) in R version 3.2.2 (R Development Core Team, 2008). Data were visualized using the ggplot2 package (Wickham, 2009) in R. The R scripts used in this analysis is available at github.com/mannfred/Lotus.    A similar experiment was conducted on 21 individuals of L. filicaulis, but rather than tracking each stage of colour change, only the number of days for PACC to initiate was recorded. The results of this experiment can be found in Supplementary Table S42.      9    2.3  Transcriptome sequencing  The entire corollas (dorsal, lateral and ventral petals) of L. filicaulis and L. sessilifolius were separated from the rest of the flower at the same developmental stage (Stage 13 of anthesis, Ojeda et al., 2012), but with two different colour stages: 1) flowers at anthesis with a yellow color, and 2) flowers initiating flower color change to red.  At least six unpollinated flowers of each type, yellow and red, were collected from one individual. Petals of each colour were removed from the flower and pooled to represent the phenotype across the entire individual. Flower petals were immediately placed on liquid nitrogen after collection and stored at 80°C until RNA extraction. RNA was extracted using the Pure LinkTM Plant RNA  Reagent from Invitrogen following the manufacturer’s protocol. RNA quantity and quality was determined using a QubitR 2.0 Invitrogen (Life Technologies) and with an Agilent 2100 Bioanalyzer, respectively. Samples with a RIN value of 7 or above were used for library construction. Paired end RNA libraries (Tables 2.1 and 2.2) were prepared for each individual sample with the Illumina kit according to the manufacturer’s protocol. All samples were sequenced (100 base pair reads) in one lane of an Illumina Hi-Seq 2000 at the NextGen Sequencing Facility at the Biodiversity Research Centre, University of British Columbia (UBC).      2.4  Sequence analysis   Aligning RNA-seq data from L. filicaulis and L. sessilifolius has not yet been reported. Therefore, I compared two methods to discover differentially expressed genes relevant to PACC. In the first method, RNA-seq reads were aligned to the reference genome of a closely related model organism, Lotus japonicus. In the second method, RNA-seq reads were assembled into contigs (putative genes) and aligned to a reference transcriptome that was assembled de novo from the reads themselves.  All scripts used in the analyses are available at github.com/mannfred/Lotus.     2.5  TopHat Alignment   RNA-seq reads from L. filicaulis were analysed by TopHat. FASTQC Galaxy v0.63 (Andrews, 2010) was executed on a public Galaxy server (Goecks et al., 2010) to check the quality of reads and to identify sequencing artifacts. Sequencing reads were mapped to the Lotus japonicus genome release 2.5 (Sato et al., 2008) using TopHat2 Galaxy v0.9 (Kim et al., 2013). Sequence 10    Alignment Map (.sam) files were processed by HTSeq (Anders et al., 2015) to count the number of RNA-seq reads that mapped to each TopHat feature (gene), and differential expression (DE) was calculated using edgeR (Robinson et al., 2010).  Raw counts per gene were used instead of FPKM transformed counts because edgeR incorporates its own dispersion-normalization procedure in its DE calculations (Robinson et al., 2010). EdgeR results were processed using a set of custom R scripts to filter significant results (Benjamini-Hochberg FDR <0.05 (Benjamini & Hochberg, 1995)) and differences in fold change.   DE genes were analyzed for ontology term enrichment (Yekutieli FDR<0.05 (Yekutieli & Benjamini, 1999)) using the Singular Enrichment Analysis tool with Lotus japonicus 2.5 genome reference in AgriGO (Du et al., 2010). Because the functional annotation of the L. japonicus genome is ongoing, genes associated with enriched GO categories were annotated for putative function by BLASTing the Glycine max (L.) Merr. CDS library (Williams 82 Assembly 2 version 1, Schmutz et al., 2010) sourced from Phytozome v10.3 (Goodstein et al., 2012).    2.6  STAR Alignment   RNA-seq reads from both L. filicaulis and L. sessilifolius were mapped to the Lotus japonicus genome Kazusa version 2.5 (Sato et al., 2008) using STAR v2.4 (Dobin et al., 2013). Successfully mapped transcripts were assembled and expression was estimated using RSEM v1.2.19 (Li & Dewey, 2011).  Differential expression of the yellow and red flower transcriptomes was calculated using the same methods as above.     2.7  De novo Assembly by Trinity  Sequenced Illumina reads from L. filicaulis and L. sessilifolius were assembled de novo into contigs in Trinity v2.0.6 (Grabherr et al., 2011). Using assembled contigs as a reference transcriptome, contigs were measured for expression using Trinity’s built-in RSEM tool (Li & Dewey, 2011) and DE was calculated as above. DE contigs were then identified by BLASTn (Altschul et al., 1997) as their top scoring Lotus japonicus homolog (expectation value of 1e-4) using the L. japonicus v2.5 CDS library sourced from Kazusa (Sato et al., 2008). Ontology term enrichment and annotation of associated genes was analysed as above.      11    2.8  Nucleotide Alignments  Trinity contigs BLASTing to L. japonicus genes of interest were investigated further by BLASTing the Legume IP database (Li et al., 2011) for Glycine max (v2.0) (Schmutz et al., 2010), Medicago truncatula Gaertn. (Mt4.0v1) (Young et al., 2011), and an outgroup Arabidopsis thaliana (L.) Heynh. (TAIR10) (Lamesch et al., 2011) to further identify homologous genes. Trinity contigs were trimmed before and after putative start and stop codons, respectively, which were identified by pairwise sequence alignment of their L. japonicus homolog using EMBOSS Needle (Rice et al., 2000). Coding sequences of putative homologs were compiled along with the relevant trimmed Trinity contig into a .fasta file and translated to amino acid sequences using TranslatorX (Abascal et al., 2010). Multiple sequence alignment was performed using the T-Coffee (Notredame et al., 2000) option in TranslatorX. The phylogenetic relationship of a given Trinity contig to its relatives was visualized using RAxML v8.2 (Stamatakis, 2014) in Mesquite (Maddison & Maddison, 2015).    2.9  Gene family composition  Trinity (de novo assembly) sequences were BLASTed (1e-4) against the L. japonicus CDS library at LegumeIP to detect gene duplicates – specifically those not expressed at the time of sampling (this approach assumes no new gene duplications have occurred between L. japonicus and our two study species). BLAST results sharing the same annotation (i.e. gene name) as the top alignment were considered to be a part of the same gene family. Certain gene families (e.g. DFR, CHS) have been especially well-studied in L. japonicus, but CDS libraries are not necessarily updated in real time in accordance to ongoing L. japonicus research. In these cases, published literature on specific gene families took precedence over LegumeIP results.          12    Table 1 Next-generation library construction for L. filicaulis.  Library no.  Library ID  (Barcode)  Yellow / Red  Plant no.  Flower no.  (pooled)   1  ATGAGC  Red  1  ≥6  2  ACTGAT  Yellow  1  ≥6        Table 2 Next-generation library construction for L. sessilifolius.  Library no.  Library ID  (Barcode)  Yellow / Red  Plant no.  Flower no.  (pooled)   1  CAAAAG  Red  1  ≥6  2  ATTCCT  Yellow  1  ≥6                                    13    3  Results      3.1  Pollination accelerates colour change in L. filicaulis  Four distinct colour stages of PACC were observed; 1) Entirely yellow, 2) blush of orange, typically at the keel, 3) systemic orange colouration across the corolla, and 4) red, onset typically at the keel (Figure 2.1). The general linear model to test the effects of time (days) and treatment on colour was:    COLOUR = CONSTANT + DAYS + TREATMENT + DAYS*TREATMENT   Among plant standard deviation was effectively zero (s= 2.664 e-05) and was omitted as a random effect.   The number of days from anthesis was a significant predictor of colour (df= 1, 39; p= 1.69 e-09), as well as the effect of treatment through time (df= 1, 39; p= 5.06 e-04). No significant effect of treatment alone was detected (df= 1, 39; p= 0.851).   The rate of progression through the four stages of PACC in the hand pollinated treatment is over twice that of the control (see Figure 2.1 caption).      3.2  Illumina HiSeq library   A total of 12.5 million and 11.9 million .fastq reads were sequenced from the yellow-stage and red-stage RNA-seq libraries of L. filicaulis, respectively.  GC content was 46% for the forward yellow-stage and red-stage reads and 45% for the reverse reads.     Similarly, 6.7 million and 12.0 million .fastq reads were sequenced from the yellow-stage and red-stage RNA-seq libraries of L. sessilifolius, respectively.  GC content was 45% for the forward yellow-stage and red-stage reads and 44% for the reverse reads.     3.3  De novo assembly of non-model organism RNA reads outperforms alignment of reads to a reference genome of a closely related species  Two methods were used to discover differential expression in PACC, 1) direct mapping of reads to the L. japonicus genome using two different splice junction mappers, and 2) de novo assembly 14    of reads to contigs followed by BLASTing these contigs to a L. japonicus CDS library. The evaluation of the two approaches is summarised in Tables 3.1 and 3.2. Direct alignment by STAR and TopHat achieved low mapping rates due to unsuccessful heterologous mapping from our study species to L. japonicus. However, the de novo assembly protocol achieved a high rate of gene identification, and produced independent contigs that could be verified for identity and biological relevance by amino acid alignment and phylogenetic methods.  Trinity contigs of interest were found to align with homologous genes of related legumes, and their known phylogenetic relationship was successfully predicted by these alignments (Supplementary Figures S1-S53).  A comparison of the most differentially expressed contigs of both stages and of both Lotus species can be found in Supplementary Tables S38-S41.    3.4               The cyanidin pathway is upregulated in the red stage of PACC in L. filicaulis and                      L. sessilifolius  Regardless of the bioinformatics protocol, upregulation of numerous genes in the anthocyanin biosynthetic pathway (ABP) was detected in the red stage of PACC in both Lotus species examined (Tables 3.3 and 3.4, Figures 3.2 and 3.3). Except for anthocyanin synthase and flavonoid 3’-hydroxylase (both apparently single copy), gene duplicates or multiple gene families were identified in the Lotus japonicus genome for each of the genes of the ABP in both species (Figures 3.2 and 3.3). As a comparison the same analysis was carried out for the lignin biosynthetic pathway (LBP), which is not expected to be associated with PACC. There is no consistent upregulation of LBP genes during PACC. Generally, LBP genes are either not expressed, downregulated during PACC, or not differentially expressed (Figures 3.2 and 3.3).   3.5  The carotenoid pathway is upregulated in the yellow stage of PACC in L. filicaulis and  L. sessilifolius  Genes annotated to the carotenoid biosynthetic process (GO:0016117) were significantly upregulated at the yellow stage (i.e. downregulated at the red stage of PACC) in both Lotus species (Supplementary Tables S35-S36). The core carotenoid pathway is represented, including the two major branches producing zeaxanthin and lutein (Figures 3.4 and 3.5).     15    3.6  Differences between L. filicaulis and L. sessilifolius   Although the cyanidin pathway is upregulated during PACC in both Lotus species, there are differences in the composition of genes expressed, specifically those genes that are a part of a gene family. For example, CHALCONE SYNTHASE (CHS) is active in both species but the gene family members expressed in L. filicaulis and L. sessilifolius are disjoint sets. Similar differences are observed in the expression of DFR. However, it is notable that of the four F3H duplicates known for L. japonicus, only one is implicated in PACC for the two Lotus species studied. The expression of transcription factors and regulators contribute to numerous other differences between PACC in L. filicaulis and L. sessilifolius (Supplementary Tables S11-S18, S27-S34).  Finally, I bring attention to the presence of FLAVONOL SYNTHASE (FLS) in L. sessilifolius (Table 6, Figure 5). I detect an upregulation of FLS during the yellow stage of PACC in this species, and an absence of activity of this gene in L. filicaulis. The ecological and evolutionary implications of this are discussed below.        16      Figure 3 Left panel: Standardization of flower colour using photographs of four distinct stages: anthesis (‘Yellow’, stage 0), onset of keel colouration (‘Blush’, stage 1), systemic orange across the corolla (‘Orange’, stage 2), and full colouration (‘Red’, stage 3). Graph: timing of stages in the hand pollinated treatment (orange line, colour stage = 0.739(Days) – 0.121) and control (unpollinated) (turquoise line, colour stage = 0.358(Days) – 0.059).   Intercepts were not fixed (anthesis) to maintain zero-mean residuals. Each point represents the mean number of days to one of four stages (mean of three replicates per treatment per individual plant). The size of points is proportional to the amount of over-plotted data.        17      Figure 4 Graphical representation of the differential expression of genes (Trinity contigs) involved in flavonoid and lignin biosynthesis throughout PACC in Lotus filicaulis. The boxes represent genes as annotated in the Lotus japonicus genome; light grey boxes represent genes that were not found in the L. filicualis transcriptome (gene may be absent or may have been undetected). Expression of putative orthologs in L. filicaulis are indicated by colour (red: upregulated at red stage; blue: upregulated at yellow stage; dark grey: not differentially expressed). 4coumaroyl-CoA and malonyl CoA are derivatives of phenylalanine and acetatepyruvate, respectively. In the flavonoid pathway CHS = chalcone synthase, CHI = chalcone isomerase, F3’H = flavonoid 3’-hydroxylase, F3H = flavnone 3hydroxylase, DFR is dihydroflavonol 4-reductase, and ANS is anthocyanidin synthase. In the lignin pathway C3’H = coumaroyl shikimate 3’-hydroxylase, CCoAOMT = caffeoyl-CoA O-methyltransferase, CCR = cinnamoyl-CoA reductase, CAD = cinnamyl alcohol dehydrogenase, POD = peroxidase, F5H = ferulate 5hydroxylase, and COMT = caffeic acid O-methyltransferase.    18        Figure 5 Graphical representation of the differential expression of genes (Trinity contigs) involved in flavonoid and lignin biosynthesis throughout PACC in Lotus sessilifolius. Conventions and acronyms are as defined in the legend to Figure 4.                           19      Figure 6 Graphical representation of the differential expression of genes (Trinity contigs) involved in carotenoid biosynthesis throughout PACC in Lotus filicaulis. The boxes represent genes as annotated in the Lotus japonicus genome; light grey boxes represent genes that were not found in the L. filicualis transcriptome (gene may be absent or may have been undetected). Expression of putative orthologs in L. filicaulis are indicated by colour (red: upregulated at red stage; blue: upregulated at yellow stage; dark grey: not differentially expressed). Geranylgeranyl diphosphate is a derivative of pyruvate and glyceraldehyde 3phosphate. In the carotenoid pathway PSY = phytoene synthase, PDS = phytoene desaturase, CIS = carotene isomerase, ζ-CDS = ζ-carotene desaturase, CRTISO = prolycopene isomerase, CYB = β-lycopene cyclase, CYE = ε-lycopene cyclase, βCHY = β-carotene hydroxylase, ε-CHY = ε-carotene hydroxylase, ZEP = zeaxanthin epoxidase, and VDE = violaxanthin de-epoxidase.     20      Figure 7 Graphical representation of the differential expression of genes (Trinity contigs) involved in carotenoid biosynthesis throughout PACC in Lotus sessilifolius. Conventions and acronyms are as defined in the legend to Figure 6.                      21    Table 3  Comparison of protocols used to detect differential expression of RNA in L. filicaulis.    Analysis type (mapping reference)  Reads mapped  yellow stage   Reads mapped red stage  DE genes yellow stage ↑  DE  genes  Red  stage  ↑  Enriched GO terms yellow stage  Enriched GO terms red stage  TopHat  (L. japonicus genome)  8,538,623  7,243,178  1419  962  107  84  STAR   (L. japonicus genome)  4,334,187  3,566,554  1398  958  128  97  Trinity   14,025,763  13,167,862  3287  1244  112  44  (L. filicaulis transcriptome)                           22      Table 4  Comparison of protocols used to detect differential expression of RNA in  L. sessilifolius.    Analysis type (mapping reference)  Reads mapped  yellow stage   Reads mapped red stage  DE genes yellow stage ↑  DE  genes  Red  stage  ↑  Enriched GO terms yellow stage  Enriched GO terms red stage   TopHat  (L. japonicus genome)  1,333,258  1,855,091  394  632  122  95  STAR   (L. japonicus genome)  1,272,184  1,976,009  610  954  151  120  Trinity   13,085,868  23,373,724  1175  1468  100  57  (L. sessilifolius transcriptome)                       23    Table 5  Selected genes for enzymes involved in flavonoid biosynthesis in L. filicaulis. The first column lists differentially expressed (FDR<0.05) Trinity contigs. The second column lists the gene names produced by BLASTing and aligning contigs to the Glycine max CDS library, followed by the function of the enzyme which it codes for. The fourth and fifth columns list raw counts per gene at  each colour stage, followed by the normalized log2 fold change. Sequences for the Trinity contigs in this table are listed in Appendix 1.      Trinity contig Glycine max homolog Function Counts per gene (Yellow stage) Counts per gene (Red stage) Normalized Log2FC (Red/Yellow) TR19889|c3_g1  Glyma.01g43880.1  CHALCONE  SYNTHASE (CHS)  Produces naringenin chalcone from phenylalanine and acetate-pyruvate derivatives.   1385    19064    -4.1392    TR48518|c0_g1  Glyma.11G011500.1  CHALCONE  SYNTHASE (CHS)  Produces naringenin chalcone from phenylalanine and acetate-pyruvate derivatives.  77  430  -2.8356  TR19889|c2_g1  Glyma.01G228700.1  CHALCONE  SYNTHASE (CHS)  Produces naringenin chalcone from phenylalanine and acetate-pyruvate derivatives.  17  207  -3.9514  TR52191|c0_g1  Glyma.06G202300.1  FLAVONOID 3’- HYDROXYLASE  (F3’H)  Catalyses naringenin to eriodictyol   1586  15638  -3.6580  TR38960|c0_g1  Glyma02g05450.1  FLAVANONE 3HYDROXYLASE (F3H)  Catalyses naringenin to dihyrdrokaempferol and eriodictyol to dihydroquercetin   1830    6123    -2.0988    TR29586|c0_g1  chr5.CM0077.210.r2.m  DIHYDROFLAVONOL  4-REDUCTASE (DFR)  Catalyses dihydroquercetin to leucocyanidin and dihydrokaempferol to leucopelargonidin   148  10163  -6.4567  24    Trinity contig Glycine max homolog Function Counts per gene (Yellow stage) Counts per gene (Red stage) Normalized Log2FC (Red/Yellow) TR50049|c0_g1  ANTHOCYANIDIN  SYNTHASE (ANS)  Catalyses leucocyanidin to cyanidin and leucopelargonidin to pelargonidin   1010  14783  -4.2278    TR23981|c0_g1  Glyma17g34530.2  MORE AXILLARY  BRANCHES (MAX1)  Positive regulator of several flavonoid biosynthesis enzymes  45  2987  -6.3605  TR11002|c0_g1  Glyma14g06750.1  PRODUCTION OF  ANTHOCYANIN  PIGMENT (PAP1)     Encodes MYB75  transcription factor involved in anthocyanin metabolism   475  54  2.7778                                 25     Table 6  Selected genes for enzymes involved in flavonoid biosynthesis in L. sessilifolius.  See Table 5 for column descriptions. Sequences for the Trinity contigs in this table are listed  in Appendix 2.      Trinity contig Glycine max homolog Function Counts per gene (Yellow stage) Counts per gene (Red stage) Normalized Log2FC (Red/Yellow) TRINITY_DN5 4168_c0_g2  Glyma.11G011500.1  CHALCONE  SYNTHASE (CHS)  Produces naringenin chalcone from phenylalanine and acetate-pyruvate derivatives.   65    4382    -6.1743    TRINITY_DN5 4168_c0_g1  Glyma.11G011500.1  CHALCONE  SYNTHASE (CHS)  Produces naringenin chalcone from phenylalanine and acetate-pyruvate derivatives.  17  1134  -6.1510  TRINITY_DN5 2652_c1_g1  Glyma.19G105100.1  CHALCONE  SYNTHASE (CHS)  Produces naringenin chalcone from phenylalanine and acetate-pyruvate derivatives.  145  660  -2.2875  TRINITY_DN5 2780_c0_g1  Glyma.06G202300.1  FLAVONOID 3’- HYDROXYLASE  (F3’H)  Catalyses naringenin to eriodictyol   7  2073  -8.2859  TRINITY_DN5 2390_c0_g1  Glyma.02G048400.1 FLAVANONE 3HYDROXYLASE (F3H)  Catalyses naringenin to dihydrokaempfero l and eriodictyol to dihydroquercetin   276  1893    -2.8794    26    Trinity contig Glycine max homolog Function Counts per gene (Yellow stage) Counts per gene (Red stage) Normalized Log2FC (Red/Yellow) TRINITY_DN5 2789_c0_g1  Glyma.17G252200.1  DIHYDROFLAVONOL  4-REDUCTASE (DFR)  Catalyses dihydroquercetin to leucocyanidin and dihydrokaempfero l to  leucopelargonidin   19  434  -4.6063  TRINITY_DN5 3094_c0_g1  Glyma.11G027700.1  ANTHOCYANIDIN  SYNTHASE (ANS)  Catalyses leucocyanidin to cyanidin and leucopelargonidin to pelargonidin   30  3761  -7.0659  TRINITY_DN3 9800_c0_g1  Glyma.13G082300.1  FLAVONOL  SYNTHASE (FLS)   Mediates production of isorhamnetin,  1601  112  3.7338  quercetin, and kaempferol from dihydroflavonol substrates                              27    4  Discussion      4.1  The significance of pollination as a trigger for PACC  I present the first experimental evidence that PACC is accelerated by pollination in L. filicaulis (Figure 2.1); these measurements support those made of Acmispon glaber (Jones & Cruzan, 1999). This is strongly suggestive that PACC is a trait that has evolved via interaction with pollinators rather than an incidental characteristic of the aging process with no fitness consequences. This is unsurprising as many floral traits are indeed driven by pollinators (Schiestl & Johnson, 2013) and it is consistent with this trait being a widespread evolutionary convergence (Weiss, 1995). Viewing PACC as an evolved adaptive trait raises the question of the precise adaptive function that it confers. Brito et al. (2015) suggest that the retention of flowers is to increase attraction of insect pollinators at long range, and then at short range increase foraging efficiency by signalling flower quality to pollinators.    The demonstration of a pollination trigger for PACC also raises the question of what the precise nature of the trigger is. Pollination involves both physical movement of the petals of the flower (thigmostimulus), deposition of pollen on the stigma, subsequent growth of pollen tubes, and fertilization of the ovules. All these processes have physiological consequences and could be transduced into signals leading to PACC. Further experimental work is required to dissect the events of pollination that lead to the acceleration of PACC. Even without pollination, colour change eventually occurs. This suggests that there is an additional, late-acting or  ‘failsafe’ trigger for the onset of PACC, one that presumably responds to late acting autogamy or to some aspect of floral aging.    4.2  De novo assembly is preferred for RNA-seq data without a reference genome The two RNA-seq aligners used in our study, STAR and TopHat, performed similarly in aligning sequenced reads to the genome of a sister species. Our results are consistent with a recently published comparison of 11 commonly used alignment protocols (Engström et al., 2013). STAR 1-pass uses gene annotation information to identify exon splice junctions before aligning to the reference genome. TopHat also uses an annotation file to identify junctions, and furthermore, uses this information to align reads directly against annotated transcripts. Therefore, both 28    alignment protocols require a well-annotated genome, which currently does not exist for L. filicaulis or L. sessilifolius. Aligning L. filicaulis and L. sessilifolius reads to the genome of L. japonicus led to a substantial loss of data as the majority of reads remained unmapped. However, the number of L. filicaulis reads mapped to L. japonicus outperformed the number of mapped L. sessilifolius reads when considering the same protocol. This underscores the importance of phylogenetic relatedness when using a model genome to study a congener.   Our results strongly support the use of a de novo assembly of RNA-seq reads into a transcriptome (then mapping reads back onto the transcriptome) as opposed to attempts at cross-species mapping in Lotus. Contigs may then be identified by reciprocal BLAST (with putative genes checked by amino acid alignment) against the most closely related species with an annotated genome.     4.3  PACC is associated with the wholesale upregulation of the flavonoid pathway  Ojeda et al. (2013) previously studied gene expression of some flavonoid biosynthesis genes involved in PACC in L. filicaulis and L. sessilifolius by PCR. Our results support and extend their findings. With the exception of chalcone isomerase, at least one copy of each gene in the flavonoid biosynthetic pathway leading to cyanidin production is differentially expressed (increased) at the red stage of PACC (Figures 3.2 and 3.3). An alternative scenario might be that most of these genes might already be expressed at the yellow stage with the exception of one critical enzyme without which pigment cannot be made. Under this scheme the upregulation of a single enzyme would be enough to initiate colour change.  Our results make it clear that this is not the case. Instead nearly the entire pathway is upregulated. Except for chalcone isomerase, all genes in the pathway have relatively low expression at anthesis and increase dramatically at the PACC stage (Tables 3.3 and 3.4; Figures 3.2 and 3.3). This implies that the genes in the pathway are co-regulated as a unit during PACC, possibly by a master regulator (see discussion below under MAX1).    4.4  PACC is associated with highly specific upregulation of members of gene families   Many of the enzymes involved are coded for by multiple genes in gene families. An example is DFR which has numerous copies (DFR1-DFR5) in Lotus japonicus (Shimada et al., 2005). However, during PACC only one paralog is upregulated. Other examples are obvious from 29    inspection of Figures 3.2 and 3.3. In these cases, the upregulation of specific copies, rather than all copies of a particular enzyme gene, seems to drive PACC. Some gene copies are apparently not expressed at either stage, but may be involved in tissue-specific expression elsewhere.    It is worth noting that different gene copies within the DFR and CHS families are expressed between L. filicaulis and L. sessilifolius. This is also the case for several gene families of the carotenoid pathway (Figures 3.4 and 3.5). This lends support to the convergent evolution of PACC between L. filicaulis and L. sessilifolius, as hypothesized by Ojeda et al (2013; Figure 2). However, it remains possible that, regardless of differences in the upregulation of gene copies, some general aspect of the flavonoid pathway (e.g. how it is regulated) is a developmental pre-pattern for PACC that is common to Lotus. In this case, the apparent convergent evolution of PACC could be considered a “latent homology” (Nagy et al., 2014).    Because anthocyanins are highly versatile and serve a wide range of functions (Kong, 2003), it may be that gene duplication is key to the evolution of novel adaptations involving anthocyanins, including PACC. This may be especially likely for the putatively tetraploid L. sessilifolius, and although L. filicaulis is a diploid, it has had numerous whole genome duplication events in its history, like many plants. Neo-functionalization, where directional selection leads to novel function following a gene duplication event, may explain why only certain copies of flavonoid biosynthesis genes were actively transcribed during PACC.  Alternatively the association of PACC with duplicated genes might be explained by escape from adaptive conflict (Des Marais & Rausher, 2008). In this scenario a single copy gene is adapted to simultaneously perform its ancestral function while performing some novel function, but is constrained in its ability to improve due to antagonistic pleiotropy. Following gene duplication both ancestral and novel functions have the potential to be better adapted. Marais and Rausher (2008) demonstrated that adaptive changes in the DFR gene of pre-duplication and post-duplication plant species are best explained as escape from adaptive conflict. In order to determine if PACC is a result of gene duplication, evidence for the ancestral and novel functions of flavonoid biosynthesis genes would need to be studied in L. filicaulis, L. sessilifolius and their relatives. In the case of L. japonicus, various duplicates of DFR differ in their responses to stress and organ specificity (Yoshida et al., 2010), but whether any such gene copies are functionally divergent in our study species, and whether any of these copies are flower-specific remains unstudied.    30      4.5  Upregulation of flavonoid biosynthesis may be antagonistic to other phenylpropanoid pathways  Expression of genes in the lignin biosynthetic pathway (LBP) suggests that a shift from the lignin branch of the phenylpropanoid pathway to the flavonoid branch may occur over the course of PACC (Figures 3.2 and 3.3). Transcriptome and metabolite profiling of Fragaria x ananassa (Duchesne) has demonstrated a metabolic interaction between flavonoid and lignin biosynthesis in strawberry fruit (Ring et al., 2013). Lignin biosynthesis competes for the same upstream substrate (coumaroyl-CoA) as flavonoid biosynthesis and, as would be expected under constant substrate availability, a shift from lignin production to flavonoid production can be measured in anthocyanin-rich tissues (Ring et al., 2013). Therefore it is plausible that in L. filicaulis and L. sessilifolius a drop in substrate availability may result in the downregulation of LBP genes observed during PACC.  Alternatively, expression of LBP genes may be reduced following flowering, regardless of PACC, due simply to developmental changes associated with aging.     4.6  Carotenoid biosynthesis is downregulated during PACC  Carotenoids are widespread in the yellow to orange flowers of numerous insectpollinated taxa, and likely play a role in the detectability of flowers by pollinators (Tananka et al. 2008, Cronk and Ojeda 2008). Consequently, the biosynthesis of carotenoids is generally well understood (Zhu et al. 2010). I detected the upregulation of the genes controlling the core carotenoid pathway at the yellow stage of PACC in both Lotus species – this is expected given the definitively yellow appearance of the flowers of both species at anthesis. To the extent that flavonoid biosynthesis may be crucial to the red stage of PACC, the coincident downregulation of carotenoid biosynthesis genes may be important in accentuating colour change. Alternatively, the downregulation of carotenoids may represent a senescence-related process separate from flavonoid production.     4.7  Is MAX1 a master regulator of PACC?  PACC in Lotus filicaulis and Lotus sessilifolius involves a wholesale upregulation of the cyanidin branch of the anthocyanin biosynthetic pathway (ABP). Given the number of genes 31    involved it is unlikely that each gene is regulated entirely independently. Instead it is possible that triggering of a top-level regulator then initiates a regulatory cascade activating the entire pathway in concert.  An obvious such potential regulator would be PRODUCTION OF ANTHOCYANIN PIGMENT 1 (PAP1), a gene known to be a powerful regulator of anthocyanin production in Arabidopsis (Borevitz et al., 2000). Perhaps strangely, the Lotus homologue of PAP1 is actually downregulated during PACC in L. filicaulis (Table 5) and therefore does not seem to be involved. However, of the other known regulators that are differentially expressed during PACC in our study, one, MORE AXILLARY BRANCHES (MAX1), is a potential candidate as a top level regulator. MAX1 is strongly upregulated at the red stage of PACC in L. filicaulis (Table 5).  The sequence recovered for MAX1 in L. filicaulis lacks a DNA-binding domain and is therefore likely to encode a Cytochrome P450 rather than a transcription factor (Figure S27). Indeed, in Arabidopsis, MAX1 encodes CYP711A1, and is known to be a positive regulator of the flavonoid pathway (Lazar & Goodman, 2006). Arabidopsis mutants max1, have been demonstrated to have 11 down-regulated flavonoid biosynthesis genes including F3H, F3’H, DFR, ANS, and UFGT (Lazar & Goodman, 2006). However, beyond what is known of Cytochrome P450s in flavonoid biosynthesis in general (Ayabe & Akashi, 2006), the exact biochemical mechanism in ABP of CYP711A1 remains unstudied. Interestingly MAX1 was first characterized for its effect on axillary bud behaviour. However the link between ABP and axillary bud formation (if any) remains obscure, although regulation of auxin transport through inhibition of PIN-function by flavonoids in the bud is one possibility that has been suggested (Lazar & Goodman, 2006). It is known that fertilized ovules are a source of auxin (Sundberg & Østergaard, 2009), and that auxin can control anthocyanin biosynthesis (Liu et al., 2014). Counter to that, it has been shown that the MAX pathway acts as an entirely independent regulator of auxin transport (Bennett et al. 2006). The versatility of MAX1 is further confirmed by the involvement of MAX1 in the strigolactone signalling pathway (Challis et al., 2013).      4.8  The role of FLS in PACC in Lotus sessilifolius  FLS forms flavonols (quercetin, kaempferol, and isorhamnetin) from dihydroflavonols, the same substrate used by DFR to produce cyanidin. FLS has been shown to be antagonistic to anthocyanin accumulation, which increases in FLS knockouts (Lee et al., 2016). Ojeda et al. (2013) found that pre-colour change (yellow flowered) Lotus species from the Canary Islands 32    contain comparatively more flavonols (mostly isorhamnetin) than anthocyanins. Flavonols are generally colourless to the human eye but absorb strongly in the UV. Despite this Ojeda et al. (2013) demonstrated a reduction in UV reflectance of the petals following PACC (Ojeda et al. 2013). Whatever the mechanism, a shift from flavonol to cyanidin production may affect the detectability of the flowers by pollinators.  It is of interest that L. sessilifolius and L. filicaulis differ markedly in the behaviour of FLS. The reduction of flavonol production with floral age in L. sessilifolius (a Canary Island species) is the basis of the suggestion made by Ojeda et al. (2013) that an FLS mediated switch to anthocyanins from flavonols may be occurring. However, the absence of FLS expression in L. filicaulis indicates that a different mechanism prevails in that species, although I cannot rule out the possibility that RNA levels were too low to be detected. The differential expression of FLS at the yellow stage of L. sessilifolius supports the notion that there is an FLS-mediated switch from flavonols to anthocyanins (which may therefore be the basis of an ecological function of PACC to signal floral viability to insect pollinators). It also implies that, in this species at least, anthocyanin biosynthesis is antagonistic to the production of flavonols. Furthermore, Ojeda et al. (2013) demonstrated that the transition from bee- to birdpollinated Lotus spp. involves the increased biosynthesis of anthocyanins at the expense of flavonol production.        4.9  The role of red in PACC  Red anthocyanin pigmentation of flowers has long been associated with bird pollination. For instance, the scarlet colouration of bird pollinated Lamiaceae species has been attributed to both cyanidin and pelargonidin (Saito & Harborne, 1992). It is thought that in bird pollination syndromes red serves not only as a bird-attractant but also a bee deterrent (Rodríguez-Gironés & Santamaría, 2004), though red-coloured objects should not be considered as invisible to bees (Chittka & Waser, 1997; Martínez-Harms et al., 2010). It is possible that in plants exhibiting ornithophily, red acts as a common signal of low nutritional status to bees. In bird pollinated plants highly dilute nectar is also thought to be an adaptation to deter bees (Bolten & Feinsinger, 1978) as this is difficult for bees to process, and energetically expensive to convert to concentrated storage forms (i.e. honey). There is an intriguing parallel here between PACC and bird pollination; in both cases red pigmentation is potentially signalling aspects of nutritional 33    status to bees (Chittka & Waser, 1997; Martínez-Harms et al., 2010). Red and related colours that are common in PACC may therefore constitute a preferred signal of foraging unsuitability, so incurring an adaptive advantage to the plant by optimizing pollinator foraging on young, newly opened, yellow flowers.                                                       34    5  Conclusion    In this thesis I 1) present an analysis of the transcriptomics of post-anthesis colour change in Lotus filicaulis and Lotus sessilifolius and 2) demonstrate that PACC is mediated by pollination in L. filicaulis.   I present the first evidence that PACC is mediated by pollination in L.  filicaulis. This is the foundation for understanding the ecological function of PACC in Lotus. Further work will need to address the physiological basis of the trigger(s) of PACC, e.g. is PACC triggered by the deposition of pollen on the stigma, or some other physical cue?    This is the first transcriptome-wide view of how gene expression is modulated through PACC for any species; the contigs listed in Appendices 1 and 2 are the first coding sequences available for these Lotus species. These sequences are hypotheses, which allows for both verification of this thesis and further work – especially if gene expression in other Lotus species is to be studied. Thus, this is an important contribution to better understand the evolution of pollination in Lotus.                                            35    Bibliography    Abascal F, Zardoya R, Telford MJ. 2010. TranslatorX: multiple alignment of nucleotide sequences guided by amino acid translations. Nucleic Acids Research 38: W7-13.    Altschul S, Madden TL, Schaffer AA, Zhang JH, Zhang Z, Miller W, Lipman DJ. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25: 3389-3402.    Anders S, Pyl PT, Huber W. 2015. 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Archives of Biochemistry and Biophysics 504: 132-141.        43    Appendix 1     Sequences for Trinity contigs listed in Table 5   >TR19889_c1_g1 [organism=Lotus filicaulis] CHS CTTGGCTGAGAACAACAAAGGTGCTCGTGTACTCGTTGTTTGTTCTGAACTTACTGCAGTTACCTTCC GTGGCCCTAGTGACACTCACCTAGACAGCCTTGTTGGGCAAGCATTGTTTGGAGACGGAGCAGCCGCA CTCATTGTTGGTTCCGATCCAGTACCAGAAGTTGAGAAGCCTTTGTTTGAGCTAGTTTGGACTGCACA GACTATTGCTCCAGATAGTGAAGGAGCCATTGATGGCCACCTTCGTGAAGTTGGATTGACATTTCATC TCCTTAAAGATGTTCCTGGG    >TR48518_c0_g1 [organism=Lotus filicaulis] CHS ATGGTGAGTGTAGCTGAGATTCGCAAGGCTCAAAGAGCTGAAGGCCCAGCAACCATCTTTGCTATTGG CACTGCAAATCCTCCTAACTGTGTTGATCAAAGCACCTATCCTGATTTCTACTTCAGAGTCACCAACA GCGAACACAAAACAGAGCTCAAGGAAAAATTTCAGCGCATGTGTGATAAGTCTATGATCAAGAAGAGA TACATGCACTTAACAGAAGATCTTTTGAAAGAGAACCCTAACATGTGCGCTTATATGGCGCCATCTTT GGATGCTAGGCAAGACATGGTGGTGGTAGAGGTACCTAGACTAGGCAAAGAAGCTGCTGTCAAGGCTA TA    >TR19889_c3_g1[organism=Lotus filicaulis] CHS ATGGTGAGTGTAGCTGAGATTCGTAAGGCTCAAAGGGCAGAAGGCCCAGCAACCATCCTGGCTATTGG CACTGCAAATCCACCAAACTGTGTGGACCAAAGCACTTATCCTGATTTCTACTTCAAAATCACAAACA GCGAGCACATGACAGAGCTTAAGGAGAAGTTTCAGCGCATGTGTGACAAGTCCATGATCAAGAAGAGA TACATGTACTTGAATGAAGAAATTTTGAAAGAGAACCCCAACCTTTGTGCTTATATGGCACCATCTTT GGATGCTAGGCAGGACATGGTGGTGGTAGAGGTACCAAGGTTAGGGAAAGAAGCTGCAACAAAGGCTA TAAAAGAATGGGGCCAGCCAAAATCTAAGATTACTCACTTAATCTTTTGCACCACAAGTGGTGTAGAC ATGCCTGGTGCTGATTATCAACTCACCAAACTCTTGGGTCTTCGCCCATCTGTGAAAAGGTACATGAT GTACCAACAAGGGTGCTTTGCAGGTGGCACGGTGCTTCGTTTGGCTAAAGATTTGGCTGAGAACAACA AAGGTGCACGTGTGCTAGTTGTTTGTTCTGAGATTACTGCGGTTACATTCCGTGGCCCTAATGACACT CACCTAGACAGCCTTGTGGGGCAAGCTTTGTTCGGAGATGGAGCAGCTGCAGTCATTGTTGGTTCTGA TCCAGTGCCTGAAATTGAGAAACCTTTGTTTGAACTAGTTTGGACTGCACAAACTATTGCTCCAGATA GTGACGGAGCCATCGATGGTCACCTTCGTGAAGTTGGATTGACGTTCCATCTCCTTAAAGATGTTCCT GGGATTGTTTCAAAGAACATTGAGAAAGCACTAATTGAGGCCTTCCAACCATTAGGCATATCTGATTA CAACTCAATTTTTTGGATTGCACACCCAGGCGGCCCAGCAATTCTGGACCAAGTTGAGCAGAAGTTGA GCTTGAAACCTGAAAAGATGAAAGCCACTAGAGAAGTGCTAAGTGAATATGGGAACATGTCAAGTGCA TGTGTCCTATTCATCTTAGATGAAATGAGAAAGAAATCAGCTCAAGATGGACTCAAAACCACTGGTGA AGGACTTGAATGGGGTGTGCTATTCGGTTTCGGACCTGGACTTACCATTGAAACTGTTGTTTTGCGTA 44    GTGTGGCTATTTAATAT    >TR52191|c0_g1 [organism=Lotus filicaulis] F3’H ATGTTCC  CATGGATGATCATTGGCTTTGCCACCATCACATTCCTCATCTTCATACACCGTGTCGTGA  AGTTCTCAACACGTCCCTCACTCCCACTCCCTCCGGGCCCAAAACCATGGCCCATAATAG  GAAACTTTCCCCATATGGGCCCCGTGCCGCACCACTCCCTCGCAGCTTTGGCCCGAGCCC  ACGGCCCGCTTATGCACCTCAAATTGGGCTTTGTGGACGTTGTGGTGGCGGCGTCAGCCG  CCGTGGCGGAGCAGTTTTTGAAGGTTCATGATGCAAATTTTAGTAGCCGGCCGCCTAATG  CCGGAGCTAAGTACATAGCTTATAACTATCAGGATCTCGTGTTCGCTCCGTACGGTGCAC  GGTGGCGATATCTCCGGAAAATCACCAACCTCCACCTCTTCTCCGGCAAGGCTTTGGATA  ATTTCAAACACTTGCGTCAGGAAGAGGTATCGAGATTGACACGCAACATATCAAAGTCCA  ACTCAAAAGCTGTGAACTTGGGACAACTACTGAATGTATGCACTACCAACGCATTATCCA  GGGTAATGATCGGAAGAAGAGTGTTCAACGACGGCGACGGTGGGTGTGATCCTAGAGCTG  ATGAGTTTAAGGCCATGGTGGTGGAGTTAATGGTGTTAGCGGGTGTTTTCAACATAGGTG  ACTTCATTCCTTCCTTGGAGTGGCTAGACCTTCAAGGGGTTCAAGCTAAGATGAAGAAAT  TGCACAATAGGTTTGATGAATTTTTAACCAGCATTATTGAGGAACACAATACTTCTTCCA  AGAGTGAGAATCATAAGGATTTATTGAGTACGTTGCTGTCGCTTAAAGATGTTCCTGATG  ATGATGGAAACAGACTCAATGATATTGAGATCAAAGCACTACTTCTGAACATGTTCACAG  CTGGGACTGACACGTCAGCTAGCACAACAGAATGGGCCATTGCAGAACTGATCCGTAGCC  CAAGAATTCTAGCCCAGGTCCAACAAGAATTGGACACAGTTGTGGGCCGAGAAAGAAACG  TGAGAGAAGATGACTTGCCTCATCTCCCATACTTGCAAGCAGTGGTGAAAGAAACCTTCC  GTCTTCACCCATCAACCCCTCTTTCACTGCCACGTGTCGCATCTGAGAGCTGTGAGGTAC  TGGGCTACCACATACCAAAGGGGTCCACTCTCTTGGTGAACGTGTGGGCCATAGCCCGTG  ACCCTAAAGAATGGGCTGAGCCATTGGAGTTCAAGCCCGAAAGGTTCCTAGAGGGTGATA  AGGTTGATGTTGATGTTAAGGGCAATGACTTTAAGGTGATACCCTTTGGTGCTGGACGTA  GGATATGTGCTGGGATGAGCCTTGGGCTTCGTATGGTTCAGCTGTTAACTGCAACTCTGG  TCCATTCGTTTAACTGGGAGCTAGAAAATGGGCTTAACCATGAAAAGCTCAACATGGATG AAGCTTATGGGCTAACATTGCAACGGGCTGTGCCTTTGTCAGTGTACTCTAGGCCCAGGC  TCTCGCCACATGTGTATGCAGCCTCTCATTGA    >TR38960|c0_g1 [organism=Lotus filicaulis]F3H ATGGCATCATTCA  AACCCAAAACTCTCACCACACTGGCTCAACAAAACACCCTTGAGTCCAGCTTCGTCCGTG  ACGAAGACGAGCGCCCAAAGGTCGCTTACAACAATTTCAGCAATGAGATCCCGGTAATTT  CCCTCGCCGGAATCGACGAGGTCGATGGCCGGAGATCCGAGATTTGTAACAAGATTGTTG  AAGCTTGTGAGAATTGGGGTATTTTTCAGGTTGTTGATCATGGGGTCGACACTGAATTGG TTTCCCACATGACCACTCTTGCTAAAGAGTTCTTTGCTCTTCCACCGGAGGAGAAGCTTC 45    GGTTTGACATGACCGGTGGCAAAAAGGGTGGTTTCATCGTCTCCAGCCACCTCCAAGGAG AATCAGTGCAGGATTGGAGAGAGATAGTGACATACTTTTCATACCCAATCAGGAACAGGG ACTACTCACGGTGGCCGGACACCCCAGCAGGGTGGAAGGCGGTGACGGAGGAGTACAGCG AGAAGCTGATGGGTTTAGCATGCAAGCTGTTGGAGGTGTTGTCTGAGGCAATGGGGTTAG  AGAAAGAGGCTCTAACAAAGGCGTGTGTGGATATGGAGCAGAAGGTTGTGGTGAATTATT  ACCCAAAATGCCCTCAACCTGACCTCACTCTTGGGCTGAAGCGACACACTGATCCTGGCA  CCATTACTCTGCTTCTTCAAGATCAGGTGGGTGGGCTTCAAGCTACTAGGGATAATGGGA  AGACATGGATCACTGTGCAGCCTCTTGAAGGAGCTTTTGTTGTCAATCTTGGTGACCATG  GTCATTATCTTAGCAATGGAAGGTTCAAGAACGCTGACCACCAAGCAGTGGTGAACTCAA  ACTCAAGCCGTTTATCGATCGCCACATTCCAGAACCCGGCACCAGATGCAACTGTATACC  CTTTGAAGGTTCGAGAGGGAGAGAAGTCAGTAATGGAAGAACCAATCACATTTGCTGAAA  TGTACAGGAGGAAGATGAGCAAGGACATTGAGCTTGCTCGGATGAAGAAGCTGGCTAAGG AGAAGAAACTGCAGGATTTGGAGAAGGCCAAACTTGAACCCAAGCCCATGAATGAGATCT  TTGCTTAAG    >TR29586|c0_g1_i1 [organism=Lotus filicaulis]DFR ATGGGTTCAGTACCAGAAACAGTTTGCGTTACCGGAGCTGCAGGTTTCATCGGGTCATGGC  TTGTCATGAGACTCATGGAACGTGGCTATATGGTTCGCGCCACTGTAAGAGACCCTGCTA  ACATGAAGAAGGTGAAGCATTTGCTGGAACTGCCAGAGGCAAAGACCAAGCTGACTCTGT  GGAAAGCTGATCTTGCTGAAGAGGGAAGCTTTGATGAAGCCATCAAAGGGTGCACTGGAG  TTTTCCATGTGGCCACACCCATGGATTTTGAGTCCAAGGACCCAGAGAATGAAGTGATTA  AACCTACCATAAACGGGGTACTAGACATCATGAAGGCATCCCAGAAGGCCAAAACTGTAC  GGAGGCTGGTTTTCACATCCTCTGCAGGTACCCTCAACGTCATAGAGCACCAAAAACAAA  TGTTTGATGAGAGCTGCTGGAGCGATGTTGAGTTCTGCCGAAGAGTAAAGATGACCGGTT  GGATGTATTTTGTTTCAAAGACACTGGCAGAGCAAGAAGCGTGGAAATTTGCCAAAGAGC  ATGGCATTGACTTCATTACAATCATTCCACCCCTTGTTGTTGGTTCCTTTCTAATGCCGA  CAATGCCACCAAGCCTAATCACTGCTCTTTCTCCTATCACCGGAAACGAGGCCCATTATT  CGATCATAAAGCAAGGCCAATATGTCCACTTGGATGATCTTTGTCTTGCCCATATTTTCC  TGTTTGAGCACCCTGAATCAGAAGGAAGGTATATCTGTAGTGCATCTGAGGCTACTATCC  ATGACATTGCAAAGTTAATCAACTCAAAGTATCCAGAGTACAATATCCCCACTAAGTTCA  AGAATATTCCAGATGAATTGGAGCTTGTCAGATTTTCATCAAAGAAGATCAAAGACATGG  GATTCGAATTTAAATACAGCTTAGAGGATATGTACACTGGAGCAATTGACACGTGCAAGG  AAAAAGGGCTTCTTCCTAAAGCTGCAGAAAATCCAAGCAATGGCAAATAA    >TR50049|c0_g1_i1 [organism=Lotus filicaulis] ANS ATGGCGCC  46    TACTGTTGTTGAGAGAGTTGAAAGCTTATCGGGCAGTGGGATACAATCTATCCCAAAGGA ATATGTGAGGCCAAAAGAAGAGCTGGCAAACATAGGCGACGTTTTTGAGGAGGAAAAGAA GGTTGGGCCTCAAGTTCCAACGATTGATTTGAAAGAGATAGACTCGCCGGACGAGTTTGT TCGGGCCAAGTGCAGGGAGAAGCTTAGGAAGGCAGCCGAGGAGTGGGGGGTGATGCACCT GGTGAATCATGGGATCCCGGATGAGCTCCTAAACCAATTGAAGAGTGCTGGGGCAGAGTT TTTCTCTTTGCCGGTCGAGGAGAAGGAGAAGTATGCAAATGACCAGGCGGCCGGGAATGT  GCAAGGGTACGGGAGCAAGCTGGCCAACAATGCCAGTGGTCAGCTTGAATGGGAAGATTA  CTTCTTCCACCTTATCTTTCCAGAAGACAAGCGTGACCTCTCCATCTGGCCCAAGACACC  ATCATATTATACGGAAGTTACCAGCGACTATGCAAGGCGACTAAGAGTGCTTGCGAGCAA  GATACTGGAAGTGCTATCTCTGGAATTGGGTCTCGAAGAAGGAAGGTTAGAGAAGGAAGT  TGGTGGAATGGAAGAGCTTCTACTTCAGATGAAAATCAACTACTACCCAAAATGCCCCCA  GCCAGAGCTAGCTCTCGGAGTCGAAGCCCACACCGACATAAGCGCACTCACTTTCCTCCT  CCACAACATGGTCCCAGGCCTGCAACTTTTCTACGAAGGAAAATGGGTCACGGCAAAATG  CGTTCCTGATTCAATCCTCATGCACATTGGTGACACCACTGAGATTTTAAGCAATGGTAA  GTTCAAGAGCATTCTTCACAGGGGTTTGGTGAACAAGGAAAAAGTTAGAATATCTTGGGC  AGTGTTCTGTGAACCCCCTAAGGAGAAAATCATCCTCAAACCACTGCCTGAGCTTGTGAC TGAGACCGAACCAGCGCGGTTTCCGCCACGCACTTTTGCTCAGCATATTCATCACAAACT  GTTCAGGAAGGACCAGGAAGCTTCAGCCCAATCCAAATGA    >TR23981|c0_g1 [organism=Lotus filicaulis] MAX1 ATGGTG  TTCATGGATTTTGAATGGTTGTTCCAAATTCCAAGTGTCCCTTGGTCTTCAGCAATGTTT  ACCTTGCTGGCTACAATAGGAGGATTCTCAGTGTATCTGTATGGGCCCTACTGGGGTGTG  AGGAAAGTTCCAGGTCCACCATCTGTGCCTCTGATAGGACACCTTCCCTTGCTGGCTAAG  TATGGCCCTGATGTCTTCTCAGTCCTTGCCAAGCAATATGGCCCAATCTACAGATTTCAT  ATGGGAAGACAACCTCTAATAATCATAGCAGATGCAGAGCTTTGCAAAGAGGCAGGCATC  AAAAAATTTAAAGATATTACAAACAGAAGCATTCCCTCTCCCATTTCTGCATCCCCCCTT  CACCAAAAGGGTCTCTTCTTCACCAAAGATTCACAATGGTCTACCATGAGAAACACCATA  TTATCACTCTACCAGCCATCACACTTATCCAGATTAGTGCCAACCATGCAGTCATTCATA  GAATCAGCAACTCAAAATCTAGATTCTCAAAAAGAAGACTTTATCTTTTCTAATCTTTCG  CTCAGCTTGGCAACTGATGTGATTGGACAAGCAGCATTTGGAGTTGATTTTGGCCTCTCG  AAGCCTCAACCAGTGTGTGATGAAATCAAGAGTGTTAACAAGGAAGTAAGGGATTCAAGT  ACTGGTAATGAAGTATCAGATTTCATTAACCAACACATTTACTCCACAACCCAACTCAAG  ATGGACCTATCTGGTTCCTTCTCCATCATACTGGGTTTACTTGTCCCAATTCTCCAGGAG  CCATTTAGGCAGGTTCTCAAGAGAATTCCAGGCACCATGGACTGGAAAATCGAGCGTACT  AACCGAAAACTAAGTGGCCGTCTTGATGAGATTGTAGAGAAGAGAATGAAAGATAGGGTC  AGAAGTTCAAAGGACTTCTTGTCACTCATTCTAAATGCAAGAGAATCAAAAACTGTTTCA  GAAAATGTGTTCACACCTGACTATATCAGTGCTGTTACTTATGAGCACCTGCTTGCAGGA  47    TCAGCAACCACATCCTTTACCTTGTCTTCCATTGTCTATTTGGTTGCTGGGCATCCAGAA GTTGAGAAAAAGATGCTTCAAGAGATTGATGGGTTTGGTCCAGTAGATCAGACACCAACT TCTCAAGATCTTCAAGAGAAGTTTCCTTACCTTGATCAGGTGATCAAAGAGGCCATGAGA TATTACACAGTCTCCCCATTGGTTGCAAGAGAAACATCAAATGAAGTAGAGATTGGAGGT TACCTTCTTCCAAAGGGGACTTGGGTCTGGTTAGCGCTTGGAGTTGTAGCAAAAGATCCC AGAAACTTTCCGGAGCCAGAAAAGTTCAAACCGGAGAGGTTTGATCCCAAATGTGAAGAA  ATGAAAAGAAGACATCCTTATGCTTTCATACCATTTGGAATTGGTCCTCGGGCTTGCATT  GGTCAGAAATTTTCCCTGCAAGAAATCAAGCTTTCTCTGATTCACTTATACAGGAAATAC  TTGTTTCGGCATTCACCTAATATGGAGAATCCCTTAGAACTTGAATATGGCATAGTGCTC  AACTTCAAGCACGGTGTCAAGGTTAGAGCCATAAAAAGAACAGAAAGGAGCTGCTAA    48    >TR11002|c0_g1_i1[organism=Lotus filicaulis] PAP1 ATTGGCTCTTACCTTTCTACAATATCTCTTTCTTTCTCTTGCTCTTGGCTTCTTTATATT TTGATATTTTCTTTCCTTCAGTGGAGCTCAACTTTGTAGTAGCAGCCTCACTGAAAACAT TTACCTTTCCTTCTGATATTTTGGTATTCTCAGAACATCTTTTATTGTTCTAGTTTCAGA ATTCATCACACTCACATCAAAATTCTGGCACACCACCATCATTTTCCTAAGATTCTCTAC TTAAAAGGTATGACTTTTATAGATTCTCAACTTTCTCTTGATCACACATAAAAGATTCTA ACTTATGTGAGAATCAAAGATCTTTGGTTTCTTCTTTTCTGTGATTCTCTTCTGTAATCA CAAGAGCACCCCTTTTCTAACTCTTATTTCCATCTCTTGAGGAACTTCATTCAAGTGATA GGAGAGTTTTGTATGCTTGTGGCTGTGGTTTAAGTTATGGATTTGGAAACACTCTATTCC CCTTGTTTTATGTCAAATTCAAATTGGTTTGTCCAAGAGAGTGCTCACAACACAGAATGG AGTAGAGAAGATAACAAGAGGTTTGAAAGTGCCCTTGCTATTTATGATAAGGACACACCA GATAGATGGCTGAATGTGGCTGCAATGATTCCTGGGAAGACTGTGCTTGATGTGATCAAG CAGTACAGGGAATTGGAAGAAGATGTGGGTGAAATTGAAGCAGGGCATGTTCCTGTCCCT GGATATCATTCCTCTTCTTTCACCTTTGAAGTTGTTGAGAACCAGAACTTTGATGGACTC AAAAGGAAGCCTGGAACAACTCTCAGGGGTTCTGATCATGAGAGGAAGAAAGGAGTACCG TGGACCGAAGAAGAACACAAACGCTTTCTGATGGGACTTCTAAAGTATGGTAAAGGGGAC TGGAGAAACATCGCTCGCAATTTTGTTATGACAAAGACTCCCACACAAGTAGCTAGCCAT GCTCAGAAGTACTACATAAGGCAAAAGGTTTCTGGAGGAAAAGATAAGAGGAGACCAAGC ATCCATGATATAACCACTGTTAATCTTACAGAAACTAGTACCACATCAGAAAACAACAAG CCACTTTCATTCAAAAGTACTAGTAACTACAACAGTGGCTCACTCATGGTTTTCAACCCA AATTGTGATGACTTGTTGATGATGCCATCTTCATCTGATATCATCACTTCCAAGACCCTT AAACTTCAAGGGCAGGATCTTTATGATTGCTCTTTACATGAGGCTTATGCTAAGTTAAAA ATTCCTAGTTTCAGGGCAGCACCTAGAAACTTCAATAAGGAAGCTGTTTTTGGTATTCAT GTACTATAAGAATGCCAACTATGACAATGTTCAAATTAAGGCAAACCAATTCCACTGCAT AGATTATTGTTCTGTAGGATTTTGATAATGTAGATGAAATGAAGCTACTTGTTTCATGAA CATCCTGAAAGGAAATGGCAATAAAGTTTCAATCCTTAAAACAAAAAAAAAAAT       49    Appendix 2    Sequences for Trinity contigs listed in Table 6   >TRINITY_DN54168_c0_g2 [organism=Lotus filicaulis] CHS ATGGTGAGTGTAGCTGAGATTCGTAAGGCTCAGAGGGCTGAAGGCCCAGCAACC  ATCCTCGCTATTGGCACTGCTAATCCACCAAACTGTGTCGACCAAAGCACTTATCCTGAT  TTTTACTTCAGAATCACTAACAGTGAACACATGACTGAGCTCAAGGAGAAGTTTCAGCGC ATGTGTGACAAGTCTATGATCAAGAAGAGATATATGCACTTAACTGAAGATCTTTTGAAA  GAGAACCCTAACATGTGCGCTTA    >TRINITY_DN54168_c0_g1 [organism=Lotus filicaulis] CHS CATCTCCTTAAAGATGTTCCTGGGATTGTTTCAAAGAACATTGATAAAGCACTAGTT  GAGGCTTTCCAACCATTGAACATATCTGACTACAACTCAATTTTTTGGATTGCACACCCA  GGTGGCCCAGCAATTCTTGACCAAGTTGAGCAGAAGTTGAGCTTGAAACCTGAAAAGATG  AGGGCCACTAGGGAAGTGCTAAGTGAATATGGTAACATGTCAAGTGCATGCGTCCTATTC  ATCTTAGACGAAATGAGAAAGAAATCAGCTCAAGATGGACTCAAAACCACCGGTGAAGGA CTTGAATGGGGTGTGTTGTTTGGTTTTGGACCTGGACTTACCATTGAAACAGTTGTTTTG  CGTAGTGTGGCTATTTAA    >TRINITY_DN52652_c1_g1 [organism=Lotus filicaulis] CHS ATGGTCACCGTCGAGGAGATCCGCAACGCACAGCGTTCCCATGGCCCCGCCACCATCCTCGCCTTCGG CACCGCCACCCCTTCCCACTGTGTCATGCAAGCTGATTACCCTGACTACTACTTCCGCATCACCAACA GTGAACACATGACTGACCTCAAAGAAAAATTCAAGCGCATGTGTGAAAAGTCCATGATCAGGAAGCGC TACATGCACCTGACGGAGGAGATTCTGAAGGAGAATCCAAACATGTGCGCGTACATGGCGCCGTCGCT GGATGCTCGTCAGGACTTGGTGGTGGTGGAGGTGCCGAAGCTTGGTAAAGACGCGGCGGCGAAGGCGA TCAAGGAGTGGGGTCAACCCAAGTCAAAGATCACTCACCTCGTTTTCTGCACCACCTCCGGCGTCGAC ATGCCCGGCGCCGATTACCAGCTCACCAAGCTTCTCGGCCTCAAACCCTCCGTGAAACGCCTCATGAT GTACCAGCAAGGCTGCTTCGCCGGCGGCACGGTGCTCCGGTTGGCCAAGGATCTCGCCGAGAACAACA AAGGCGCAAGAGTTCTCGTCGTCTGCTCTGAAATCACCGCCGTCACGTTCCGCGGCCCCTCCGACACT CACCTCGACTCGCTCGTCGGGCAAGCCCTCTTCGGCGACGGCGCCGCCGCGATGATTGTGGGATCCGA CCCTGACCTCGCCGTGGAGCGTCCGATTTTCGAATTGGTCTCCGCCGCGCAGACGATTCTCCCTGATT CCGACGGCGCAATCGACGGCCACCTCCGTGAGGTGGGGCTGACATTCCACCTTCTGAAGGACGTTCCG GGGATCATCTCGAAGCACATTGAGAAGAGCTTGACGGAGGCGTTTGCGCCGATTGGGGTTTCTGATTG GAACTCGCTGTTCTGGATCGCGCATCCGGGTGGACCGGCGATTTTGGACCAGGTTGAAGTGAAACTCC GGTTGAAAGAGGAGAAACTCCGGTCGACCCGGCACGTGCTGAGTGAGTACGGAAACATGTCAAGCGCG 50    TGCGTGCTGTTTATCTTGGATGAGGTGAGGAGGAGGTCGAAGGAGGAAGGGAAGGAAACCACCGGAGA AGGGTTGGAGTGGGGGGTGCTATTCGGGTTCGGGCCGGGTCTCACCGTCGAGACCGTCGTGTTGCACA GTGTTCCC    >TRINITY_DN52780_c0_g1 [organism=Lotus filicaulis] F3’H ATGTTCCAATGGATGATCATTGGCTTCGCC  ACCATCACAGTCATCATCTTCATACACCGTGTCGTGAAGTTCGCAACACGTCCCTCACTC  CCACTCCCTCCGGGTCCAAAACCATGGCCCATAATAGGAAACCTCCCCCACATGGGTCCG  GTGCCGCACCACTCACTCGCAGCCTTAGCCCAAACACACGGCCCGCTTATGCACCTTAAA  TTGGGCTTTGTGGATGTCGTGGTGGCGGCATCAGCCACCGTGGCGGAGCAGTTTTTAAAA  GTTCATGATGCGAATTTTAGTAGCCGGCCCCCCAACGCCGGAGCTAAGTACATAGCTTAT  AACTATCAGGATCTCGTGTTTGCTCCCTACGGCGCACGGTGGCGATATCTCCGAAAAATC  ACCTCTCTTCATCTCTTCTCTGGCAAGGCCTTGGATAATTTTAAGCACTTGCGTCAGGAA  GAAGTATCAAGATTGACACGCAACATATCAAAGTCCAACTCAAAAGTTGTCAACTTGGGA  CAACTACTAAATGTGTGCACTACCAACGCATTATCAAGGGTAATGATTGGAAGGAGAGTG  TTCAACGACGGCGACGGTGGGTGTGATCCTAGAGCTGATGAGTTTAAGGCCATGGTGGTG  GAGTTAATGGTGTTAGCGGGTGTTTTCAACATAGGTGACTTCATTCCTTCCTTGGAGTGG  TTAGACCTTCAAGGGGTGCAAGCTAAGATGAAGAATTTGCACAATAGGTTTGATGAATTT  TTAACCAGCATAATTGAGGAACACGATACATCTTCAAAGAGTGAGAATCATAAAGATTTG  TTAAGCACATTATTGTCGCTTAAAGAGGTTGCCGATGATGATGGAAATAAACTCAATGAT  ATCGAGATCAAAGCACTACTTCTGAACATGTTCACAGCTGGGACAGACACGTCAGCAAGC  ACAACAGAATGGGCCATTGCAGAACTCATCCGCAACCCAAAAATTCTGGCCCAAGTCCAA  CAAGAATTGGACACAGTCGTGGGCCGAGACAGAAACGTGAGAGAAGATGACTTGCCTCAT  CTCCCATACTTACAAGCAGTGGTGAAAGAAACCTTTCGTCTTCATCCATCAACCCCTCTT  TCACTGCCACGTGTCGCATCTGAGAGCTGTGAGGTACTTGGCTACCACATCCCAAAGGGT  TCCACACTCTTGGTGAACGTGTGGGCCATAGCCCGTGACCCTAAAGAATGGGCTGACCCA  TTGGAGTTCAAGCCTGAAAGGTTCTTGCAGGGTGGTGGTGATAAGGTTCATGTTGATGTT  AAGGGCAATGACTTTGAGGTCATACCCTTTGGTGCTGGACGTAGGATATGTGCTGGGATG  AGCCTTGGGCTTCGTATGGTTCAGCTGTTAACTGCAACTCTGGCTCATTCGTTTAACTGG  GAGCTTGAAAATGGGCTTAACCATGAAAAGCTCAACATGGATGAAGCATATGGGTTAACA  TTGCAACGGGCTGTGCCTTTGTCAGTGTACTCTAGGCCAAGGCTCTCGCCACATGTGTA    >TRINITY_DN52390_c0_g1 [organism=Lotus filicaulis] F3H CTCACCACTCTAGCCCAACAAAACACCCTTGAGTCCAGCTTCGTCCGTGACGAAGACGAGCGCCCAAA GGTCGCTTACAACAATTTCAGCAACGAGATCCCGGTCATTTCCCTCGCCGGAATCGACGAGGTCGATG GCCGGAGATCCGAGATATGTAACAAGATTGTTGAGGCTTGTGAGAACTGGGGAATCTTCCAGGTTGTC AACCACGGCGTCGACACTGAATTGGTTTCCCATATGACCACTCTTGCCAAAGAGTTCTTTGCTCTTCC CCCTGAAGAGAAGCTTCGCTTTGACATGTCCGGTGGCAAAAAGGGTGGTTTCATCGTCTCCAGCCACC 51    TCCAAGGAGAATCAGTGCAGGATTGGAGAGAGATAGTGACATACTTTTCATACCCAATAAGGCAGAGG GACTACTCACGGTGGCCAGACACCCCGGCGGGGTGGAAGGCGGTGACGGAGGAGTACAGCGAGAAGCT GATGGGTTTAGCATGCAAGCTATTGGAGGTGTTATCAGAGGCAATGGGGTTAGAGAAAGAGGCTCTAA CAAAGGCATGTGTTGATATGGACCAGAAAGTTGTGGTGAATTATTACCCAAAATGCCCTCAGCCTGAC CTCACACTTGGACTGAAGAGACACACTGATCCTGGCACCATTACTCTGCTGCTTCAGGATCAGGTGGG TGGGCTTCAAGCAACTAGGGATAATGGGAAGACATGGATCACTGTGCAGCCTGTTGAAGGAGCTTTCG TTGTCAATCTTGGTGACCATGGTCATTATCTGAGCAATGGGAGGTTCAAGAACGCTGATCACCAAGCA GTGGTGAACTCAAACTCAAGCCGTTTATCGATCGCCACATTCCAGAACCCAGCACCAGATGCAACTGT GTACCCATTGAAGGTTCGAGAGGGAGAGAAGTCGGTCCTAGATGAACCAATCACATTCGCTGAAATGT ACAGGAGGAAGATGAGCAAGGACATTGAGCTTGCTCGGATGAAGAAGCTGGCTAAGGAGAAGAAACTG CAGGATTTGGAGAAGGCCAAACTTGAGCCTAAGCCCATGAATGAGATCTTTGCTTA    >TRINITY_DN52789_c0_g1 [organism=Lotus filicaulis]DFR GAAACGGTTTGCGTTACAGGGGCTGCTGGTTTCATCGGGTCATGGCTCGT  CATGAGGCTCATCGAGCGTGGCTACACGGTTCGAGCCACCGTACGTGACCCAGCAAACAT  GAAGAAGGTGAAGCATTTGCTGGAACTGCCAAATGCAAAGACCAAGCTGACTCTGTGGAA  AGCTGATCTTGCTGAAGAGGGAAGCTTTGATGAAGCCATCAAAGGGTGCACTGGAGTTTT  CCATGTGGCCACACCCATGGATTTTGAGTCCAAGGACCCTGAGAATGAAGTAATAAAGCC  AACAATAAATGGATTGTTAGACATCTTGAAAGCATGTGAGAAGGCCAAAAGTGTAAGAAG  GTTGGTATTCACATCCTCAGCAGGGACTGTGGATGTTACAGAGCAACAAAAGCCTGTTAT  TGATGAAACATGCTGGAGTGACATTGAATTCTGCCTCAGAGTCAAGATGACTGGCTGGAT  GTATTTCGTTTCCAAGACACGAGCAGAACAAGAAGCGTGGAAATATGCAAAAGAGCACAA  CATAGACTTCGTCTCAGTCATTCCACCTCTTGTGGTTGGCCCATTTCTCATGCCAACAAT  GCCACCTAGCCTAATCACTGCTCTCTCACTCATCACAGGAAATGAGGCCCATTACTCAAT  CATAAAGCAAGGCCAATATGTCCACTTAGATGACCTTTGTCTAGCTCATATATTCTTGTT  TGAGAACCCAAAAGCCCAAGGGAGATACATGTGCTCTGCATATGAGGCAACCATTCATGA  AGTTGCAAGAATGATCAACAAGAAGTACCCAGAGTTCAATGTCCCCACAAAGTTCAAGGA  TATTCCCGATGAATTGGACATCATTAAATTTTCTTCAAAGAAGATCACAGACTTGGGGTT  CAAATTTAAGTACAGCTTAGAGGACATGTACACAGGAGCCATTGAAACCTGCAGAGAAAA  GGGGCTTCTTCCTAAAACTTCTGAAACTCCAGTTACTAATGGCACAACTCAGAAATAA    >TRINITY_DN53094_c0_g1 [organism=Lotus filicaulis] ANS ATGGCGCCTACTGTTGTTGAGAGGGTTGAAAGCTTATCAACCAGTGGGATACAATCTATCCCAAAGGA ATATGTGAGGCCGAAAGAGGAGCTGGAAAACATAGGCAACGTTTTTGAGGAGGAAAAGAAGGATGGGC CTCAAGTTCCAACAATTGACTTGAAAGAGATAGACTCTCCCGACGAGTTTGTTCGGGCCAATTGCAGG GAGAAGCTGAGGAAGGCGGCGGAGGAGTGGGGGGTGATGCACCTGGTGAACCATGGGATCCCGGACGA GCTTCTGAACCAATTGAAGACAGCTGGAGCCGAGTTTTTCTCTTTGCCTGTCGAGGAGAAGGAGAAGT ACGCGAATGATCAGACGACCGGAAATGTGCAAGGGTACGGGAGCAAGCTGGCTAACAATGCCAGTGGC 52    CAGCTTGAATGGGAAGATTACTTCTTCCATCTTATCTTTCCAGAAGACAAACGTGACCTCTCTATCTG GCCCAAGACACCATCATATTATACAGAGGTTACCAGCGACTATGCAAGGCGACTGAGAGTCCTTGCGA GCAAGGTGCTGGAGGTGCTATCTCTGGAATTGGGTCTGGAAGAAGGAAGGTTAGAGAAGGAAGTTGGT GGAATGGAAGAGCTTCTACTTCAGATGAAGATCAACTACTACCCAAAATGCCCCCAGCCAGAGCTTGC TCTAGGAGTTGAAGCCCACACCGATGTAAGCTCACTCACTTTCCTCGTCCACAACATGGTCCCAGGCC TGCAGCTTTTCTACCAAGATAAATGGATCACAGCAAAATGTGTCCCTGATTCAATTCTCATGCACATT GGTGACACCATTGAGATCCTGAGCAATGGCAAGTTCAAGAGCATTCTTCACAGGGGGTTGGTGAACAA GGAAAAAGTTAGAATATCTTGGGCTGTGTTCTGTGAACCCCCTAAGGAGAAAATCATCCTCAAGCCAC TTCCTGAGCTTGTGACTGAGACCGAACCGGCGCTGTTTCCGCCGCGCACTTTTGCTCAGCATCTTCAT CACAAATTGTTCAGGAAGGACCAGGAAGCTGCTGCTCAATCCAAATGA    >TRINITY_DN39800_c0_g1 [organism=Lotus filicaulis] FLS TGGAG  GTGCTAAGGGTGCAAACCGTGGCAACCCAGTCAAAAGACGCTTCCATCCCCTCGATGTTC  GTGAGGCCGAAGACAGAGCAGCCTGGCCTCACGACCGTTCGCGGGGTGGAACTGGAGGTG  CCGATCATTGATTTGAGCGGCTCCGATGAAGAAAAGGTGCTGCGTGAGATCGTGGAGGCG  AGTAAGGAGTGGGGGATGTTTCAAGTTGTGAATCATGAGATACCTGTTGAAGTTATAGCC  AAATTGCAGAGTGTGGGGAAACATTTCTTTGAGTTGCCACAAGGGGAAAAAGAGGTGTGT  GGTAAGATTGATGGGTCTGATTCTGTGGAAGGTTATGGGACTAAACTTCAGAAGGAGGTG  AATGGGAAGAAAGGTTGGGTGGATCATTTGTTTCATATTATTTGGCCAACTTCTGCCATT  AACTACCGATTCTGGCCTAAGAATCCTCCTTCTTACAGGGAGGTGAATGAGGAATATGGC  AAGTACCTGCGTAGTGTGGGGGACAAACTTTTCAAAAGCTTGTCAATTGGGTTGGGCCTT  GAAGAAAATGAGCTAAAAGAAGCTGCAGGTGGAGATGACATGATTCACCTATTAAAGATC  AACTACTACCCACCATGTCCATGTCCTGAACTGGTTCTGGGTGTGCCACCACACACAGAC  ATGTCATTCATGACCATTCTAGTTCCCAACGAGGTGCAGGGCCTCCAAGCCTTCAGGGAT  GGTCACTGGTACGATGTTAAGTATGTCCCCAATGCCCTCGTCATTCACATTGGAGACCAA  ATGGAGATAGTGAGCAATGGGAAATATAAGGCTGTCCTGCACAGAACAACAGTGAACAAA  GAGGAGACAAGAATGTCATGGCCAGTGTTCATAGAGCCCCAAGGACACCATGAAGTTGGT CCTCACTCCAAGTTGGTTAACCACGACAATCCACCAAAATACAAGACCAAGAAATATAAG  GACTATGCCTACTGCAAGCTTAATAAGATCCCTCAGTAA            53    Appendix 3   Supplementary Tables S1-S54   Table S1. Tophat Results: Significantly enriched GO terms corresponding to the yellow stage of a L. filicaulis flower.  Enriched GO Term: Description Number of associated genes FDR GO:0015979photosynthesis 42 1.70E-05 GO:0009628response to abiotic stimulus 179 1.70E-05 GO:0042221response to chemical stimulus 243 2.50E-05 GO:0050896response to stimulus 376 0.00014 GO:0010033response to organic substance 159 0.00014 GO:0009725response to hormone stimulus 124 0.00014 GO:0044281small molecule metabolic process 207 0.00018 GO:0044283small molecule biosynthetic process 107 0.00024 GO:0009719response to endogenous stimulus 131 0.00024 GO:0019684photosynthesis, light reaction 26 0.00024 GO:0046394carboxylic acid biosynthetic process 69 0.0024 GO:0016053organic acid biosynthetic process 69 0.0024 GO:0006091generation of precursor metabolites and energy 44 0.0037 GO:0009416response to light stimulus 81 0.0041 GO:0008152metabolic process 796 0.0041 GO:0005976polysaccharide metabolic process 40 0.0041 GO:0009314response to radiation 81 0.0044 GO:0010218response to far red light 15 0.0044 GO:0043436oxoacid metabolic process 106 0.0044 GO:0006950response to stress 233 0.0044 GO:0010608posttranscriptional regulation of gene expression 25 0.0044 GO:0006082organic acid metabolic process 106 0.0044 GO:0019752carboxylic acid metabolic process 106 0.0044 GO:0009409response to cold 52 0.0047 GO:0009607response to biotic stimulus 102 0.0056 GO:0042180cellular ketone metabolic process 108 0.0056 GO:0044264cellular polysaccharide metabolic process 35 0.0064 GO:0009637response to blue light 16 0.008 GO:0006519cellular amino acid and derivative metabolic process 87 0.008 54    Enriched GO Term: Description Number of associated genes FDR    GO:0032501multicellular organismal process 207 0.0086 GO:0016072rRNA metabolic process 16 0.011 GO:0009639response to red or far red light 29 0.011 GO:0051707response to other organism 96 0.011 GO:0008610lipid biosynthetic process 58 0.011 GO:0006073cellular glucan metabolic process 29 0.011 GO:0019748secondary metabolic process 58 0.011 GO:0016441posttranscriptional gene silencing 17 0.012 GO:0009733response to auxin stimulus 44 0.012 GO:0033692cellular polysaccharide biosynthetic process 24 0.012 GO:0044042glucan metabolic process 29 0.012 GO:0051704multi-organism process 115 0.012 GO:0043648dicarboxylic acid metabolic process 22 0.014 GO:0007275multicellular organismal development 197 0.014 GO:0000271polysaccharide biosynthetic process 24 0.014 GO:0055114oxidation reduction 133 0.015 GO:0005975carbohydrate metabolic process 114 0.016 GO:0008652cellular amino acid biosynthetic process 33 0.018 GO:0009791post-embryonic development 119 0.02 GO:0070887cellular response to chemical stimulus 80 0.023 GO:0006720isoprenoid metabolic process 25 0.024 GO:0009617response to bacterium 55 0.024 GO:0006520cellular amino acid metabolic process 55 0.024 GO:0009250glucan biosynthetic process 18 0.024 GO:0006364rRNA processing 14 0.024 GO:0042214terpene metabolic process 13 0.026 GO:0009415response to water 41 0.028 GO:0016246RNA interference 12 0.028 GO:0009765photosynthesis, light harvesting 10 0.029 GO:0009414response to water deprivation 40 0.031 GO:0009308amine metabolic process 64 0.037 GO:0032502developmental process 204 0.038 GO:0006629lipid metabolic process 91 0.044 GO:0006721terpenoid metabolic process 20 0.044 GO:0030422production of siRNA involved in RNA interference 11 0.046 GO:0009309amine biosynthetic process 34 0.048 55    Enriched GO Term: Description Number of associated genes FDR    GO:0044106cellular amine metabolic process 57 0.048 GO:0003824catalytic activity 688 0.002 GO:0009507chloroplast 311 3.60E-35 GO:0044435plastid part 232 3.60E-35 GO:0044434chloroplast part 228 1.00E-34 GO:0009536plastid 319 1.90E-33 GO:0009532plastid stroma 145 7.50E-26 GO:0009570chloroplast stroma 139 2.80E-24 GO:0044464cell part 910 2.10E-19 GO:0043227membrane-bounded organelle 630 2.10E-19 GO:0044444cytoplasmic part 570 2.10E-19 GO:0005623cell 910 2.10E-19 GO:0043231intracellular membrane-bounded organelle 628 2.10E-19 GO:0009526plastid envelope 123 6.00E-19 GO:0043226organelle 656 1.00E-18 GO:0043229intracellular organelle 655 1.20E-18 GO:0005737cytoplasm 598 1.80E-18 GO:0044422organelle part 370 4.00E-18 GO:0044446intracellular organelle part 368 4.50E-18 GO:0009941chloroplast envelope 118 5.70E-18 GO:0009579thylakoid 106 6.70E-17 GO:0031976plastid thylakoid 92 1.30E-16 GO:0009534chloroplast thylakoid 92 1.30E-16 GO:0031984organelle subcompartment 92 1.50E-16 GO:0044436thylakoid part 86 2.70E-16 GO:0044424intracellular part 720 3.50E-16 GO:0055035plastid thylakoid membrane 78 2.10E-15 GO:0042651thylakoid membrane 80 2.80E-15 GO:0005622intracellular 727 3.50E-15 GO:0009535chloroplast thylakoid membrane 77 4.20E-15 GO:0031975envelope 136 5.50E-15 GO:0031967organelle envelope 134 1.90E-14 GO:0034357photosynthetic membrane 80 3.50E-14 GO:0048046apoplast 71 3.00E-08 GO:0005576extracellular region 92 1.30E-07 GO:0010287plastoglobule 20 6.20E-06 56    Enriched GO Term: Description Number of associated genes FDR GO:0030312external encapsulating structure 87 6.30E-06 GO:0016020membrane 441 8.20E-06 GO:0005618cell wall 85 1.40E-05 GO:0031090organelle membrane 148 2.70E-05 GO:0031977thylakoid lumen 20 4.60E-05 GO:0005886plasma membrane 234 7.00E-05 GO:0043232intracellular non-membrane-bounded organelle 121 7.10E-05 GO:0043228non-membrane-bounded organelle 121 7.10E-05 GO:0010319stromule 15 0.00015 GO:0009505plant-type cell wall 40 0.00029 GO:0031974membrane-enclosed lumen 80 0.00042 GO:0070013intracellular organelle lumen 79 0.00042 GO:0043233organelle lumen 79 0.00044 GO:0005730nucleolus 48 0.0026 GO:0009295nucleoid 10 0.01 GO:0044459plasma membrane part 104 0.012 GO:0031978plastid thylakoid lumen 11 0.015 GO:0009543chloroplast thylakoid lumen 11 0.015 GO:0009521photosystem 13 0.016 GO:0042646plastid nucleoid 8 0.025 GO:0009506plasmodesma 91 0.036 GO:0055044symplast 91 0.036 GO:0030054cell junction 91 0.038 GO:0005911cell-cell junction 91 0.038 GO:0000229cytoplasmic chromosome 6 0.043 GO:0009508plastid chromosome 6 0.043 GO:0046658anchored to plasma membrane 12 0.044            57    Table S2. Tophat Results: Significantly enriched GO terms corresponding to the red stage of a L. filicaulis flower.  Enriched GO Term: Description Number of associated genes FDR GO:0050896response to stimulus 266 0.00056 GO:0055085transmembrane transport 70 0.00056 GO:0005975carbohydrate metabolic process 92 0.0012 GO:0051179localization 152 0.0014 GO:0009605response to external stimulus 56 0.0015 GO:0042221response to chemical stimulus 164 0.0015 GO:0009991response to extracellular stimulus 27 0.0016 GO:0051234establishment of localization 145 0.0016 GO:0006810transport 144 0.0016 GO:0009932cell tip growth 23 0.0017 GO:0071704organic substance metabolic process 139 0.0017 GO:0009813flavonoid biosynthetic process 19 0.0029 GO:0035295tube development 24 0.0029 GO:0048868pollen tube development 24 0.0029 GO:0031667response to nutrient levels 23 0.0029 GO:0031668cellular response to extracellular stimulus 25 0.0029 GO:0071496cellular response to external stimulus 25 0.0031 GO:0006575cellular amino acid derivative metabolic process 41 0.0048 GO:0042398cellular amino acid derivative biosynthetic process 32 0.0049 GO:0009725response to hormone stimulus 82 0.0051 GO:0010033response to organic substance 105 0.0059 GO:0009812flavonoid metabolic process 20 0.0074 GO:0006811ion transport 53 0.0074 GO:0009719response to endogenous stimulus 87 0.0074 GO:0031669cellular response to nutrient levels 21 0.0077 GO:0009699phenylpropanoid biosynthetic process 22 0.01 GO:0009860pollen tube growth 18 0.011 GO:0042594response to starvation 19 0.011 GO:0009826unidimensional cell growth 28 0.011 58    Enriched GO Term: Description Number of associated genes FDR GO:0060560developmental growth involved in morphogenesis 28 0.011 GO:0016036cellular response to phosphate starvation 14 0.011 GO:0009737response to abscisic acid stimulus 43 0.014 GO:0032989cellular component morphogenesis 36 0.016 GO:0048588developmental cell growth 21 0.016 GO:0006725cellular aromatic compound metabolic process 93 0.016 GO:0015698inorganic anion transport 14 0.016 GO:0044282small molecule catabolic process 37 0.016 GO:0006576cellular biogenic amine metabolic process 13 0.017 GO:0044262cellular carbohydrate metabolic process 59 0.018 GO:0009267cellular response to starvation 18 0.019 GO:0019438aromatic compound biosynthetic process 70 0.019 GO:0051704multi-organism process 81 0.022 GO:0048610reproductive cellular process 18 0.026 GO:0009856pollination 25 0.03 GO:0016137glycoside metabolic process 16 0.033 GO:0009718anthocyanin biosynthetic process 11 0.034 GO:0008361regulation of cell size 34 0.037 GO:0010200response to chitin 17 0.037 GO:0032502developmental process 144 0.038 GO:0006519cellular amino acid and derivative metabolic process 59 0.038 GO:0019748secondary metabolic process 40 0.038 GO:0000902cell morphogenesis 31 0.043 GO:0032501multicellular organismal process 140 0.044 GO:0006595polyamine metabolic process 7 0.046 GO:0044248cellular catabolic process 61 0.046 GO:0022892substrate-specific transporter activity 72 0.00021 GO:0005215transporter activity 90 0.00021 GO:0022857transmembrane transporter activity 77 0.00021 GO:0015291secondary active transmembrane transporter activity 30 0.0005 59    Enriched GO Term: Description Number of associated genes FDR    GO:0022891substrate-specific transmembrane transporter activity 65 0.00072 GO:0022804active transmembrane transporter activity 45 0.00072 GO:0016791phosphatase activity 27 0.0017 GO:0016798hydrolase activity, acting on glycosyl bonds 44 0.0017 GO:0004553hydrolase activity, hydrolyzing O-glycosyl compounds 42 0.0017 GO:0015075ion transmembrane transporter activity 50 0.0017 GO:0003779actin binding 13 0.0047 GO:0015103inorganic anion transmembrane transporter activity 15 0.0047 GO:0015297antiporter activity 19 0.0053 GO:0042578phosphoric ester hydrolase activity 29 0.0058 GO:0008509anion transmembrane transporter activity 18 0.024 GO:0016020membrane 305 0.0014 GO:0005886plasma membrane 164 0.0033                     60    Table S3. Yellow Stage TopHat Results: Genes for enzymes annotated to GO:0009813 (Flavonoid biosynthetic process, FDR=1). The first column lists differentially expressed (FDR<0.005) assembled RNA-seq reads that have mapped to the Lotus japonicus genome. The second column is their soybean (Glycine max) BLAST homolog, followed by expression at two stages of flower development.   L. japonicus ID G. max BLAST ID G. max description Counts per gene (Yellow) Counts per gene (Red) Log2FC chr2.CM0018.1300.r2.m Glyma.01G228700.1 Naringenin-chalcone synthase 143 11 2.87090 chr5.CM1077.590.r2.m Glyma.03G187000.1 UDP-glucosyl transferase 73C 44 1 4.52042 chr1.CM0122.1600.r2.m Glyma.03G222100.1 Nitrogen regulatory protein P-II 134 18 2.07132 chr1.CM0012.990.r2.m Glyma.04G254200.1 B3 DNA binding domain // Auxin response factor 119 12 2.48162 chr1.CM0064.910.r2.m Glyma.05G076300.1 NA 1067 169 1.83858 chr3.LjT40P18.80.r2.m Glyma.07G021600.1 Shikimate O-hydroxycinnamoyltransferase 350 25 2.98347 chr3.CM0452.240.r2.d Glyma.07G021600.1 Shikimate O-hydroxycinnamoyltransferase 8669 1189 2.04670 chr3.LjT10E18.60.r2.m Glyma.10G210600.1 B3 DNA binding domain // Auxin response factor 230 26 2.32171 chr3.CM0136.10.r2.m Glyma.11G145500.1 AUX/IAA family // B3 DNA binding domain // Auxin response factor 28 2 2.93242 chr4.CM0432.2880.r2.m Glyma.13G173300.1 O-METHYLTRANSFERASE 2443 223 2.63371 chr3.CM0106.330.r2.m Glyma.13G344700.1 B-box zinc finger 712 37 3.44375 chr1.LjT35H06.80.r2.m NA NA 51 0 8.32466               61    Table S4. Red Stage TopHat Results: Genes for enzymes annotated to GO:0009813 (Flavonoid biosynthetic process, FDR = 0.0029) .  The first column lists differentially expressed (FDR<0.005) assembled RNA-seq reads that have mapped to the Lotus japonicus genome. The second column is their soybean (Glycine max) BLAST homolog, followed by expression at two stages of flower development.  L. japonicus ID G. max BLAST ID G. max description Counts per gene (Yellow) Counts per gene (Red) Log2FC chr2.CM0608.560.r2.m Glyma.01G048200.1 RIBONUCLEASE T2 872 8349 -4.07828 chr2.CM0124.30.r2.m Glyma.01G088300.1 STEROL REGULATORY ELEMENT-BINDING PROTEIN 8 26 -2.49628 chr3.LjB14O06.120.r2.a Glyma.01G166200.1 naringenin 3-dioxygenase 228 542 -2.06782 chr4.CM0119.240.r2.m Glyma.01G166200.1 naringenin 3-dioxygenase 1043 3498 -2.56494 chr1.CM0284.250.r2.m Glyma.01G228700.1 Naringenin-chalcone synthase 7 144 -5.15029 chr2.CM0018.760.r2.m Glyma.01G228700.1 Naringenin-chalcone synthase 115 905 -3.79376 chr2.CM0018.730.r2.m Glyma.01G228700.1 Naringenin-chalcone synthase 48 170 -2.63974 chr2.CM0018.1200.r2.m Glyma.01G228700.1 Naringenin-chalcone synthase 3 61 -5.09247 chr5.CM0077.110.r2.m Glyma.02G158700.1 bifunctional dihydroflavonol 4-reductase/flavanone 4-reductase 32 929 -5.67183 chr5.CM0077.210.r2.m Glyma.02G158700.1 bifunctional dihydroflavonol 4-reductase/flavanone 4-reductase 150 10354 -6.92689 chr1.CM0105.1870.r2.m Glyma.03G113400.1 Proton-exporting ATPase 304 1049 -2.60557 chr1.CM0133.560.r2.m Glyma.04G052100.1 Cytochrome P450 CYP3/CYP5/CYP6/CYP9 subfamilies 40 2793 -6.9393 chr1.LjT39K18.30.r2.m Glyma.04G147700.1 HEME OXYGENASE 636 1935 -2.42427 chr2.CM0191.680.r2.m Glyma.04G227700.1 Quercetin 3-O-methyltransferase 4 21 -3.16141 chr1.CM0410.430.r2.m Glyma.08G247100.1 Transferase family 2403 7930 -2.54173 chr6.CM0013.1540.r2.a Glyma.09G038900.1 MYB-LIKE DNA-BINDING PROTEIN MYB 7 125 -4.94628 62    L. japonicus ID G. max BLAST ID G. max description Counts per gene (Yellow) Counts per gene (Red) Log2FC chr2.CM0021.2820.r2.m Glyma.11G027700.1 Leucocyanidin oxygenase 1001 15407 -4.76318 chr5.CM0077.120.r2.m Glyma.14G072700.1 bifunctional dihydroflavonol 4-reductase/flavanone 4-reductase 11 61 -3.27199 chr5.CM0180.690.r2.m Glyma.20G241700.1 chalcone isomerase 529 1254 -2.06419                         63    Table S5. Yellow Stage TopHat Results: Genes for enzymes annotated to GO:0009809 (Lignin biosynthetic process, FDR=0.24). The first column lists differentially expressed (FDR<0.005) assembled RNA-seq reads that have mapped to the Lotus japonicus genome. The second column is their soybean (Glycine max) BLAST homolog, followed by expression at two stages of flower development.   L. japonicus ID G. max BLAST ID G. max description Counts per gene (Yellow) Counts per gene (Red) Log2FC chr6.LjT35D18.30.r2.m Glyma.03G223000.1 Methionine adenosyltransferase 17391 2734 1.849893404 chr3.LjT40P18.80.r2.m Glyma.07G021600.1 Shikimate O-hydroxycinnamoyltransferase 350 25 2.983475586 chr3.CM0452.240.r2.d Glyma.07G021600.1 Shikimate O-hydroxycinnamoyltransferase 8669 1189 2.046705844 chr3.LjT45M09.50.r2.d Glyma.07G023700.1 cinnamoyl-CoA reductase 1692 266 1.849549234 chr4.CM0387.960.r2.m Glyma.07G258700.1 beta-glucosidase 131 1 6.090954877 chr6.CM0385.240.r2.d Glyma.09G038500.1 CHITINASE 1204 8 6.398266711 chr5.CM0200.3250.r2.m Glyma.10G262400.1 cinnamyl-alcohol dehydrogenase 4676 705 1.910116099        *Lignin biosynthetic process was not found to be enriched at the red stage of floral colour change in L. filicaulis, given the TopHat results.                  64    Table S6. STAR Results: Significantly enriched GO terms corresponding to the yellow stage of a L. filicaulis flower.   Enriched GO Term: Description Number of associated genes FDR GO:0009628response to abiotic stimulus 184 1.00E-06 GO:0050896response to stimulus 379 1.80E-05 GO:0042221response to chemical stimulus 241 1.80E-05 GO:0044281small molecule metabolic process 206 0.00017 GO:0009416response to light stimulus 87 0.00024 GO:0015979photosynthesis 37 0.00024 GO:0009314response to radiation 87 0.00032 GO:0010033response to organic substance 153 0.00059 GO:0044283small molecule biosynthetic process 103 0.00086 GO:0006950response to stress 237 0.00086 GO:0005976polysaccharide metabolic process 42 0.00089 GO:0008610lipid biosynthetic process 63 0.00097 GO:0009725response to hormone stimulus 117 0.00097 GO:0008152metabolic process 789 0.0018 GO:0044264cellular polysaccharide metabolic process 37 0.0018 GO:0009607response to biotic stimulus 104 0.0018 GO:0046394carboxylic acid biosynthetic process 68 0.0018 GO:0016053organic acid biosynthetic process 68 0.0018 GO:0009719response to endogenous stimulus 123 0.0021 GO:0019684photosynthesis, light reaction 23 0.0023 GO:0009637response to blue light 17 0.0027 GO:0033692cellular polysaccharide biosynthetic process 26 0.0027 GO:0000271polysaccharide biosynthetic process 26 0.0028 GO:0051707response to other organism 98 0.0028 GO:0010218response to far red light 15 0.0028 GO:0009409response to cold 52 0.0028 GO:0043436oxoacid metabolic process 105 0.0028 GO:0032501multicellular organismal process 208 0.0028 GO:0010608posttranscriptional regulation of gene expression 25 0.0028 GO:0006082organic acid metabolic process 105 0.0028 GO:0019752carboxylic acid metabolic process 105 0.0028 GO:0007275multicellular organismal development 200 0.0033 GO:0042214terpene metabolic process 15 0.0035 GO:0005975carbohydrate metabolic process 117 0.0037 GO:0019748secondary metabolic process 59 0.0045 65    Enriched GO Term: Description Number of associated genes FDR    GO:0009791post-embryonic development 122 0.0047 GO:0006720isoprenoid metabolic process 27 0.0051 GO:0042180cellular ketone metabolic process 106 0.0051 GO:0055114oxidation reduction 134 0.0067 GO:0006073cellular glucan metabolic process 29 0.0075 GO:0032502developmental process 208 0.0085 GO:0044042glucan metabolic process 29 0.0095 GO:0071554cell wall organization or biogenesis 45 0.011 GO:0006091generation of precursor metabolites and energy 40 0.012 GO:0051704multi-organism process 113 0.014 GO:0009639response to red or far red light 28 0.014 GO:0044255cellular lipid metabolic process 70 0.015 GO:0006629lipid metabolic process 93 0.016 GO:0071555cell wall organization 32 0.017 GO:0006519cellular amino acid and derivative metabolic process 83 0.017 GO:0048856anatomical structure development 168 0.018 GO:0044262cellular carbohydrate metabolic process 80 0.018 GO:0006721terpenoid metabolic process 21 0.018 GO:0009266response to temperature stimulus 62 0.019 GO:0009250glucan biosynthetic process 18 0.019 GO:0016441posttranscriptional gene silencing 16 0.019 GO:0005982starch metabolic process 14 0.025 GO:0008299isoprenoid biosynthetic process 22 0.025 GO:0009617response to bacterium 54 0.025 GO:0048731system development 105 0.027 GO:0048513organ development 105 0.027 GO:0034637cellular carbohydrate biosynthetic process 37 0.03 GO:0016051carbohydrate biosynthetic process 38 0.031 GO:0071669plant-type cell wall organization or biogenesis 24 0.031 GO:0009414response to water deprivation 39 0.038 GO:0010154fruit development 53 0.038 GO:0006633fatty acid biosynthetic process 31 0.039 GO:0009658chloroplast organization 20 0.04 GO:0016072rRNA metabolic process 14 0.041 GO:0010035response to inorganic substance 75 0.044 GO:0032787monocarboxylic acid metabolic process 56 0.048 66    Enriched GO Term: Description Number of associated genes FDR    GO:0003824catalytic activity 689 0.00013 GO:0009507chloroplast 312 1.80E-36 GO:0044435plastid part 232 2.90E-36 GO:0044434chloroplast part 228 1.10E-35 GO:0009536plastid 321 2.20E-35 GO:0009532plastid stroma 148 7.90E-28 GO:0009570chloroplast stroma 142 3.30E-26 GO:0044464cell part 920 2.20E-24 GO:0005623cell 920 2.20E-24 GO:0043227membrane-bounded organelle 639 3.00E-23 GO:0044444cytoplasmic part 579 3.00E-23 GO:0043231intracellular membrane-bounded organelle 637 3.50E-23 GO:0005737cytoplasm 608 3.10E-22 GO:0043226organelle 662 7.90E-22 GO:0043229intracellular organelle 661 9.00E-22 GO:0044424intracellular part 732 2.60E-20 GO:0044422organelle part 371 1.90E-19 GO:0009579thylakoid 111 2.10E-19 GO:0044446intracellular organelle part 369 2.10E-19 GO:0009526plastid envelope 122 3.10E-19 GO:0005622intracellular 738 4.60E-19 GO:0031976plastid thylakoid 96 8.40E-19 GO:0009534chloroplast thylakoid 96 8.40E-19 GO:0031984organelle subcompartment 96 1.00E-18 GO:0009941chloroplast envelope 117 3.60E-18 GO:0044436thylakoid part 89 4.60E-18 GO:0055035plastid thylakoid membrane 82 1.30E-17 GO:0042651thylakoid membrane 84 1.80E-17 GO:0009535chloroplast thylakoid membrane 81 2.80E-17 GO:0034357photosynthetic membrane 84 3.10E-16 GO:0031975envelope 136 1.70E-15 GO:0031967organelle envelope 134 6.10E-15 GO:0048046apoplast 75 6.60E-10 GO:0005576extracellular region 97 2.20E-09 GO:0016020membrane 443 8.30E-07 GO:0030312external encapsulating structure 88 1.90E-06 67    Enriched GO Term: Description Number of associated genes FDR    GO:0031090organelle membrane 152 2.10E-06 GO:0031977thylakoid lumen 22 3.50E-06 GO:0005618cell wall 86 4.40E-06 GO:0010287plastoglobule 19 1.60E-05 GO:0010319stromule 16 3.60E-05 GO:0005886plasma membrane 232 4.80E-05 GO:0009505plant-type cell wall 41 0.0001 GO:0043232intracellular non-membrane-bounded organelle 118 0.00013 GO:0043228non-membrane-bounded organelle 118 0.00013 GO:0031974membrane-enclosed lumen 81 0.00016 GO:0070013intracellular organelle lumen 80 0.00016 GO:0043233organelle lumen 80 0.00016 GO:0031978plastid thylakoid lumen 13 0.0014 GO:0009543chloroplast thylakoid lumen 13 0.0014 GO:0005730nucleolus 46 0.0056 GO:0009295nucleoid 10 0.0089 GO:0044459plasma membrane part 103 0.01 GO:0042646plastid nucleoid 8 0.023 GO:0009506plasmodesma 90 0.035 GO:0055044symplast 90 0.035 GO:0030054cell junction 90 0.037 GO:0005911cell-cell junction 90 0.037 GO:0000229cytoplasmic chromosome 6 0.041 GO:0009508plastid chromosome  6 0.041          68    Table S7. STAR Results: Significantly enriched GO terms corresponding to the red stage of a L. filicaulis flower.   Enriched GO Term: Description Number of associated genes FDR GO:0050896response to stimulus 278 3.30E-06 GO:0042221response to chemical stimulus 175 2.40E-05 GO:0009605response to external stimulus 60 0.00014 GO:0005975carbohydrate metabolic process 95 0.00014 GO:0009991response to extracellular stimulus 29 0.00025 GO:0009725response to hormone stimulus 89 0.00033 GO:0009813flavonoid biosynthetic process 21 0.00042 GO:0010033response to organic substance 111 0.00054 GO:0031668cellular response to extracellular stimulus 27 0.00054 GO:0051179localization 151 0.00054 GO:0009719response to endogenous stimulus 93 0.00054 GO:0071496cellular response to external stimulus 27 0.00054 GO:0071704organic substance metabolic process 141 0.00054 GO:0031667response to nutrient levels 24 0.0009 GO:0055085transmembrane transport 66 0.0009 GO:0051234establishment of localization 144 0.00093 GO:0042398cellular amino acid derivative biosynthetic process 34 0.00093 GO:0006575cellular amino acid derivative metabolic process 43 0.00094 GO:0006810transport 143 0.00094 GO:0009812flavonoid metabolic process 22 0.00099 GO:0009699phenylpropanoid biosynthetic process 24 0.0017 GO:0009932cell tip growth 22 0.0023 GO:0031669cellular response to nutrient levels 22 0.0029 GO:0006725cellular aromatic compound metabolic process 97 0.0035 GO:0042594response to starvation 20 0.0041 GO:0035295tube development 23 0.0041 GO:0048868pollen tube development 23 0.0041 GO:0009718anthocyanin biosynthetic process 13 0.0041 GO:0044282small molecule catabolic process 39 0.0048 69    Enriched GO Term: Description Number of associated genes FDR    GO:0019438aromatic compound biosynthetic process 73 0.0051 GO:0009737response to abscisic acid stimulus 44 0.0067 GO:0044262cellular carbohydrate metabolic process 61 0.0067 GO:0006519cellular amino acid and derivative metabolic process 63 0.0067 GO:0032989cellular component morphogenesis 37 0.0071 GO:0009267cellular response to starvation 19 0.0077 GO:0019748secondary metabolic process 43 0.0077 GO:0006950response to stress 161 0.0079 GO:0016036cellular response to phosphate starvation 14 0.0086 GO:0046283anthocyanin metabolic process 14 0.012 GO:0016137glycoside metabolic process 17 0.013 GO:0006811ion transport 51 0.013 GO:0015698inorganic anion transport 14 0.014 GO:0009698phenylpropanoid metabolic process 27 0.014 GO:0006576cellular biogenic amine metabolic process 13 0.014 GO:0009826unidimensional cell growth 27 0.015 GO:0060560developmental growth involved in morphogenesis 27 0.015 GO:0009628response to abiotic stimulus 109 0.015 GO:0009415response to water 31 0.017 GO:0009860pollen tube growth 17 0.018 GO:0000902cell morphogenesis 32 0.022 GO:0048588developmental cell growth 20 0.024 GO:0009714chalcone metabolic process 7 0.024 GO:0042181ketone biosynthetic process 7 0.024 GO:0009715chalcone biosynthetic process 7 0.024 GO:0044281small molecule metabolic process 131 0.025 GO:0044248cellular catabolic process 62 0.026 GO:0006012galactose metabolic process 6 0.027 GO:0006796phosphate metabolic process 93 0.029 GO:0006793phosphorus metabolic process 93 0.032 70    Enriched GO Term: Description Number of associated genes FDR    GO:0006066alcohol metabolic process 38 0.032 GO:0034656nucleobase, nucleoside and nucleotide catabolic process 15 0.032 GO:0034655nucleobase, nucleoside, nucleotide and nucleic acid catabolic process 15 0.032 GO:0006595polyamine metabolic process 7 0.04 GO:0044283small molecule biosynthetic process 65 0.04 GO:0006855multidrug transport 11 0.042 GO:0009856pollination 24 0.042 GO:0048610reproductive cellular process 17 0.042 GO:0051704multi-organism process 78 0.045 GO:0008361regulation of cell size 33 0.046 GO:0009611response to wounding 26 0.046 GO:0015893drug transport 11 0.046 GO:0046395carboxylic acid catabolic process 15 0.047 GO:0016054organic acid catabolic process 15 0.047 GO:0000904cell morphogenesis involved in differentiation 21 0.048 GO:0044270cellular nitrogen compound catabolic process 17 0.048 GO:0005215transporter activity 89 0.00069 GO:0022857transmembrane transporter activity 75 0.00092 GO:0022892substrate-specific transporter activity 69 0.0011 GO:0015291secondary active transmembrane transporter activity 29 0.0012 GO:0016798hydrolase activity, acting on glycosyl bonds 44 0.0026 GO:0004553hydrolase activity, hydrolyzing O-glycosyl compounds 42 0.0026 GO:0022891substrate-specific transmembrane transporter activity 62 0.0034 GO:0016791phosphatase activity 26 0.0035 GO:0022804active transmembrane transporter activity 42 0.0035 GO:0003824catalytic activity 466 0.0035 GO:0015103inorganic anion transmembrane transporter activity 15 0.0046 71    Enriched GO Term: Description Number of associated genes FDR    GO:0042578phosphoric ester hydrolase activity 29 0.0065 GO:0015075ion transmembrane transporter activity 46 0.012 GO:0015297antiporter activity 18 0.013 GO:0003779actin binding 12 0.013 GO:0016210naringenin-chalcone synthase activity 7 0.02 GO:0008509anion transmembrane transporter activity 18 0.02 GO:0015238drug transmembrane transporter activity 11 0.044 GO:0016787hydrolase activity 183 0.047 GO:0016020membrane 302 0.0026 GO:0005886plasma membrane 159 0.016                            72    Table S8. Yellow Stage STAR Results: Genes for enzymes annotated to GO:0009813 (Flavonoid biosynthetic process, FDR=1). The first column lists differentially expressed (FDR<0.005) assembled RNA-seq reads that have mapped to the Lotus japonicus genome. The second column is their soybean (Glycine max) BLAST homolog, followed by expression at two stages of flower development.   L. japonicus ID G. max BLAST ID G. max description Counts per gene (Yellow) Counts per gene (Red) Log2FC chr2.CM0018.1300.r2.m Glyma.01G228700.1 Naringenin-chalcone synthase. 237.49 21.67 2.60689 chr5.CM1077.590.r2.m Glyma.03G187000.1 UDP-glucosyl transferase 73C 81.84 1.67 4.479036 chr1.CM0122.1600.r2.m Glyma.03G222100.1 Nitrogen regulatory protein P-II 118 15 2.151554 chr1.CM0012.990.r2.m Glyma.04G254200.1 B3 DNA binding domain // Auxin response factor 111 12 2.383216 chr1.CM0064.910.r2.m Glyma.05G076300.1 NA 725 113 1.863352 chr3.LjT40P18.80.r2.m Glyma.07G021600.1 Shikimate O-hydroxycinnamoyltransferase. 329 23 3.015946 chr3.CM0452.240.r2.d Glyma.07G021600.1 Shikimate O-hydroxycinnamoyltransferase. 8006 1066 2.091311 chr3.LjT10E18.60.r2.m Glyma.10G210600.1 B3 DNA binding domain // Auxin response factor 192 19 2.513908 chr4.CM0432.2880.r2.m Glyma.13G173300.1 O-METHYLTRANSFERASE 1884 169 2.660585 chr3.CM0106.330.r2.m Glyma.13G344700.1 B-box zinc finger 603 34 3.327629 chr1.LjT35H06.80.r2.m NA NA 40 0 7.976076              73    Table S9. Red Stage STAR Results: Genes for enzymes annotated to GO:0009813 (Flavonoid biosynthetic process, FDR = 0.00042) .  The first column lists differentially expressed (FDR<0.005) assembled RNA-seq reads that have mapped to the Lotus japonicus genome. The second column is their soybean (Glycine max) BLAST homolog, followed by expression at two stages of flower development.  L. japonicus ID G. max BLAST ID G. max description Counts per gene (Yellow) Counts per gene (Red) Log2FC chr2.CM0608.560.r2.m Glyma.01G048200.1 RIBONUCLEASE T2 792 7451 -4.05106 chr3.LjB14O06.120.r2.a Glyma.01G166200.1 naringenin 3-dioxygenase 263.79 764.34 -2.3498 chr4.CM0119.240.r2.m Glyma.01G166200.1 naringenin 3-dioxygenase 2001.21 6829.66 -2.58854 chr3.CM0590.770.r2.d Glyma.01G228700.1 Naringenin-chalcone synthase 21.17 687.21 -5.83859 chr1.CM0284.250.r2.m Glyma.01G228700.1 Naringenin-chalcone synthase 11.33 274.05 -5.43578 chr2.CM0018.760.r2.m Glyma.01G228700.1 Naringenin-chalcone synthase 327.66 1550.24 -3.05735 chr2.CM0018.730.r2.m Glyma.01G228700.1 Naringenin-chalcone synthase 86.27 323.5 -2.72878 chr3.CM0590.840.r2.m Glyma.01G228700.1 Naringenin-chalcone synthase 21.17 687.21 -5.83859 chr2.CM0018.1190.r2.m Glyma.01G228700.1 Naringenin-chalcone synthase 176.4 432.25 -2.11192 chr2.CM0018.1200.r2.m Glyma.01G228700.1 Naringenin-chalcone synthase. 5.93 97.27 -4.79591 chr5.CM0077.110.r2.m Glyma.02G158700.1 bifunctional dihydroflavonol 4-reductase/flavanone 4-reductase 29 799 -5.59379 chr5.CM0077.210.r2.m Glyma.02G158700.1 bifunctional dihydroflavonol 4-reductase/flavanone 4-reductase 140 9266 -6.86429 chr1.CM0105.1870.r2.m Glyma.03G113400.1 Proton-exporting ATPase. 285 949 -2.55225 chr1.CM0133.560.r2.m Glyma.04G052100.1 Cytochrome P450 CYP3/CYP5/CYP6/CYP9 subfamilies 40 2680 -6.87787 chr1.LjT39K18.30.r2.m Glyma.04G147700.1 HEME OXYGENASE 546 1688 -2.44547 chr2.CM0191.680.r2.m Glyma.04G227700.1 Quercetin 3-O-methyltransferase. 3 20 -3.48622 74    L. japonicus ID G. max BLAST ID G. max description Counts per gene (Yellow) Counts per gene (Red) Log2FC       chr1.CM0410.430.r2.m Glyma.08G247100.1 Transferase family 2201 7151 -2.51737 chr6.CM0013.1540.r2.a Glyma.09G038900.1 MYB-LIKE DNA-BINDING PROTEIN MYB 7 114 -4.81165 chr2.CM0021.2820.r2.m Glyma.11G027700.1 Leucocyanidin oxygenase. 929 14061 -4.73711 chr5.CM0077.120.r2.m Glyma.14G072700.1 bifunctional dihydroflavonol 4-reductase/flavanone 4-reductase 14 60 -2.90284 chr5.CM0180.690.r2.m Glyma.20G241700.1 chalcone isomerase 484 1165 -2.08436                    75    Table S10. Yellow Stage STAR Results: Genes for enzymes annotated to GO:0009809 (Lignin biosynthetic process, FDR=0.41). The first column lists differentially expressed (FDR<0.005) assembled RNA-seq reads that have mapped to the Lotus japonicus genome. The second column is their soybean (Glycine max) BLAST homolog, followed by expression at two stages of flower development.   L. japonicus ID G. max BLAST ID G. max description Counts per gene (Yellow) Counts per gene (Red) Log2FC chr6.LjT35D18.30.r2.m Glyma.03G223000.1 Methionine adenosyltransferase 16493 2557 1.87182 chr3.LjT40P18.80.r2.m Glyma.07G021600.1 cinnamoyl-CoA reductase 329 23 3.01594 chr3.CM0452.240.r2.d Glyma.07G021600.1 cinnamoyl-CoA reductase 8006 1066 2.09131 chr3.LjT45M09.50.r2.d Glyma.07G023700.1 Shikimate O-hydroxycinnamoyltransferase. 1426 220 1.87849 chr4.CM0387.960.r2.m Glyma.07G258700.1 beta-glucosidase 106 1 5.78762 chr6.CM0385.240.r2.d Glyma.09G038500.1 CHITINASE 1058 11 5.75861 chr5.CM0200.3250.r2.m Glyma.10G262400.1 cinnamyl-alcohol dehydrogenase 3805 583 1.88869   *Lignin biosynthetic process was not found to be expressed at the red stage of floral colour change in L. filicaulis, given the STAR results.                 76    Table S11. Trinity Results: Significantly enriched GO terms corresponding to the yellow stage of a L. filicaulis flower. Enriched GO category:Description Number of associated genes FDR  GO:0009790embryonic development 162 1.60E-16 GO:0032501multicellular organismal process 557 6.60E-16 GO:0032502developmental process 566 1.30E-15 GO:0007275multicellular organismal development 532 5.60E-15 GO:0009628response to abiotic stimulus 399 1.90E-10 GO:0009791post-embryonic development 314 2.80E-10 GO:0009987cellular process 1908 1.10E-09 GO:0044237cellular metabolic process 1498 1.10E-09 GO:0048856anatomical structure development 434 1.20E-09 GO:0000003reproduction 319 1.30E-09 GO:0050896response to stimulus 859 1.70E-09 GO:0008152metabolic process 1871 1.80E-09 GO:0006396RNA processing 140 2.40E-09 GO:0044281small molecule metabolic process 462 5.30E-08 GO:0009793embryonic development ending in seed dormancy 101 7.00E-08 GO:0009058biosynthetic process 821 4.60E-07 GO:0022414reproductive process 296 1.00E-06 GO:0010467gene expression 548 1.30E-06 GO:0044238primary metabolic process 1517 1.40E-06 GO:0003006reproductive developmental process 260 2.30E-06 GO:0010154fruit development 136 2.30E-06 GO:0040007growth 142 2.70E-06 GO:0044085cellular component biogenesis 161 4.10E-06 GO:0042254ribosome biogenesis 46 4.40E-06 GO:0009653anatomical structure morphogenesis 203 5.10E-06 GO:0048316seed development 126 6.00E-06 GO:0065007biological regulation 739 6.10E-06 GO:0022613ribonucleoprotein complex biogenesis 49 7.00E-06 GO:0044249cellular biosynthetic process 783 7.90E-06 GO:0009416response to light stimulus 178 1.10E-05 GO:0048731system development 254 1.10E-05 GO:0009314response to radiation 180 1.30E-05 GO:0008610lipid biosynthetic process 133 1.40E-05 GO:0034470ncRNA processing 58 2.60E-05 GO:0034660ncRNA metabolic process 73 3.50E-05 GO:0048608reproductive structure development 226 3.60E-05 GO:0050789regulation of biological process 641 4.00E-05 GO:0006950response to stress 519 4.90E-05 77    Enriched GO category:Description Number of associated genes FDR     GO:0048518positive regulation of biological process 128 4.90E-05 GO:0042221response to chemical stimulus 496 5.30E-05 GO:0009607response to biotic stimulus 224 6.20E-05 GO:0048513organ development 247 8.00E-05 GO:0009887organ morphogenesis 73 8.80E-05 GO:0015979photosynthesis 67 9.30E-05 GO:0009409response to cold 110 9.60E-05 GO:0044283small molecule biosynthetic process 211 0.00023 GO:0005976polysaccharide metabolic process 80 0.00025 GO:0051707response to other organism 210 0.00026 GO:0051704multi-organism process 255 0.00026 GO:0006412translation 130 0.00026 GO:0008219cell death 64 0.00029 GO:0016265death 64 0.00029 GO:0009266response to temperature stimulus 140 0.0003 GO:0043436oxoacid metabolic process 225 0.0003 GO:0019752carboxylic acid metabolic process 225 0.0003 GO:0006082organic acid metabolic process 225 0.0003 GO:0010033response to organic substance 317 0.00031 GO:0016043cellular component organization 331 0.00035 GO:0040008regulation of growth 46 0.00038 GO:0007166cell surface receptor linked signaling pathway 33 0.00043 GO:0016072rRNA metabolic process 32 0.00044 GO:0048869cellular developmental process 165 0.0006 GO:0042180cellular ketone metabolic process 228 0.00078 GO:0009451RNA modification 33 0.00082 GO:0000271polysaccharide biosynthetic process 49 0.00093 GO:0044264cellular polysaccharide metabolic process 69 0.00095 GO:0006364rRNA processing 29 0.00097 GO:0006629lipid metabolic process 205 0.00099 GO:0033692cellular polysaccharide biosynthetic process 48 0.00099 GO:0009725response to hormone stimulus 237 0.0011 GO:0009719response to endogenous stimulus 256 0.0011 GO:0019748secondary metabolic process 120 0.0016 GO:0023033signaling pathway 67 0.002 GO:0044255cellular lipid metabolic process 150 0.002 GO:0009657plastid organization 55 0.0029 GO:0008380RNA splicing 37 0.0032 78    Enriched GO category:Description Number of associated genes FDR     GO:0009640photomorphogenesis 28 0.0037 GO:0046394carboxylic acid biosynthetic process 131 0.0037 GO:0016053organic acid biosynthetic process 131 0.0037 GO:0010608posttranscriptional regulation of gene expression 44 0.0037 GO:0019684photosynthesis, light reaction 39 0.0041 GO:0006996organelle organization 204 0.0042 GO:0006457protein folding 51 0.0045 GO:0010025wax biosynthetic process 20 0.0046 GO:0044267cellular protein metabolic process 474 0.0053 GO:0050794regulation of cellular process 534 0.0058 GO:0006519cellular amino acid and derivative metabolic process 176 0.0062 GO:0019538protein metabolic process 556 0.0062 GO:0048646anatomical structure formation involved in morphogenesis 57 0.0062 GO:0023052signaling 255 0.0067 GO:0009733response to auxin stimulus 86 0.0069 GO:0009637response to blue light 27 0.0069 GO:0012501programmed cell death 51 0.0074 GO:0006779porphyrin biosynthetic process 29 0.0078 GO:0006631fatty acid metabolic process 80 0.0096 GO:0005982starch metabolic process 26 0.01 GO:0065008regulation of biological quality 214 0.011 GO:0032787monocarboxylic acid metabolic process 120 0.011 GO:0009639response to red or far red light 53 0.011 GO:0046148pigment biosynthetic process 51 0.012 GO:0019953sexual reproduction 38 0.013 GO:0023034intracellular signaling pathway 33 0.014 GO:0030154cell differentiation 120 0.015 GO:0033014tetrapyrrole biosynthetic process 30 0.015 GO:0000375RNA splicing, via transesterification reactions 23 0.016 GO:0007389pattern specification process 53 0.018 GO:0003002regionalization 47 0.02 GO:0008283cell proliferation 32 0.02 GO:0009606tropism 27 0.02 GO:0006633fatty acid biosynthetic process 62 0.02 GO:0023060signal transmission 200 0.023 GO:0006778porphyrin metabolic process 34 0.024 79    Enriched GO category:Description Number of associated genes FDR     GO:0023046signaling process 200 0.025 GO:0043067regulation of programmed cell death 24 0.026 GO:0042440pigment metabolic process 57 0.027 GO:0016071mRNA metabolic process 40 0.031 GO:0000377RNA splicing, via transesterification reactions with bulged adenosine as nucleophile 22 0.031 GO:0033013tetrapyrrole metabolic process 35 0.032 GO:0006073cellular glucan metabolic process 50 0.041 GO:0022621shoot system development 94 0.043 GO:0048367shoot development 94 0.043 GO:0032989cellular component morphogenesis 91 0.044 GO:0006875cellular metal ion homeostasis 16 0.044 GO:0048569post-embryonic organ development 58 0.048 GO:0003824catalytic activity 1566 6.40E-06 GO:0009055electron carrier activity 60 2.10E-05 GO:0005198structural molecule activity 108 0.0096 GO:0016740transferase activity 596 0.0096 GO:0004872receptor activity 38 0.019 GO:0005623cell 2324 2.10E-90 GO:0044464cell part 2324 2.10E-90 GO:0043226organelle 1659 5.60E-66 GO:0043229intracellular organelle 1656 9.90E-66 GO:0044424intracellular part 1838 1.20E-65 GO:0005622intracellular 1866 2.20E-65 GO:0043227membrane-bounded organelle 1581 1.60E-64 GO:0043231intracellular membrane-bounded organelle 1576 3.10E-64 GO:0005737cytoplasm 1511 1.00E-62 GO:0044444cytoplasmic part 1406 5.50E-57 GO:0009536plastid 696 8.80E-55 GO:0009507chloroplast 659 4.80E-53 GO:0044435plastid part 414 1.10E-32 GO:0044434chloroplast part 398 9.00E-30 GO:0044446intracellular organelle part 814 1.90E-28 GO:0044422organelle part 817 2.40E-28 GO:0012505endomembrane system 148 1.80E-23 GO:0031976plastid thylakoid 188 2.40E-23 GO:0009534chloroplast thylakoid 188 2.40E-23 GO:0031984organelle subcompartment 188 3.10E-23 80    Enriched GO category:Description Number of associated genes FDR     GO:0044436thylakoid part 176 4.20E-23 GO:0009579thylakoid 212 3.90E-22 GO:0042651thylakoid membrane 164 1.00E-21 GO:0055035plastid thylakoid membrane 159 1.00E-21 GO:0009535chloroplast thylakoid membrane 158 1.50E-21 GO:0034357photosynthetic membrane 165 3.20E-20 GO:0005739mitochondrion 261 4.30E-13 GO:0009532plastid stroma 206 2.00E-12 GO:0031975envelope 243 9.20E-12 GO:0009526plastid envelope 193 1.60E-11 GO:0031967organelle envelope 240 2.40E-11 GO:0009570chloroplast stroma 192 2.40E-10 GO:0009941chloroplast envelope 183 2.90E-10 GO:0031974membrane-enclosed lumen 197 1.00E-09 GO:0070013intracellular organelle lumen 194 1.20E-09 GO:0043233organelle lumen 194 1.40E-09 GO:0043232intracellular non-membrane-bounded organelle 280 3.80E-09 GO:0043228non-membrane-bounded organelle 280 3.80E-09 GO:0031090organelle membrane 332 1.60E-08 GO:0009505plant-type cell wall 92 1.00E-07 GO:0010287plastoglobule 36 1.10E-06 GO:0016020membrane 971 1.40E-06 GO:0030529ribonucleoprotein complex 142 2.30E-06 GO:0044428nuclear part 183 4.20E-06 GO:0031977thylakoid lumen 37 8.10E-06 GO:0005634nucleus 524 9.50E-06 GO:0032991macromolecular complex 360 5.30E-05 GO:0031978plastid thylakoid lumen 26 0.0001 GO:0009543chloroplast thylakoid lumen 26 0.0001 GO:0030312external encapsulating structure 164 0.00011 GO:0031224intrinsic to membrane 250 0.00016 GO:0005618cell wall 159 0.00039 GO:0005730nucleolus 100 0.00051 GO:0005576extracellular region 155 0.00074 GO:0005840ribosome 104 0.00097 GO:0031981nuclear lumen 134 0.0017 GO:0031225anchored to membrane 44 0.0063 GO:0009706chloroplast inner membrane 22 0.013 81    Enriched GO category:Description Number of associated genes FDR     GO:0009295nucleoid 17 0.015 GO:0042170plastid membrane 34 0.016 GO:0005794Golgi apparatus 162 0.017 GO:0016021integral to membrane 204 0.02 GO:0009528plastid inner membrane 22 0.022 GO:0010319stromule 19 0.022 GO:0042646plastid nucleoid 14 0.023 GO:0031969chloroplast membrane 30 0.03 GO:0005887integral to plasma membrane 13 0.035 GO:0005874microtubule 24 0.043 GO:0009521photosystem 22 0.043 GO:0044425membrane part 429 0.048                     82    Table 12. Trinity Results: Significantly enriched GO terms corresponding to the red stage of a L. filicaulis flower. Enriched GO category:Description Number of associated genes FDR  GO:0050896response to stimulus 426 2.60E-23 GO:0006950response to stress 280 1.20E-17 GO:0023052signaling 168 1.60E-17 GO:0065007biological regulation 368 1.60E-17 GO:0032502developmental process 256 2.80E-16 GO:0050794regulation of cellular process 289 1.80E-15 GO:0007242intracellular signaling cascade 111 8.50E-15 GO:0032501multicellular organismal process 245 8.50E-15 GO:0050789regulation of biological process 318 1.10E-14 GO:0009607response to biotic stimulus 134 1.40E-14 GO:0023060signal transmission 133 2.90E-14 GO:0023046signaling process 133 3.00E-14 GO:0051707response to other organism 127 9.80E-14 GO:0007275multicellular organismal development 232 1.70E-13 GO:0007165signal transduction 128 2.30E-13 GO:0009987cellular process 780 9.60E-13 GO:0048856anatomical structure development 202 2.50E-12 GO:0042221response to chemical stimulus 246 2.90E-12 GO:0051179localization 226 8.80E-12 GO:0006575cellular amino acid derivative metabolic process 74 1.00E-11 GO:0023034intracellular signaling pathway 34 1.00E-11 GO:0051704multi-organism process 141 1.10E-11 GO:0042398cellular amino acid derivative biosynthetic process 60 1.10E-11 GO:0009699phenylpropanoid biosynthetic process 45 2.50E-11 GO:0023033signaling pathway 51 2.70E-11 GO:0010033response to organic substance 167 4.30E-11 GO:0009719response to endogenous stimulus 142 4.60E-11 GO:0009790embryonic development 69 4.70E-11 GO:0009751response to salicylic acid stimulus 42 5.60E-11 GO:0009653anatomical structure morphogenesis 106 3.60E-10 GO:0051234establishment of localization 211 4.40E-10 GO:0009605response to external stimulus 86 8.00E-10 GO:0006810transport 208 1.20E-09 GO:0019748secondary metabolic process 74 1.30E-09 GO:0044238primary metabolic process 626 2.30E-09 GO:0009698phenylpropanoid metabolic process 50 3.50E-09 GO:0007166cell surface receptor linked signaling pathway 26 4.60E-09 83    Enriched GO category:Description Number of associated genes FDR     GO:0012501programmed cell death 39 5.00E-09 GO:0006519cellular amino acid and derivative metabolic process 101 5.00E-09 GO:0008219cell death 42 6.30E-09 GO:0016265death 42 6.30E-09 GO:0009813flavonoid biosynthetic process 32 1.00E-08 GO:0048518positive regulation of biological process 70 1.40E-08 GO:0048731system development 123 2.10E-08 GO:0009725response to hormone stimulus 122 4.40E-08 GO:0009056catabolic process 109 5.80E-08 GO:0009624response to nematode 32 6.70E-08 GO:0000003reproduction 136 7.10E-08 GO:0048513organ development 120 1.20E-07 GO:0009628response to abiotic stimulus 163 2.00E-07 GO:0009812flavonoid metabolic process 33 2.30E-07 GO:0044237cellular metabolic process 589 2.80E-07 GO:0009723response to ethylene stimulus 34 4.50E-07 GO:0009753response to jasmonic acid stimulus 43 4.50E-07 GO:0022414reproductive process 131 5.00E-07 GO:0009620response to fungus 48 7.50E-07 GO:0040007growth 67 1.10E-06 GO:0031323regulation of cellular metabolic process 169 2.40E-06 GO:0007610behavior 15 2.60E-06 GO:0002376immune system process 55 2.90E-06 GO:0009058biosynthetic process 332 4.50E-06 GO:0042493response to drug 21 4.90E-06 GO:0044283small molecule biosynthetic process 99 6.50E-06 GO:0061024membrane organization 27 7.00E-06 GO:0016044cellular membrane organization 27 7.00E-06 GO:0003006reproductive developmental process 111 1.40E-05 GO:0080090regulation of primary metabolic process 159 1.40E-05 GO:0006955immune response 52 2.70E-05 GO:0006952defense response 100 2.80E-05 GO:0031326regulation of cellular biosynthetic process 152 2.90E-05 GO:0050832defense response to fungus 36 3.10E-05 GO:0009889regulation of biosynthetic process 152 3.20E-05 GO:0048869cellular developmental process 78 3.70E-05 GO:0044281small molecule metabolic process 183 3.80E-05 GO:0015893drug transport 19 4.20E-05 84    Enriched GO category:Description Number of associated genes FDR     GO:0044248cellular catabolic process 89 5.60E-05 GO:0044249cellular biosynthetic process 314 7.90E-05 GO:0006873cellular ion homeostasis 20 0.0001 GO:0055082cellular chemical homeostasis 20 0.0001 GO:0048608reproductive structure development 97 0.00011 GO:0008152metabolic process 710 0.00011 GO:0051171regulation of nitrogen compound metabolic process 145 0.00011 GO:0019222regulation of metabolic process 173 0.00012 GO:0009308amine metabolic process 67 0.00012 GO:0045449regulation of transcription 138 0.00014 GO:0010556regulation of macromolecule biosynthetic process 142 0.00014 GO:0009793embryonic development ending in seed dormancy 40 0.00018 GO:0019219regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolic process 142 0.00018 GO:0009887organ morphogenesis 34 0.0002 GO:0048646anatomical structure formation involved in morphogenesis 31 0.0002 GO:0009611response to wounding 39 0.0002 GO:0009737response to abscisic acid stimulus 58 0.00021 GO:0009408response to heat 32 0.00023 GO:0016042lipid catabolic process 19 0.00023 GO:0016043cellular component organization 141 0.00023 GO:0031667response to nutrient levels 28 0.00024 GO:0045087innate immune response 45 0.00027 GO:0043067regulation of programmed cell death 16 0.00027 GO:0044242cellular lipid catabolic process 16 0.00027 GO:0007568aging 23 0.00031 GO:0009991response to extracellular stimulus 31 0.00033 GO:0019953sexual reproduction 22 0.00039 GO:0009266response to temperature stimulus 62 0.00042 GO:0006350transcription 145 0.00053 GO:0009791post-embryonic development 113 0.00054 GO:0043069negative regulation of programmed cell death 11 0.00056 GO:0000278mitotic cell cycle 17 0.00062 GO:0060255regulation of macromolecule metabolic process 151 0.00069 GO:0009863salicylic acid mediated signaling pathway 14 0.00076 GO:0040008regulation of growth 22 0.00078 GO:0016192vesicle-mediated transport 36 0.00084 GO:0048522positive regulation of cellular process 42 0.00085 85    Enriched GO category:Description Number of associated genes FDR     GO:0042594response to starvation 24 0.00088 GO:0046942carboxylic acid transport 20 0.00088 GO:0010468regulation of gene expression 146 0.00089 GO:0006855multidrug transport 16 0.0009 GO:0010646regulation of cell communication 25 0.0009 GO:0006928cellular component movement 15 0.0011 GO:0060548negative regulation of cell death 11 0.0011 GO:0071554cell wall organization or biogenesis 43 0.0011 GO:0006022aminoglycan metabolic process 8 0.0013 GO:0009966regulation of signal transduction 24 0.0013 GO:0023051regulation of signaling process 24 0.0013 GO:0010941regulation of cell death 16 0.0014 GO:0015849organic acid transport 20 0.0014 GO:0006979response to oxidative stress 50 0.0015 GO:0071446cellular response to salicylic acid stimulus 14 0.0015 GO:0071495cellular response to endogenous stimulus 54 0.0019 GO:0015837amine transport 15 0.0021 GO:0006629lipid metabolic process 86 0.0027 GO:0009733response to auxin stimulus 40 0.0029 GO:0019932second-messenger-mediated signaling 10 0.003 GO:0048878chemical homeostasis 29 0.003 GO:0009415response to water 39 0.0031 GO:0009414response to water deprivation 38 0.0037 GO:0048583regulation of response to stimulus 38 0.0037 GO:0006865amino acid transport 14 0.004 GO:0009814defense response, incompatible interaction 30 0.004 GO:0006796phosphate metabolic process 120 0.004 GO:0009739response to gibberellin stimulus 20 0.0044 GO:0006793phosphorus metabolic process 120 0.0045 GO:0000272polysaccharide catabolic process 13 0.0047 GO:0044106cellular amine metabolic process 54 0.0047 GO:0065008regulation of biological quality 92 0.0049 GO:0071310cellular response to organic substance 63 0.0049 GO:0009888tissue development 50 0.0049 GO:0009409response to cold 44 0.0052 GO:0022603regulation of anatomical structure morphogenesis 14 0.0058 GO:0042439ethanolamine and derivative metabolic process 7 0.006 GO:0007267cell-cell signaling 7 0.006 86    Enriched GO category:Description Number of associated genes FDR     GO:0048523negative regulation of cellular process 44 0.006 GO:0048316seed development 47 0.0065 GO:0007010cytoskeleton organization 26 0.0065 GO:0043687post-translational protein modification 127 0.0066 GO:0006576cellular biogenic amine metabolic process 15 0.0066 GO:0006464protein modification process 137 0.0066 GO:0030036actin cytoskeleton organization 18 0.0071 GO:0009057macromolecule catabolic process 48 0.0071 GO:0030029actin filament-based process 19 0.0072 GO:0010118stomatal movement 16 0.0072 GO:0009642response to light intensity 23 0.008 GO:0030154cell differentiation 53 0.0083 GO:0005975carbohydrate metabolic process 100 0.009 GO:0070887cellular response to chemical stimulus 71 0.0093 GO:0042538hyperosmotic salinity response 13 0.01 GO:0009873ethylene mediated signaling pathway 13 0.01 GO:0009416response to light stimulus 66 0.011 GO:0009755hormone-mediated signaling pathway 43 0.012 GO:0030003cellular cation homeostasis 13 0.013 GO:0009612response to mechanical stimulus 7 0.014 GO:0000902cell morphogenesis 38 0.014 GO:0007626locomotory behavior 8 0.014 GO:0006972hyperosmotic response 14 0.014 GO:0006811ion transport 60 0.015 GO:0010154fruit development 48 0.016 GO:0016567protein ubiquitination 20 0.016 GO:0009314response to radiation 66 0.016 GO:0043436oxoacid metabolic process 87 0.016 GO:0010054trichoblast differentiation 14 0.016 GO:0006082organic acid metabolic process 87 0.016 GO:0019752carboxylic acid metabolic process 87 0.016 GO:0016310phosphorylation 102 0.017 GO:0048585negative regulation of response to stimulus 16 0.018 GO:0032870cellular response to hormone stimulus 45 0.018 GO:0022610biological adhesion 7 0.019 GO:0007155cell adhesion 7 0.019 GO:0051094positive regulation of developmental process 14 0.019 GO:0046395carboxylic acid catabolic process 18 0.02 87    Enriched GO category:Description Number of associated genes FDR     GO:0016054organic acid catabolic process 18 0.02 GO:0044255cellular lipid metabolic process 60 0.021 GO:0048468cell development 35 0.022 GO:0043412macromolecule modification 140 0.023 GO:0071555cell wall organization 27 0.023 GO:0006026aminoglycan catabolic process 6 0.024 GO:0006030chitin metabolic process 6 0.024 GO:0006032chitin catabolic process 6 0.024 GO:0019722calcium-mediated signaling 7 0.026 GO:0032446protein modification by small protein conjugation 20 0.027 GO:0010119regulation of stomatal movement 11 0.027 GO:0015692lead ion transport 5 0.028 GO:0009963positive regulation of flavonoid biosynthetic process 5 0.028 GO:0007584response to nutrient 5 0.028 GO:0048645organ formation 10 0.028 GO:0032989cellular component morphogenesis 40 0.028 GO:0070882cellular cell wall organization or biogenesis 17 0.029 GO:0010053root epidermal cell differentiation 16 0.03 GO:0050801ion homeostasis 21 0.031 GO:0016049cell growth 38 0.032 GO:0009867jasmonic acid mediated signaling pathway 13 0.032 GO:0007264small GTPase mediated signal transduction 17 0.033 GO:0006754ATP biosynthetic process 9 0.033 GO:0019725cellular homeostasis 24 0.033 GO:0048519negative regulation of biological process 55 0.034 GO:0032535regulation of cellular component size 42 0.035 GO:0090066regulation of anatomical structure size 42 0.035 GO:0042180cellular ketone metabolic process 87 0.035 GO:0006644phospholipid metabolic process 18 0.035 GO:0048532anatomical structure arrangement 12 0.035 GO:0007389pattern specification process 23 0.035 GO:0071395cellular response to jasmonic acid stimulus 13 0.037 GO:0023057negative regulation of signaling process 11 0.037 GO:0009968negative regulation of signal transduction 11 0.037 GO:0010648negative regulation of cell communication 11 0.037 GO:0008361regulation of cell size 39 0.038 GO:0023056positive regulation of signaling process 8 0.038 GO:0009967positive regulation of signal transduction 8 0.038 88    Enriched GO category:Description Number of associated genes FDR     GO:0002218activation of innate immune response 10 0.039 GO:0002253activation of immune response 10 0.039 GO:0006839mitochondrial transport 9 0.039 GO:0048584positive regulation of response to stimulus 16 0.041 GO:0070647protein modification by small protein conjugation or removal 21 0.044 GO:0022403cell cycle phase 17 0.045 GO:0006970response to osmotic stress 53 0.046 GO:0000087M phase of mitotic cell cycle 10 0.046 GO:0007067mitosis 10 0.046 GO:0009055electron carrier activity 56 1.10E-19 GO:0003824catalytic activity 688 7.80E-17 GO:0030528transcription regulator activity 119 1.40E-14 GO:0022804active transmembrane transporter activity 76 2.40E-11 GO:0005215transporter activity 135 3.30E-11 GO:0003700transcription factor activity 97 4.90E-10 GO:0022857transmembrane transporter activity 107 3.90E-08 GO:0015291secondary active transmembrane transporter activity 45 3.90E-08 GO:0015294solute:cation symporter activity 19 1.30E-07 GO:0043169cation binding 268 1.60E-07 GO:0022892substrate-specific transporter activity 97 2.30E-07 GO:0042578phosphoric ester hydrolase activity 47 3.20E-07 GO:0015293symporter activity 19 3.70E-07 GO:0004091carboxylesterase activity 36 1.40E-06 GO:0016787hydrolase activity 266 1.80E-06 GO:0015295solute:hydrogen symporter activity 14 7.40E-06 GO:0016298lipase activity 24 9.40E-06 GO:0022891substrate-specific transmembrane transporter activity 85 1.20E-05 GO:0004497monooxygenase activity 26 2.30E-05 GO:0005509calcium ion binding 40 2.50E-05 GO:0000287magnesium ion binding 25 2.70E-05 GO:0016791phosphatase activity 36 2.90E-05 GO:0004722protein serine/threonine phosphatase activity 18 5.20E-05 GO:0008324cation transmembrane transporter activity 51 6.40E-05 GO:0016563transcription activator activity 11 8.70E-05 GO:0019787small conjugating protein ligase activity 31 8.80E-05 GO:0015075ion transmembrane transporter activity 64 8.80E-05 GO:0016788hydrolase activity, acting on ester bonds 105 0.00011 GO:0046872metal ion binding 247 0.00012 89    Enriched GO category:Description Number of associated genes FDR     GO:0051119sugar transmembrane transporter activity 14 0.00013 GO:0016874ligase activity 51 0.00014 GO:0016301kinase activity 122 0.00015 GO:0016879ligase activity, forming carbon-nitrogen bonds 41 0.00018 GO:0016638oxidoreductase activity, acting on the CH-NH2 group of donors 10 0.0002 GO:0016881acid-amino acid ligase activity 34 0.00023 GO:0046943carboxylic acid transmembrane transporter activity 19 0.00024 GO:0004842ubiquitin-protein ligase activity 29 0.00034 GO:0005342organic acid transmembrane transporter activity 19 0.00044 GO:0005506iron ion binding 59 0.00044 GO:0008134transcription factor binding 15 0.0006 GO:0016798hydrolase activity, acting on glycosyl bonds 53 0.0006 GO:0004553hydrolase activity, hydrolyzing O-glycosyl compounds 50 0.00087 GO:0015297antiporter activity 23 0.0013 GO:0004672protein kinase activity 100 0.0014 GO:0015144carbohydrate transmembrane transporter activity 14 0.0014 GO:0016740transferase activity 242 0.0014 GO:0016773phosphotransferase activity, alcohol group as acceptor 113 0.0014 GO:0004721phosphoprotein phosphatase activity 19 0.0018 GO:0015662ATPase activity, coupled to transmembrane movement of ions, phosphorylative mechanism 9 0.0028 GO:0008194UDP-glycosyltransferase activity 24 0.0047 GO:0005275amine transmembrane transporter activity 14 0.0052 GO:0015171amino acid transmembrane transporter activity 13 0.0065 GO:0015179L-amino acid transmembrane transporter activity 6 0.0078 GO:0015197peptide transporter activity 6 0.0078 GO:0004674protein serine/threonine kinase activity 80 0.0092 GO:0008081phosphoric diester hydrolase activity 11 0.0095 GO:0015300solute:solute antiporter activity 13 0.011 GO:0015238drug transmembrane transporter activity 13 0.017 GO:0030246carbohydrate binding 27 0.017 GO:0004568chitinase activity 6 0.019 GO:0030234enzyme regulator activity 27 0.029 GO:0008415acyltransferase activity 22 0.03 GO:0015399primary active transmembrane transporter activity 22 0.033 GO:0005337nucleoside transmembrane transporter activity 5 0.037 GO:0012505endomembrane system 126 5.00E-52 GO:0044464cell part 882 5.80E-40 90    Enriched GO category:Description Number of associated genes FDR     GO:0005623cell 882 5.80E-40 GO:0031224intrinsic to membrane 166 3.80E-20 GO:0016021integral to membrane 141 7.10E-16 GO:0009505plant-type cell wall 47 1.70E-07 GO:0005622intracellular 600 5.40E-07 GO:0044424intracellular part 578 1.30E-05 GO:0005887integral to plasma membrane 13 1.80E-05 GO:0044425membrane part 201 4.00E-05 GO:0005739mitochondrion 94 0.00012 GO:0031410cytoplasmic vesicle 23 0.00012 GO:0008287protein serine/threonine phosphatase complex 10 0.00013 GO:0031982vesicle 25 0.00014 GO:0031988membrane-bounded vesicle 23 0.00078 GO:0016023cytoplasmic membrane-bounded vesicle 21 0.00078 GO:0043226organelle 482 0.0073 GO:0043229intracellular organelle 481 0.0077 GO:0043227membrane-bounded organelle 450 0.023 GO:0043231intracellular membrane-bounded organelle 449 0.023 GO:0005634nucleus 197 0.023 GO:0045177apical part of cell 7 0.036 GO:0005856cytoskeleton 30 0.043                 91    Table S13. Red Stage Trinity Results: Genes for enzymes annotated to the over-represented GO:0009813 (Flavonoid biosynthetic process, FDR=1.0e-08). The first two columns list differentially expressed (FDR<0.05) Trinity contigs and gene names as annotated by BLASTing the Lotus japonicus genome. The third column is their soybean BLAST homolog, followed by expression at two stages of flower development. Trinity contig L. japonicus ID G. max BLAST ID Gene description   Counts per gene (Yellow) Counts per gene (Red) Log2FC TR29586|c1_g3 chr5.CM0077.790.nc Glyma.17G252200.1 bifunctional dihydroflavonol 4-reductase/flavanone 4-reductase (DFR1) 0 78.33 -9.4766 TR29586|c0_g1 chr5.CM0077.690.nc Glyma.14G072700.1 bifunctional dihydroflavonol 4-reductase/flavanone 4-reductase (DFR3) 148 10163 -6.4567 TR17543|c0_g1 chr5.CM0077.790.nc Glyma.14G072700.1 bifunctional dihydroflavonol 4-reductase/flavanone 4-reductase (DFR5) 0 28 -8.0022 TR29586|c1_g4 chr5.CM0077.790.nc Glyma.17G252200.1 bifunctional dihydroflavonol 4-reductase/flavanone 4-reductase (DFR5) 0 65.1 -9.2140 TR29586|c2_g1 chr5.CM0077.790.nc Glyma.17G252200.1 bifunctional dihydroflavonol 4-reductase/flavanone 4-reductase (DFR5) 9 195 -4.7724 TR29586|c1_g1 chr5.CM0077.790.nc Glyma.17G252200.1 bifunctional dihydroflavonol 4-reductase/flavanone 4-reductase (DFR5) 6 38.56 -3.0276 TR52576|c0_g1 chr5.CM0077.780.nc Glyma.17G252200.1 bifunctional dihydroflavonol 4-reductase/flavanone 4-reductase (DFR5) 7 33 -2.56972 TR43444|c0_g2 chr1.CM0133.170.nc Glyma.17G227500.1 Cytochrome P450 CYP3/CYP5/CYP6/CYP9 subfamilies 6 98.36 -4.3545 TR43444|c0_g1 chr1.CM0133.170.nc Glyma.17G227500.1 Cytochrome P450 CYP3/CYP5/CYP6/CYP9 subfamilies 0 13.64 -7.0078 92    Trinity contig L. japonicus ID G. max BLAST ID Gene description   Counts per gene (Yellow) Counts per gene (Red) Log2FC        TR23263|c0_g1 LjSGA_060583.1 Glyma.17G227500.1 Cytochrome P450 CYP3/CYP5/CYP6/CYP9 subfamilies 13 182 -4.1492 TR23981|c0_g1 chr1.CM0133.170.nc Glyma.04G052100.1 Cytochrome P450 CYP3/CYP5/CYP6/CYP9 subfamilies (putative MAX1)  45 2897 -6.3605 TR38960|c0_g1 chr4.CM0119.240.nc Glyma.02G048400.1 Flavanone 3-dioxygenase (F3H1) 1830 6123 -2.09884 TR61355|c0_g2 LjSGA_043399.2 Glyma.20G009300.2 Flavanone 3-dioxygenase (F3H1) 117.49 453.22 -2.3081 TR13112|c0_g1 chr2.CM0124.280.nd Glyma.02G124700.1 Flavanone 3-dioxygenase (F3H2) 6 33 -2.7873 TR53383|c0_g1 chr4.CM0387.340.nc Glyma.08G092800.1 Flavanone 3-dioxygenase (F3H5) 1 50 -5.8140 TR50359|c0_g2 LjSGA_025000.1 Glyma.12G235300.1 Flavanone 3-dioxygenase (F3H8) 4 40 -3.6326 TR46116|c0_g1* chr2.CM0124.160.nd Glyma.02G124700.1 Flavanone 3-dioxygenase * 1 21 -4.5668 TR52191|c0_g1 chr4.CM0429.360.nc Glyma.06G202300.1 Flavonoid 3'-monooxygenase (F3’H1) 1586 15638 -3.6580 TR38633|c0_g1 chr6.CM0055.290.nc Glyma.09G146300.1 Kinesin motor domain // PPR repeat // Trehalose-phosphatase 1219 3586 -1.9130 TR50049|c0_g2 chr2.CM0304.350.nc Glyma.11G027700.1 Leucocyanidin oxygenase (ANS1) 0 165.97 -10.565 TR50049|c0_g1 chr2.CM0304.350.nc Glyma.11G027700.1 Leucocyanidin oxygenase (ANS1) 1010 14783.03 -4.2278 TR42413|c0_g1 chr2.LjT36E17.150.nd Glyma.14G032400.1 MATE EFFLUX FAMILY PROTEIN 0 53 -8.9201 TR32220|c0_g1 LjT02E24.110.nc Glyma.09G038900.1 MYB-LIKE DNA-BINDING PROTEIN MYB 7 111 -4.31638 93    Trinity contig L. japonicus ID G. max BLAST ID Gene description   Counts per gene (Yellow) Counts per gene (Red) Log2FC        TR19889|c3_g1 chr1.CM0591.390.nd Glyma.11G011500.1 Naringenin-chalcone synthase (CHS1) 1385 19064 -4.1392 TR48518|c0_g1 chr2.CM0018.700.nc Glyma.11G011500.1 Naringenin-chalcone synthase (CHS2) 77 430 -2.8356 TR19889|c2_g1 chr2.CM0018.710.nc Glyma.01G228700.1 Naringenin-chalcone synthase (CHS9) 17 207 -3.9514 TR51814|c0_g1 chr1.CM0105.880.nc Glyma.03G262600.1 Proton-exporting ATPase. 272 951 -2.1617 TR1381|c0_g1 chr1.CM0105.600.nc Glyma.07G048700.1 Quercetin 3-O-methyltransferase (3-OMT) 4 126 -5.2853 TR57984|c0_g1 chr2.CM0641.550.nc Glyma.01G048400.1 Ribonuclease T(2) 383 12922 -5.4323 TR57984|c0_g2 chr2.CM0641.560.nc Glyma.01G048200.1 RIBONUCLEASE T2 850 8077 -3.6046 TR45786|c0_g1 chr3.LjT46L11.20.nc Glyma.12G238900.1 Thaumatin family 1 261 -8.1955 TR14130|c0_g1 chr1.CM0063.200.nc Glyma.08G010500.1 Transferase family 1 29 -5.0304 TR45798|c0_g1 chr1.CM0371.40.nc Glyma.16G039500.1 Transferase family 21 432 -4.7098 TR33284|c0_g1 chr1.CM0410.130.nc Glyma.18G268100.1 Transferase family 2499 8136 -2.05944 TR34067|c0_g2 LjSGA_053613.2 Glyma.10G062200.1 UDP-glucosyl transferase 73C (UGT) 0 16.8 -7.2859 TR48968|c0_g1 chr1.CM0012.680.nd Glyma.19G187000.1 UDP-glucosyl transferase 73C (UGT) 164 5940 -5.5340              94    Table S14. Yellow Stage Trinity Results: Genes for enzymes annotated to GO:0009813 (Flavonoid biosynthetic process, FDR=1). The first two columns list differentially expressed (FDR<0.05) Trinity contigs and gene names as annotated by BLASTing the Lotus japonicus genome. The third column is their soybean BLAST equivalent, followed by expression at two stages of flower development.  Trinity contig L. japonicus ID G. max BLAST ID Gene description   Counts per gene (Yellow) Counts per gene (Red) Log2FC TR27395|c0_g1 chr1.CM0122.1600.r2.m Glyma.03G222100.2 Nitrogen regulatory protein P-II 119 14 2.7213 TR4932|c0_g1 chr1.CM0476.410.r2.a Glyma.04G227700.1 Quercetin 3-O-methyltransferase        (3-OMT) 5616 1315 1.7378 TR4801|c6_g1 LjSGA_035363.1 Glyma.05G052000.1 Cytochrome P450 57 0 8.6691 TR31484|c0_g1 chr1.CM0064.910.r2.m Glyma.05G076300.1 NA 1020 163 2.2883 TR35194|c0_g1 chr3.CM0452.240.r2.d Glyma.08G220200.1 Shikimate O-hydroxycinnamoyltransferase  (HCT) 8894 1190 2.5452 TR34244|c0_g1 LjSGA_050386.1 Glyma.09G186300.1 Cytochrome P450 CYP2 subfamily 74 14 2.0370 TR32422|c0_g1 chr5.CM1125.790.r2.a Glyma.10G026000.1 EGL3 (ENHANCER OF GLABRA3), DNA BINDING / TRANSCRIPTION FACTOR (EGL3) 131.18 0 9.8676 TR66030|c0_g1 chr1.CM0012.990.r2.m Glyma.10G053500.1 AUX/IAA family // B3 DNA binding domain // Auxin response factor 169 23 2.5151 TR34067|c0_g1 chr5.CM1077.590.r2.m Glyma.10G062200.1 UDP-glucosyl transferase 73C (UGT73C) 170 0.2 10.2433 TR34449|c0_g1 chr3.LjT10E18.60.r2.m Glyma.12G076200.1 AUX/IAA family // B3 DNA binding domain // Auxin response factor 265 29 2.8306 TR46051|c0_g2 LjSGA_013806.1 Glyma.12G109800.1 Quercetin 3-O-methyltransferase        (3-OMT) 4923.93 244.53 3.9718 95    Trinity contig L. japonicus ID G. max BLAST ID Gene description   Counts per gene (Yellow) Counts per gene (Red) Log2FC        TR28709|c0_g1 chr4.CM0432.2880.r2.m Glyma.13G173300.1 O-METHYLTRANSFERASE (OMT) 2026.03 168.18 3.2347        TR28709|c0_g2 chr4.CM0432.2880.r2.m Glyma.13G173300.1 O-METHYLTRANSFERASE (OMT) 231.97 25.82 2.7958 TR39181|c0_g1 chr3.CM0106.330.r2.m Glyma.13G344700.1 B-box zinc finger 625 32 3.9265 TR30560|c0_g1 chr3.LjT40P18.80.r2.m Glyma.13G371000.1 Shikimate O-hydroxycinnamoyltransferase (HCT) 259.39 14.83 3.7436 TR30560|c0_g2 chr3.LjT40P18.80.r2.m Glyma.13G371000.1 Shikimate O-hydroxycinnamoyltransferase (HCT) 80.61 7.17 3.1560 TR3240|c0_g1 LjSGA_097658.1 Glyma.15G154100.1 Flavanone 3-hydroxylase (F3H)  1520 83 3.8365 TR11418|c0_g1 LjSGA_027597.1 Glyma.15G221300.1 Flavonol 3-O-glucosyltransferase (UFGT) 1126 23 5.2502 TR24510|c0_g2 LjSGA_071841.2 Glyma.15G221300.1 Flavonol 3-O-glucosyltransferase (UFGT) 134 0 9.9003 TR20072|c0_g1 chr6.CM0013.1540.r2.a Glyma.16G073000.1 myb proto-oncogene protein, plant 315 0 11.1325 TR4801|c4_g1 LjSGA_035363.1 Glyma.17G134100.1 Cytochrome P450 CYP2 subfamily 77 0 9.1021 TR26322|c0_g1 chr1.CM0410.460.r2.m Glyma.18G268100.1 Transferase family 534 104 2.0025 TR50011|c0_g1 chr1.CM0104.1140.r2.m Glyma.19G105100.1 Naringenin-chalcone synthase (CHS) 24259 5817 1.7036 TR19685|c0_g1 LjSGA_045968.2 Glyma.20G180000.1 B3 DNA binding domain // Auxin response factor 26.01 0 7.5409   96    Table S15. Similarly Expressed Trinity Results: Genes for enzymes annotated to GO:0009813 (Flavonoid biosynthetic process, FDR=1). The first two columns list Trinity contigs expresand gene names as annotated by BLASTing the Lotus japonicus genome. The third column is their soybean BLAST homolog, followed by expression at two stages of flower development. Trinity contig L. japonicus ID G. max BLAST ID Gene description   Counts per gene (Yellow) Counts per gene (Red) Log2FC TR43354|c0_g1 chr5.CM0180.690.r2.m Glyma.20G241700.1 chalcone isomerase (CHI)  61.37 168.88 -1.6533 TR20726|c0_g1 LjSGA_018608.1 Glyma.18G048300.1 Flavanone 3-hydroxylase (F3H2) 14.78 10.7 0.2720 TR20726|c0_g2 LjSGA_018608.1 Glyma.18G048300.1 Flavanone 3-hydroxylase (F3H2) 4.18 2.31 0.6411 TR9870|c1_g1 LjSGA_041422.1 Glyma.08G149800.1 Flavanone 3-hydroxylase (F3H3) 2.73 0 4.4836 TR31785|c0_g1 LjSGA_067411.1 Glyma.02G136200.1 Flavanone 3-dioxygenase (F3H4) 1218.06 1201.12 0.7287 TR123|c0_g1 chr2.CM0191.680.r2.m Glyma.04G227700.1 Quercetin 3-O-methyltransferase  (3-OMT) 0.27 1.84 -2.7781 TR31193|c0_g1 chr4.CM0227.690.r2.m Glyma.02G104700.1 Flavonol 3-O-glucosyltransferase (UFGT) 3.44 1.08 1.5431 TR45767|c0_g1 chr4.CM1616.680.r2.m Glyma.08G062000.1 Anthocyanidin reductase (ANR)  0.46 2.7 -2.5202 TR3325|c1_g1 chr2.CM0021.2950.r2.m Glyma.01G215400.1 3,5-epimerase/4-reductase (GME) 9.01 12.07 -0.6159 TR18411|c0_g1 LjSGA_047295.1 Glyma.01G096600.1 transcription factor MYC2 24.74 7.74 1.4663 TR38249|c0_g1 chr4.CM0007.1140.r2.m Glyma.05G127300.2 26S proteasome regulatory subunit N12 62.23 63.42 -0.2245 97    Trinity contig L. japonicus ID G. max BLAST ID Gene description   Counts per gene (Yellow) Counts per gene (Red) Log2FC        TR34052|c0_g1 chr2.CM0608.560.r2.m Glyma.02G107900.1 RIBONUCLEASE T2 0 5.01 -5.5424 TR17827|c0_g1 LjSGA_052002.1 Glyma.19G187500.1 UDP-glucosyl transferase 73C (UGT73C) 3.21 3.48 -0.3123 TR62356|c1_g1 chr5.CM0052.810.r2.m Glyma.02G081000.1 Sterol 3-beta-glucosyltransferase 13.47 31.35 -1.4669 TR62356|c1_g3 chr5.CM0052.810.r2.m Glyma.10G224000.1 SPHINGOMYELIN SYNTHETASE // SUBFAMILY NOT NAMED 13.29 14.18 -0.3049 TR35590|c0_g1 chr2.CM0124.30.r2.m Glyma.02G100700.1 STEROL REGULATORY ELEMENT-BINDING PROTEIN 1.18 1.53 -0.3171 TR5549|c0_g1 chr2.CM0124.30.r2.m Glyma.02G100700.1 STEROL REGULATORY ELEMENT-BINDING PROTEIN 0.42 2.39 -2.5963 TR26623|c0_g1 chr2.CM0177.810.r2.m Glyma.09G204500.1 transcription factor MYC2 24.75 22.14 -0.0484 TR61787|c0_g1 LjSGA_091313.0.1 Glyma.02G136000.1 ATP-dependent Clp protease adaptor protein ClpS 5.78 16.45 -1.5641 TR32422|c0_g2 chr5.CM1125.790.r2.a Glyma.10G026000.1 EGL3 (ENHANCER OF GLABRA3), DNA BINDING / TRANSCRIPTION FACTOR 49.62 66.36 -0.6287 98    Trinity contig L. japonicus ID G. max BLAST ID Gene description   Counts per gene (Yellow) Counts per gene (Red) Log2FC        TR40782|c0_g1 chr3.CM0136.10.r2.m Glyma.11G145500.1 AUX/IAA family // B3 DNA binding domain // Auxin response factor 5.23 0 4.8824 TR7528|c0_g1 chr3.CM0136.10.r2.m Glyma.11G145500.1 AUX/IAA family // B3 DNA binding domain // Auxin response factor 4.6 0 4.4836 TR20842|c1_g1 LjSGA_014979.1 Glyma.18G104100.1 Transferase family 1731.14 1737.77 1.4098 TR19685|c0_g2 LjSGA_045968.2 Glyma.20G180000.1 B3 DNA binding domain // Auxin response factor 6.87 3.12 0.9419 TR14793|c0_g1 LjSGA_111580.1 Glyma.13G082700.1 Thaumatin family 3.54 0.6 2.3293 TR14793|c1_g1 LjSGA_111580.1 Glyma.14G163700.1 Thaumatin family 4.99 0 3.9305 TR47026|c0_g1 chr1.CM1911.100.r2.m Glyma.03G122000.1 coumaroylquinate(coumaroylshikimate) 3'-monooxygenase 38.65 16.98 0.9787 TR47026|c0_g2 chr1.CM1911.100.r2.m Glyma.03G122000.1 coumaroylquinate(coumaroylshikimate) 3'-monooxygenase 182.35 58.95 1.4244 TR38719|c0_g1 LjSGA_024944.1 Glyma.10G210600.1 B3 DNA binding domain // Auxin response factor 1.24 0 4.4836 TR38719|c0_g2 LjSGA_024944.1 Glyma.10G210600.1 B3 DNA binding domain // Auxin response factor 13.28 5.31 1.1199 TR52576|c1_g1  chr5.CM0077.120.r2.m NA NA 2.08 1.34 0.5912 TR65913|c1_g1 chr2.CM0191.680.r2.m NA NA 1.2 9.4 -2.7781 99    Trinity contig L. japonicus ID G. max BLAST ID Gene description   Counts per gene (Yellow) Counts per gene (Red) Log2FC        TR38780|c0_g2 chr5.CM0180.670.r2.m NA NA 4.1 8.72 -1.2662 TR55653|c1_g1 LjSGA_022904.1 NA NA 4.66 2.09 1.1443 TR62356|c0_g1 chr5.CM0052.810.r2.m NA NA 13.82 12.13 0.0737 TR42787|c0_g1 chr1.CM0104.1140.r2.m NA NA 2.72 2.5 0.0564 TR17090|c0_g1 LjSGA_020776.1.1 NA NA 2.29 0 4.4836 TR41909|c0_g1 LjSGA_020776.1.1 NA NA 6.82 0 3.9305 TR31193|c1_g1 chr4.CM0227.690.r2.m NA NA 2.89 0 3.9305 TR37424|c0_g1 chr4.CM0227.690.r2.m NA NA 2.8 1.11 1.1847 TR13846|c0_g1 chr3.CM0136.10.r2.m NA NA 7.76 0 6.1747 TR27061|c0_g1 chr6.LjT09C09.100.r2.a NA NA 1.64 1.03 0.5912 TR30767|c0_g1 chr6.LjT09C09.100.r2.a Glyma.20G207700.1 NA 4.24 0 4.4836 TR40275|c0_g1 chr6.LjT09C09.100.r2.a Glyma.02G004200.1 NA 3.84 0 3.93057 TR19889|c1_g1 chr2.CM0018.760.r2.m NA NA 52.86 99.33 -0.8652 TR25290|c0_g1 chr5.CM0180.690.r2.m NA NA 1.11 3.35 -1.6119 TR12068|c0_g1 chr5.CM0052.810.r2.m NA NA 0.6 4.36 -2.7781 TR48553|c0_g1 chr5.CM1125.790.r2.a NA NA 1.45 3.57 -1.2976 TR46080|c0_g1 LjSGA_040150.1 NA NA 1.33 3.88 -1.7486 TR46080|c0_g2 LjSGA_040150.1 NA NA 5.24 5.38 -0.2436 100    Trinity contig L. japonicus ID G. max BLAST ID Gene description   Counts per gene (Yellow) Counts per gene (Red) Log2FC        TR39677|c1_g1 LjSGA_092302.1 NA NA 1.44 1.4 -0.1316 TR28458|c1_g1 LjSGA_054002.1 NA NA 1.82 0 3.9305 TR51898|c0_g1 chr2.CM0124.30.r2.m NA NA 0.46 2.38 -2.4460 TR3728|c0_g1 chr1.CM0476.410.r2.a NA NA 2.45 3.5 -0.31712 TR61647|c0_g1 chr5.CM0200.3220.r2.m NA NA 68.47 112.77 -0.9246 TR5553|c0_g1 chr3.CM0452.240.r2.d NA NA 2.23 3.14 -0.3171 TR60161|c0_g1 chr1.CM0104.1140.r2.m NA NA 2.64 5.23 -0.8954                     101    Table S16. Yellow Stage Trinity Results: Genes for enzymes annotated to the over-represented GO:000 (Lignin biosynthetic process, FDR=). The first two columns list differentially expressed (FDR<0.05) Trinity contigs and gene names as annotated by BLASTing the Lotus japonicus genome. The third column is their soybean BLAST homolog, followed by expression at two stages of flower development.  Trinity contig L. japonicus ID G. max BLAST ID Gene description   Counts per gene (Yellow) Counts per gene (Red) Log2FC TR4932|c0_g1 chr1.CM0476.410.r2.a Glyma.04G227700.1 Quercetin 3-O-methyltransferase (-3-OMT) 5616 1315 1.7378 TR4801|c6_g1 LjSGA_035363.1 Glyma.05G052000.1 Cytochrome P450 57 0 8.6691 TR37863|c1_g1 LjSGA_024849.1 Glyma.07G023700.1 cinnamoyl-CoA reductase (CCR) 24 1 4.0867 TR47015|c0_g1 LjSGA_040039.1 Glyma.07G026300.1 NAD DEPENDENT EPIMERASE/DEHYDRATASE // SUBFAMILY NOT NAMED 239 22 3.0785 TR25023|c0_g1 LjSGA_027787.1 Glyma.07G050600.1 No apical meristem (NAM) protein 69 0 8.9441 TR8127|c0_g1 chr4.CM0387.960.r2.m Glyma.07G258700.1 beta-glucosidase 118 1 6.3777 TR35194|c0_g1 chr3.CM0452.240.r2.d Glyma.08G220200.1 Shikimate O-hydroxycinnamoyltransferase (HCT) 8894 1190 2.5452 TR5179|c0_g1 chr1.CM0017.470.r2.a Glyma.08G362900.1 Transaldolase. 4763 939 1.9860 TR46051|c0_g2 LjSGA_013806.1 Glyma.12G109800.1 caffeoyl-CoA O-methyltransferase (CCoAOMT) 536 10 5.3722 TR30560|c0_g1 chr3.LjT40P18.80.r2.m Glyma.13G371000.1 Quercetin 3-O-methyltransferase (-3-OMT) 4923.93 244.53 3.9718 TR30560|c0_g2 chr3.LjT40P18.80.r2.m Glyma.13G371000.1 Shikimate O-hydroxycinnamoyltransferase (HCT) 259.39 14.83 3.7436 TR34012|c0_g1 chr3.LjT45M09.50.r2.d Glyma.15G003600.1 Shikimate O-hydroxycinnamoyltransferase (HCT) 80.61 7.17 3.1560 102    Trinity contig L. japonicus ID G. max BLAST ID Gene description   Counts per gene (Yellow) Counts per gene (Red) Log2FC        TR49520|c0_g1 chr6.CM0385.240.r2.d Glyma.15G143600.1 cinnamoyl-CoA reductase (CCR) 2562 398 2.3295 TR36532|c0_g1 chr6.LjT35D18.30.r2.m Glyma.15G190500.1 CHITINASE 1112 7 6.9328 TR4801|c4_g1 LjSGA_035363.1 Glyma.17G134100.1 Methionine adenosyltransferase (MAT)  19644 3001 2.3539 TR38742|c0_g1 LjSGA_021886.2 Glyma.17G171100.1 Cytochrome P450 CYP2 subfamily 77 0 9.1021 TR61337|c0_g1 chr5.CM0200.3250.r2.m Glyma.20G128600.1 cinnamyl-alcohol dehydrogenase (CAD) 138.5 20.92 2.3536 TR61337|c0_g3 chr5.CM0200.3250.r2.m Glyma.20G128600.1 cinnamyl-alcohol dehydrogenase (CAD) 47.87 0 8.4218 TR61337|c0_g2 chr5.CM0200.3250.r2.m Glyma.20G128600.1 cinnamyl-alcohol dehydrogenase (CAD) 4056.51 588.62 2.4273                  103    Table S17. Red Stage Trinity Results: Genes for enzymes annotated to the over-represented GO:000 (Lignin biosynthetic process, FDR=). The first two columns list differentially expressed (FDR<0.05) Trinity contigs and gene names as annotated by BLASTing the Lotus japonicus genome. The third column is their soybean BLAST homolog, followed by expression at two stages of flower development. Trinity contig L. japonicus ID G. max BLAST ID Gene description   Counts per gene (Yellow) Counts per gene (Red) Log2FC TR24303|c0_g1 LjSGA_128268.1 Glyma.01G177900.1 coniferyl-alcohol glucosyltransferase (CAGT) 60 761 -4.0182 TR13393|c0_g1 LjSGA_030253.1 Glyma.03G184000.1 Methionine adenosyltransferase (MAT) 1894 5353 -1.8553 TR1381|c0_g1 chr1.CM0105.600.nc Glyma.07G048700.1 Quercetin 3-O-methyltransferase (3-OMT) 4 126 -5.2853 TR14130|c0_g1 chr1.CM0063.200.nc Glyma.08G010500.1 Transferase family 1 29 -5.0304 TR4013|c1_g3 LjSGA_011490.1 Glyma.12G019700.1 Cinnamyl-alcohol dehydrogenase (CAD) 8 172 -4.7586 TR45798|c0_g1 chr1.CM0371.40.nc Glyma.16G039500.1 Transferase family 21 432 -4.7098                   104    Table S18. Similarly Expressed Trinity Results: Genes for enzymes annotated to GO:0009813 (Lignin  process, FDR=1). The first two columns list Trinity contigs expresand gene names as annotated by BLASTing the Lotus japonicus genome. The third column is their soybean BLAST homolog, followed by expression at two stages of flower development. Trinity contig L. japonicus ID G. max BLAST ID Gene description   Counts per gene (Yellow) Counts per gene (Red) Log2FC TR4638|c1_g1 chr2.CM0249.1510.r2.m Glyma.11G069600.1 LACCASE 0.49 0.28 0.5912 TR13756|c0_g1 chr6.CM0084.30.r2.m Glyma.18G065100.1 LACCASE 8.82 0 10.251 TR2439|c0_g1 chr1.CM2049.30.r2.m Glyma.18G193400.1 LACCASE 3.37 0.6 2.2824 TR35330|c1_g1 chr1.CM2049.30.r2.m Glyma.18G193200.1 LACCASE 3.99 0.59 2.5184 TR48670|c0_g2 chr1.CM2049.30.r2.m Glyma.07G142400.1 LACCASE 2.73 0 5.6687 TR34461|c0_g1 chr1.CM0147.370.r2.m Glyma.08G359100.1 LACCASE 7.9 0 6.4344 TR34461|c1_g1 chr1.CM0147.370.r2.m Glyma.18G177400.1 LACCASE 4.28 0 8.2967 TR34461|c2_g1 chr1.CM0147.370.r2.m Glyma.07G133900.1 LACCASE 12.03 0 5.6687 TR13756|c0_g2 chr6.CM0084.30.r2.m Glyma.18G065100.1 LACCASE 0 1.77 -6.5269 TR65076|c0_g1 chr6.CM0084.30.r2.m Glyma.02G224800.1 LACCASE 0.59 6.62 -3.6477 TR62643|c0_g1 chr3.CM0243.390.r2.m Glyma.07G054200.1 LACCASE 0.59 2.87 -2.2060 TR36532|c0_g1 chr6.LjT35D18.30.r2.m Glyma.15G190500.1 Methionine adenosyltransferase (MAT) 1483.98 252.36 2.3539 TR45709|c0_g1 chr6.LjT35D18.30.r2.m Glyma.17G039000.1 Methionine adenosyltransferase (MAT) 112.08 58.48 0.7446 TR64116|c0_g1 LjSGA_030253.1 Glyma.13G141600.1 Methionine adenosyltransferase (MAT) 40.05 69.47 -0.6148 TR13393|c0_g1 LjSGA_030253.1 Glyma.03G184000.1 Methionine adenosyltransferase (MAT) 101.29 317.51 -1.8553 105    Trinity contig L. japonicus ID G. max BLAST ID Gene description   Counts per gene (Yellow) Counts per gene (Red) Log2FC        TR24590|c0_g1 LjSGA_140570.1 Glyma.13G141600.1 Methionine adenosyltransferase (MAT) 21.61 58.23 -1.6092 TR13516|c0_g1 LjSGA_035751.3 Glyma.07G089700.1 Cytochrome P450 CYP2 subfamily 25.96 16.48 0.4589 TR15440|c0_g1 LjSGA_035751.3 Glyma.09G186400.1 Cytochrome P450 CYP2 subfamily 6.11 10.04 -0.7962 TR37032|c0_g1 LjSGA_021748.1.1 Glyma.07G023700.1 cinnamoyl-CoA reductase (CCR) 7.23 1.2 2.4086 TR4013|c1_g1 LjSGA_073761.1 Glyma.12G019700.1 Cinnamyl-alcohol dehydrogenase (CAD) 24.28 21.27 0.0141 TR39650|c0_g1 chr2.CM0249.890.r2.m Glyma.11G072000.1 Peroxidase (POD) 5.78 1.47 1.7657 TR39650|c0_g2 chr2.CM0249.890.r2.m Glyma.11G072000.1 Peroxidase (POD) 0.52 0.01 5.4510 TR10667|c0_g1 chr4.CM0131.580.r2.m Glyma.02G052700.1 Peroxidase (POD) 7.12 0 4.4836 TR47026|c0_g1 chr1.CM1911.100.r2.m Glyma.03G122000.1 coumaroylquinate(coumaroylshikimate) 3'-monooxygenase (C3’H) 38.65 16.98 0.9787 TR47026|c0_g2 chr1.CM1911.100.r2.m Glyma.03G122000.1 coumaroylquinate(coumaroylshikimate) 3'-monooxygenase (C3’H) 182.35 58.95 1.4244 TR31562|c0_g1 chr1.CM0017.470.r2.a Glyma.08G362900.1 Transaldolase. 3.1 0 4.4836   chr1.CM0017.470.r2.a Glyma.08G362900.1 Transaldolase. 318.62 69.82 1.9860 TR123|c0_g1 chr2.CM0191.680.r2.m Glyma.04G227700.1 Quercetin 3-O-methyltransferase  (3-OMT) 0.27 1.84 -2.7781 106    Trinity contig L. japonicus ID G. max BLAST ID Gene description   Counts per gene (Yellow) Counts per gene (Red) Log2FC        TR39944|c0_g1 chr3.CM0574.80.r2.d Glyma.19G182300.1 Phenylalanine ammonia-lyase (PAL) 1.33 2.63 -1.0588 TR49219|c1_g1 chr3.CM0574.80.r2.d Glyma.03G181600.1 Phenylalanine ammonia-lyase (PAL) 0 16.69 -7.1991 TR36182|c0_g1 chr2.CM0177.130.r2.d Glyma.01G020900.1 L-THREONINE 3-DEHYDROGENASE (TDH) 27.5 43.99 -0.8770 TR50128|c0_g2 chr6.CM0055.420.r2.m Glyma.15G059500.1 L-THREONINE 3-DEHYDROGENASE (TDH) 1.62 3.31 -1.2317 TR50128|c0_g1 LjSGA_070619.1 Glyma.15G059500.1 L-THREONINE 3-DEHYDROGENASE (TDH) 15.98 4.57 1.6040 TR34200|c0_g1 chr6.CM0055.420.r2.m Glyma.15G059500.1 L-THREONINE 3-DEHYDROGENASE (TDH) 5.23 3.64 0.3340 TR63600|c0_g2 chr6.CM0139.260.r2.m Glyma.15G106000.1 GLYCOSYL HYDROLASE 2.21 2.27 -0.2523 TR63600|c0_g1 chr6.CM0139.260.r2.m Glyma.15G106000.1 GLYCOSYL HYDROLASE 1.45 0.87 0.5441 TR53088|c0_g1 chr1.CM0104.480.r2.a Glyma.16G050200.1 V-type H+-transporting ATPase subunit C 108.06 64.97 0.5319 TR20842|c1_g1 LjSGA_014979.1 Glyma.18G104100.1 Transferase family 1731.14 1737.77 1.4098 TR40987|c0_g1 chr6.LjT34E09.150.r2.m Glyma.13G073200.1 elongation factor EF-1 beta subunit 114.07 34.18 1.5444 TR12259|c0_g1 LjSGA_075859.1 Glyma.20G175500.1 No apical meristem (NAM) protein 2.65 6.65 -1.5147 107           Trinity contig L. japonicus ID G. max BLAST ID Gene description   Counts per gene (Yellow) Counts per gene (Red) Log2FC        TR21604|c0_g2 chr2.CM0249.600.r2.d Glyma.01G169200.1 ferulate-5-hydroxylase 268.84 239.65 -0.0414 TR60250|c0_g1 chr6.LjT35D18.30.r2.m NA  5.93 0.89 2.5390 TR40104|c1_g1 LjSGA_035655.2 NA  7.01 2.59 1.3203 TR13073|c0_g1 chr3.CM0792.180.r2.d NA  3.01 0 5.6687 TR32183|c1_g1 chr3.CM0792.180.r2.d NA  3.99 1.27 1.5431 TR54702|c0_g1 chr3.CM0792.180.r2.d NA  6.21 0 4.8824 TR42774|c0_g1 chr2.CM0249.600.r2.d NA  5.72 3.81 0.6162 TR13250|c0_g1 chr6.CM0139.240.r2.m NA  2.3 0 5.6687 TR26810|c0_g1 chr6.CM0139.240.r2.m NA  9.59 0 5.1945 TR26810|c1_g1 chr6.CM0139.240.r2.m NA  4.9 0 3.9305 TR35810|c0_g1 chr6.CM0139.240.r2.m NA  3.96 0.52 2.6856 TR8127|c0_g2 chr6.CM0139.240.r2.m NA  10.45 0 5.4510 TR34627|c0_g1 chr1.CM0023.30.r2.d NA  1.62 0.95 0.6162 TR61337|c0_g2 chr5.CM0200.3250.r2.m Glyma.20G128600.1  11.69 1.97 2.3536 TR61337|c0_g4 chr5.CM0200.3250.r2.m Glyma.20G128600.1  5.23 4.36 0.0739 TR60726|c0_g1 chr4.CM0042.2100.r2.m NA  5.45 0 5.19459 TR34461|c3_g1 chr1.CM0147.370.r2.m NA  6.89 0 4.4836 108    Trinity contig L. japonicus ID G. max BLAST ID Gene description   Counts per gene (Yellow) Counts per gene (Red) Log2FC        TR10653|c0_g1 chr3.LjT45M09.50.r2.d NA  2.36 1.12 0.9283 TR19271|c0_g1 chr3.LjT45M09.50.r2.d NA  5.23 0 4.8824 TR65913|c1_g1 chr2.CM0191.680.r2.m NA  1.2 9.4 -2.7781 TR36166|c0_g1 LjSGA_035751.3 NA  0 4.07 -4.2662 TR5553|c0_g1 chr3.CM0452.240.r2.d NA  2.23 3.14 -0.3171 TR37416|c0_g1 chr6.CM0084.30.r2.m NA  0 6.27 -5.2282 TR3728|c0_g1 chr1.CM0476.410.r2.a NA  2.45 3.5 -0.3171 TR45016|c0_g1 LjSGA_073761.1 NA  1.36 3.22 -1.3166                      109    Table S19. Tophat Results: Significantly enriched GO terms corresponding to the red stage of a L. sessilifolius flower.  Enriched GO Term: Description Number of associated genes FDR GO:0034654nucleobase, nucleoside, nucleotide and nucleic acid biosynthetic process 21 5.00E-22 GO:0010035response to inorganic substance 36 1.80E-14 GO:0055114oxidation reduction 41 1.10E-12 GO:0034641cellular nitrogen compound metabolic process 52 2.20E-12 GO:0046686response to cadmium ion 20 4.00E-12 GO:0010038response to metal ion 29 4.60E-12 GO:0055085transmembrane transport 42 5.40E-12 GO:0018130heterocycle biosynthetic process 25 5.30E-11 GO:0019438aromatic compound biosynthetic process 36 1.90E-09 GO:0006725cellular aromatic compound metabolic process 47 3.30E-09 GO:0009611response to wounding 28 4.80E-08 GO:0009718anthocyanin biosynthetic process 12 5.60E-08 GO:0044271cellular nitrogen compound biosynthetic process 36 6.50E-08 GO:0031540regulation of anthocyanin biosynthetic process 8 4.50E-07 GO:0046283anthocyanin metabolic process 12 7.70E-07 GO:0031537regulation of anthocyanin metabolic process 8 6.70E-06 GO:0034655nucleobase, nucleoside, nucleotide and nucleic acid catabolic process 6 9.10E-06 GO:0034656nucleobase, nucleoside and nucleotide catabolic process 6 9.10E-06 GO:0042221response to chemical stimulus 104 1.10E-05 GO:0046483heterocycle metabolic process 40 1.40E-05 GO:0009962regulation of flavonoid biosynthetic process 9 2.00E-05 GO:0048610reproductive cellular process 9 5.90E-05 GO:0048868pollen tube development 12 6.50E-05 GO:0009605response to external stimulus 42 6.70E-05 GO:0009753response to jasmonic acid stimulus 23 0.00012 GO:0009651response to salt stress 27 0.00015 GO:0006970response to osmotic stress 31 0.00019 GO:0046148pigment biosynthetic process 16 0.00059 GO:0042440pigment metabolic process 17 0.00085 GO:0010039response to iron ion 6 0.001 GO:0031668cellular response to extracellular stimulus 13 0.0016 GO:0005984disaccharide metabolic process 9 0.0023 GO:0016036cellular response to phosphate starvation 8 0.0025 GO:0006811ion transport 35 0.0033 110    Enriched GO Term: Description Number of associated genes FDR    GO:0006814sodium ion transport 6 0.0033 GO:0035295tube development 12 0.0035 GO:0016137glycoside metabolic process 11 0.0035 GO:0042398cellular amino acid derivative biosynthetic process 23 0.0037 GO:0009813flavonoid biosynthetic process 15 0.0041 GO:0043455regulation of secondary metabolic process 9 0.0042 GO:0031669cellular response to nutrient levels 11 0.0043 GO:0009860pollen tube growth 9 0.0043 GO:0009719response to endogenous stimulus 57 0.0051 GO:0006820anion transport 13 0.0058 GO:0060918auxin transport 10 0.0058 GO:0009812flavonoid metabolic process 15 0.0066 GO:0046700heterocycle catabolic process 8 0.0085 GO:0015698inorganic anion transport 9 0.0087 GO:0010224response to UV-B 9 0.01 GO:0010033response to organic substance 64 0.011 GO:0006595polyamine metabolic process 5 0.012 GO:0042742defense response to bacterium 17 0.012 GO:0009311oligosaccharide metabolic process 9 0.013 GO:0009310amine catabolic process 9 0.013 GO:0006979response to oxidative stress 25 0.013 GO:0009737response to abscisic acid stimulus 26 0.013 GO:0009914hormone transport 10 0.013 GO:0009932cell tip growth 10 0.018 GO:0009267cellular response to starvation 9 0.019 GO:0009926auxin polar transport 9 0.019 GO:0019439aromatic compound catabolic process 5 0.023 GO:0009856pollination 12 0.027 GO:0005991trehalose metabolic process 5 0.028 GO:0009699phenylpropanoid biosynthetic process 17 0.032 GO:0006575cellular amino acid derivative metabolic process 25 0.035 GO:0009991response to extracellular stimulus 14 0.035 GO:0009116nucleoside metabolic process 8 0.035 GO:0009725response to hormone stimulus 50 0.035 GO:0048588developmental cell growth 10 0.038 GO:0009063cellular amino acid catabolic process 8 0.039 111    Enriched GO Term: Description Number of associated genes FDR    GO:0032549ribonucleoside binding 32 2.40E-34 GO:0016210naringenin-chalcone synthase activity 8 6.70E-06 GO:0016791phosphatase activity 20 0.0073 GO:0022891substrate-specific transmembrane transporter activity 43 0.021 GO:0042578phosphoric ester hydrolase activity 21 0.021 GO:0015103inorganic anion transmembrane transporter activity 9 0.021 GO:0008509anion transmembrane transporter activity 11 0.049 GO:0005773vacuole 55 5.40E-23 GO:0005911cell-cell junction 24 6.70E-23 GO:0030054cell junction 24 4.50E-22 GO:0005774vacuolar membrane 28 1.40E-09 GO:0044437vacuolar part 28 2.10E-09 GO:0009941chloroplast envelope 17 3.40E-06 GO:0005886plasma membrane 68 4.50E-06 GO:0009705plant-type vacuole membrane 12 1.00E-05 GO:0000325plant-type vacuole 15 1.20E-05 GO:0048046apoplast 18 7.40E-05 GO:0009526plastid envelope 17 9.00E-05 GO:0016020membrane 158 0.00033 GO:0044459plasma membrane part 27 0.0004 GO:0009570chloroplast stroma 13 0.015 GO:0005777peroxisome 10 0.016 GO:0042579microbody 10 0.016 GO:0005802trans-Golgi network 7 0.017 GO:0005618cell wall 25 0.048             112    Table S20. Tophat Results: Significantly enriched GO terms corresponding to the yellow stage of a L. sessilifolius flower.  Enriched GO Term: Description Number of associated genes FDR GO:0055114oxidation reduction  43 2.10E-20 GO:0034654nucleobase, nucleoside, nucleotide and nucleic acid biosynthetic process 15 1.80E-16 GO:0034641cellular nitrogen compound metabolic process 44 4.80E-14 GO:0018130heterocycle biosynthetic process 24 8.80E-14 GO:0044271cellular nitrogen compound biosynthetic process 34 3.10E-11 GO:0046686response to cadmium ion 15 8.40E-10 GO:0016053organic acid biosynthetic process 34 4.00E-09 GO:0046394carboxylic acid biosynthetic process 34 4.00E-09 GO:0019438aromatic compound biosynthetic process 28 8.00E-09 GO:0006725cellular aromatic compound metabolic process 35 4.70E-08 GO:0043648dicarboxylic acid metabolic process 14 2.80E-07 GO:0043436oxoacid metabolic process 46 3.70E-07 GO:0006082organic acid metabolic process 46 3.70E-07 GO:0019752carboxylic acid metabolic process 46 3.70E-07 GO:0010035response to inorganic substance 20 4.20E-07 GO:0042180cellular ketone metabolic process 46 5.10E-07 GO:0010038response to metal ion 17 1.90E-06 GO:0046483heterocycle metabolic process 32 3.20E-06 GO:0009073aromatic amino acid family biosynthetic process 10 3.20E-06 GO:0046417chorismate metabolic process 10 3.20E-06 GO:0006633fatty acid biosynthetic process 17 3.30E-06 GO:0009072aromatic amino acid family metabolic process 11 8.40E-06 GO:0006631fatty acid metabolic process 20 1.30E-05 GO:0008610lipid biosynthetic process 28 1.60E-05 GO:0032787monocarboxylic acid metabolic process 28 2.20E-05 GO:0030244cellulose biosynthetic process 8 0.00015 GO:0010025wax biosynthetic process 7 0.00018 GO:0010102lateral root morphogenesis 9 0.00026 113    Enriched GO Term: Description Number of associated genes FDR    GO:0010101post-embryonic root morphogenesis 9 0.00026 GO:0009886post-embryonic morphogenesis 11 0.00042 GO:0010311lateral root formation 6 0.00042 GO:0010166wax metabolic process 7 0.00042 GO:0019439aromatic compound catabolic process 6 0.00046 GO:0006073cellular glucan metabolic process 14 0.00059 GO:0030243cellulose metabolic process 8 0.00062 GO:0006629lipid metabolic process 36 0.00071 GO:0044255cellular lipid metabolic process 31 0.00079 GO:0044042glucan metabolic process 14 0.0011 GO:0009309amine biosynthetic process 15 0.0015 GO:0008652cellular amino acid biosynthetic process 14 0.0017 GO:0044262cellular carbohydrate metabolic process 26 0.0026 GO:0009250glucan biosynthetic process 10 0.0042 GO:0008299isoprenoid biosynthetic process 10 0.0042 GO:0000162tryptophan biosynthetic process 5 0.0046 GO:0009664plant-type cell wall organization 8 0.0046 GO:0046219indolalkylamine biosynthetic process 5 0.0046 GO:0009416response to light stimulus 27 0.0046 GO:0044264cellular polysaccharide metabolic process 15 0.0057 GO:0009314response to radiation 27 0.007 GO:0016109tetraterpenoid biosynthetic process 5 0.0095 GO:0016117carotenoid biosynthetic process 5 0.0095 GO:0042401cellular biogenic amine biosynthetic process 6 0.012 GO:0006586indolalkylamine metabolic process 5 0.014 GO:0016114terpenoid biosynthetic process 8 0.014 GO:0006568tryptophan metabolic process 5 0.014 GO:0010015root morphogenesis 12 0.014 GO:0009555pollen development 10 0.014 GO:0042221response to chemical stimulus 63 0.014 114    Enriched GO Term: Description Number of associated genes FDR    GO:0051273beta-glucan metabolic process 5 0.014 GO:0051274beta-glucan biosynthetic process 5 0.014 GO:0044106cellular amine metabolic process 20 0.015 GO:0010218response to far red light 6 0.016 GO:0048527lateral root development 9 0.019 GO:0009628response to abiotic stimulus 48 0.02 GO:0005976polysaccharide metabolic process 16 0.02 GO:0006720isoprenoid metabolic process 10 0.021 GO:0033692cellular polysaccharide biosynthetic process 11 0.023 GO:0048528post-embryonic root development 9 0.024 GO:0042435indole derivative biosynthetic process 5 0.029 GO:0034285response to disaccharide stimulus 5 0.029 GO:0006520cellular amino acid metabolic process 19 0.032 GO:0000271polysaccharide biosynthetic process 11 0.036 GO:0016108tetraterpenoid metabolic process 5 0.037 GO:0016116carotenoid metabolic process 5 0.037 GO:0042434indole derivative metabolic process 5 0.039 GO:0042430indole and derivative metabolic process 5 0.039 GO:0030258lipid modification 6 0.039 GO:0019321pentose metabolic process 5 0.048 GO:0048229gametophyte development 13 0.048 GO:0034440lipid oxidation 5 0.049 GO:0032549ribonucleoside binding 27 3.90E-32 GO:0051213dioxygenase activity 10 5.30E-05 GO:0016835carbon-oxygen lyase activity 11 0.0075 GO:0005507copper ion binding 11 0.0077 GO:0016705oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen 13 0.025 GO:0043167ion binding 81 0.025 GO:0016760cellulose synthase (UDP-forming) activity 5 0.025 115    Enriched GO Term: Description Number of associated genes FDR    GO:0044434chloroplast part 69 1.80E-35 GO:0009941chloroplast envelope 41 5.80E-35 GO:0044435plastid part 70 4.20E-33 GO:0009526plastid envelope 43 5.30E-33 GO:0009570chloroplast stroma 43 1.10E-32 GO:0005911cell-cell junction 27 4.30E-32 GO:0030054cell junction 27 4.90E-31 GO:0009532plastid stroma 43 9.40E-30 GO:0005886plasma membrane 70 3.20E-15 GO:0031967organelle envelope 44 2.00E-14 GO:0031975envelope 44 8.00E-14 GO:0009507chloroplast 81 2.40E-12 GO:0009536plastid 83 4.50E-11 GO:0009534chloroplast thylakoid 26 8.10E-11 GO:0031976plastid thylakoid 26 3.80E-10 GO:0031984organelle subcompartment 26 5.50E-10 GO:0044459plasma membrane part 30 2.00E-09 GO:0048046apoplast 20 3.60E-09 GO:0009579thylakoid 28 5.00E-07 GO:0016020membrane 121 9.10E-07 GO:0009535chloroplast thylakoid membrane 18 4.00E-06 GO:0055035plastid thylakoid membrane 18 1.10E-05 GO:0044446intracellular organelle part 95 1.20E-05 GO:0044422organelle part 95 1.20E-05 GO:0005576extracellular region 28 2.00E-05 GO:0042651thylakoid membrane 18 4.00E-05 GO:0044436thylakoid part 19 4.70E-05 GO:0005618cell wall 23 0.00072 GO:0005802trans-Golgi network 7 0.0012 GO:0005773vacuole 17 0.0019 116    Enriched GO Term: Description Number of associated genes FDR    GO:0030312external encapsulating structure 23 0.002 GO:0034357photosynthetic membrane 18 0.0025 GO:0044444cytoplasmic part 135 0.0026 GO:0010287plastoglobule 6 0.0081 GO:0022626cytosolic ribosome 9 0.02                               117    Table S21. Red Stage TopHat Results: Genes for enzymes annotated to GO:0009813 (Flavonoid biosynthetic process, FDR = 0.0066) .  The first column lists differentially expressed (FDR<0.005) assembled RNA-seq reads that have mapped to the Lotus japonicus genome. The second column is their soybean (Glycine max) BLAST homolog, followed by expression at two stages of flower development.  L. japonicus ID G. max BLAST ID G. max description Counts per gene (Yellow) Counts per gene (Red) Log2FC chr2.CM0021.2820.r2.m Glyma.11G027700.1 Ribonuclease T(2) / Ribonuclease T2 11 303 -4.87472 chr2.CM0608.560.r2.m Glyma.01G048200.1 NARINGENIN,2-OXOGLUTARATE 3-DIOXYGENASE 4 53 -3.79248 chr2.CM0018.1190.r2.m Glyma.01G228700.1  Naringenin-chalcone synthase / Flavonone synthase 0 16 -7.06546 chr2.CM0018.760.r2.m Glyma.01G228700.1  Naringenin-chalcone synthase / Flavonone synthase 1 103 -6.61962 chr1.LjT39K18.30.r2.m Glyma.04G147700.1  Naringenin-chalcone synthase / Flavonone synthase 78 686 -3.24183 chr2.CM0018.730.r2.m Glyma.01G228700.1  Naringenin-chalcone synthase / Flavonone synthase 0 83 -9.43178 chr2.CM0018.1300.r2.m Glyma.01G228700.1  Naringenin-chalcone synthase / Flavonone synthase 0 43 -8.48495 chr5.CM0077.110.r2.m Glyma.02G158700.1  Naringenin-chalcone synthase / Flavonone synthase 6 108 -4.248 chr1.CM0284.240.r2.m Glyma.01G228700.1  Naringenin-chalcone synthase / Flavonone synthase 7 316 -5.57781 chr1.CM0104.1140.r2.m Glyma.08G109300.1 Dihydrokaempferol 4-reductase / NADPH-dihydromyricetin reductase 27 108 -2.10201 chr2.CM0018.1150.r2.m Glyma.01G228700.1 Dihydrokaempferol 4-reductase / NADPH-dihydromyricetin reductase 0 20 -7.38524 chr4.CM0119.240.r2.m Glyma.01G166200.1 Thromboxane-A synthase / Thromboxane synthetase 11 82 -2.99064 chr1.CM0133.560.r2.m Glyma.04G052100.1 Heme oxygenase (biliverdin-producing) / Heme oxygenase (decyclizing) 0 31 -8.01444 chr5.CM0077.210.r2.m Glyma.02G158700.1  Naringenin-chalcone synthase / Flavonone synthase 1 27 -4.69274 chr2.CM0018.1200.r2.m Glyma.01G228700.1 LEUCOANTHOCYANIDIN DIOXYGENASE-RELATED 1 83 -6.30857  118     *Flavonoid biosynthetic process was not found to be enriched at the yellow stage of floral colour change in L. sessilifolius, given the TopHat results.  *Lignin biosynthetic process was not found to be expressed at the yellow or red stages of floral colour change in L. sessilifolius, given the TopHat results.  Table S22. STAR Results: Significantly enriched GO terms corresponding to the yellow stage of a L. sessilifolius  flower.   Enriched GO Term: Description Number of associated genes FDR GO:0044281small molecule metabolic process  105 4.90E-11 GO:0008610lipid biosynthetic process 40 1.10E-08 GO:0044283small molecule biosynthetic process 57 1.30E-08 GO:0006629lipid metabolic process 57 1.30E-08 GO:0046394carboxylic acid biosynthetic process 41 4.80E-08 GO:0043436oxoacid metabolic process 58 4.80E-08 GO:0016053organic acid biosynthetic process 41 4.80E-08 GO:0006082organic acid metabolic process 58 4.80E-08 GO:0019752carboxylic acid metabolic process 58 4.80E-08 GO:0042180cellular ketone metabolic process 59 5.60E-08 GO:0044255cellular lipid metabolic process 41 1.50E-06 GO:0006633fatty acid biosynthetic process 22 9.80E-06 GO:0032787monocarboxylic acid metabolic process 34 1.30E-05 GO:0006631fatty acid metabolic process 25 2.00E-05 GO:0050896response to stimulus 144 3.20E-05 GO:0055114oxidation reduction 63 3.20E-05 GO:0046417chorismate metabolic process 11 3.90E-05 GO:0009073aromatic amino acid family biosynthetic process 11 3.90E-05 GO:0043648dicarboxylic acid metabolic process 15 4.30E-05 GO:0009628response to abiotic stimulus 70 6.20E-05 GO:0042221response to chemical stimulus 93 7.70E-05 GO:0008152metabolic process 288 0.00014 GO:0009072aromatic amino acid family metabolic process 12 0.00037 GO:0008299isoprenoid biosynthetic process 14 0.00046 GO:0010025wax biosynthetic process 8 0.00047 GO:0010166wax metabolic process 8 0.00047 GO:0009058biosynthetic process 135 0.00065 119    Enriched GO Term: Description Number of associated genes FDR    GO:0044262cellular carbohydrate metabolic process 38 0.0008 GO:0006720isoprenoid metabolic process 14 0.0021 GO:0006950response to stress 90 0.0021 GO:0044264cellular polysaccharide metabolic process 17 0.0025 GO:0044249cellular biosynthetic process 128 0.003 GO:0005976polysaccharide metabolic process 18 0.0041 GO:0009416response to light stimulus 33 0.006 GO:0030244cellulose biosynthetic process 7 0.006 GO:0016114terpenoid biosynthetic process 10 0.0063 GO:0006519cellular amino acid and derivative metabolic process 36 0.0063 GO:0016126sterol biosynthetic process 5 0.0063 GO:0033692cellular polysaccharide biosynthetic process 12 0.0063 GO:0005975carbohydrate metabolic process 47 0.0063 GO:0006073cellular glucan metabolic process 14 0.0063 GO:0009314response to radiation 33 0.0068 GO:0044042glucan metabolic process 14 0.0071 GO:0000271polysaccharide biosynthetic process 12 0.0071 GO:0030243cellulose metabolic process 7 0.0071 GO:0016109tetraterpenoid biosynthetic process 6 0.012 GO:0009607response to biotic stimulus 40 0.012 GO:0016117carotenoid biosynthetic process 6 0.012 GO:0046148pigment biosynthetic process 13 0.012 GO:0010038response to metal ion 28 0.013 GO:0009657plastid organization 13 0.013 GO:0006520cellular amino acid metabolic process 24 0.013 GO:0010218response to far red light 7 0.013 GO:0010102lateral root morphogenesis 9 0.014 GO:0010101post-embryonic root morphogenesis 9 0.014 GO:0044237cellular metabolic process 220 0.014 GO:0071669plant-type cell wall organization or biogenesis 12 0.014 GO:0051707response to other organism 38 0.014 GO:0042214terpene metabolic process 7 0.014 GO:0008652cellular amino acid biosynthetic process 15 0.014 GO:0006066alcohol metabolic process 23 0.015 GO:0019748secondary metabolic process 24 0.015 GO:0034641cellular nitrogen compound metabolic process 58 0.015 120    Enriched GO Term: Description Number of associated genes FDR    GO:0009309amine biosynthetic process 16 0.015 GO:0007275multicellular organismal development 74 0.015 GO:0009250glucan biosynthetic process 9 0.015 GO:0009719response to endogenous stimulus 46 0.015 GO:0044106cellular amine metabolic process 25 0.015 GO:0010015root morphogenesis 15 0.015 GO:0016108tetraterpenoid metabolic process 6 0.015 GO:0016116carotenoid metabolic process 6 0.015 GO:0032502developmental process 78 0.015 GO:0010035response to inorganic substance 32 0.016 GO:0044238primary metabolic process 227 0.016 GO:0000162tryptophan biosynthetic process 5 0.016 GO:0010214seed coat development 5 0.016 GO:0046219indolalkylamine biosynthetic process 5 0.016 GO:0007017microtubule-based process 12 0.016 GO:0032501multicellular organismal process 76 0.016 GO:0006721terpenoid metabolic process 10 0.017 GO:0046246terpene biosynthetic process 6 0.017 GO:0042335cuticle development 5 0.019 GO:0051704multi-organism process 44 0.02 GO:0019438aromatic compound biosynthetic process 38 0.021 GO:0044271cellular nitrogen compound biosynthetic process 43 0.022 GO:0009834secondary cell wall biogenesis 5 0.024 GO:0046686response to cadmium ion 22 0.027 GO:0010033response to organic substance 53 0.029 GO:0016125sterol metabolic process 5 0.029 GO:0048528post-embryonic root development 10 0.029 GO:0009110vitamin biosynthetic process 7 0.031 GO:0009308amine metabolic process 26 0.032 GO:0006586indolalkylamine metabolic process 5 0.032 GO:0051273beta-glucan metabolic process 5 0.032 GO:0051274beta-glucan biosynthetic process 5 0.032 GO:0042440pigment metabolic process 13 0.032 GO:0006568tryptophan metabolic process 5 0.032 GO:0071704organic substance metabolic process 67 0.033 GO:0006766vitamin metabolic process 7 0.033 121    Enriched GO Term: Description Number of associated genes FDR    GO:0071554cell wall organization or biogenesis 18 0.034 GO:0009886post-embryonic morphogenesis 12 0.039 GO:0016051carbohydrate biosynthetic process 16 0.041 GO:0009611response to wounding 15 0.047 GO:0003824catalytic activity 281 1.20E-11 GO:0016491oxidoreductase activity 70 6.70E-05 GO:0005506iron ion binding 27 0.019 GO:0004506squalene monooxygenase activity 5 0.019 GO:0004497monooxygenase activity 11 0.025 GO:0016740transferase activity 100 0.033 GO:0046527glucosyltransferase activity 9 0.042 GO:0016760cellulose synthase (UDP-forming) activity 5 0.047 GO:0016759cellulose synthase activity 5 0.047 GO:0044435plastid part 91 1.20E-19 GO:0044434chloroplast part 90 1.20E-19 GO:0009507chloroplast 113 6.70E-17 GO:0009536plastid 115 8.30E-16 GO:0009526plastid envelope 56 8.90E-15 GO:0009570chloroplast stroma 57 8.90E-15 GO:0009532plastid stroma 57 2.30E-14 GO:0009941chloroplast envelope 53 1.20E-13 GO:0031967organelle envelope 57 1.90E-10 GO:0031975envelope 57 2.00E-10 GO:0044464cell part 311 2.00E-09 GO:0005623cell 311 2.00E-09 GO:0044444cytoplasmic part 197 3.00E-09 GO:0043229intracellular organelle 223 3.00E-08 GO:0043226organelle 223 3.20E-08 GO:0005737cytoplasm 204 3.20E-08 GO:0043231intracellular membrane-bounded organelle 212 3.60E-08 GO:0043227membrane-bounded organelle 212 4.50E-08 GO:0044446intracellular organelle part 126 7.30E-08 GO:0044422organelle part 126 9.90E-08 GO:0009579thylakoid 36 7.30E-07 122    Enriched GO Term: Description Number of associated genes FDR    GO:0044424intracellular part 242 8.90E-07 GO:0005622intracellular 243 4.10E-06 GO:0005618cell wall 39 6.30E-06 GO:0030312external encapsulating structure 39 7.20E-06 GO:0031976plastid thylakoid 29 9.80E-06 GO:0009534chloroplast thylakoid 29 9.80E-06 GO:0031984organelle subcompartment 29 1.00E-05 GO:0005576extracellular region 36 6.00E-05 GO:0048046apoplast 26 0.0003 GO:0044436thylakoid part 23 0.00082 GO:0016020membrane 154 0.0015 GO:0005886plasma membrane 83 0.0059 GO:0031977thylakoid lumen 8 0.0061 GO:0009535chloroplast thylakoid membrane 19 0.0061 GO:0055035plastid thylakoid membrane 19 0.0062 GO:0010287plastoglobule 7 0.01 GO:0042651thylakoid membrane 19 0.011 GO:0034357photosynthetic membrane 19 0.02                  123    Table S23. STAR Results: Significantly enriched GO terms corresponding to the red stage of a L. sessilifoliuss flower.   Enriched GO Term: Description Number of associated genes FDR GO:0009605response to external stimulus 60 1.60E-09 GO:0042221response to chemical stimulus 143 7.20E-08 GO:0009611response to wounding 34 4.90E-07 GO:0050896response to stimulus 209 9.70E-07 GO:0006810transport 123 9.70E-07 GO:0051234establishment of localization 123 1.00E-06 GO:0051179localization 126 1.60E-06 GO:0009753response to jasmonic acid stimulus 30 1.60E-06 GO:0010033response to organic substance 94 2.20E-06 GO:0009714chalcone metabolic process 11 2.70E-06 GO:0042181ketone biosynthetic process 11 2.70E-06 GO:0009715chalcone biosynthetic process 11 2.70E-06 GO:0009719response to endogenous stimulus 79 3.40E-06 GO:0031540regulation of anthocyanin biosynthetic process 11 2.50E-05 GO:0009725response to hormone stimulus 71 2.90E-05 GO:0009718anthocyanin biosynthetic process 14 3.30E-05 GO:0009962regulation of flavonoid biosynthetic process 12 4.00E-05 GO:0055085transmembrane transport 55 4.40E-05 GO:0006950response to stress 133 5.00E-05 GO:0009813flavonoid biosynthetic process 18 5.10E-05 GO:0043455regulation of secondary metabolic process 14 7.30E-05 GO:0009991response to extracellular stimulus 23 0.00011 GO:0031537regulation of anthocyanin metabolic process 11 0.00025 GO:0046283anthocyanin metabolic process 14 0.00047 GO:0006979response to oxidative stress 35 0.00054 GO:0009699phenylpropanoid biosynthetic process 20 0.00059 GO:0031668cellular response to extracellular stimulus 21 0.00059 GO:0009812flavonoid metabolic process 18 0.00059 GO:0042398cellular amino acid derivative biosynthetic process 27 0.00059 124    Enriched GO Term: Description Number of associated genes FDR    GO:0071496cellular response to external stimulus 21 0.00059 GO:0016137glycoside metabolic process 16 0.00088 GO:0005975carbohydrate metabolic process 67 0.0012 GO:0060918auxin transport 13 0.0012 GO:0009914hormone transport 13 0.0014 GO:0009056catabolic process 57 0.0014 GO:0009926auxin polar transport 12 0.0015 GO:0009628response to abiotic stimulus 87 0.0018 GO:0009629response to gravity 13 0.0018 GO:0031667response to nutrient levels 18 0.0018 GO:0044248cellular catabolic process 52 0.0019 GO:0046148pigment biosynthetic process 18 0.0028 GO:0016139glycoside catabolic process 7 0.0033 GO:0016036cellular response to phosphate starvation 12 0.0033 GO:0010224response to UV-B 14 0.0034 GO:0006811ion transport 41 0.0037 GO:0044283small molecule biosynthetic process 54 0.0038 GO:0019748secondary metabolic process 34 0.0038 GO:0006527arginine catabolic process 5 0.0051 GO:0051704multi-organism process 63 0.0074 GO:0042440pigment metabolic process 19 0.0082 GO:0042594response to starvation 15 0.0083 GO:0031669cellular response to nutrient levels 16 0.0093 GO:0006814sodium ion transport 7 0.0098 GO:0009737response to abscisic acid stimulus 33 0.01 GO:0009311oligosaccharide metabolic process 11 0.01 GO:0009733response to auxin stimulus 25 0.012 GO:0005984disaccharide metabolic process 10 0.012 GO:0009698phenylpropanoid metabolic process 21 0.013 GO:0009607response to biotic stimulus 53 0.016 125    Enriched GO Term: Description Number of associated genes FDR    GO:0006575cellular amino acid derivative metabolic process 29 0.016 GO:0046395carboxylic acid catabolic process 13 0.016 GO:0016054organic acid catabolic process 13 0.016 GO:0044282small molecule catabolic process 28 0.017 GO:0009267cellular response to starvation 14 0.018 GO:0009411response to UV 15 0.018 GO:0016052carbohydrate catabolic process 17 0.019 GO:0008216spermidine metabolic process 5 0.024 GO:0008295spermidine biosynthetic process 5 0.024 GO:0006595polyamine metabolic process 6 0.024 GO:0051707response to other organism 50 0.024 GO:0030001metal ion transport 23 0.024 GO:0005991trehalose metabolic process 6 0.024 GO:0006970response to osmotic stress 35 0.026 GO:0010817regulation of hormone levels 19 0.026 GO:0009065glutamine family amino acid catabolic process 5 0.031 GO:0009310amine catabolic process 10 0.032 GO:0044275cellular carbohydrate catabolic process 15 0.039 GO:0006820anion transport 15 0.039 GO:0015698inorganic anion transport 10 0.041 GO:0009414response to water deprivation 22 0.045 GO:0010035response to inorganic substance 41 0.047 GO:0030036actin cytoskeleton organization 11 0.047 GO:0005215transporter activity 79 2.20E-07 GO:0022857transmembrane transporter activity 69 2.20E-07 GO:0022891substrate-specific transmembrane transporter activity 59 1.40E-06 GO:0022892substrate-specific transporter activity 62 1.40E-06 GO:0016210naringenin-chalcone synthase activity 11 1.90E-06 GO:0016798hydrolase activity, acting on glycosyl bonds 40 2.10E-05 GO:0004553hydrolase activity, hydrolyzing O-glycosyl compounds 38 2.70E-05 126    Enriched GO Term: Description Number of associated genes FDR    GO:0015291secondary active transmembrane transporter activity 26 3.20E-05 GO:0016791phosphatase activity 24 0.00012 GO:0042578phosphoric ester hydrolase activity 27 0.00015 GO:0016787hydrolase activity 152 0.00017 GO:0022804active transmembrane transporter activity 36 0.00021 GO:0003779actin binding 12 0.00066 GO:0015144carbohydrate transmembrane transporter activity 11 0.00073 GO:0003824catalytic activity 346 0.00073 GO:0015075ion transmembrane transporter activity 37 0.0041 GO:0015103inorganic anion transmembrane transporter activity 11 0.018 GO:0004650polygalacturonase activity 10 0.024 GO:0016746transferase activity, transferring acyl groups 26 0.024 GO:0008509anion transmembrane transporter activity 14 0.029 GO:0015294solute:cation symporter activity 7 0.031 GO:0015295solute:hydrogen symporter activity 6 0.031 GO:0008092cytoskeletal protein binding 15 0.031 GO:0008234cysteine-type peptidase activity 12 0.031 GO:0008324cation transmembrane transporter activity 26 0.031 GO:0051119sugar transmembrane transporter activity 7 0.031 GO:0046943carboxylic acid transmembrane transporter activity 10 0.031 GO:0015297antiporter activity 13 0.035 GO:0005342organic acid transmembrane transporter activity 10 0.041 GO:0015293symporter activity 7 0.041 GO:0005773vacuole 77 6.50E-07 GO:0000325plant-type vacuole 20 0.00014 GO:0016020membrane 229 0.00017 GO:0009705plant-type vacuole membrane 17 0.00075 GO:0044437vacuolar part 41 0.022 GO:0005774vacuolar membrane 41 0.022 GO:0044464cell part 397 0.023 127    Table S24. Yellow Stage STAR Results: Genes for enzymes annotated to GO:0009813 (Flavonoid biosynthetic process, FDR=1). The first column lists differentially expressed (FDR<0.005) assembled RNA-seq reads that have mapped to the Lotus japonicus genome. The second column is their soybean (Glycine max) BLAST homolog, followed by expression at two stages of flower development.   L. japonicus ID G. max BLAST ID G. max description Counts per gene (Yellow) Counts per gene (Red) Log2FC chr5.CM1077.590.r2.m Glyma.03G187000.1 Trans-zeatin O-beta-D-glucosyltransferase / Zeatin O-glucosyltransferase 11.82 0 6.526304 chr3.LjT10E18.60.r2.m Glyma.10G210600.1 B3 DNA binding domain (B3) // Auxin response factor (Auxin_resp) 43 6 2.664685 chr1.CM0410.460.r2.m Glyma.18G268100.1 Isoflavone-7-O-beta-glucoside 6''-O-malonyltransferase / Flavone/flavonol 7-O-beta-D-glucoside malonyltransferase 24.83 0 7.577035 chr3.CM0106.330.r2.m Glyma.13G344700.1 B-BOX TYPE ZINC FINGER-CONTAINING PROTEIN-RELATED 157 18 2.963609 chr1.CM0476.410.r2.a Glyma.04G227700.1 FLAVONE 3'-O-METHYLTRANSFERASE 1 495 98 2.182379                   128    Table S25. Red Stage STAR Results: Genes for enzymes annotated to GO:0009813 (Flavonoid biosynthetic process, FDR = 0.00059) .  The first column lists differentially expressed (FDR<0.005) assembled RNA-seq reads that have mapped to the Lotus japonicus genome. The second column is their soybean (Glycine max) BLAST homolog, followed by expression at two stages of flower development.  L. japonicus ID G. max BLAST ID G. max description Counts per gene (Yellow) Counts per gene (Red) Log2FC chr2.CM0608.560.r2.m Glyma.01G048200.1 Ribonuclease T(2) / Ribonuclease T2 11 128 -3.67758 chr4.CM0119.240.r2.m Glyma.01G166200.1 NARINGENIN,2-OXOGLUTARATE 3-DIOXYGENASE 155.05 921.11 -2.72273 chr3.LjB14O06.120.r2.a Glyma.01G166200.1 NARINGENIN,2-OXOGLUTARATE 3-DIOXYGENASE 2.95 51.89 -4.20965 chr3.CM0590.770.r2.d Glyma.01G228700.1 Naringenin-chalcone synthase / Flavonone synthase 0 39.5 -8.40463 chr1.CM0284.250.r2.m Glyma.01G228700.1 Naringenin-chalcone synthase / Flavonone synthase 0 48.1 -8.66696 chr3.CM0590.840.r2.m Glyma.01G228700.1 Naringenin-chalcone synthase / Flavonone synthase 0 39.5 -8.40463 chr2.CM0018.1190.r2.m Glyma.01G228700.1 Naringenin-chalcone synthase / Flavonone synthase 8.81 182.32 -4.4707 chr2.CM0018.760.r2.m Glyma.01G228700.1 Naringenin-chalcone synthase / Flavonone synthase 2.85 307.57 -6.77327 chr2.CM0018.730.r2.m Glyma.01G228700.1 Naringenin-chalcone synthase / Flavonone synthase 8.15 367.98 -5.65332 chr2.CM0018.1300.r2.m Glyma.01G228700.1 Naringenin-chalcone synthase / Flavonone synthase 0 72.45 -9.25074 chr1.CM0284.240.r2.m Glyma.01G228700.1 Naringenin-chalcone synthase / Flavonone synthase 11.42 767.23 -6.25955 chr2.CM0018.1150.r2.m Glyma.01G228700.1 Naringenin-chalcone synthase / Flavonone synthase 5.77 256.13 -5.53724 chr2.CM0018.1200.r2.m Glyma.01G228700.1 Naringenin-chalcone synthase / Flavonone synthase 9.38 468.71 -5.83578 chr5.CM0077.110.r2.m Glyma.02G158700.1 Dihydrokaempferol 4-reductase / NADPH-dihydromyricetin reductase 20 321 -4.14841 chr1.CM0133.560.r2.m Glyma.04G052100.1 Thromboxane-A synthase / Thromboxane synthetase 0 41 -8.44015 chr1.LjT39K18.30.r2.m Glyma.04G147700.1 Heme oxygenase (biliverdin-producing) / Heme oxygenase (decyclizing) 115 1030 -3.31429 129    L. japonicus ID G. max BLAST ID G. max description Counts per gene (Yellow) Counts per gene (Red) Log2FC       chr1.CM0104.1140.r2.m Glyma.08G109300.1 Naringenin-chalcone synthase / Flavonone synthase 70 354 -2.48893 chr2.CM0021.2820.r2.m Glyma.11G027700.1 LEUCOANTHOCYANIDIN DIOXYGENASE-RELATED 14 833 -6.03436                          130    Table S26. Yellow Stage STAR Results: Genes for enzymes annotated to GO:0009808 (Lignin metabolic process, FDR=0.92). The first column lists differentially expressed (FDR<0.005) assembled RNA-seq reads that have mapped to the Lotus japonicus genome. The second column is their soybean (Glycine max) BLAST homolog, followed by expression at two stages of flower development.   L. japonicus ID G. max BLAST ID G. max description Counts per gene (Yellow) Counts per gene (Red) Log2FC chr6.CM0385.240.r2.d Glyma.09G038500.1 CHITINASE-LIKE PROTEIN 1 159 16 3.15060 chr6.CM0084.30.r2.m Glyma.02G224800.1 LACCASE-16 51 3 3.88250 chr4.CM0387.960.r2.m Glyma.07G258700.1 BETA-GLUCOSIDASE 45-RELATED 16 0 6.93742 chr2.CM0249.1520.r2.m Glyma.01G173500.1 LACCASE-11 46 1 5.21342 chr1.CM0476.410.r2.a Glyma.04G227700.1 FLAVONE 3'-O-METHYLTRANSFERASE 1 495 98 2.18237   *Lignin biosynthetic process was not found to be expressed at the red stage of floral colour change in L. sessilifolius, given the STAR results.                   131    Table S27. Trinity Results: Significantly enriched GO terms corresponding to the yellow stage of a L. sessilifolius flower.  Enriched GO category Number of associated genes FDR  GO:0034641cellular nitrogen compound metabolic process 80 4.90E-07 GO:0009791post-embryonic development 138 4.90E-07 GO:0009628response to abiotic stimulus 183 4.90E-07 GO:0043436oxoacid metabolic process 119 2.30E-06 GO:0006082organic acid metabolic process 119 2.30E-06 GO:0019752carboxylic acid metabolic process 119 2.30E-06 GO:0042180cellular ketone metabolic process 120 2.40E-06 GO:0006629lipid metabolic process 111 1.70E-05 GO:0009416response to light stimulus 87 2.80E-05 GO:0048610reproductive cellular process 15 2.80E-05 GO:0032787monocarboxylic acid metabolic process 71 3.50E-05 GO:0009606tropism 29 4.00E-05 GO:0009314response to radiation 88 4.10E-05 GO:0018130heterocycle biosynthetic process 28 0.0002 GO:0009734auxin mediated signaling pathway 28 0.0002 GO:0044262cellular carbohydrate metabolic process 77 0.0002 GO:0046394carboxylic acid biosynthetic process 64 0.0002 GO:0016053organic acid biosynthetic process 64 0.0002 GO:0046417chorismate metabolic process 16 0.0002 GO:0009073aromatic amino acid family biosynthetic process 16 0.0002 GO:0016114terpenoid biosynthetic process 23 0.00024 GO:0009072aromatic amino acid family metabolic process 19 0.00029 GO:0016108tetraterpenoid metabolic process 15 0.00039 GO:0016116carotenoid metabolic process 15 0.00039 GO:0009886post-embryonic morphogenesis 24 0.00043 GO:0046148pigment biosynthetic process 29 0.0006 GO:0016109tetraterpenoid biosynthetic process 12 0.00071 GO:0016117carotenoid biosynthetic process 12 0.00071 GO:0008299isoprenoid biosynthetic process 25 0.00073 GO:0010382cellular cell wall macromolecule metabolic process 8 0.00077 GO:0006631fatty acid metabolic process 41 0.0009 GO:0042440pigment metabolic process 31 0.00098 GO:0009629response to gravity 22 0.001 GO:0043648dicarboxylic acid metabolic process 20 0.0012 GO:0010876lipid localization 8 0.002 GO:0006721terpenoid metabolic process 25 0.0021 GO:0006720isoprenoid metabolic process 28 0.0021 132    Enriched GO category Number of associated genes FDR     GO:0046246terpene biosynthetic process 11 0.0031 GO:0044264cellular polysaccharide metabolic process 39 0.0037 GO:0008610lipid biosynthetic process 61 0.0038 GO:0019748secondary metabolic process 71 0.004 GO:0044271cellular nitrogen compound biosynthetic process 50 0.0046 GO:0006073cellular glucan metabolic process 30 0.0046 GO:0009605response to external stimulus 75 0.0063 GO:0019438aromatic compound biosynthetic process 44 0.0078 GO:0016051carbohydrate biosynthetic process 48 0.0079 GO:0009630gravitropism 19 0.0082 GO:0010166wax metabolic process 11 0.011 GO:0044042glucan metabolic process 30 0.012 GO:0010025wax biosynthetic process 10 0.013 GO:0009733response to auxin stimulus 50 0.014 GO:0009637response to blue light 15 0.014 GO:0050896response to stimulus 362 0.018 GO:0044255cellular lipid metabolic process 78 0.023 GO:0005976polysaccharide metabolic process 43 0.028 GO:0042214terpene metabolic process 12 0.028 GO:0006633fatty acid biosynthetic process 26 0.034 GO:0009266response to temperature stimulus 66 0.035 GO:0030244cellulose biosynthetic process 11 0.035 GO:0009638phototropism 11 0.038 GO:0005975carbohydrate metabolic process 106 0.038 GO:0009834secondary cell wall biogenesis 12 0.04 GO:0042546cell wall biogenesis 24 0.045 GO:0033692cellular polysaccharide biosynthetic process 27 0.05 GO:0016829lyase activity 55 0.017 GO:0016491oxidoreductase activity 152 0.017 GO:0035251UDP-glucosyltransferase activity 26 0.017 GO:0009055electron carrier activity 69 0.02 GO:0016860intramolecular oxidoreductase activity 12 0.02 GO:0008194UDP-glycosyltransferase activity 35 0.02 GO:0046527glucosyltransferase activity 28 0.02 GO:0016835carbon-oxygen lyase activity 23 0.028 GO:0016760cellulose synthase (UDP-forming) activity 10 0.029 GO:0008757S-adenosylmethionine-dependent methyltransferase activity 21 0.029 133    Enriched GO category Number of associated genes FDR     GO:0004497monooxygenase activity 37 0.038 GO:0016758transferase activity, transferring hexosyl groups 46 0.038 GO:0009507chloroplast 222 2.30E-12 GO:0009536plastid 234 1.50E-11 GO:0044434chloroplast part 80 2.50E-08 GO:0044435plastid part 85 2.30E-07 GO:0005618cell wall 74 5.00E-07 GO:0009505plant-type cell wall 65 1.00E-06 GO:0030312external encapsulating structure 76 1.70E-06 GO:0009535chloroplast thylakoid membrane 42 1.90E-06 GO:0055035plastid thylakoid membrane 42 1.10E-05 GO:0044444cytoplasmic part 485 1.50E-05 GO:0009534chloroplast thylakoid 42 3.40E-05 GO:0005737cytoplasm 542 9.40E-05 GO:0010287plastoglobule 17 9.40E-05 GO:0042651thylakoid membrane 42 9.80E-05 GO:0031976plastid thylakoid 42 0.00016 GO:0031984organelle subcompartment 42 0.00023 GO:0044436thylakoid part 42 0.001 GO:0044464cell part 901 0.001 GO:0005623cell 901 0.001 GO:0009941chloroplast envelope 21 0.0057 GO:0031225anchored to membrane 27 0.0064 GO:0009514glyoxysome 5 0.033 GO:0034357photosynthetic membrane 43 0.04 GO:0043227membrane-bounded organelle 561 0.049          134    Table S28. Trinity Results: Significantly enriched GO terms corresponding to the red stage of a L. sessilifolius flower. Enriched GO category: Description Number of associated genes FDR  GO:0050896response to stimulus 503 3.40E-08 GO:0010035response to inorganic substance 54 5.50E-08 GO:0048610reproductive cellular process 20 4.00E-07 GO:0042221response to chemical stimulus 284 6.20E-07 GO:0009414response to water deprivation 57 4.40E-06 GO:0009415response to water 58 6.00E-06 GO:0010033response to organic substance 197 6.00E-06 GO:0006950response to stress 325 1.40E-05 GO:0009719response to endogenous stimulus 168 1.50E-05 GO:0034641cellular nitrogen compound metabolic process 85 6.20E-05 GO:0009737response to abscisic acid stimulus 75 1.10E-04 GO:0010876lipid localization 11 1.10E-04 GO:0009624response to nematode 48 1.10E-04 GO:0006970response to osmotic stress 73 0.00015 GO:0009628response to abiotic stimulus 202 0.00019 GO:0009651response to salt stress 61 0.00019 GO:0009725response to hormone stimulus 151 0.00039 GO:0009607response to biotic stimulus 137 0.0013 GO:0005975carbohydrate metabolic process 140 0.0016 GO:0030001metal ion transport 52 0.0022 GO:0044262cellular carbohydrate metabolic process 87 0.0024 GO:0051707response to other organism 126 0.0024 GO:0042398cellular amino acid derivative biosynthetic process 54 0.0056 GO:0010038response to metal ion 32 0.01 GO:0043436oxoacid metabolic process 121 0.01 GO:0046174polyol catabolic process 8 0.01 GO:0006082organic acid metabolic process 121 0.01 GO:0009611response to wounding 41 0.01 GO:0019752carboxylic acid metabolic process 121 0.01 GO:0051704multi-organism process 152 0.012 GO:0006813potassium ion transport 19 0.017 GO:0042180cellular ketone metabolic process 121 0.017 GO:0006811ion transport 83 0.02 GO:0009605response to external stimulus 88 0.021 GO:0006575cellular amino acid derivative metabolic process 65 0.026 GO:0006519cellular amino acid and derivative metabolic process 107 0.026 GO:0009751response to salicylic acid stimulus 39 0.026 135    Enriched GO category Number of associated genes FDR     GO:0010107potassium ion import 6 0.034 GO:0044106cellular amine metabolic process 66 0.034 GO:0009991response to extracellular stimulus 33 0.049 GO:0016791phosphatase activity 50 0.00063 GO:0042578phosphoric ester hydrolase activity 56 0.0011 GO:0022891substrate-specific transmembrane transporter activity 116 0.0063 GO:0022857transmembrane transporter activity 140 0.008 GO:0015291secondary active transmembrane transporter activity 54 0.012 GO:0004553hydrolase activity, hydrolyzing O-glycosyl compounds 62 0.012 GO:0022892substrate-specific transporter activity 132 0.013 GO:0009055electron carrier activity 83 0.013 GO:0016798hydrolase activity, acting on glycosyl bonds 65 0.015 GO:0005506iron ion binding 85 0.017 GO:0022804active transmembrane transporter activity 98 0.017 GO:0005215transporter activity 173 0.023 GO:0015075ion transmembrane transporter activity 86 0.026 GO:0008374O-acyltransferase activity 18 0.031 GO:0020037heme binding 51 0.043 GO:0016021integral to membrane 199 0.0099 GO:0031224intrinsic to membrane 224 0.03 GO:0050896response to stimulus 503 3.40E-08 GO:0010035response to inorganic substance 54 5.50E-08 GO:0048610reproductive cellular process 20 4.00E-07 GO:0042221response to chemical stimulus 284 6.20E-07 GO:0009414response to water deprivation 57 4.40E-06 GO:0009415response to water 58 6.00E-06 GO:0010033response to organic substance 197 6.00E-06 GO:0006950response to stress 325 1.40E-05 GO:0009719response to endogenous stimulus 168 1.50E-05 GO:0034641cellular nitrogen compound metabolic process 85 6.20E-05 GO:0009737response to abscisic acid stimulus 75 1.10E-04 GO:0010876lipid localization 11 1.10E-04 GO:0009624response to nematode 48 1.10E-04 GO:0006970response to osmotic stress 73 0.00015 GO:0009628response to abiotic stimulus 202 0.00019 GO:0009651response to salt stress 61 0.00019 GO:0009725response to hormone stimulus 151 0.00039 GO:0009607response to biotic stimulus 137 0.0013 136    Enriched GO category Number of associated genes FDR     GO:0005975carbohydrate metabolic process 140 0.0016 GO:0030001metal ion transport 52 0.0022 GO:0044262cellular carbohydrate metabolic process 87 0.0024 GO:0051707response to other organism 126 0.0024 GO:0042398cellular amino acid derivative biosynthetic process 54 0.0056 GO:0010038response to metal ion 32 0.01 GO:0043436oxoacid metabolic process 121 0.01 GO:0046174polyol catabolic process 8 0.01 GO:0006082organic acid metabolic process 121 0.01 GO:0009611response to wounding 41 0.01 GO:0019752carboxylic acid metabolic process 121 0.01 GO:0051704multi-organism process 152 0.012 GO:0006813potassium ion transport 19 0.017 GO:0042180cellular ketone metabolic process 121 0.017 GO:0006811ion transport 83 0.02 GO:0009605response to external stimulus 88 0.021 GO:0006575cellular amino acid derivative metabolic process 65 0.026 GO:0006519cellular amino acid and derivative metabolic process 107 0.026 GO:0009751response to salicylic acid stimulus 39 0.026 GO:0010107potassium ion import 6 0.034 GO:0044106cellular amine metabolic process 66 0.034 GO:0009991response to extracellular stimulus 33 0.049 GO:0016791phosphatase activity 50 0.00063 GO:0042578phosphoric ester hydrolase activity 56 0.0011 GO:0022891substrate-specific transmembrane transporter activity 116 0.0063 GO:0022857transmembrane transporter activity 140 0.008 GO:0015291secondary active transmembrane transporter activity 54 0.012 GO:0004553hydrolase activity, hydrolyzing O-glycosyl compounds 62 0.012 GO:0022892substrate-specific transporter activity 132 0.013 GO:0009055electron carrier activity 83 0.013 GO:0016798hydrolase activity, acting on glycosyl bonds 65 0.015 GO:0005506iron ion binding 85 0.017 GO:0022804active transmembrane transporter activity 98 0.017 GO:0005215transporter activity 173 0.023 GO:0015075ion transmembrane transporter activity 86 0.026 GO:0008374O-acyltransferase activity 18 0.031 GO:0020037heme binding 51 0.043 GO:0016021integral to membrane 199 0.0099 137    Enriched GO category Number of associated genes FDR     GO:0031224intrinsic to membrane 224 0.03 GO:0022857transmembrane transporter activity 140 0.008 GO:0015291secondary active transmembrane transporter activity 54 0.012 GO:0004553hydrolase activity, hydrolyzing O-glycosyl compounds 62 0.012 GO:0022892substrate-specific transporter activity 132 0.013 GO:0009055electron carrier activity 83 0.013 GO:0016798hydrolase activity, acting on glycosyl bonds 65 0.015 GO:0005506iron ion binding 85 0.017 GO:0022804active transmembrane transporter activity 98 0.017 GO:0005215transporter activity 173 0.023 GO:0015075ion transmembrane transporter activity 86 0.026 GO:0008374O-acyltransferase activity 18 0.031 GO:0020037heme binding 51 0.043 GO:0016021integral to membrane 199 0.0099 GO:0031224intrinsic to membrane 224 0.03                        138    Table S29. Red Stage Trinity Results: Genes for enzymes annotated to GO:0009813 (Flavonoid biosynthetic process, FDR=0.11). The first two columns list differentially expressed (FDR<0.05) Trinity contigs and gene names as annotated by BLASTing the Lotus japonicus genome. The third column is their soybean BLAST homolog, followed by expression at two stages of flower development. Trinity contig L. japonicus ID G. max BLAST ID Gene description   Counts per gene (Yellow) Counts per gene (Red) Log2FC TRINITY_DN51676_c1_g2 chr2.CM0641.560.nc Glyma.01G048200.1 Ribonuclease T(2) / Ribonuclease T2 11 353 -5.0898 TRINITY_DN51676_c1_g1 chr2.CM0641.550.nc Glyma.01G048400.1 Ribonuclease T(2) / Ribonuclease T2 19 1992 -6.8045 TRINITY_DN50283_c0_g1 chr2.CM0124.40.nc Glyma.01G067100.1 F11M15.8 PROTEIN-RELATED 17 300 -4.2331 TRINITY_DN52390_c0_g1 chr4.CM0119.240.nc Glyma.02G048400.1 NARINGENIN,2-OXOGLUTARATE 3-DIOXYGENASE (F3H) 276 1893 -2.8794 TRINITY_DN52643_c1_g1 chr2.CM0124.280.nd Glyma.02G124700.1 Codeine 3-O-demethylase / Codeine O-demethylase 180 884 -2.3973 TRINITY_DN44592_c1_g1 chr1.CM0133.170.nc Glyma.04G052100.1 Thromboxane-A synthase / Thromboxane synthetase 0 38 -8.3044 TRINITY_DN52292_c0_g1 LjT09A12.100.nd Glyma.06G103200.1 CRYPTOCHROME-1 365 4597 -3.7563 TRINITY_DN52780_c0_g1 chr4.CM0429.360.nc Glyma.06G202300.1 FLAVONOID 3'-MONOOXYGENASE 7 2073 -8.2859 TRINITY_DN36546_c0_g1 chr3.CM0208.350.nc Glyma.07G048600.1 F3H7.17 PROTEIN 93 520 -2.5836 TRINITY_DN45435_c0_g1 chr4.CM0387.330.nc Glyma.08G092800.1 2-OXOGLUTARATE (2OG) AND FE(II)-DEPENDENT OXYGENASE SUPERFAMILY PROTEIN-RELATED 0 2374 -14.265 TRINITY_DN46871_c0_g2 LjSGA_066111.1 Glyma.08G110000.1 Quinate O-hydroxycinnamoyltransferase / Hydroxycinnamoyl coenzyme A-quinate transferase 5 35 -2.8775 139    Trinity contig L. japonicus ID G. max BLAST ID Gene description   Counts per gene (Yellow) Counts per gene (Red) Log2FC        TRINITY_DN45808_c0_g1 LjSGA_081858.1 Glyma.10G138800.1 TRANSCRIPTION FACTOR PIF3 0 47 -8.6102 TRINITY_DN54168_c0_g1 chr1.CM0284.240.nc Glyma.11G011500.1 Naringenin-chalcone synthase / Flavonone synthase 17 1134 -6.1510 TRINITY_DN54168_c0_g2 chr1.CM0591.360.nd Glyma.11G011500.1 Naringenin-chalcone synthase / Flavonone synthase 65 4382 -6.1743 TRINITY_DN53094_c0_g1 chr2.CM0304.350.nc Glyma.11G027700.1 LEUCOANTHOCYANIDIN DIOXYGENASE-RELATED (ANS) 30 3761 -7.0659 TRINITY_DN47758_c0_g1 LjT35E03.200.nd Glyma.12G194000.1 MULTIDRUG RESISTANCE PROTEIN 50 664 -3.8298 TRINITY_DN53202_c0_g1 chr3.CM0711.140.nd Glyma.12G199800.1 Transferase family (Transferase) 12 456 -5.3349 TRINITY_DN53800_c0_g1 chr6.CM0055.290.nc Glyma.13G255800.1 Anthocyanidin 3-O-glucosyltransferase / Uridine diphosphoglucose-anthocyanidin 3-O-glucosyltransferase // Flavonol 3-O-glucosyltransferase / UDP-glucose flavonol 3-O-glucosyltransferase 262 3390 -3.7951 TRINITY_DN50775_c0_g1 chr3.CM0711.100.nd Glyma.13G302300.1 COUMAROYL-COA:ANTHOCYANIDIN 3-O-GLUCOSIDE-6''-O-COUMAROYLTRANSFERASE 1-RELATED 0 6587.81 -15.737 TRINITY_DN49460_c0_g1 LjSGA_019127.1 Glyma.17G061000.1 SPERMIDINE HYDROXYCINNAMOYL TRANSFERASE 1 786 -9.5451 TRINITY_DN52789_c0_g1 chr5.CM0077.790.nc Glyma.17G252200.1 Dihydrokaempferol 4-reductase / NADPH-dihydromyricetin reductase 19 434 -4.6063 140    Trinity contig L. japonicus ID G. max BLAST ID Gene description   Counts per gene (Yellow) Counts per gene (Red) Log2FC        TRINITY_DN52652_c1_g1 chr1.CM0593.380.nc Glyma.19G105100.1 Naringenin-chalcone synthase / Flavonone synthase 145 660 -2.2875 TRINITY_DN49414_c0_g1 chr1.CM0012.680.nd Glyma.19G187000.1 UDP-GLYCOSYLTRANSFERASE 73C7 136 1143 -3.1720                               141    Table S30. Yellow Stage Trinity Results: Genes for enzymes annotated to GO:0009813 (Flavonoid biosynthetic process, FDR=1). The first two columns list differentially expressed (FDR<0.05) Trinity contigs and gene names as annotated by BLASTing the Lotus japonicus genome. The third column is their soybean BLAST equivalent, followed by expression at two stages of flower development. Trinity contig L. japonicus ID G. max BLAST ID Gene description   Counts per gene (Yellow) Counts per gene (Red) Log2FC TRINITY_DN45233_c0_g1 chr4.CM0227.650.nc Glyma.02G104600.1 GLUCOSYL/GLUCURONOSYL TRANSFERASES 65 9 2.7339 TRINITY_DN45432_c0_g1 chr2.CM0124.350.nd Glyma.02G125100.1 2-OXOGLUTARATE (2OG) AND FE(II)-DEPENDENT OXYGENASE SUPERFAMILY PROTEIN-RELATED 319 45 2.7201 TRINITY_DN51740_c2_g2 LjSGA_017175.2 Glyma.04G227700.1 FLAVONE 3'-O-METHYLTRANSFERASE 1 1247 281 2.0472 TRINITY_DN45545_c0_g1 chr3.LjT10E18.60.nc Glyma.12G174100.1 AUXIN RESPONSE FACTOR 10-RELATED 23 2.4 3.3450 TRINITY_DN52633_c4_g1 LjSGA_025000.1 Glyma.12G235100.1 OXIDOREDUCTASE, 2OG-FE II OXYGENASE FAMILY PROTEIN 962 155 2.5307 TRINITY_DN39800_c0_g1 chr1.LjB17A22.120.nc Glyma.13G082300.1 FLAVONOL SYNTHASE 3-RELATED 1601 112 3.7338 TRINITY_DN51578_c0_g1 LjSGA_065630.1 Glyma.18G208600.1 GLUCOSYL/GLUCURONOSYL TRANSFERASES 1413 162 3.0216             142    Table S31. Similarly Expressed Trinity Results: Genes for enzymes annotated to GO:0009813 (Flavonoid biosynthetic process, FDR=). The first two columns list Trinity contigs expresand gene names as annotated by BLASTing the Lotus japonicus genome. The third column is their soybean BLAST homolog, followed by expression at two stages of flower development. Trinity contig L. japonicus ID G. max BLAST ID Gene description   Counts per gene (Yellow) Counts per gene (Red) Log2FC TRINITY_DN41192_c0_g1 chr5.CM0077.690.nc Glyma.02G158700.1 Dihydrokaempferol 4-reductase / NADPH-dihydromyricetin reductase 2 0 4.0402 TRINITY_DN25538_c0_g1 chr1.CM0122.460.nc Glyma.03G222100.2 NITROGEN REGULATORY PROTEIN P-II 38 14 1.3309 TRINITY_DN48324_c0_g1 chr1.CM0476.340.nd Glyma.04G228000.1 PROTEIN TRANSPARENT TESTA GLABRA 1 493 570 -0.3114 TRINITY_DN49922_c0_g1 chr4.CM0007.970.nc Glyma.05G127300.2 26S PROTEASOME NON-ATPASE REGULATORY SUBUNIT 8 214 247 -0.3088 TRINITY_DN41679_c0_g1 chr4.CM0042.2280.nc Glyma.05G245800.1 PATHOGENESIS-RELATED THAUMATIN-LIKE PROTEIN-RELATED 3 0 4.5957 TRINITY_DN36305_c0_g1 chr1.CM0033.750.nc Glyma.10G053500.1 B3 DNA binding domain (B3) // Auxin response factor (Auxin_resp) 6 0 5.5655 TRINITY_DN38832_c0_g1 LjSGA_024944.1 Glyma.10G210600.1 B3 DNA binding domain (B3) // Auxin response factor (Auxin_resp) 28 4 2.6690 TRINITY_DN31599_c0_g1 chr3.LjT10E18.60.nc Glyma.11G145500.1 AUXIN RESPONSE FACTOR 10-RELATED 8 0 5.9729 TRINITY_DN18931_c0_g1 LjSGA_078422.1 Glyma.11G145500.1 AUXIN RESPONSE FACTOR 10-RELATED 1 2 -1.0110 TRINITY_DN45545_c0_g1 chr3.LjT10E18.60.nc Glyma.12G174100.1 AUXIN RESPONSE FACTOR 10-RELATED 23 2.4 3.3450 TRINITY_DN56369_c0_g1 chr3.CM0590.460.nd Glyma.13G279600.1 COP1-INTERACTING PROTEIN 7 (CIP7) 4 0 4.9957 143    Trinity contig L. japonicus ID G. max BLAST ID Gene description   Counts per gene (Yellow) Counts per gene (Red) Log2FC        TRINITY_DN50775_c0_g1 chr3.CM0711.100.nd Glyma.13G302300.1 COUMAROYL-COA:ANTHOCYANIDIN 3-O-GLUCOSIDE-6''-O-COUMAROYLTRANSFERASE 1-RELATED 0 6587.81 -15.7376 TRINITY_DN45545_c2_g1 chr3.LjT10E18.60.nc Glyma.13G325200.1 AUXIN RESPONSE FACTOR 10-RELATED 4 0 4.9957 TRINITY_DN52789_c0_g2 chr5.CM0077.690.nc Glyma.14G072700.1 Dihydrokaempferol 4-reductase / NADPH-dihydromyricetin reductase 18 3 2.4363 TRINITY_DN55140_c0_g1 chr5.CM0077.690.nc Glyma.14G072700.1 Dihydrokaempferol 4-reductase / NADPH-dihydromyricetin reductase 0 2 -4.1364 TRINITY_DN53766_c0_g2 chr1.CM0410.100.nc Glyma.18G268100.1 Isoflavone-7-O-beta-glucoside 6''-O-malonyltransferase / Flavone/flavonol 7-O-beta-D-glucoside malonyltransferase 187 206 -0.2415 TRINITY_DN33124_c0_g1 chr1.CM0410.130.nc Glyma.18G268100.1 Isoflavone-7-O-beta-glucoside 6''-O-malonyltransferase / Flavone/flavonol 7-O-beta-D-glucoside malonyltransferase 10 6 0.6246 TRINITY_DN57182_c0_g1 chr1.CM0410.130.nc Glyma.18G268100.1 Isoflavone-7-O-beta-glucoside 6''-O-malonyltransferase / Flavone/flavonol 7-O-beta-D-glucoside malonyltransferase 12 7 0.6663                      144    Trinity contig L. japonicus ID G. max BLAST ID Gene description   Counts per gene (Yellow) Counts per gene (Red) Log2FC        TRINITY_DN6325_c0_g1 chr1.CM0410.130.nc Glyma.18G268100.1 Isoflavone-7-O-beta-glucoside 6''-O-malonyltransferase / Flavone/flavonol 7-O-beta-D-glucoside malonyltransferase 5 1 2.0924 TRINITY_DN53766_c0_g2 chr1.CM0410.80.nc Glyma.18G268100.1 Isoflavone-7-O-beta-glucoside 6''-O-malonyltransferase / Flavone/flavonol 7-O-beta-D-glucoside malonyltransferase 187 206 -0.2415 TRINITY_DN54421_c2_g4 chr1.CM0410.40.nc Glyma.18G268600.1 Isoflavone-7-O-beta-glucoside 6''-O-malonyltransferase / Flavone/flavonol 7-O-beta-D-glucoside malonyltransferase 407 211.72 0.8384 TRINITY_DN53649_c3_g1 chr1.CM0222.40.nc Glyma.19G106900.1 WD REPEAT CONTAINING PROTEIN 211 148 0.4092 TRINITY_DN41800_c0_g1 LjSGA_045968.2 Glyma.20G180000.1 Glyma.20G180000.1.p - (M=20) PF02362//PF06507 -  4 0 4.9957 TRINITY_DN45569_c1_g1 LjSGA_045968.2 Glyma.20G180000.1 Glyma.20G180000.1.p - (M=20) PF02362//PF06507 -  1 0 0         145    Table S32. Yellow Stage Trinity Results: Genes for enzymes annotated to the over-represented GO:0009809 (Lignin biosynthetic process, FDR=1). The first two columns list differentially expressed (FDR<0.05) Trinity contigs and gene names as annotated by BLASTing the Lotus japonicus genome. The third column is their soybean BLAST homolog, followed by expression at two stages of flower development. Trinity contig L. japonicus ID G. max BLAST ID Gene description   Counts per gene (Yellow) Counts per gene (Red) Log2FC TRINITY_DN45532_c2_g1 chr2.CM0249.1210.nc Glyma.01G173600.1 LACCASE-11 23 0 7.4814 TRINITY_DN45532_c4_g1 chr2.CM0249.1210.nc Glyma.01G173600.1 LACCASE-11 17 0 7.0482 TRINITY_DN46194_c0_g1 chr4.CM0227.500.nc Glyma.02G103500.1 O-METHYLTRANSFERASE-RELATED 8146 826 3.1995 TRINITY_DN51740_c2_g2 LjSGA_017175.2 Glyma.04G227700.1 FLAVONE 3'-O-METHYLTRANSFERASE 1 1247 281 2.0472 TRINITY_DN44781_c0_g1 LjSGA_035751.3 Glyma.07G089700.1 Abieta-7,13-dien-18-ol hydroxylase / CYP720B1 8110 225 5.0688 TRINITY_DN43710_c0_g1 LjSGA_035751.3 Glyma.07G089700.1 Abieta-7,13-dien-18-ol hydroxylase / CYP720B1 91.32 12 2.8083 TRINITY_DN44818_c0_g1 chr4.CM0042.560.nd Glyma.07G089800.1 Cytochrome P450 (p450) 40 2 4.1399 TRINITY_DN45532_c0_g1 chr2.CM0249.1210.nc Glyma.11G069500.1 LACCASE-11 25 0 7.6011 TRINITY_DN52386_c0_g1 LjSGA_011490.1 Glyma.12G019700.1 ALCOHOL DEHYDROGENASE-RELATED 76 11 2.6730 TRINITY_DN34978_c0_g2 chr1.LjB20I01.110.nd Glyma.14G062300.1 MULTI-COPPER OXIDASE 11 0 6.4261 TRINITY_DN45845_c0_g1 CM0385.150.nd Glyma.15G143600.1  CHITINASE-LIKE PROTEIN 1 225 15 3.7940           146    Table S33. Red Stage Trinity Results: Genes for enzymes annotated to the over-represented GO:0009809 (Lignin biosynthetic process, FDR=). The first two columns list differentially expressed (FDR<0.05) Trinity contigs and gene names as annotated by BLASTing the Lotus japonicus genome. The third column is their soybean BLAST homolog, followed by expression at two stages of flower development. Trinity contig L. japonicus ID G. max BLAST ID Gene description   Counts per gene (Yellow) Counts per gene (Red) Log2FC TRINITY_DN49460_c0_g1 LjSGA_019127.1 Glyma.17G061000.1 SPERMIDINE HYDROXYCINNAMOYL TRANSFERASE 1 786 -9.5451 TRINITY_DN45071_c0_g1 LjSGA_021886.2 Glyma.17G171100.1 O-METHYLTRANSFERASE-RELATED 2 72 -5.1840 TRINITY_DN49973_c0_g1 LjSGA_030253.1 Glyma.13G141600.1 S-ADENOSYLMETHIONINE SYNTHASE 3 206 707 -1.8805                           147    Table S34. Similarly Expressed Trinity Results: Genes for enzymes annotated to GO:0009813 (Lignin  process, FDR=1). The first two columns list Trinity contigs expresand gene names as annotated by BLASTing the Lotus japonicus genome. The third column is their soybean BLAST homolog, followed by expression at two stages of flower development. Trinity contig L. japonicus ID G. max BLAST ID Gene description   Counts per gene (Yellow) Counts per gene (Red) Log2FC TRINITY_DN53597_c0_g1 chr2.CM0249.290.nd Glyma.01G169200.1 Cytochrome P450 CYP2 subfamily 170 92 0.7828 TRINITY_DN41837_c0_g1 LjSGA_021886.2 Glyma.01G187700.1 O-METHYLTRANSFERASE-RELATED 1831 863 0.9829 TRINITY_DN52969_c0_g1 LjSGA_096258.1 Glyma.03G122000.1  CYTOCHROME P450 98A3-RELATED 1429 1471 -0.1439 TRINITY_DN49973_c2_g1 LjSGA_030253.1 Glyma.03G184000.1 S-ADENOSYLMETHIONINE SYNTHASE 3 4222 10944 -1.4762 TRINITY_DN16999_c0_g1 chr1.CM0063.200.nc Glyma.05G203000.1 OMEGA-HYDROXYPALMITATE O-FERULOYL TRANSFERASE 0 10 -6.3911 TRINITY_DN49267_c1_g2 LjT16L08.100.nd Glyma.05G233200.1 Cytochrome P450 CYP2 subfamily 144 441 -1.7159 TRINITY_DN17837_c0_g1 chr3.LjT40P18.60.nc Glyma.07G021600.1 SHIKIMATE O-HYDROXYCINNAMOYLTRANSFERASE 5 6 -0.3570 TRINITY_DN53428_c1_g1 LjSGA_040039.1 Glyma.07G026300.1 CINNAMOYL-COA:NADP OXIDOREDUCTASE-LIKE 1-RELATED 156 46 1.6571 TRINITY_DN33065_c0_g1 LjSGA_027787.1 Glyma.07G050600.1 NAC DOMAIN-CONTAINING PROTEIN 43-RELATED 3 0 4.59573 TRINITY_DN33065_c0_g2 LjSGA_027787.1 Glyma.07G050600.1 NAC DOMAIN-CONTAINING PROTEIN 43-RELATED 0 0 0 TRINITY_DN66560_c0_g1 LjSGA_027787.1 Glyma.07G050600.1 NAC DOMAIN-CONTAINING PROTEIN 43-RELATED 1 0 0 TRINITY_DN17072_c0_g1 LjSGA_035751.3 Glyma.07G089700.1 Abieta-7,13-dien-18-ol hydroxylase / CYP720B1 102 31 1.61231 TRINITY_DN49973_c3_g1 LjSGA_140570.1 Glyma.07G233800.1  S-ADENOSYLMETHIONINE SYNTHASE 4 13 15 -0.3058 TRINITY_DN51745_c0_g1 chr3.CM0452.240.nd Glyma.08G220200.1 SHIKIMATE O-HYDROXYCINNAMOYLTRANSFERASE 568 908 -0.7788 148    Trinity contig L. japonicus ID G. max BLAST ID Gene description   Counts per gene (Yellow) Counts per gene (Red) Log2FC        TRINITY_DN42496_c1_g1 chr1.CM0410.190.nc Glyma.08G246100.1 LRR RECEPTOR-LIKE SERINE/THREONINE-PROTEIN KINASE RPK2 10 7 0.406346 TRINITY_DN47225_c0_g1 chr1.CM0410.190.nc Glyma.08G246100.1 LRR RECEPTOR-LIKE SERINE/THREONINE-PROTEIN KINASE RPK2 24 5 2.13427 TRINITY_DN37023_c0_g2 LjSGA_030670.1 Glyma.08G311800.1 SPERMIDINE HYDROXYCINNAMOYL TRANSFERASE 4 1 1.77955 TRINITY_DN37010_c0_g1 chr1.CM0147.770.nc Glyma.08G359100.1 LACCASE-17 1 1 -0.0908 TRINITY_DN32484_c0_g1 LjSGA_011490.1 Glyma.12G019700.1 ALCOHOL DEHYDROGENASE-RELATED 5 4 0.21381 TRINITY_DN41325_c0_g1 LjSGA_011490.1 Glyma.12G019700.1 ALCOHOL DEHYDROGENASE-RELATED 5 1 2.09241 TRINITY_DN41325_c0_g2 LjSGA_011490.1 Glyma.12G019700.1 ALCOHOL DEHYDROGENASE-RELATED 4 6 -0.6698 TRINITY_DN49071_c2_g3 LjSGA_015763.2 Glyma.12G019700.1 ALCOHOL DEHYDROGENASE-RELATED 2 2 -0.0961 TRINITY_DN54121_c0_g1 LjSGA_013806.1 Glyma.12G109800.1 O-METHYLTRANSFERASE FAMILY PROTEIN 124 228 -0.9800 TRINITY_DN49611_c0_g1 LjT45M09.100.nd Glyma.13G369800.1 NAD DEPENDENT EPIMERASE/DEHYDRATASE 1434 375 1.83260 TRINITY_DN35253_c1_g1 chr1.LjB20I01.110.nd Glyma.14G062300.1 MULTI-COPPER OXIDASE 3 0 4.59573 TRINITY_DN50309_c0_g1 LjSGA_070619.1 Glyma.15G059500.1 ALCOHOL DEHYDROGENASE RELATED 48 24 0.89451 TRINITY_DN51988_c1_g1 LjT35D18.30.nc Glyma.15G190500.1 S-ADENOSYLMETHIONINE SYNTHASE 4 8441 2869 1.45468        149    Trinity contig L. japonicus ID G. max BLAST ID Gene description   Counts per gene (Yellow) Counts per gene (Red) Log2FC        TRINITY_DN2300_c0_g1 chr1.CM0593.100.nd Glyma.16G047300.1 Protein kinase domain (Pkinase) // Leucine rich repeat N-terminal domain (LRRNT_2) // Leucine rich repeat (LRR_8) 6 4 0.47076 TRINITY_DN38374_c0_g1 chr1.CM0593.100.nd Glyma.16G047300.1 Protein kinase domain (Pkinase) // Leucine rich repeat N-terminal domain (LRRNT_2) // Leucine rich repeat (LRR_8) 0 10 -6.3911 TRINITY_DN38272_c0_g1 chr1.CM0593.40.nd Glyma.16G047300.1 Protein kinase domain (Pkinase) // Leucine rich repeat N-terminal domain (LRRNT_2) // Leucine rich repeat (LRR_8) 10 0 6.29031 TRINITY_DN49071_c1_g1 LjT48C16.160.nd Glyma.18G091500.1  ALCOHOL DEHYDROGENASE-RELATED 93 70 0.30726 TRINITY_DN37010_c1_g1 chr1.CM0147.770.nc Glyma.18G177200.1 MULTI-COPPER OXIDASE 4 0 4.99577 TRINITY_DN47225_c1_g1 chr1.CM0410.190.nc Glyma.18G267000.1 LRR RECEPTOR-LIKE SERINE/THREONINE-PROTEIN KINASE RPK2 61.37 47 0.27337 TRINITY_DN47225_c1_g2 chr1.CM0410.190.nc Glyma.18G267000.1 LRR RECEPTOR-LIKE SERINE/THREONINE-PROTEIN KINASE RPK2 1.63 0 4.04029 TRINITY_DN50519_c0_g1 chr5.CM0200.1180.nc Glyma.20G128600.1 CINNAMYL ALCOHOL DEHYDROGENASE 4-RELATED 423 183 1.1061 TRINITY_DN58801_c0_g1 LjSGA_075859.1 Glyma.20G175500.1 No apical meristem (NAM) protein (NAM) 4 1 1.7795      150    Table S35. L. filicaulis Yellow Stage Trinity Results: Genes for enzymes annotated to GO:00016117 (Carotenoid biosynthetic process, FDR=0.11). The first two columns list differentially expressed (FDR<0.05) Trinity contigs and gene names as annotated by BLASTing the Lotus japonicus genome. The third column is their soybean BLAST homolog, followed by expression at two stages of flower development. Trinity contig L. japonicus ID G. max BLAST ID Gene description   Counts per gene (Yellow) Counts per gene (Red) Log2FC TR50146|c0_g1 chr3.CM0996.270.r2.a Glyma.01G028900.1 ATP-CITRATE SYNTHASE 34.96 2 3.7015 TR45451|c0_g1 chr4.CM0570.280.r2.a Glyma.01G064200.1 AGAMOUS-LIKE MADS-BOX PROTEIN AGL8-RELATED 1414 151 2.8697 TR18321|c0_g1 LjSGA_059500.1 Glyma.01G186200.1 ZEAXANTHIN EPOXIDASE, CHLOROPLASTIC 310 63 1.9404 TR32448|c0_g2 chr5.CM1125.160.r2.m Glyma.02G149400.1 Not Available 10.54 0.64 3.1901 TR52234|c0_g2 LjSGA_013562.2 Glyma.02G188200.1 Prolycopene isomerase / CRTISO 6.01 18.45 2.2958 TR33198|c0_g1 chr2.LjT08I01.60.r2.a Glyma.02G240200.1 PHYTOENE SYNTHASE, CHLOROPLASTIC 4765 1049 1.8268 TR64960|c0_g1 chr3.CM0282.920.r2.m Glyma.06G238100.1 SQUAMOSA PROMOTER-BINDING-LIKE PROTEIN 4-RELATED 117 1 6.3654 TR22838|c0_g1 chr5.CM0456.560.r2.m Glyma.09G132200.1 Beta-carotene 3-hydroxylase / Beta-carotene 3,3'-monooxygenase 4 0 0 TR60004|c0_g2 chr5.CM1077.670.r2.m Glyma.10G062900.1 Carotene epsilon-monooxygenase / LUT1 33.2 8 7.9261 TR3908|c0_g1 chr5.CM1323.150.r2.m Glyma.10G160500.1 9,9'-di-cis-zeta-carotene desaturase / Zeta-carotene desaturase 21.7 5.36 1.9696 TR53492|c0_g1 chr4.CM0044.50.r2.m Glyma.11G252500.1 Zeta-carotene isomerase / 15-cis-zeta-carotene isomerase 446 18 4.2661 TR15422|c0_g1 chr1.CM0104.3370.r2.m Glyma.16G036100.1 POLYRIBONUCLEOTIDE NUCLEOTIDYLTRANSFERASE 1, CHLOROPLASTIC 27.03 0 7.5950 TR18321|c1_g1 LjSGA_078276.1 NA zeaxanthin epoxidase 4274.46 305.41 3.4517 TR18321|c2_g1 LjSGA_024159.1 NA zeaxanthin epoxidase 366 77 1.8908 151    Table S36. L. filicaulis Similarly Expressed Trinity Results: Genes for enzymes annotated to GO:00016117 (Carotenoid biosynthetic process, FDR=1). The first two columns list Trinity contigs expresand gene names as annotated by BLASTing the Lotus japonicus genome. The third column is their soybean BLAST homolog, followed by expression at two stages of flower development. Trinity contig L. japonicus ID G. max BLAST ID Gene description   Counts per gene (Yellow) Counts per gene (Red) Log2FC TR52234|c0_g1 LjSGA_013562.2 Glyma.02G188200.1 ATP-CITRATE SYNTHASE 6.01 18.45 -1.9168 TR15191|c0_g1 chr6.CM0013.1810.r2.d Glyma.09G066200.1 Not Available 143 46 1.27775 TR7103|c0_g1 LjSGA_075584.1 Glyma.07G273600.1 Prolycopene isomerase / CRTISO 129.46 16 3.93057 TR32448|c0_g1 chr5.CM1125.160.r2.m Glyma.02G149400.1 Glutathione transferase / S-(hydroxyalkyl)glutathione lyase 10.54 0.64 2.97110 TR15422|c0_g1 chr1.CM0104.3370.r2.m Glyma.16G036100.1 LYCOPENE BETA CYCLASE, CHLOROPLASTIC 27.03 0 1.93647 TR51506|c0_g1 chr6.CM0367.660.r2.d Glyma.09G008500.1 Prolycopene isomerase / CRTISO 2279 594 1.58315 TR42882|c0_g1 LjSGA_050034.1 Glyma.09G000600.1 MONOOXYGENASE 4 0 4.88247 TR64376|c0_g1 LjSGA_046377.1 Glyma.02G283400.1 UBIQUINOL OXIDASE 4, CHLOROPLASTIC/CHROMOPLASTIC 222 571 -1.7188 TR61986|c0_g1 LjSGA_045520.1 Glyma.03G128900.1 LYCOPENE EPSILON CYCLASE, CHLOROPLASTIC 635 164 1.59588 TR60004|c0_g1 chr5.CM1077.670.r2.m Glyma.10G062900.1 Beta-carotene 3-hydroxylase / Beta-carotene 3,3'-monooxygenase 33.2 8 1.67434 TR13886|c0_g1 chr5.CM1323.150.r2.m Glyma.10G160500.1  PROTEIN LUTEIN DEFICIENT 5, CHLOROPLASTIC 21.7 5.36 1.75885 TR33253|c0_g1 chr1.CM1413.250.r2.a Glyma.09G252800.1 Carotene epsilon-monooxygenase / LUT1 963 463 0.69985 TR61205|c0_g1 chr6.LjT13N05.10.r2.m Glyma.11G253000.1 9,9'-di-cis-zeta-carotene desaturase / Zeta-carotene desaturase 1488 691 0.7499        152    Trinity contig L. japonicus ID G. max BLAST ID Gene description   Counts per gene (Yellow) Counts per gene (Red) Log2FC        TR6924|c0_g1 chr2.CM0545.170.r2.m Glyma.14G046600.1 15-cis-phytoene desaturase / Plant-type phytoene desaturase 81 23 1.45539 TR24925|c0_g1 LjSGA_031176.1 Glyma.17G174500.1  LYCOPENE BETA/EPSILON CYCLASE PROTEIN 241 58 1.6964 TR22838|c0_g1 chr5.CM0456.560.r2.m Glyma.09G132200.1 POLYRIBONUCLEOTIDE NUCLEOTIDYLTRANSFERASE 1, CHLOROPLASTIC 4 0 4.88247 TR50146|c2_g1 chr2.CM0008.330.r2.m Glyma.01G028900.1 ZEAXANTHIN EPOXIDASE, CHLOROPLASTIC 840 625 0.06998                        153    Table S37. L. sessilifolius Yellow Stage Trinity Results: Genes for enzymes annotated to GO:00016117 (Carotenoid biosynthetic process, FDR=0.00027). The first two columns list differentially expressed (FDR<0.05) Trinity contigs and gene names as annotated by BLASTing the Lotus japonicus genome. The third column is their soybean BLAST homolog, followed by expression at two stages of flower development. Trinity contig L. japonicus ID G. max BLAST ID Gene description   Counts per gene (Yellow) Counts per gene (Red) Log2FC TRINITY_DN53748_c0_g1 LjSGA_045520.1 Glyma.03G128900.1  LYCOPENE BETA CYCLASE, CHLOROPLASTIC 38.81 0 8.240016 TRINITY_DN50287_c0_g1 LjSGA_046377.1 Glyma.08G306200.1 Glutathione transferase / S-(hydroxyalkyl)glutathione lyase // 15-cis-phytoene synthase / PSase 102 10 3.232895 TRINITY_DN49175_c0_g1 LjT17B03.90.nd Glyma.09G008500.1 UBIQUINOL OXIDASE 4, CHLOROPLASTIC/CHROMOPLASTIC 1268 232 2.347616 TRINITY_DN50928_c1_g1 LjSGA_055879.1 Glyma.09G233300.1 Protein disulfide-isomerase / S-S rearrangase 187 42 2.0493 TRINITY_DN48832_c0_g1 LjSGA_013562.2 Glyma.10G086700.1 Prolycopene isomerase / CRTISO 249 43 2.428307 TRINITY_DN50878_c0_g1 chr2.CM0018.70.nc Glyma.11G016200.1 CYTOCHROME P450 97B3, CHLOROPLASTIC 170 13 3.59459 TRINITY_DN50044_c0_g1 chr6.LjT13N05.100.nc Glyma06g11820.1 15-cis-phytoene desaturase / Plant-type phytoene desaturase 2187 425 2.260955 TRINITY_DN53304_c1_g1 LjT01G11.200.nd Glyma.17G174500.1 ZEAXANTHIN EPOXIDASE, CHLOROPLASTIC 73 14 2.270513 TRINITY_DN38311_c0_g2 LjT40E15.150.nc Glyma.20G227900.1 9,9'-di-cis-zeta-carotene desaturase / Zeta-carotene desaturase 8.33 7.97 -0.10057 TRINITY_DN53720_c2_g1 LjSGA_021331.1 Glyma10g07210.1 carotene epsilon-monooxygenase  204 21 3.170623 TRINITY_DN53304_c3_g1 LjSGA_078276.1 Glyma11g05960.1 zeaxanthin epoxidase 185 12 3.830864 TRINITY_DN53304_c5_g1 LjSGA_024159.1 Glyma17g20020.1| zeaxanthin epoxidase 2027 321 2.556112  154    Table S38 Red stage L. filicaulis: 19 contigs with greatest magnitude of log fold-change recovered from entire transcriptome.  Trinity_ID LogFC Lotus1.0_ID Lotus2.5_ID Annotation (UniProt and AnnoMine) TR7064|c2_g1 -16.5797 NA LjT31N05.100.r2.d SJCHGC01014 protein (Fragment) OS=Schistosoma japonicum TR34298|c0_g1 -13.9764 NA chr4.CM0026.290.r2.m Putative senescence-associated protein (Fragment) OS=Pisum sativum TR50987|c0_g1 -13.3631 LjSGA_061080.1.1 chr4.CM0026.290.r2.m Putative senescence-associated protein (Fragment) OS=Pisum sativum TR3216|c0_g3 -13.3391 LjSGA_089366.0.1 LjSGA_089366.0.1 NA TR29498|c0_g1 -13.3057 chr4.CM0042.1410.nc chr4.CM0042.1350.r2.m Dynamin OS=Cucumis melo subsp. melo TR48381|c3_g1 -13.1261 chr5.CM1439.180.nd chr5.CM1439.390.r2.m RING/U-box superfamily protein TR42412|c0_g1 -13.1231 LjT03L03.160.nd chr5.CM0696.1060.r2.a NA TR53102|c0_g1 -13.1113 chr4.CM0229.70.nc chr4.CM0229.70.r2.m Probable polygalacturonase At2g43860 TR40587|c0_g1 -13.0933 chr3.CM0129.380.nd chr3.CM0129.50.r2.d NA TR2085|c0_g1 -13.0644 LjSGA_078330.2 chr4.CM0026.290.r2.m NA TR1538|c0_g2 -12.7012 LjSGA_125503.1 LjT31N05.100.r2.d SJCHGC01014 protein (Fragment) OS=Schistosoma japonicum TR3124|c0_g3 -12.6853 chr5.CM0040.150.nd chr5.CM0040.150.r2.d Protein of unknown function DUF1677, plant TR4260|c0_g1 -12.5775 chr4.CM0288.1020.nd chr4.CM0288.1020.r2.d 1-aminocyclopropane-1-carboxylate oxidase OS=Trifolium repens TR14717|c0_g3 -12.558 LjSGA_070603.1 chr3.CM0213.540.r2.m ATP-binding cassette transporter, putative OS=Ricinus communis TR20264|c0_g1 -12.4303 LjSGA_025923.1 LjSGA_025923.1 NA 155    Trinity_ID LogFC Lotus1.0_ID Lotus2.5_ID Annotation (UniProt and AnnoMine)      TR48491|c0_g1 -12.3818 chr1.LjT16O03.100.nd chr1.LjT16O03.100.r2.d Legumain-like protease (Precursor) OS=Ixodes ricinus TR46724|c0_g3 -12.3162 chr5.CM0200.550.nc chr5.CM0200.2620.r2.m Zinc finger protein OS=Cicer arietinum TR49591|c0_g1 -12.3111 LjT07J01.20.nd chr5.CM0052.370.r2.a GmCK2p OS=Glycine max TR23809|c0_g6 -12.1554 LjSGA_022872.2 LjSGA_022872.2 Elongation factor 1-alpha OS=Ricinus communis                            156    Table S39 Yellow stage L. filicaulis: 19 contigs with greatest magnitude of log fold-change recovered from entire transcriptome.  Trinity_ID LogFC Lotus1.0_ID Lotus2.5_ID Annotation (Annomine and UniProt) TR24089|c0_g1 14.89616 chr1.CM0088.370.r2.d NA NA TR15262|c0_g2 14.61414 chr5.CM0072.330.r2.d NA Flowering-promoting factor 1-like protein 1 TR43132|c0_g1 14.19693 LjSGA_013861.2 LjSGA_013861.2 Putative auxin efflux carrier protein 6 OS=Medicago truncatula TR55706|c0_g1 14.16986 chr6.CM0679.380.r2.m NA member of Alpha-Expansin Gene Family TR47113|c0_g4 13.92623 LjSGA_045866.1 LjSGA_045866.1 Early-responsive to dehydration OS=Medicago truncatula TR33662|c0_g2 13.54451 LjSGA_020750.1 LjSGA_020750.1 ribonuclease T2 TR16126|c0_g1 13.45947 LjSGA_032186.1 LjSGA_032186.1 NA TR33992|c0_g1 13.24861 chr1.LjT44L17.240.r2.d NA NA TR50837|c0_g1 13.23161 LjSGA_071404.1 LjSGA_071404.1 Lachrymatory-factor synthase, putative OS=Ricinus communis TR56525|c0_g2 13.20029 chr3.CM0152.240.r2.m NA Beta-amylase TR17308|c2_g2 13.02148 chr6.CM0037.300.r2.m NA Cellulose synthase 3 OS=Eucalyptus grandis TR63082|c0_g1 12.93094 LjSGA_009749.1 LjSGA_009749.1 Putative ripening related protein OS=Cicer arietinum TR50996|c0_g1 12.92962 chr3.CM0129.210.r2.d NA TPR domain protein OS=Arthrospira platensis NIES-39 TR48869|c0_g3 12.91239 LjSGA_012731.1 LjSGA_012731.1 DEAD (Asp-Glu-Ala-Asp) box polypeptide 5 OS=Mus musculus 157    Trinity_ID LogFC Lotus1.0_ID Lotus2.5_ID Annotation (UniProt and AnnoMine)      TR3230|c0_g2 12.8773 chr5.CM0052.240.r2.d NA RANBP2-like and GRIP domain-containing protein 5/6 TR16751|c0_g1 12.83714 chr6.CM0066.630.r2.m NA NA TR53791|c0_g1 12.81876 LjSGA_020451.1 LjSGA_020451.1 WD repeat-containing protein OS=Medicago truncatula TR8063|c0_g1 12.76956 chr4.CM0126.800.r2.d NA Glucose-methanol-choline oxidoreductase OS=Halococcus salifodinae DSM 8989 TR49365|c1_g1 12.73229 chr3.CM0136.330.r2.m NA Fasciclin-like AGP 11 OS=Populus canescens                    158    Table S40 Red stage L. sessilifolius 19 contigs with greatest magnitude of log fold-change recovered from entire transcriptome. Trinity_ID LogFC Lotus1.0_ID Lotus2.5_ID Annotation (Annomine and Uniprot) TRINITY_DN50775_c0_g1 -15.7376 chr3.CM0711.100.nd chr3.CM0711.100.r2.d Malonyl-coenzyme:anthocyanin 5-O-glucoside-6'''-O-malonyltransferase TRINITY_DN45435_c0_g1 -14.2652 chr4.CM0387.330.nc chr4.CM0387.1100.r2.m Thebaine 6-O-demethylase TRINITY_DN11784_c0_g2 -13.6968 LjT33L17.60.nc chr2.CM0660.220.r2.m Bark storage protein A TRINITY_DN54303_c0_g1 -12.8579 LjSGA_138785.1 LjSGA_138785.1 Raffionse synthase 2 OS=Glycine max TRINITY_DN49759_c0_g1 -12.802 LjSGA_010954.1 LjSGA_010954.1 Catalytic, putative OS=Ricinus communis TRINITY_DN50868_c0_g1 -12.5777 chr2.CM1150.550.nd chr2.CM0028.320.r2.d Metalloendoproteinase 1 TRINITY_DN52783_c0_g1 -12.3214 chr4.CM0307.20.nc chr4.CM0307.20.r2.m DNA polymerase III PolC-type TRINITY_DN40888_c0_g2 -11.9258 chr1.CM0141.350.nd chr1.CM0141.360.r2.a R2R3 MYB related transcription factor OS=Ipomoea batatas TRINITY_DN16795_c0_g1 -11.8435 LjT03L03.160.nd chr5.CM0696.1060.r2.a Peroxidase OS=Populus trichocarpa TRINITY_DN47218_c0_g1 -11.5483 chr1.LjT43O05.150.nd chr1.LjT43O05.70.r2.a pathogenesis-related family protein TRINITY_DN42182_c0_g1 -11.5201 LjSGA_034431.0.1 LjSGA_034431.0.1 TIFY Transcription factor TRINITY_DN50876_c1_g1 -11.4787 LjT03E06.40.nd chr1.CM0012.70.r2.d Protein of unknown function DUF3741 TRINITY_DN48639_c0_g2 -11.4787 chr3.CM0164.80.nd chr3.CM0164.80.r2.d dichloro-2,5-cyclohexadiene-1,4-diol dehydrogenase TRINITY_DN49642_c0_g1 -11.4448 LjSGA_111612.1.2 chr4.CM0007.970.r2.a Putative senescence-associated protein (Fragment) OS=Ipomoea nil      159    Trinity_ID LogFC Lotus1.0_ID Lotus2.5_ID Annotation (UniProt and AnnoMine)      TRINITY_DN34011_c0_g1 -11.4362 LjSGA_053993.1 LjSGA_053993.1 Beta-fructofuranosidase OS=Citrus sinensis TRINITY_DN48126_c0_g1 -11.3968 LjT45L18.200.nd LjT45L18.200.r2.d Proteinase inhibitor I3, Kunitz legume TRINITY_DN50442_c0_g1 -11.3789 LjSGA_147445.1.1 chr4.LjT13G24.170.r2.a SJCHGC01393 protein OS=Schistosoma japonicum TRINITY_DN49028_c1_g1 -11.3333 chr1.CM0295.60.nd chr1.CM0295.140.r2.m Phosphate transporter OS=Lotus japonicus TRINITY_DN53079_c2_g1 -11.3287 LjSGA_008068.1 LjSGA_008068.1 D-galactose transporter GalP OS=Edwardsiella tarda                         160    Table S41 Yellow stage L. sessilifolius: 19 contigs with greatest magnitude of log fold-change recovered from entire transcriptome.  Trinity_ID LogFC Lotus1.0_ID Lotus2.5_ID Annotation (Annomine and UniProt) TRINITY_DN11722_c0_g1 11.10024 chr1.CM0141.260.nc chr1.CM0141.260.r2.m Expansin-A15 TRINITY_DN51824_c2_g1 10.13103 LjSGA_043454.1 LjSGA_043454.1 Ribose-phosphate pyrophosphokinase 3 OS=Capsaspora owczarzaki (strain ATCC 30864) TRINITY_DN48232_c0_g1 10.02803 chr1.CM0088.1030.nc chr1.CM0088.1030.r2.a Homeodomain-related (Fragment) OS=Medicago truncatula TRINITY_DN43262_c0_g2 9.784361 chr6.LjT10I04.70.nd chr6.CM0778.70.r2.d Calmodulin-binding protein OS=Medicago truncatula TRINITY_DN51657_c0_g1 9.652068 chr1.CM0398.130.nd chr1.CM0398.250.r2.d Gibberellin induced protein OS=Medicago truncatula TRINITY_DN43721_c0_g2 9.624089 LjSGA_011684.1 LjSGA_011684.1 Glucan 1,3-beta-glucosidase OS=Medicago truncatula TRINITY_DN47886_c0_g1 9.595556 LjSGA_025651.1.1 chr3.CM2163.270.r2.m Disease resistance response protein 1 OS=Glycine max TRINITY_DN52257_c0_g4 8.907039 chr6.CM1829.200.nc chr6.CM0139.1580.r2.m NA TRINITY_DN46161_c0_g1 8.907039 chr4.CM0219.190.nc chr4.CM0219.190.r2.m energized vacuolar membrane proton pump TRINITY_DN40465_c0_g3 8.811031 chr1.CM0009.350.nc chr1.CM0009.350.r2.m CER1-like 2 TRINITY_DN34483_c0_g2 8.760519 LjT26P12.80.nd LjT26P12.100.r2.a NA TRINITY_DN47039_c0_g2 8.708175 chr4.CM0219.380.nd chr4.CM0219.380.r2.d Heparanase-like protein OS=Medicago truncatula TRINITY_DN47671_c0_g1 8.708175 LjSGA_043568.1 LjSGA_043568.1 NA 161    Trinity_ID LogFC Lotus1.0_ID Lotus2.5_ID Annotation (UniProt and AnnoMine)      TRINITY_DN40925_c0_g3 8.681273 LjSGA_031888.1.1 chr1.CM0104.3590.r2.m alpha/beta-Hydrolases superfamily protein TRINITY_DN42547_c0_g1 8.681273 LjSGA_027078.2 LjSGA_027078.2 PMR5 N-terminal domain TRINITY_DN36333_c0_g1 8.65386 LjSGA_092888.1 LjSGA_092888.1 Major facilitator superfamily domain, general substrate transporter TRINITY_DN25191_c0_g1 8.625916 chr5.CM0239.470.nd chr5.CM0239.440.r2.d xylosidase/alpha-L-arabinofuranosidase 2 TRINITY_DN44884_c1_g1 8.625916 CM0466.10.nd chr5.CM0466.460.r2.d NA TRINITY_DN46831_c0_g2 8.568349 LjSGA_031037.1 chr4.CM0387.700.r2.a NA                       162    Table S42 Results of hand pollination experiment. Hand pollinated treatments are denoted by P, controls are denoted by N.  Treatment  Plant number Days to PACC from anthesis (mean of 3 reps) P 1 3.33333 P 2 3.33333 P 3 3 P 4 3 P 5 2.66667 P 6 3.33333 P 7 2.33333 P 8 2.66667 P 9 2.33333 P 10 2.33333 P 11 2.33333 P 12 2.66667 P 13 2 P 14 2 P 15 2 P 16 2.66667 P 17 2.33333 P 18 2 P 19 2 P 20 2 P 21 2 N 1 5 N 2 5.33333 N 3 4.6667 N 4 4.66667 N 5 4.66667 N 6 5 163    Treatment  Plant number Days to PACC from anthesis (mean of 3 reps)    N 7 4.66667 N 8 4.33333 N 9 4.33333 N 10 4 N 11 4.33333 N 12 4.66667 N 13 4 N 14 4 N 15 4.66667 N 16 4 N 17 5 N 18 5 N 19 4.33333 N 20 5 N 21 4.66667     Mean number of days to observe colour change in hand pollinated treatment = 2.5 ± 0.2 days Mean number of days to observe colour change in control                  = 4.6 ± 0.2 days p = 2.2 x 10-16 n  = 21        164    Appendix 4  Supplementary Figures S1 – S10 Amino acid alignments for non-differentially expressed carotenoid genes in Lotus filicaulis  Figure S1. Amino acid alignment for β-CHY  Lfil    1 ------------KPYPLTLTVKSSINKNSTKPSSWVSPDWLTSLSRSLTAGKNDDSGIPI Ljap    1 SLSLSPLSLFPPKPYPLTLSVKSSINKNSTKPSSWVSPDWLTSLSRSLTAGKNDDSGIPI Gmax    1 SLSLTPLSVFPPTPSPKRLSVKSSINKPTTKPSSWLSPDWLTSLSRSLTAG-NDVSGIPV Mtru    1 SLPLSHLSLSSFKRYPLRILTKSSTNKNTTKSTSWVSPDWLTSLSKSLTTSKNDDSNIPI Atha    1 SSSYSSLFTAKPSPKPKTFSIRSSIEKPSSKSQSWVSPDWLTTLTRTLSSGKNDESGIPI   Lfil   49 ASAKLDDVSDLLGGALFLPLFKWMKEYGPIYRLAAGPRNFVVVSDPAIAKHVLKNYGKYG Ljap   61 ASAKLDDVSDLLGGALFLPLFKWMKEYGPIYRLAAGPRNFVVVSDPAIAKHVLKNYGKYG Gmax   60 ASAKLDDVSDLLGGALFLPLFKWMQDYGPIYRLAAGPRNFVVVSDPAIAKHVLRNYGKYA Mtru   61 ASAKLDDVSDLLGGALFLPLFKWMNEYGPIYRLAAGPRNFVVVSDPAIAKHVLKNYGKYG Atha   61 ANAKLDDVADLLGGALFLPLYKWMNEYGPIYRLAAGPRNFVIVSDPAIAKHVLRNYPKYA   Lfil  109 KGLVAEVSEFLFGSGFAIAEGPLWTARRRAVVPSLHKRYLSVIVDRVFCRCAERLVEKLQ Ljap  121 KGLVAEVSEFLFGSGFAIAEGPLWTARRRAVVPSLHKRYLSVIVDRVFCRCAERLVEKLQ Gmax  120 KGLVAEVSEFLFGSGFAIAEGPLWTARRRAVVPSLHKRYLSVIVDRVFCRCAERLVEKLQ Mtru  121 KGLVAEVSEFLFGDGFAIAEGPLWTARRRAVVPSLHKRYLSIMVDRVFCKCAERLVEKLQ Atha  121 KGLVAEVSEFLFGSGFAIAEGPLWTARRRAVVPSLHRRYLSVIVERVFCKCAERLVEKLQ   Lfil  169 PDALNGTAVNMEDKFSQLTLDVIGLSVFNYDFDSLNADSPVIGAVYTALKEAEARSTDLL Ljap  181 PDALNGTAVNMEDKFSQLTLDVIGLSVFNYDFDSLNADSPVIGAVYTALKEAEARSTDLL Gmax  180 PDALNGTAVNMEAKFSQLTLDVIGLSVFNYNFDSLNTDSPVIEAVYTALKEAEARSTDLL Mtru  181 ADAVNGTAVNMEDKFSQLTLDVIGLSVFNYNFDALNSDSPVIEAVYTALKEAEARSTDLL Atha  181 PYAEDGSAVNMEAKFSQMTLDVIGLSLFNYNFDSLTTDSPVIEAVYTALKEAELRSTDLL   Lfil  229 PYWQVEFLRKIIPRQIKAEKAVSIIRKTVENLIEKCKEIVESEGERIDGDEYVNDSDPSI Ljap  241 PYWQVEFLRKIIPRQIKAENAVTIIRKTVEDLIEKCKEIVESEGERIDGDEYVNDSDPSI Gmax  240 PYWKFKFLCKIIPRQIKAEEAVSVIRKTVEDLIEKCREIVESEGERIDVEEYVNDSDPSI Mtru  241 PYWKIDFLCKIIPRQIKAENAVTVIRKTVEDLIEQCKEIVESEGERIDADEYVNDADPSI Atha  241 PYWKIDALCKIVPRQVKAEKAVTLIRETVEDLIAKCKEIVEREGERINDEEYVNDADPSI   Lfil  289 LRFLLASREEVSSDQLRDDLLSMLVAGHETTGSVLTWTLYLLSKDSSSLAKVQEEVDRVL Ljap  301 LRFLLASREEVSSDQLRDDLLSMLVAGHETTGSVLTWTLYLLSKDSSSLAKVQEEVDRVL Gmax  300 LRFLLASREEVSSVQLRDDLLSLLVAGHETTGSVLTWTLYLLSKDSSSLAKAQEEVDRVL Mtru  301 LRFLLASREEVSSVQLRDDLLSMLVAGHETTGSVLTWTLYLLSKDSSSLAKAQEEVDRVL Atha  301 LRFLLASREEVSSVQLRDDLLSMLVAGHETTGSVLTWTLYLLSKNSSALRKAQEEVDRVL   Lfil  349 QGRRPTFEDMKSLKFLTRCITESLRLYPHPPVLIRRAQVPDELPGAYKVNAGQDIMISVY Ljap  361 QGRRPTFEDMKNLKFLTRCITESLRLYPHPPVLIRRAQVPDELPGAYKVNAGQDIMISVY 165    Gmax  360 QGRRPTYEDIKDLKFLTRCIIESLRLYPHPPVLIRRAQVPDELPGGYKLDAGQDIMISVY Mtru  361 QGRRPTYEDMKDLKFLNRCIIESLRLYPHPPVLIRRSQIPDELPGDYKIDAGQDIMISVY Atha  361 EGRNPAFEDIKELKYITRCINESMRLYPHPPVLIRRAQVPDILPGNYKVNTGQDIMISVY   Lfil  409 NIHHSSEVWDRAEEFMPERFDLDGPMPNETNTDFRFIPFSGGPRKCVGDQFALLEATVSL Ljap  421 NIHHSSEVWDRAEEFLPERFDLDGPMPNETNTDFRFIPFSGGPRKCVGDQFALLEATVSL Gmax  420 NIHRSSEVWDRAEEFVPERFDLDGPVPNETNTDFRFIPFSGGPRKCVGDQFALMEAIVAL Mtru  421 NIHHSSKVWDRAEEFLPERFDLDGPVPNETNTDFRFIPFSGGPRKCVGDQFALLEATVAL Atha  421 NIHRSSEVWEKAEEFLPERFDIDGAIPNETNTDFKFIPFSGGPRKCVGDQFALMEAIVAL   Lfil  469 AIFLQHMNFELVPDQNISMTTGATIHTTNGLYMKLSQRVK Ljap  481 AIFLQHMNFELVPDQNISMTTGATIHTTNGLYMKLSQRVK Gmax  480 AIFLQHMNFELVPDQNISMTTGATIHTTNGLYMKLSRRLK Mtru  481 AVFLQHMNFELVPDQNIGMTTGATIHTTNGLYMKLSQRLK Atha  481 AVFLQRLNVELVPDQTISMTTGATIHTTNGLYMKVSQR--       166    Figure S2. Amino acid alignment for CDS  Lfil    1 MAGCGILSAVNLNYHGGARNTIKLCSSSSSTDSSISLSFGGSESLRLG--PSTRARLRNH Ljap    1 MAGCGILSAVNLNYHAGARNTIKLGSSSSSTDSSISLSFGGSESLRLG--PSTRARLRNH Gmax    1 MAACGYISAANFNYLVGARNISKFAS----SDATISFSFGGSDSMGLTLRA----PKRNH Mtru    1 MALYGSISSPNLNWQIGPKTISKSTSSSMNSYTTISLSFSSSVSMGLNLRPSTRARLRNH Atha    1 MVVFGNVSAANLPYQNGFLE---------------ALSSGGCELMGHSFRPTSQARRRST   Lfil   59 GSPLRVVCIDYPRPQLENTVNFLEAAYLSSTFRASPRPTKPLKVVIAGAGLAGLSTAKYL Ljap   59 GSPFRVVCIDYPRPQLENTVNFLEAAYLSSTFRASPRPAKPLKVVIAGAGLAGLSTAKYL Gmax   53 FSPLRVVCVDYPRPELENTVNFVEAAYLSSTFRASPRPLKPLNIVIAGAGLAGLSTAKYL Mtru   61 GSPLKVVCIDYPRPELDDTVNFIEASYLSSTFRASPRPTKPLKVVIAGAGLAGLSTAKYL Atha   46 AGPLQVVCVDIPRPELENTVNFLEAASLSASFRSAPRPAKPLKVVIAGAGLAGLSTAKYL   Lfil  119 ADAGHKPILLEARDVLGGKVAAWKDEDGDWYETGLHIFFGAYPNVQNLFGELGINDRLQW Ljap  119 ADAGHKPILLEARDVLGGKVAAWKDEDGDWYETGLHIFFGAYPNVQNLFGELGINDRLQW Gmax  113 ADAGHKPILLEARDVLGGKVAAWKDKDGDWYETGLHIFFGAYPNVQNLFGELGINDRLQW Mtru  121 ADAGHKPILLEARDVLGGKVAAWKDEDGDWYETGLHIFFGAYPNVQNLFGELGINDRLQW Atha  106 ADAGHKPLLLEARDVLGGKIAAWKDEDGDWYETGLHIFFGAYPNVQNLFGELGINDRLQW   Lfil  179 KEHSMIFAMPSKPGEFSRFDFAEVLPAP Ljap  179 KEHSMIFAMPSKPGEFSRFDFAEVLPAP Gmax  173 KEHSMIFAMPNKPGEFSRFDFPEVLPSP Mtru  181 KEHSMIFAMPSKPGEFSRFDFPEVLPSP Atha  166 KEHSMIFAMPSKPGEFSRFDFPDVLPAP       167    Figure S3. Amino acid alignment for CRTISO-2  Lfil    1 EADVVVIGSGLGGLCCAALLARYEQNVLVLESHDLPGGAAHSFDIKGYKFDSGPSLFSGL Ljap    1 EADVVVIGSGLGGLCCAALLARYEQNVLVLESHDLPG----------------------- Gmax    1 EADVVVIGSGIGGLCCGALLARYEEDVLVLESHDQPGGAAHSFDIKGYNFDSGPSLFSGL Mtru    1 ETDVIVIGSGIGGLSCAALLARYEQDVVVFESHDHAGGAAHSFDVKGYKFDSGPSLFSGL Atha    1 EADVVVIGSGIGGLCCGALLARYDQDVIVLESHDHPGGAAHSFEIKGYKFDSGPSLFSGL   Lfil   61 QSRGPQANPLAQVLDALGESLPCATYDSWKVYLPEGEFLSRIGPTEFSKDLEKYGGPNAV Ljap   38 ---------------------------SWMRWVSHSHALLM------------------- Gmax   61 QSRGPQANPLAQVLDALGESLPCATYDSWMVHIPEGQFLSRIGPTEFFKDLQQYAGPNAV Mtru   61 QSRGPQANPLAQVLDALGESVPCATYDSWMVHVPEADFLSRIGPTEFLKDLHNYAGPEAV Atha   61 QSRGPQANPLAQVLDALGESFPCKKYDSWMVYLPEGDFLSRIGPTDFFKDLEKYAGPSAV   Lfil  121 QEWRKLLDAVLPLSTAAMALPPLSIRGDLGVLYTAAARYAPSLLKSFLQMGPQGALGATK Ljap   52 ------------------------------------------------------------ Gmax  121 LEWRKLLDAVLPLSTAAMALPPLSIRGDLGVLSTAAARYAPSLFKSFLQMGPRASLRATK Mtru  121 QEWQKLLDAVLPLSTAAMALPPLSVRGDFGVLYTAAARYAPSLFNTFFQMGPQAALRSTQ Atha  121 QEWEKLLGAILPLSSAAMALPPLSIRGDLGVLSTAAARYAPSLLKSFIKMGPKGALGATK   Lfil  181 LLRPFKEILDGLELKDPFLRNWIDLLSFLLAG---------------------------- Ljap   52 ------------------------------------------------------------ Gmax  181 LLRPFSEILDDLQLNDPFIRNWIDLLSFLLAGVKANAVLSAEMIYMFAEWYKPGCCLEYP Mtru  181 LLSPFSQILDSLQLKNPFIRNWIDLLSFLLAGVKSDSILSAEMVYMFAEWYKPGGCLEYP Atha  181 LLRPFSEIVDSLELKDPFIRNWIDLLAFLLAGVKSDGILSAEMIYMFAEWYKPGCTLEYP   Lfil      -------------------------------------------------------- Ljap   52 -------------------------------------------IHGRFIYLKVNLV Gmax  241 LHGSAAIIDALVRGLEKFGGRISLQSHVENIVVENDRAIGVKLGSGQFIRAKKAVV Mtru  241 LDGAAGIVDALIRGLEKFGGRISLQSHVEKIVVENDRAIGVKLRSGQFIRAKKAVV Atha  241 IDGTGAVVEALVRGLEKFGGRLSLKSHVENIVIENGKAVGVKLRNGQFVRARKAVV      168    Figure S4. Amino acid alignment for CRTISO-3  Lfil    1 SSSIFSSLNKFNPLSLRSPLRIRATVVKDTEGKVVVEKTFPSKIPLQTGAEEDSDSSPPT Ljap    1 SSSIFSSLNKFSPLSLRSPLRVRATVVKDTEGKVVVEKTFPSKIPLQTGAEEDSDSSPPT Gmax    1 SSSLFSPLPK----HTAPQLRARSSLLQDNEKKVIVHDSFPSKTPLHTADK-GGNSINTS Mtru    1 CDSSSSLFTPLNKFRPLSFRPPLRIRSSLLQDKEDKVITQNTFPSKTPLDSVTENDDDTS Atha    1 SSGTLRPLVTHLPLSSRRLLRVQATILQDDEEKVVVEESFKAETSTGTEPLENMSSSSTS   Lfil   61 AFELGVIKVEQSINIFLTDSVIKILDALYRDRNYARFFVLETIARVPYFAFMSVLHMYES Ljap   61 AFELGVIKVEQSINIFLTDSVIKILDALYRDRNYARFFVLETIARVPYFAFMSVLHMYES Gmax   56 AFEKRIIKVEQSVNIFLTDSVIKILDTLYHDRHYARFFVLETIARVPYFAFMSVLHMYES Mtru   61 STSAWEKGVIKVEQSVNIFLTDSVIKILDALYRDRNYARFFVLETIARVPYFAFMSILHM Atha   61 AFETWIIKLEQGVNVFLTDSVIKILDTLYRDRTYARFFVLETIARVPYFAFMSVLHMYET   Lfil  121 FGWWRRADYLKVHFAESWNEMHHLLIMEELGGNAWWFDRFLAQHIAIFYYFMTALMYTIS Ljap  121 FGWWRRADYLKVHFAESWNEMHHLLIMEELGGNAWWFDRFLAQHIAIFYYFMTALMYTIS Gmax  116 FGWWRRADYLKVHFAESWNEMHHLLIMEELGGNAWWFDRFLAQHIAIFYYIMTVLMYAVS Mtru  121 YESFGWWRRADYLKVHFAESWNEMHHLLIMEELGGNAWWFDRFLAQHIAIFYYFMTALMY Atha  121 FGWWRRADYLKVHFAESWNEMHHLLIMEELGGNSWWFDRFLAQHIATFYYFMTVFLYILS   Lfil  181 PRMAYHFSECVENHAFETYDKFIKDQ--- Ljap  181 PRMAYHFSECVENHAFETYDKFIKDQ--- Gmax  176 PRMAYHFSECVESHAFETYDKFIKVQ--- Mtru  181 LISPRMAYHFSECVESHAFETYDKFIKEQ Atha  181 PRMAYHFSECVESHAYETYDKFLKAS---                 169    Figure S5. Amino acid alignment for CYB-1 Lfil    1 ------------------------------------------------------------ Ljap    1 ILVETDHIGRQASGSVTVTDGETIVYTTVCLDDVPSEPSDFFPLSVNYQERFSAAGRTSG Gmax    1 ILVETAHIGRQASGSVTVTDGETIVYTTVCLDDVPCEPSDFFPLSVIYQERFSAAGRTSG Mtru    1 ILVETGHMGRQASGSVTVTDGETIVYTTVCLDDIPSEPSDFFPLSVNYQERFSAAGRTSG Atha    1 ILVETGLMGRQASSAVTVTDGETIVYTSVCLADVPSEPSDFLPLYVHYQERFSAVGRTSG   Lfil    1 ------------------------------------------------------------ Ljap   61 GFFKREGKTKDHEVLICRLIDRPLRPSMPKGFYHETQILSWVLSYDGLHSPDSLAVTAAG Gmax   61 GFFKREGKIKDHEVLICRLIDRPLRPTMPKGFYHETQILSWVLSYDGLHSPDSLAITAAG Mtru   61 GFFKREGKTKDHEVLICRLIDRPLRPTMPKGFYHETQILSWVLSYDGLHIPDSLAVTAAG Atha   61 GFFKREGRTKDHEVLICRLIDRPLRPTMPKGFYNETQILSWVLSYDGLHAPDALAVTSAG   Lfil    1 ------------------------------------------------------------ Ljap  121 IAVALSEVPMSKTVAGVRIGLVGDKFLVNPTTEEMENSELDLLLAGTDSAILMIEGYSNF Gmax  121 IAVALSEVPMSKAVAGVRVGLVGDKYIVNPTTEEMEHSELDLLLAGTDNAILMIEGYSNF Mtru  121 IALALSEVPTSKAVAGVRVGLVGDKYIVNPTSEEMENSELDLMLAGTDSAILMIEGYGNF Atha  121 IAVALSEVPNAKAIAGVRVGLIGGEFIVNPTVKEMEESQLDLFLAGTDTAILTIEGYSNF   Lfil    1 ------------------------------------------------------------ Ljap  181 LPEEKLLEAVEVGQDAVRAICKEVAALVKKCGKPKMFDAIKLPPPELYKHVEEIAGDELV Gmax  181 LPEEKLLKAVEVGQNAVRAICNEVEALVKKCGKPKMLDAIKLPPPELYEHVEAIASDELV Mtru  181 LPEEKLLKAVEVGQDAVRAICNEVEALVKKCGKPKMVDAIKLPPPELYKHVEEIAGDELV Atha  181 LPEEMLLQAVKVGQDAVQATCIAIEVLAKKYGKPKMLDAIRLPPPELYKHVKELAGEELT   Lfil    1 ------------------------------------------------------------ Ljap  241 KVLQIRNKIPRRKALSSLEEKVIKILTENGVVTNDATQRSNTETIAEILEDEDEDEEVIV Gmax  241 KVLQIRNKIPRRKALSSLEEKVLKILTENGFVSNDSTLRSNSETIAEILEVEDEDEEVIV Mtru  241 KVLQIRNKIPRRKALSSLEEKVLKILSENGFVTNDTAPRNNAETIAEILEDEDEDEEVIV Atha  241 KALQIKSKISRRKAISSLEEKVLTILTEKGYVIDEVA-FGTIEAQPDLLEDEDEDEEVVP   Lfil    1 -------------------------------VTSKFLRKRIVEGGKRSDGRKPNEIRPIN Ljap  301 DGEVDEGDVHIKPTARKPLFSEVDVKLVFKEVTSKFLRKRIVEGGKRSDGRKPNEIRPIN Gmax  301 DGEVDEGDVHIKPTPRKPLFSEVDVKLVFKEVTSKYWRKRMVEGGKRSDGRTPDGIRPIY Mtru  301 DGEVDEGDVHIKPTPRKPLFSEVDVKLVFKDVTSKFLRKRIVEGGKRSDGRTPNEIRPIN Atha  300 EGEVDQGDVHIRPIPRKPLFSEVDVKLVFKEVSSKLLRRRIVEGGKRSDGRTLDEIRPIN   Lfil   30 SRCGLLPRAHGSTLFTRGETQSLAVVTLGDKQMAQRIDNLMGVDEFKKFYLQYSFPPSCV Ljap  361 SRCGLLPRAHGSTLFTRGETQSLAVVTLGDKQMAQRIDNLMGVDEFKKFYLQYSFPPSCV Gmax  361 SRCGLLPRAHGSTLFTRGETQALAVVTLGDKQMAQRIDSLDGVDEFKRFYLQYSFPPSCV Mtru  361 SSCGLLPRAHGSALFTRGETQSLAVATLGDNKMAQRIDNLMDVDDYKRFYLQYSFPPSCV Atha  360 SRCGLLPRAHGSTLFTRGETQALAVVTLGDKQMAQRIDNLEGSDEYKRFYLQYTFPPSSV   Lfil   90 GEVGRIGAPSRREIGHGMLAERSLEPILPSDKDFPYTIRVESTITESNGSSSMASVCGGC Ljap  421 GEVGRIGAPSRREIGHGMLAERSLEPILPSDKDFPYTIRVESTITESNGSSSMASVCGGC Gmax  421 GEVGRIGAPSRREIGHGMLAERSLEPILPSEDDFPYTIRVESTITESNGSSSMASVCGGC Mtru  421 GEVGRIGAPSRREIGHGMLAERSLETILPSDKDFPYTIRVESTITESNGSSSMASVCGGC Atha  420 GEVGRIGAPSRREIGHGTLAERALETILPSDDDFPYTIRVESTVIESNGSSSMASVCGGC  170     Lfil  150 LALQDAGVPIKSAIAGIAMGMVLDTKEFGGDGTPLILSDITGSEDASGDMDFKVAGNEHG Ljap  481 LALQDAGVPIKSAIAGIAMGMVLDTKEFGGDGTPLILSDITGSEDASGDMDFKVAGNEHG Gmax  481 LALQDAGVPIKGSIAGIAMGMVLDTKEFGGDGTPLILSDITGSEDASGDMDFKVAGNEDG Mtru  481 LALQDAGVPIKSSIAGIAMGLVLDTKEFGGDGTPLILSDITGSEDASGDMDFKVAGNEDG Atha  480 LALQDAGVPVKCSVAGIAMGMVWDTEEFGGDGSPLILSDITGAEDASGDMDFKVAGNEDG   Lfil  210 ITAFQMDIKVGGITLPVMREALLQARDGRKHILGEMMNCSPPPAKILSKYAPLIHIMKVR Ljap  541 ITAFQMDIKVGGITLPVMREALLQARDGRKHILGEMMNCSPPPAKILSKYAPLIHIMKVR Gmax  541 ITAFQMDIKVGGITLPIMRKALLQARDGRKHILGAMMNSSPPPAKRLSKYAPLIHVMKVR Mtru  541 ITAFQMDIKVGGITLAIMREALLQAKDGRKHILGEMMKCLPPPAKRLSKYAPLIHVMKVR Atha  540 VTAFQMDIKVGGITLEIMEKALIQAKAGRRHILAEMAKCSPPPTLSLSKYAPLILIMKVH Lfil  270 PDKVNLIIGSGGKKVKSIIEESGIEAIDTDDNGTVKIFARDLSSLERSKAMISNLTMVPA Ljap  601 PDKVNLIIGSGGKKVKSIIEESGIEAIDTDDNGTVKIFARDLSSLERSKAMISNLTMVPA Gmax  601 PDRINLIIGSGGKKVKSIIEESGVDTIDTEDDGTVKIFAKDLPSLEKSKAIINSLTMVPT Mtru  601 PDKINLIIGSGGKKVKSIIEQSGIEAIDTEDNGTVKIFARDLASLEMSKAIISNLTMVPT Atha  600 PSKVYSLIGSGGKKVKSIIEESGVEAIDMQDDGTVKIMAIDVASLERAKAIISGLTMVPS   Lfil  330 IGDIYRNCEIKSIAPYGVFVEIAPGREGLCHISELSSGWLPKAEDVFKVGDRIDVKLIEI Ljap  661 IGDIYRNCEIKSIAPYGVFVEIAPGREGLCHISELSSGWLPKAEDVFKVGDRIDVKLIEI Gmax  661 IGDIYRSCEIKSIASYGVFVEIAPGREGLCHISELSSGWLPKAEDVFKVGDRIDVKLIEI Mtru  661 IGDIYRNCEIKSIVPYGAFVEIAPGREGLCHISELSSAWLAKAEDAFKVGDRIDVKLIEI Atha  660 VGDIYRNCEIKSMAPYGAFVEIAPGREGLCHISELSAEWLAKPEDAYKVGDRIDVKLIEV   Lfil  390 NDKGQLRLSHRALLPDTDPDGIAEEKIEQPKDKINTPKGDSSKRSSRDDSALPSKKFIRR Ljap  721 NDKGQLRLSHRALLPDTDPDGIAEEKIEQPKDKINTPKGDSSKRSSRDDSALPSKKFIRR Gmax  721 NEKGQLRLSHRALLPDTEASGLVEEKTEQSKDKSSTPNVTSSKRSSEDDSVLPSKKFVRR Mtru  721 NDKGQLRLSHRALLPDADPDGLVEEKTEQN-AKVNTRKGRSSKRSSEDNSALPSKKFIRR Atha  720 NEKGQLRLSVRALLPESETDDSTKDKSSQR-KYVNTSSKDASKVSSGDELVL-KKKDVRR   Lfil  450 MVSPSQDKPVTNKDKIKRSSNKASDKDESSLVGEEA Ljap  781 MVSPSQDKPVTNKDKIKRSSNKASDKDESSLVSEEA Gmax  781 LVSSSLDKPFTNKDKTKKSSNKASGKDESSLVSGEA Mtru  780 S---SEDKPVTNKDKIKKSDN----KDESSLVSEEA Atha  778 ATGGSSDKTMNS-----------SSTNEESLVNGEA       171    Figure S6. Amino acid alignment for CYB-2 Lfil    1 LRASSSALLELVPEFKKENLDFELPLYDSTKGAVVNLAVVGGGPAGLAVAQQVSEAGLSV Ljap    1 LRASSSALLELVPEVKKENLDFELPLYDSMKGAVVDLAVVGGGPAGLAVAQQVSEAGLSV Gmax    1 LRASSSALLELVPEFKKENLDFELPLYDSSKGAMVDLAVVGGGPAGLAVAQQVSEAGLSV Mtru    1 LRASSSALLELVPEFKKENLDFELPLYDSTKGTVVDLVVVGGGPAGLAVAQQVSEAGLSV Atha    1 VVSGSAALLDLVPETKKENLDFELPLYDTSKSQVVDLAIVGGGPAGLAVAQQVSEAGLSV   Lfil   61 CAIDPNPRLIWPNNYGVWVDEFEAMDLLDCLDTTWSGAVVYIDDKTKKYLDRPYGRVNRK Ljap   61 CAIDPNPRLIWPNNYGVWVDEFEAMDLLDCLDTTWSGAVVYIDDKTKKYLDRPYGRVNRK Gmax   61 CAIDPNPRLIWPNNYGVWVDEFEAMDLLDCLDTIWSGAVVFIDDKTKKDLDRPYGRVNRK Mtru   61 CAIDPNPRLIWPNNYGVWVDEFEAMDLLDCLDKTWSGAVVYIDDKTKKDLDRPYGRVNRK Atha   61 CSIDPSPKLIWPNNYGVWVDEFEAMDLLDCLDTTWSGAVVYVDEGVKKDLSRPYGRVNRK   Lfil  121 LLKSKMLQKCISNGVKFHQAKVIKVIHEESKSMLICNDGVTIQATVVLDATGFSRSLVQY Ljap  121 LLKSKMLQKCISNGVKFHQAKVIKVIHEESKSMLICNDGVTIQATVVLDATGFSRSLVQY Gmax  121 LLKSKMLQKCISNGVKFHQAKVIKVIHEEAKSLLICNDGVTVHATVVLDATGFSRCLVQY Mtru  121 LLKSKMLQKCIANGVKFHQAKVIKVIHEESKSMLICNDGVTVQATVVLDATGFSRCLVQY Atha  121 QLKSKMLQKCITNGVKFHQSKVTNVVHEEANSTVVCSDGVKIQASVVLDATGFSRCLVQY   Lfil  181 DKPYNPGYQVAYGILAEVEEHPFDVDKMLFMDWRDSHLDNNMELKEKNSRIPTFLYAMPF Ljap  181 DKPYNPGYQVAYGILAEVEEHPFDVDKMLFMDWRDSHLDNNMELKEKNSRIPTFLYAMPF Gmax  181 DKPYNPGYQVAYGILAEVDEHPFDVDKMLFMDWRDSHLDNDMELKQRNSRIPTFLYAMPF Mtru  181 DKPYNPGYQVAYGILAEVEEHPFDVDKMLFMDWRDSHLDNDLKLKERNSKIPTFLYAMPF Atha  181 DKPYNPGYQVAYGIVAEVDGHPFDVDKMVFMDWRDKHLDSYPELKERNSKIPTFLYAMPF   Lfil  241 SSTKIFLEETSLVARPGLRMDDIQDRMVARLKHLGIKVKSIEEDEHCVIPMGGPLPVLPQ Ljap  241 SSTKIFLEETSLVARPGLRMDDIQDRMVARLKHLGIKVKSIEEDEHCVIPMGGPLPVLPQ Gmax  241 SSTKIFLEETSLVARPGLRMDDIQERMAARLKHLGIRVKSIEEDEHCVIPMGGPLPVLPQ Mtru  241 SSTKIFLEETSLVARPGLRMDDIQDRMVARLKHLGINVKSIEEDEQCVIPMGGPLPVLPQ Atha  241 SSNRIFLEETSLVARPGLRMEDIQERMAARLKHLGINVKRIEEDERCVIPMGGPLPVLPQ   Lfil  301 RVIGIGGTAGMVHPSTGYMVARTLAAAPIVANAIVQYLGSDRGISGDGLSSLVWKDLWPI Ljap  301 RVIGIGGTAGMVHPSTGYMVARTLAAAPIVANAIVQYLGSDRGISGDGLSSLVWKDLWPI Gmax  301 RVVGIGGTAGMVHPSTGYMVARTLAAAPIVANSIVQCLGSDRGFSGDEISAQVWKDLWPI Mtru  301 RVVGIGGTAGMVHPSTGYMVARTLAAAPIVANAIVQYLGSDRGLLGDEVSARVWKDLWPI Atha  301 RVVGIGGTAGMVHPSTGYMVARTLAAAPIVANAIVRYLGSPNSLRGDQLSAEVWRDLWPI   Lfil  361 ERRRQREFFCFGMDILLKLDLPGTRRFFDAFFDLEPHYWHGFLSSRLFLPELLVFGLSLF Ljap  361 ERRRQREFFCFGMDILLKLDLPGTRRFFDAFFDLEPHYWHGFLSSRLFLPELLVFGLSLF Gmax  361 QRRRQREFFCFGMDILLKLDLPGTKRFFDAFFDLEPHYWHGFLSSRLFLHELLFFGLSLF Mtru  361 ERRRQREFFCFGMDVLLKLDLPGTRRFFDAFFNLEPHYWHGFLSSRLYLPELFTFGLSLF Atha  361 ERRRQREFFCFGMDILLKLDLDATRRFFDAFFDLQPHYWHGFLSSRLFLPELLVFGLSLF   Lfil  421 SYASNTSRIEIMAKGTVP Ljap  421 SYASNTSRIEIMAKGTVP Gmax  421 SYASNTSRVEIMAKGTLP Mtru  421 SYASNTSRLEIMAKGTLP Atha  421 SHASNTSRLEIMTKGTVP  172    Figure S7. Amino acid alignment for CYE  Lfil    1 MLLLQLQPFNGSHLTVGTPFPLKKCSRRTLLLRPQAVPSRTQRIMESVSVSGEVGGAGGA Ljap    1 MLLLQLQPFNGSHLTVGTPFPQKKCSRRTLLLRPQAVPSRTQRIMESVSVSGEVGGAGGA Gmax    1 MLLLRLQPINGSQLSVGTSFPHKKCSRRPLLSRPQAVPSRTQRIMESVSVSGEVGGAGGA Mtru    1 MSLLQLQPINGSQLLV-----QKKCSRKAFRFRAQAVPSKTQRIMESVSVSGEVGGAGGA Atha    1 MVVLQIHQFNGSSVSRAQTNQGRRRASTTVCVQTQLAPSLTQKIMESISVGGEAGGAGGA   Lfil   61 YSYEALKRLDQLWSSLCSTQEVVQEPQQVVSSIPSLFRSSDLANKAEGSYDVVVCGGTLG Ljap   61 YSYEALKRLDQLWSSLCSAQEVVQEPQQVVSSIPSLFRSSDLANKAEGSYDVVVCGGTLG Gmax   61 YSYEALKRLDQQWSSICSSQEVVQEPQQVVSTIPSLFGSSDLADKAEGSYDVLVCGGTLG Mtru   56 YSYEALKRLDQLWSNICSPQEVVQEPQQIVSTIPSLFTSSDLADKIEGSYDVLVCGGTLG Atha   61 YSYNALKRLDNIWSNICTQPTGPQETQQIVSRVSGFSQDYSMGNNLVGTFDIVVCGGTLG   Lfil  121 IFIATALCARGLKVAIVERNVLKGREQDWNISRKELLELVEVGVLEEDDIERATSINFNP Ljap  121 IFIATALCARGLKVAIVERNVLKGREQDWNISRKELLELVEVGVLEEDDIERATSINFNP Gmax  121 IFIATALCARGLRVAIVERNVLKGRDQEWNISTKELLELVEVGVLEEDDIERATAVKFNP Mtru  116 IFIATALCARGLRVAIVERNVLKGREQEWNISRKELLELVEVGILEEDDIERVTTAKFNP Atha  121 IFLATALCAKGLRVAVVERNAIKGRDQEWNISRKEMKELTEVRVLTEDEIEEVIAAKFNP   Lfil  181 NRCGFESKGDIWVNNILHLGVSPVKLLEIVKERFISLGGVIFEGCSVSCINIYEDAAVLK Ljap  181 NRCGFESKGDIWVNNILHLGVSPVKLIEIVKERFISLGGVIFEGCSVSCINIYEDAAVLK Gmax  181 NRCGFERKGDIWVNDILNLGVSPAKLIEIVKTRFISLGGVIFEGCGVSCINVYEDAAVLK Mtru  176 NRCGFESKGDIWVNNILNLGVSPVRLIEIVKKRFISLGGVFFEGYSVSFINVYEDAAVLK Atha  181 NRCGFENLGDIWVEDILNLGVSPAKLVETVKQRFISLGGVILEDSSLSSIVIYNDLAVMQ   Lfil  241 LSGDKILSSRLIIDAMGNFSPIVKQIRRGRKPDGVCLVVGTCARGFESNSTSDVIFSSSS Ljap  241 LSGDKILSSRLIIDAMGNFSPIVKQIRRGRKPDGVCLVVGTCARGFESNSTSDVIFSSSS Gmax  241 LSGDKILSSRLIIDAMGNFSPVVKQIRGGRKPDGVCLVVGTCARGFETNSTSDVIFSSSS Mtru  236 LSGGKVLSSRLIIDAMGNFSPVVKQIRRGRKPDGVCLVVGTCARGFENNSNSDVIFSSSS Atha  241 LSKGDTLSSRLVIDAMGNFSPILKQIKRGRKPDGMCLVVGSCAHGFKENSSSDVIYSSSS   Lfil  301 VKKVGDSQAQYFWEAFPAASGPLDRTTYMFTYVEAQPGSPKLEELLEAYWDLMPEYQGVS Ljap  301 VKKVGDSQAQYFWEAFPAASGPLDRTTYMFTYVEAQPGSPKLEELLEAYWDLMPEYQGVS Gmax  301 IKKVGDSKAQLFWEAFPAGSGPLDRTTYLFTYVEPQPASPKLEELLEAYWDLMPEYQGVS Mtru  296 VKRVGDSKAQYFWEAFPAGSGPLDRTTYMFTYVEPQPGSPKLEELLEEYWNLMPEYQGVP Atha  301 VTRVADSNVQLFWEAFPAGSGPLDRTTYMFTYTEPQSTSPSLEDLLEEYWKLMPKYQGVS   Lfil  361 LDNLEILRVIYGIFPTYRESPLPAAFSRVLQFGDASGIQSPVSFGGFGSLTRHLGRISAG Ljap  361 LDNLEILRVIYGIFPTYRESPLPAAFCRVLQFGDASGIQSPVSFGGFGSLTRHLGRISAG Gmax  361 LDNLEILRVIYGIFPTYRQSPLPAAFSRVLQFGDASGIQSPVSFGGFGSLTRHLGRLSAG Mtru  356 LDNLEILRVIYGIFPTYRESPLPAAFSRVLQFGDASGIQSPVSFGGFGSLTRHLGRLSAG Atha  361 LDELEILRVVYGIFPTYRNSPLPAAFDRVLQFGDASGIQSPVSFGGFGSLTRHLGRLSNG   Lfil  421 IYEAINGDYLDSYNLSLLNPYMPNLSASWLFQRAMSAKKQSNVPPDFINELLYANFSCMQ Ljap  421 IYEAINGDYLDSYNLSLLNPYMPNLSASWLFQRAMSAKKQSNVPPDFINELLFANFSCMQ Gmax  421 IHEAINGDYLDSYSLSLLNPYMPNLSASWLFQRAMSAKKQSNVPADFINELLYTNFSCMQ Mtru  416 IQEAIDGDYLDSYNLSLLNPYMPNLSASWLFQRAMSAKQQSDVPEDFINELLYANFSCMQ Atha  421 IYDAIDGDLLDSDSLSKLNPYMPNLSASWLFQRAMSAKQQLDVSRGFTNELLHVNFSCMQ 173      Lfil  481 RLGDPVLRPFLQDVVQFGPLSKTLGLVMLTKPQILPSIFK Ljap  481 RLGDPVLRPFLQDVVQFGPLSKTLGLVMLTKPQILPSIFK Gmax  481 RLGDPVLRPFLQDVVQFGPLSKTLGLVMLTNPKILPSIFK Mtru  476 RLGDPVLRPFLQDVVQFGALSKTLGLVMLTRPQILPSIFK Atha  481 RLGDPVLRPFLQDIIQFGPLAKTLGLVMLTKPQIIPSIFR       174    Figure S8. Amino acid alignment for E-CHY  Lfil    1 MASHAALLHVPPPLSIPTQRFYHKQICINRLKPSTTSSSSSWFPCSSSSVIVCSSSNGKS Ljap    1 MASHAALLHVPPPLSIPTQRFYHKQICINRLKPSTTSSSSSWFPCSSSSVIVCSSSNGKS Gmax    1 MASHVALLRVPPPLSISTQRFHAKQICINGLKLTNTSSSSSCFPCSCSSVITCSSSNGRD Mtru    1 MASHLTLLHAPPPLSLQTKTFHSKYITIKPLKPTTTSSSCSLFPCSCSSFIACSSSNGRS Atha    1 MAMAFPL-SYTPTI------------TVKP--------------V-SNFVVFSSSSNGRD   Lfil   61 SDSGDDG------IKSKQRAELSARIAKGEFTVNKQSGFPSDLKKSLSQVGVPNEILDFL Ljap   61 SDSGDDG------IKSKQRAELSARIAKGEFTVNKQSGFPSDLKKSLSQVGVPNEILDFL Gmax   61 PNSVDEEKQVERILEEKRRAALSAKIASGEFTVKQKSGLLSI-MEGLAKVGVPNEVLEFL Mtru   61 PDSVDDGKSADQLLEEKRRAELSAKIASGEFTVKQESGLPSILKKSLSNLGVSNEILEFL Atha   33 PNSVPNGKSLEKLQEEKRRAELSARIASGAFTVRKS-SFPSTVKNGLSKIGIPSNVLDFM   Lfil  115 FGWIEGGGYYPKVPEAKGSINAVRNEAFFIPLYELYLTYGGIFRLTFGPKSFLIVSDPSI Ljap  115 FGWIEGGGYYPKVPEAKGSINAVRNEAFFIPLYELYLTYGGIFRLTFGPKSFLIVSDPSI Gmax  120 FGWFEGGGEHPKIPEAKGSIKAVRSVAFFIPLYELYLTYGGIFRLTFGPKSFLIVSDPSI Mtru  121 FG------LYPKIPEAKGSISAIRSEAFFIPLYELYITYGGIFRLNFGPKSFLIVSDPAI Atha   92 FDWTGSDQDYPKVPEAKGSIQAVRNEAFFIPLYELFLTYGGIFRLTFGPKSFLIVSDPSI   Lfil  175 AKHILKDNAKGYSKGILAEILDFVMGKGLIPADGEIWRVRRRAIVPALHQKYVAAMIGLF Ljap  175 AKHILKDNAKGYSKGILAEILDFVMGKGLIPADGEIWRVRRRAIVPALHQKYVAAMIGLF Gmax  180 AKHILRENSKAYSKGILAEILDFVMGKGLIPADGEIWRVRRRAIVPALHQKYVAAMIGLF Mtru  175 AKHILKDNSKAYSKGILAEILDFVMGKGLIPADGEIWRVRRRTIVPALHLKFVAAMIGLF Atha  152 AKHILKDNAKAYSKGILAEILDFVMGKGLIPADGEIWRRRRRAIVPALHQKYVAAMISLF   Lfil  235 GQATDRLCKKLDTAASDGEDVEMESLFSRLTLDVIGKAVFNYDFDSLSNDTGIIEAVYNV Ljap  235 GQATDRLCKKLDTAASDGEDVEMESLFSRLTLDVIGKAVFNYDFDSLSNDTGIIEAVYNV Gmax  240 GQAADRLCQKLDAAASDGEDVEMESLFSRLTLDIIGKAVFNYDFDSLSNDTGIVEAVYTV Mtru  235 GQATDRLCQKLDTAASDGEDVEMESLFSRLTLDVIGKAVFNYDFDSLSNDTGIIEAVYTV Atha  212 GEASDRLCQKLDAAALKGEEVEMESLFSRLTLDIIGKAVFNYDFDSLTNDTGVIEAVYTV   Lfil  295 LREAEDRSVSPIPTWEIPVWKDISPRQKKVNAALKLVNDTLDDLIAICKRMVDEEELQFH Ljap  295 LREAEDRSVSPIPTWEIPIWKDISPRQKKVNAALKLVNDTLDDLIAICKRMVDEEELQFH Gmax  300 LREAEDRSVAPIPVWEIPIWKDVSPRLRKVNAALKLINDTLDDLIAICKRMVDEEELQFH Mtru  295 LREAEDRSISPIPVWDLPIWKDISPRQRKVTAALKLVNDTLNNLIAICKRMVDEEELQFH Atha  272 LREAEDRSVSPIPVWDIPIWKDISPRQRKVATSLKLINDTLDDLIATCKRMVEEEELQFH   Lfil  355 EEYMNEQDPSILHFLLAAGDDVSSKQLRDDLMTLLIAGHETSAAVLTWTFYLLSKEPSVM Ljap  355 EEYMNEQDPSILHFLLAAGDDVSSKQLRDDLMTLLIAGHETSAAVLTWTFYLLSKEPSVM Gmax  360 EEYMNEQDPSILHFLLASGDDVSSKQLRDDLMTMLIAGHETSAAVLTWTFYLLSKEPRVM Mtru  355 EEYMNEQDPSILHFLLASGDDVTSKQLRDDLMTMLIAGHETSAAVLTWTFYLLSKEPSVM Atha  332 EEYMNERDPSILHFLLASGDDVSSKQLRDDLMTMLIAGHETSAAVLTWTFYLLTTEPSVV   Lfil  415 SKLQEEVDSVLGDRFPTIEDMKKLKYTTRVINESLRLYPQPPVLIRRSIDDDVLGEYPIK Ljap  415 SKLQEEVDSVLGDRFPTIEDMKKLKYTTRVINESLRLYPQPPVLIRRSIDDDVLGEYPIK Gmax  420 SKLQEEVDSVLGDQYPTIEDMKKLKYTTRVINESLRLYPQPPVLIRRSLEDDVLGEYPIK Mtru  415 SKLQEEVDSVLGDRFPTIEDMKKLKYTTRVINESLRLYPQPPVLIRRSIEDDVLGEYPIK Atha  392 AKLQEEVDSVIGDRFPTIQDMKKLKYTTRVMNESLRLYPQPPVLIRRSIDNDILGEYPIK 175      Lfil  475 KGEDIFISLWNLHRSPHLWDDADKFEPERWPLDGPNPNETNQNFRYLPFGGGQRKCIGDM Ljap  475 KGEDIFISLWNLHRSPHLWDDADKFEPERWPLDGPNPNETNQNFRYLPFGGGQRKCIGDM Gmax  480 RNEDIFISVWNLHRSPKLWDDADKFEPERWALDGPSPNETNQNFKYLPFGGGPRKCVGDL Mtru  475 RGEDIFISVWNLHRSPTLWNDADKFEPERWPLDGPNPNETNQGFKYLPFGGGPRKCIGDM Atha  452 RGEDIFISVWNLHRSPLHWDDAEKFNPERWPLDGPNPNETNQNFSYLPFGGGPRKCIGDM   Lfil  535 FATFETVVALAMLVRRFNFQMAIGAPPVVMTTGATIHTTQGLNMTVTRRIQPPIVPSLHM Ljap  535 FATFETVVALAMLVRRFNFQMAIGAPPVVMTTGATIHTTQGLNMTVTRRIQPPIVPSLHM Gmax  540 FASYETVVALAMLVRRFNFQIAVGAPPVEMTTGATIHTTQGLKMTVTHRIKPPIVPSLQM Mtru  535 FASYEVVVALAMLVRRFNFQMAVGAPPVVMTTGATIHTTQGLNMTVTRRIKPPIVPSLQM Atha  512 FASFENVVAIAMLIRRFNFQIAPGAPPVKMTTGATIHTTEGLKLTVTKRTKPLDIPSVPI  Lfil  595 STVEVDQKDQVYQAQ Ljap  595 STVEVDQKDQVYQAQ Gmax  600 STLEVDQKGEVYQAQ Mtru  595 STLEVDQKDQVYQAQ Atha  572 LPMD--SRDEVSSAL          176    Figure S9. Amino acid alignment for ZEP-1  Lfil    1 LCYNSLNPSTAAFSRTNFSVPSNKDHSLETSPFSYVHRLGRATKQGKKVTHVKAAVAERE Ljap    1 LCYNSLNPSTSAFSRTNFSVPSNKDHSLETSPFSYVHRLGRATKQGKKVTHVKAAVAERE Gmax    1 LCYNSLNPSTTVFSRTHFSVPLNKELPLDASPFGYNCGVGRTRKQRKKVMHVKCAVV--E Mtru    1 LSHKCLSPSMTTFSRTHFSNP-----------FSYGN---RTTKQRRKLMQVKATVM-HE Atha    1 FCY-SINPSKLDFTRTHVFSPVSKQFYLDLSSFGKPGGV-SGFRSRRALLGVKAALVEKE   Lfil   61 APKKQLRVLVAGGGIGGLVFALAAKKKGFEVVVFEKDLSAVRGEGQYRGPIQIQSNALAA Ljap   61 APKKQLRVLVAGGGIGGLVFALAAKKKGFEVVVFEKDLSAVRGEGQYRGPIQIQSNALAA Gmax   59 APKKQLRILVAGGGIGGLVFALAAKRKGFEVMVFEKDLSAIRGEGQYRGPIQIQSNALAA Mtru   46 APKKQLKVLVAGGGIGGLVFALAAKRKGFEVVVFEKDLSAIRGEGQYRGPIQIQSNALAA Atha   59 EKKKKSRVLVAGGGIGGLVFALAAKKKGFDVLVFEKDLSAIRGEGKYRGPIQIQSNALAA   Lfil  121 LEAID--------------------- Ljap  121 LEAIDPDVADEVMRLWCITGDRINGL Gmax  119 LEAIDSEVADEVMRVGCITGDRINGL Mtru  106 LEAIDMNVADEVMRVGCITGDRINGL Atha  119 LEAIDIEVAEQVMEAGCITGDRINGL       177    Figure S10. Amino acid alignment for ZEP-2  Lfil    1 ----KIREYDSKRLVKEHFKRLSGKELPIKSAQVTGSTDFSELQDKEPWLSSSKLVVKPD Ljap    1 MARKKIREYDSKRLVKEHFKRLSGKELPIKSAQVTGSTDFSELQDKEPWLSSSKLVVKPD Gmax    1 MARKKIREYDSKRLLKEHFKRISGQELPIKSAQVTESTNFSELAEKEPWLLSSKLVVKPD Mtru    1 MARKKIREYDSKRLLKEHFKRLSGKDLPIKSAQVTEATDFTELQDKEQWLSSSKLVVKPD Atha    1 MARKKIREYDSKRLVKEHFKRLSGQELPIRSVQINQETDLNELVEREPWLSSEKLVVKPD   Lfil   57 MLFGKRGKSGLVALNLDFAQVASFVKERLGKEVEMGGCKGPITTFIVEPFIPHNEEFYLN Ljap   61 MLFGKRGKSGLVALNLDFAQVASFVKERLGKEVEMGGCKGPITTFIVEPFIPHNEEFYLN Gmax   61 MLFGKRGKSGLVALNLDLAEVDSFVKERLGKEVEMGGCKGPITTFIVEPFIPHNEEFYLN Mtru   61 MLFGKRGKSGLVALNLDLAQVASFVKERLGKEVEMGGCKGPITTFIVEPFIPHNEEYYLN Atha   61 MLFGKRGKSGLVALNLDFADVATFVKERLGKEVEMSGCKGPITTFIVEPFVPHNEEFYLN   Lfil  117 IVSERLGNSISFSECGGIDIEENWDKVKTVFIPTGVSLTSEIVAPLVATLPLEIKGEIEE Ljap  121 IVSERLGNSISFSECGGIDIEENWDKVKTVFIPTGVSLTSEIVAPLVATLPLEIKGEIEE Gmax  121 IVSERLGNSISFSECGGIEIEENWDKVKTVFMPTGVSLTSESIAPLVATLPLEIKGEIEE Mtru  121 IVSDRLGNSISFSECGGIDIEENWDKVKTVFIPTGVSLTSEIIAPLVATLPLEIKGEIEE Atha  121 IVSDRLGCSISFSECGGIDIEENWDKVKTITIPTGASLTFEICAPLVATLPLEIKGELED   Lfil  177 FLKVIFTLFQDLDFTFLEMNPFTLVDGKPYPLDMRGELDDTAAFKNFKKWGNIEFPLPFG Ljap  181 FLKVIFTLFQDLDFTFLEMNPFTLVDGKPCPLDMRGELDDTAAFKNFKKWGNIEFPLPFG Gmax  181 FLKVIFTLFQDLDFTFLEMNPFTLVNGKPYPLDMRGELDDTAAFKNFKKWGNIEFPLPFG Mtru  181 FLKVIFTLFQDLDFTFLEMNPFTLVDGKPYPLDMRGELDDTAAFKNFKKWGNIEFPLPFG Atha  181 FIQVIFTLFEDLDFTFLEMNPFTLVDGKPYPLDMRGELDDTAAFKNFKKWGDIEFPMPFG   Lfil  237 RVMSATESFVHELDEKTSASLKFTVLNPEGRIWTMVAGGGASVIYADTVGDLGYAAELGN Ljap  241 RVMSATESFVHELDEKTSASLKFTVLNPKGRIWTMVAGGGASVIYADTVGDLGFASELGN Gmax  241 RVMSTTEAFIHGLDEKTSASLKFTVLNPMGRIWTMVAGGGASVIYADTVGDLGYAPELGN Mtru  241 RVMSATESFIHGLDEKTSASLKFTVLNPKGRIWTMVAGGGASVIYADTVGDLGFANELGN Atha  241 RVMSSTESFIHGLDEKTSASLKFTVLNPKGRIWTMVAGGGASVIYADTVGDLGYASELGN   Lfil  297 YAEYSGAPKEDEVLQYARVVIDCATANPDGQKRALVIGGGIANFTDVAATFSGIIRALKE Ljap  301 YAEYSGAPKEDEVLQYARVVIDCATANPDGQKRALVIGGGIANFTDVAATFSGIIRALKE Gmax  301 YAEYSGAPKEDEVLQYARVVIDCATSNPDGQKRALVVGGGIANFTDVAATFSGIIRALKE Mtru  301 YAEYSGAPNEEEVLQYARVVIDCATANPDGQKRALVIGGGIANFTDVAATFSGIIRALKE Atha  301 YAEYSGAPKEDEVLQYARVVIDCATANPDGKSRALVIGGGIANFTDVAATFNGIIRALKE   Lfil  357 KEQKLKEAKMHLYVRRGGPNYQKGLAKMRALGEEIGIPIEVYGPEATMTGICKEAIQCIT Ljap  361 KESKLKAARMHIYVRRGGPNYQKGLALMRALGEEIGIPIEVYGPEATMTGICKEAIQCIT Gmax  361 KEQKLKEAKMHIYVRRGGPNYQKGLAKMRALGEEIGIPIEVYGPEATMTGICKQAIQYIT Mtru  361 KESKLKAARMHLYVRRGGPNYQKGLEKMRALGEEIGIPIEVYGPEATMTGICKQAIQCIT Atha  361 KEAKLKAARMHIFVRRGGPNYQKGLAKMRSLGDEIGVPIEVYGPEATMTGICKEAIQYIT   Lfil  417 AAA Ljap  421 AAA Gmax  421 AAA Mtru  421 ASA Atha  421 AAA 178    Appendix 5  Supplementary Figures S11 – S17 Amino acid alignments for yellow-differentially expressed carotenoid genes in Lotus filicaulis  Figure S11. Amino acid alignment for CDS-1  Lfil    1 M--ASLIHCPAATSLSATR-ADSVGFLVLPSSSRFIKTHKCRVRCSLDSNVSDMSVNAPK Ljap    1 M--ASLIHCPAATSLSATR-GDSVGFLV-PSSSRFIKTHKCRVRCSLDSNVSDMSVNAPK Gmax    1 MAMASLIQCS-ATSLSAV-----------PITTRFTRTHKSRLRCSLDANVSDMSVNAPK Mtru    1 MM-TSLIHCP-ATSLSTTRPGDSVAFFV-PSRFTKTRSYKPRVRCSLDSNVSDMSTNAPK Atha    1 M--ASSVVFAATGSLSVP-----------P-----LKSRRFYVNSSLDSDVSDMSVNAPK   Lfil   58 GLFPPEPEHYRGPKLKVAIIGAGLAGMSTAVELLDQGHEVDIYESRPFIGGKVGSFVDKR Ljap   57 GLFPPEPEHYRGPKLKVAIIGAGLAGMSTAVELLDQGHEVDIYESRPFIGGKVGSFVDKR Gmax   49 GLFPPEPEHYRGPKLKVAIIGAGLAGMSTAVELLDQGHEVDIYESRPFIGGKVGSFVDKG Mtru   58 GLFPPEPEHYRGPKLKVAIIGAGLAGMSTAVELLDQGHEVDIYESRTFIGGKVGSFVDKR Atha   43 GLFPPEPVPYKGPKLKVAIIGAGLAGMSTAVELLDQGHEVDIYDSRTFIGGKVGSFVDRR   Lfil  118 GNHIEMGLHVFFGCYNNLFRLMKKVGAENNLLVKDHTHTFVNTGGQIGELDFRFPIGAPL Ljap  117 GNHIEMGLHVFFGCYNNLFRLMKKVGAENNLLVKDHTHTFVNTGGQIGELDFRFPIGAPL Gmax  109 GNHIEMGLHVFFGCYNNLFRLLKKVGAENNLLVKDHTHTFVNKGGQIGELDFRFPIGAPI Mtru  118 GNHIEMGLHVFFGCYNNLFRLLKKVGAENNLLVKDHTHTFVNKGGQIGELDFRFPVGAPL Atha  103 GNHIEMGLHVFFGCYNNLFRLMKKVGAEKNLLVKDHTHTFINKDGTIGELDFRFPVGAPI   Lfil  178 HGITAFLTTNQLKTYDKARNALALALSPVVRALVDPDGALRDIRNLDSVSFSDWFLSKGG Ljap  177 HGITAFLTTNQLKTYDKARNALALALSPVVRALVDPDGALRDIRNLDSVSFSDWFLSKGG Gmax  169 HGIRAFLTTNQLNTYDKARNAVALALSPVVRALVDPDGALRDIRNLDSISFSDWFLSKGG Mtru  178 HGINAFLTTNQLKTYDKARNAVALALSPVVRALVDPDGALRDIRNLDSVSFSDWFMSKGG Atha  163 HGIRAFLVTNQLKPYDKLRNSLALALSPVVKALVDPDGAMRDIRNLDSISFSDWFLSKGG   Lfil  238 TRTSIQKMWDPVAYALGFIDCDNISARCMLTIFALFATKTEASLLRMLKGSPDVYLSGPI Ljap  237 TRTSIQKMWDPVAYALGFIDCDNISARCMLTIFALFATKTEASLLRMLKGSPDVYLSGPI Gmax  229 TRMSITKMWDPVAYALGFIDCDNISARCMLTIFALFATKTEASLLRMLKGSPDVYLSGPI Mtru  238 TRTSIKKMWDPVAYALGFIDCDNISARCMLTIFALFATKTEASLLRMLKGSPDVYLSGPI Atha  223 TRASIQRMWDPVAYALGFIDCDNMSARCMLTIFSLFATKTEASLLRMLKGSPDVYLSGPI   Lfil  298 RKYITDRGGRFHLRWGCREILYDESADGSTYVKGLSMSKATAKQIVKADAYVAACDVPGI Ljap  297 RKYITDRGGRFHLRWGCREILYDESADGSTYVKGLSMSKATAKQIVKADAYVAACDVPGI Gmax  289 RKYIMDRGGRFHLRWGCRELLYDKSADGSIYVRGLSMSKATAKKIVKADAYVAACDVPGI Mtru  298 RKYITDRGGRFHLRWGCREVLYDKSADGSTYVTGLSLSKATEKKIVEADAYVAACDVPGI Atha  283 KQYITDRGGRIHLRWGCREILYDKSADGETYVTGLAISKATNKKIVKADVYVAACDVPGI   Lfil  358 KRLIPSEWRENEFFNNIYELVGVPVVTVQLRYNGWVTELQNLEKSRQLKKATGLDNLLYT Ljap  357 KRLIPSEWRENEFFNNIYELVGVPVVTVQLRYNGWVTELQNLEKSRQLKKATGLDNLLYT 179    Gmax  349 KRLLPSEWREQEFFNNIYELVGVPVVTVQLRYNGWVTELQDLEKSRRLGKAVGLDNLLYT Mtru  358 KRLLPSEWREKEFFNNIYELVGVPVVTVQLRYNGWVTELQNLELSRQLKKATGLDNLLYT Atha  343 KRLLPKEWRESRFFNDIYELEGVPVVTVQLRYNGWVTELQDIELARQLKRAVGLDNLLYT   Lfil  418 PDADFSCFADLALTSPEDYYIEGQGSLLQCVLTPGDPYMPLPNEEIIARVAKQ------- Ljap  417 PDADFSCFADLALTSPEDYYIEGQGSLLQCVLTPGDPYMPLPNEEIIARVAKQ------- Gmax  409 PDADFSCFADLALSSPEDYYIEGQGSLLQCVLTPGDPYMPLPNDEIIARVAKQVLALFPS Mtru  418 PDADFSCFADLALASPEDYYIEGQGSLLQCVLTPGDPYMPLPNEEIISRVAKQVISLFPS Atha  403 PDADFSCFADLALASPADYYIEGQGTLLQCVLTPGDPYMRMPNDKIIEKVAMQVTELFPS   Lfil      ------------------------------------------------------------ Ljap      ------------------------------------------------------------ Gmax  469 SQGLEVTWSSVVKIGQSLYREGPGKDPYRPDQKTPVRNFFLAGSYTKQDYIDSMEGATLS Mtru  478 SQGLEVTWSSVVKIGQSLYREGPGKDPFRPDQKTPVKNFFLSGSYTKQDYIDSMEGATLS Atha  463 SRGLEVTWSSVVKIAQSLYREAPGKDPFRPDQKTPIKNFFLAGSYTKQDYIDSMEGATLS   Lfil      ------------------------------------------- Ljap      ------------------------------------------- Gmax  529 GRQASAYICDAGEELVALRKKLDAEFKDDLKISNTKDELSLVX Mtru  538 GRQTSAYICDAGEELVALRKELLAQSKDDIKFTNTKDELSLVX Atha  523 GRQASSYICDAGEELAELNKKLSSS------ATAVPDELSLVX    180    Figure S12. Amino acid alignment for CIS  Lfil    1 MASSPLVFSAPFFSHRTSSTKTHLSLPLSSSRSVFPSKPLLLSRKLLAPASVAEVGEDSA Ljap    1 MASSPLVFSAPFSSHRTSSTKTHLSLPLSSSRSVFPSKPLLLSRKLLAPASVAEVGEDSA Gmax    1 MAASVVV-STSLSVYGY-HRRPSISITKTNLKSNSNSVAPIYCRKFVARTSIGEVGEDSA Mtru    1 MASTPLVFSTSISSHLL-HHHP--SLPSSSLCQYFSSNPLLLSHKLVTRVSSSEVGEDSA Atha    1 MAVYHLLLSSPPSLLPPSPRRPNLRIPAH-PRSLLSSSSPV-IRKILVRSTLREIGEDSA   Lfil   61 AFDLQSQKVSSWVYFTVILGVVLFALNVVWIDPSTGFGKDFVDAVSGISDSPEVVMLLLI Ljap   61 VFDLQSQKISSWVYFTVILGVVLFALNVVWIDPSTGFGKDFVDAVSGISDSPEVVMLLLI Gmax   59 VFELGKQKVSSWIYFTAILGVVLYVLNVAWIDNSTGYGKAFIDAVSTLSDSSEVVMLILI Mtru   58 TFDIQNQKLSSWFYFTAVLGVVLSVLNVIWIDDSTGFGKAFVDSISGISDSHEVVMLVLV Atha   59 AFELGKQKLVSWVYFGVVLGVVLFILNVVWIDNSTGFGKSFIDAVSNISGSPEVAMLMLI   Lfil  121 LIFAGVHSGLASFRDTGEKLIGERAFRVLFAGTSLPLALTTIVYFINHRYDGLQLWQLQN Ljap  121 LIFAGVHSGLASFRDTGEKLIGERAFRVLFAGTSLPLALTTIVYFINHRYDGFQLWQLQN Gmax  119 LIFAGVHSGLASFRNTGEKLIGERPFRVLFAGISLPLAVSTVVYFINHRYDGLQLWQLQD Mtru  118 LIFAGVHSGLASFRDTGEKLIGERAYRVLFAGTSLPLALTMIVYFINHRYDGLQLWQLQD Atha  119 LIFAIVHSGLASLRDIGEKLIGERAFRVLFAGISLPLAMSTIVYFINHRYDGSQLWQLQG   Lfil  181 VPGLHQLLWLSNFISFYFLYPSTFNLLEVAAVDKPKLHLWETGIMRITRHPQLVGQVIWC Ljap  181 VPGLHQLLWLSNFISFYFLYPSTFNLLEVAAVDKPKLHLWETGIMRITRHPQLVGQVIWC Gmax  179 APGLHQLLWLSNFISFFFLYPSTFNLLEVAAVDKPKLHLWETGIIRITRHPQMVGQVIWC Mtru  178 VPGVHQLVWISNFISFLFLYPATFNLLEVAAVDKPKLHLYETGIMRITRHPQMVGQVMWC Atha  179 VPGVHEAIWVANFVSFFFLYPSTFNLLEVAAVDKPKMHLWETGIMRITRHPQVLNISLMC   Lfil  241 LAHTIWIGNSVAVAASVGLIGHHLFGAWNGDRRLAKRYGEDFDLVKERTSIVPFAAILDG Ljap  241 LAHTIWIGNSVAVAASVGLIGHHLFGAWNGDRRLAKRYGEDFDLVKERTSIVPFAAILDG Gmax  239 LAHTIWIGNSVAVAASIGLIAHHLFGVWNGDRRLAIRYGEDFELVKSRTSVVPFAAILDG Mtru  238 LAHTVWIGNSVAVAASFALIAHHLFGVWNGDRKLAERYGEDFEIVKGRTSVVPFAAILDG Atha  239 LNMKT----------------------------------------------I--------   Lfil  301 RQRLPKDFYKEFIRLPYLTITFLTLGAYFAHPLMQSASFNLHW Ljap  301 RQRLPKDFYKEFIRLPYLTITFLTLGAYFAHPLMQSASFNLHW Gmax  299 RQELPKDFYKEFIRLPYLTVTAITLGAYFAHPLMQAASFNLHW Mtru  298 RQKLPKDFYKEFIRLPYFTITALTLGAYFAHPLMRAASLKLHW Atha  245 --------SFIFVFQKYRKIGFL--------------------       181    Figure S13. Amino acid alignment for CRTISO  Lfil    1 VKGARVLVLEKYVIPGGSSGFYQRDGYTFDVGSSVMFGFSDKGNLNLITQALEAVGCKMQ Ljap    1 VKGARVLVLEKYVIPGGSSGFYQRDGYTFDVGSSVMFGFSDKGNLNLITQALEAVGCKMQ Gmax    1 VKGARVLVLEKYVIPGGSSGFYQRDGYTFDVGSSVMFGFSDKGNLNLITQALEAVGCRMQ Mtru    1 VKGAKVLVLEKYVIPGGSSGFYHREGYTFDVGSSVMFGFSDKGNLNLITQALEAVGCRMQ Atha    1 VKEARVLVLEKYLIPGGSSGFYERDGYTFDVGSSVMFGFSDKGNLNLITQALKAVGRKME   Lfil   61 VVPDPTTVHFHLPNNLSVRVHREYDKFIEELTCYFPHEKEGILKFYGECWKIFNALNSLE Ljap   61 VVPDPTTVHFHLPNNLSVRVHREYDKFIEELTCYFPHEKEGILKFYGECWKIFNALNLLE Gmax   61 VVPDPTTVHFHLPNNLSVRVHKEYDKFIEELTSYFPHEKEGILKFYGECWKIFNALNSLE Mtru   61 VIPDPTTVHFHLPNHLSVRVHREYDKFIEELTSYFPHEKDGILKFYGECWKIFNALNSLE Atha   61 VIPDPTTVHFHLPNNLSVRIHREYDDFIAELTSKFPHEKEGILGFYGDCWKIFNSLNSLE   Lfil  121 LKSLEEPLYLFGQFFQKPLECLTLAYYLPQNAGAIARKYIQDPQLLSFIDAECFIVSTVN Ljap  121 LKSLEEPLYLFGQFFQKPLECLTLAYYLPQNAGAIARKYIQDPQLLSFIDAECFIVSTVN Gmax  121 LKSLEEPLYLFGQFFRRPLECLTLAYYLPQNAGAIARKYIQDPQLLSFIDAECFIVSTVN Mtru  121 LKSLEEPLYLFGQFFQKPLECLTLAYYLPQNAGAIARKYIQDPQLLSFIDAECFIVSTVN Atha  121 LKSLEEPIYLFGQFFQKPLECLTLAYYLPQNAGAIARKYIKDPQLLSFIDAECFIVSTVN   Lfil  181 ALQTPMINAAMVLCDRHFGGINYPLGGVGGIAKSLAKGLADQGSEILYKANVTSVIIEQG Ljap  181 ALQTPMINAAMVLCDRHFGGINYPLGGVGGIAKSLAKGLADQGSEILYKANVTSVIIEQG Gmax  181 ALQTPMINAAMVLCDRHFGGINYPLGGVGGIAKSLAKGLVDQGSEIVYKANVTSIIIEQG Mtru  181 ALQTPMINASMVLCDRHFGGINYPLGGVGGIAKSLAKGLVDQGSQILYKANVTSIITEQG Atha  181 ALQTPMINASMVLCDRHYGGINYPVGGVGGIAKSLAEGLVDQGSEIQYKANVKSIILDHG   Lfil  241 KAVGVRLSDGREFFAKTIISNATRWDT--------------------------------- Ljap  241 KAVGVRLSDGREFFAKTIISNATRWDTFG------------------------------- Gmax  241 KAVGVRLSNGREFFAKTIISNATRWDTFGKLLKGVPLPKEEENFQKVYVKAPSFLSIHMG Mtru  241 KAVGVRLSDGREFFAKTIISNATRWDTFGKLVKGESLPKEEENFQKVYVKAPSFLSIHMG Atha  241 KAVGVRLADGREFFAKTIISNATRWDTFGKLLKGEKLPKEEENFQKVYVKAPSFLSIHMG   Lfil      ------------------------------------------------------------ Ljap  270 ------------------------------------------------------------ Gmax  301 VKAEVLPPDTDCHHFVLESNWSKLEEPYGSIFLSIPTVLDSSLAPEGRHILHIFTTSSME Mtru  301 VKAEVLPPDTDCHHFVLENNWTSLEEPYGSIFLSIPTILDSSLAPEGRHILHIFTTSSMD Atha  301 VKAEVLPPDTDCHHFVLEDDWKNLEEPYGSIFLSIPTILDSSLAPDGRHILHIFTTSSIE   Lfil      ------------------------------------------------------------ Ljap  270 ------------------------------------------------------------ Gmax  361 DWEGLSRVEYEAKKQLVADEITSRLENKLFPGLRSSIDFMEVGTPKTHRRYLARDEGTYG Mtru  361 DWQGLSRIDYEAKKQVVADEIISRLEKKLFPGLRSSIDFIEVGTPKTHRRYLARDEGTYG Atha  361 DWEGLPPKEYEAKKEDVAARIIQRLEKKLFPGLSSSITFKEVGTPRTHRRFLARDKGTYG   Lfil      ------------------------------------------------------------ Ljap  270 ------------------------------------------------------------ Gmax  421 PMPRRIPKGLLGMPFNTTGIDGLYCVGDSCFPGQGVIAVAFSGVMCAHRVAADIGLEKKS Mtru  421 PMPRSIPKGLLGMPFNTTSIDGLYCVGDSCFPGQGVIAVAFSGVMCAHRVAADIGLEKKS 182    Atha  421 PMPRGTPKGLLGMPFNTTAIDGLYCVGDSCFPGQGVIAVAFSGVMCAHRVAADIGLEKKS   Lfil      --------------- Ljap  270 ------------WFR Gmax  481 PVLDSMLLRLLGWLR Mtru  481 PVLDGMLLGLLGWLR Atha  481 RVLDVGLLGLLGWLR       183    Figure S14. Amino acid alignment for E-CHY  Lfil    1 --------------------------TLWWLVTLPLPSMC------------------SR Ljap    1 -LSLSPLSLFPPSVKSSINKKPSSWVSPDWLTSLSRSLTAGKNDDSGIPIASAKLDDVSD Gmax    1 SLSLTPLSVFPPSVKSSINKKPSSWLSPDWLTSLSRSLTAG-NDVSGIPVASAKLDDVSD Mtru    1 SLPLSHLSLSSFLTKSSTNKKSTSWVSPDWLTSLSKSLTTSKNDDSNIPIASAKLDDVSD Atha    1 SSSYSSLFTAKPSIRSSIEKKSQSWVSPDWLTTLTRTLSSGKNDESGIPIANAKLDDVAD   Lfil   17 IMG---------------------------------NMARVLLLRFRSFCLGLVLQLLRV Ljap   60 LLGGALFLPLFKWMKEYGPIYRLAAGPRNFVVVSDPAIAKHVLKNYGKYGKGLVAEVSEF Gmax   60 LLGGALFLPLFKWMQDYGPIYRLAAGPRNFVVVSDPAIAKHVLRNYGKYAKGLVAEVSEF Mtru   61 LLGGALFLPLFKWMNEYGPIYRLAAGPRNFVVVSDPAIAKHVLKNYGKYGKGLVAEVSEF Atha   61 LLGGALFLPLYKWMNEYGPIYRLAAGPRNFVIVSDPAIAKHVLRNYPKYAKGLVAEVSEF   Lfil   44 HSGGTCLL---XWIGY-----------FVDVLRDXWRSYNLMH---------------LM Ljap  120 LFGGFAIAEGPLWTARRRAVVPSLHKRYLSVIVDRVFCRCAERLVEKLQPDALNGTAVNM Gmax  120 LFGGFAIAEGPLWTARRRAVVPSLHKRYLSVIVDRVFCRCAERLVEKLQPDALNGTAVNM Mtru  121 LFGGFAIAEGPLWTARRRAVVPSLHKRYLSIMVDRVFCKCAERLVEKLQADAVNGTAVNM Atha  121 LFGGFAIAEGPLWTARRRAVVPSLHRRYLSVIVERVFCKCAERLVEKLQPYAEDGSAVNM   Lfil   75 ELQLTWFHSXLLMLLVYLYSTMTLTQTVLLSVLFTLLXKRQARSTDLLPYWQVEFLRKII Ljap  180 EDKFSQLTLDVIGLSVFNYDFDSLNDSPVIGAVYTALKEAEARSTDLLPYWQVEFLRKII Gmax  180 EAKFSQLTLDVIGLSVFNYNFDSLNDSPVIEAVYTALKEAEARSTDLLPYWKFKFLCKII Mtru  181 EDKFSQLTLDVIGLSVFNYNFDALNDSPVIEAVYTALKEAEARSTDLLPYWKIDFLCKII Atha  181 EAKFSQMTLDVIGLSLFNYNFDSLTDSPVIEAVYTALKEAELRSTDLLPYWKIDALCKIV   Lfil  135 PRQIKAEKAVSIIRKTVENLIEKCKEIVESEGERIDGDEYVNDSDPSILRFLLASREEVS Ljap  240 PRQIKAENAVTIIRKTVEDLIEKCKEIVESEGERIDGDEYVNDSDPSILRFLLASREEVS Gmax  240 PRQIKAEEAVSVIRKTVEDLIEKCREIVESEGERIDVEEYVNDSDPSILRFLLASREEVS Mtru  241 PRQIKAENAVTVIRKTVEDLIEQCKEIVESEGERIDADEYVNDADPSILRFLLASREEVS Atha  241 PRQVKAEKAVTLIRETVEDLIAKCKEIVEREGERINDEEYVNDADPSILRFLLASREEVS   Lfil  195 SDQLRDDLLSMLVAGHETTGSVLTWTLYLLSKDSSSLAKVQEEVDRVLQGRRPTFEDMKS Ljap  300 SDQLRDDLLSMLVAGHETTGSVLTWTLYLLSKDSSSLAKVQEEVDRVLQGRRPTFEDMKN Gmax  300 SVQLRDDLLSLLVAGHETTGSVLTWTLYLLSKDSSSLAKAQEEVDRVLQGRRPTYEDIKD Mtru  301 SVQLRDDLLSMLVAGHETTGSVLTWTLYLLSKDSSSLAKAQEEVDRVLQGRRPTYEDMKD Atha  301 SVQLRDDLLSMLVAGHETTGSVLTWTLYLLSKNSSALRKAQEEVDRVLEGRNPAFEDIKE   Lfil  255 LKFLTRCITESLRLYPHPPVLIRRAQVPDELPGAYKVNAGQDIMISVYNIHHSSEVWDRA Ljap  360 LKFLTRCITESLRLYPHPPVLIRRAQVPDELPGAYKVNAGQDIMISVYNIHHSSEVWDRA Gmax  360 LKFLTRCIIESLRLYPHPPVLIRRAQVPDELPGGYKLDAGQDIMISVYNIHRSSEVWDRA Mtru  361 LKFLNRCIIESLRLYPHPPVLIRRSQIPDELPGDYKIDAGQDIMISVYNIHHSSKVWDRA Atha  361 LKYITRCINESMRLYPHPPVLIRRAQVPDILPGNYKVNTGQDIMISVYNIHRSSEVWEKA   Lfil  315 EEFMPERFDLDGPMPNETNTDFRFIPFSGGPRKCVGDQFALLEATVSLAIFLQHMNFELV Ljap  420 EEFLPERFDLDGPMPNETNTDFRFIPFSGGPRKCVGDQFALLEATVSLAIFLQHMNFELV Gmax  420 EEFVPERFDLDGPVPNETNTDFRFIPFSGGPRKCVGDQFALMEAIVALAIFLQHMNFELV Mtru  421 EEFLPERFDLDGPVPNETNTDFRFIPFSGGPRKCVGDQFALLEATVALAVFLQHMNFELV 184    Atha  421 EEFLPERFDIDGAIPNETNTDFKFIPFSGGPRKCVGDQFALMEAIVALAVFLQRLNVELV   Lfil  375 PDQNISMTTGATIHTTNGLYMKLSQRVK Ljap  480 PDQNISMTTGATIHTTNGLYMKLSQRVK Gmax  480 PDQNISMTTGATIHTTNGLYMKLSRRLK Mtru  481 PDQNIGMTTGATIHTTNGLYMKLSQRLK Atha  481 PDQTISMTTGATIHTTNGLYMKVSQR--       185    Figure S15. Amino acid alignment for PSY Lfil    1 VTLFWVASYPNLEVSNSTGSLHSTRDVRLFDSSKFMLPDFRSIRAKKDRKKRWRFCSLST Ljap    1 VTLFWVASFPNLQVSNSTRLLDSIRDVRLFDSSKFMSRDFRSIRAKKDRKKRWRFCSLST Gmax    1 VTFLWFVSSPGLEVSHSTGLLDSVRHVKFLDSSKVMSRDFGSIRVEKDKEKGWRLCSLST Mtru    1 VTLLRVASSPSLEVSCSNGLLDSFGVVKLVDSSKFLSRDFVSIRAKKDKKKTRRFCSLST Atha    1 VAVLWVATSS-----LNPDPMNNCGLVRVLESSRLFSP-CQNQRLNKGKKKQI--PTWSS   Lfil   61 GVKYACIGQSGLESASSFPMISSVLANPAAAEVAVSSEQKVYDVVLKQASLIKRKLSAGD Ljap   61 GVKYACVGQSGLESASSFPMISNVLANPAAAEVAVSSEQKVYDVVLKQASLIKRKLSAGD Gmax   61 DMKYACVGRSGLESASNFPLIANVLANPAAGEVAVSSEQKVYDVVLKQASLVKRKLGAGE Mtru   61 DIKYACVGQPGLESASNFPLLSNVLANPTAGEVTVSSEQKVYDVVLKQASLVKRKLSSGE Atha   53 SFVRNRSRR--------IGVVSSSLVASPSGEIALSSEEKVYNVVLKQAALVNKQLRSYD   Lfil  121 LGVKQNVFLPGNLSLLSEAYDRCGEICAEYAKTFYLGTLLMTPERRRAIWAIYVWCRRTD Ljap  121 HGVKQNVFLPGNLSLLSEAYDRCGEICAEYAKTFYLGTLLMTPERRRAIWAIYVWCRRTD Gmax  121 LDAKPDIALPGNLSLLNEAYDRCGEICAEYAKTFYLGTLLMTPERRRAIWAIYVWCRRTD Mtru  121 LEVKPEIVLPGNLSLLSEAYDRCGEICAEYAKTFYLGTLLMTPERRRAIWAIYVWCRRTD Atha  105 LDVKQDVVLPGSLSLLGEAYDRCGEVCAEYAKTFYLGTLLMTPERRKAIWAIYVWCRRTD   Lfil  181 ELVDGPNASHITPTALDRWESRLEELFQGRPFDMLDAAVSDTVTKFPVDIQPFKDMIEGM Ljap  181 ELVDGPNASHITPTALDRWESRLEELFQGRPFDMLDAAVSDTVTKFPVDIQPFKDMIEGM Gmax  181 ELVDGPNASQITPTALDRWESRLEELFQGRPFDMLDAALADTVAKFPVDIQPFKDMIEGM Mtru  181 ELVDGPNASHITATAMDRWESRLDELFQGRPFDMLDAALSDTVNRFPVDIQPFKDMIEGM Atha  165 ELVDGPNASHITPMALDRWEARLEDLFRGRPFDMLDAALADTVARYPVDIQPFRDMIEGM   Lfil  241 RTDLRKSRYKNFDELYLYCYYVAGTVGLMSVPIMGISPDSQTSTESVYNAALALGIANQL Ljap  241 RTDLRKSRYKNFDELYLYCYYVAGTVGLMSVPIMGISPDSQTSTESVYNAALALGIANQL Gmax  241 RLDLKKPRYKNFDELYLYCYYVAGTVGIMSVPIMGISPNSEATTESVYNAALALGIANQL Mtru  241 RMDLKKSRYKSFDELYLYCYYVAGTVGLMSVPVMGISTHSQATTESVYNAALALGIANQL Atha  225 RMDLKKSRYQNFDDLYLYCYYVAGTVGLMSVPVMGIDPKSKATTESVYNAALALGIANQL   Lfil  301 TNILRDVGEDASRGRVYLPQDELAQAGLSEDDIFAGKVTDKWRNFMKSQIKRARMFFDEA Ljap  301 TNILRDVGEDASRGRVYLPQDELAQAGLSEDDIFAGKVTDKWRNFMKSQIKRARMFFDEA Gmax  301 TNILRDVGEDASRGRVYLPQDELAQAGLSDEDIFAGKVTDKWRNFMKSQIKRARMFFDEA Mtru  301 TNILRDVGEDARRGRVYLPQDELTLAGLSDDDIFAGKVTDKWRNFMKSQIKRARTFFDEA Atha  285 TNILRDVGEDARRGRVYLPQDELAQAGLSDEDIFAGKVTDKWRNFMKMQLKRARMFFDEA   Lfil  361 EKGVMELNKASRWPVLASLLLYRQILDEIEANDYNNFTKRAYVSKTKKLLSLPAAYARTM Ljap  361 EKGVMELNKASRWPVLASLLLYRQILDEIEANDYNNFTKRAYVSKTKKLLSLPAAYARTM Gmax  361 EKGVTELNEASRWPVWASLLLYRQILDEIEANDYNNFTRRAYVSKAKKFLSLPAAYARSI Mtru  361 EKGVTELNEQSRWPVWASLLLYRQILDEIEANDYNNFTKRAYVSKTKKLLSLPLAYARSM Atha  345 EKGVTELSAASRWPVWASLLLYRRILDEIEANDYNNFTKRAYVGKVKKIAALPLAYAKSV   Lfil  421 VAPPRKLSHV Ljap  421 VAPPRKLSHV Gmax  421 VPPSRKLSPV Mtru  421 VPPSKKLSHV Atha  405 LKTSSSRLSI  186    Figure S16. Amino acid alignment for ZEP-1  Lfil    1 -VXLCLVILSMPCSQIWAKED--------------------------------------- Ljap    1 GVXLCLVILSMPCSQIWAKED--------------------------------------- Gmax    1 GLPVTRVISRMVLQEILARAVGEDIIMNASNVVNFVDDGNKVTVELENGQKYEGDVLVGA Mtru    1 GLPVTRVISRMALQEILARAVGDDVIMNGSNVVDFIDHETKVTVVLDNGQKYDGDLLVGA Atha    1 GLPVTRVISRMTLQQILARAVGEDVIRNESNVVDFEDSGDKVTVVLENGQRYEGDLLVGA   Lfil   21 ------------------------------------------------------------ Ljap   22 ------------------------------------------------------------ Gmax   61 DGIWSKVRKQLFGLTEAVYSGYTCYTGIADFVPADIETVGYRVFLGHKQYFVSSDVGAGK Mtru   61 DGIWSKVRTKLFGSTEATYSGYTCYTGIADFVPPDIESVGYRVFLGHKQYFVSSDVGAGK Atha   61 DGIWSKVRNNLFGRSEATYSGYTCYTGIADFIPADIESVGYRVFLGHKQYFVSSDVGGGK   Lfil   21 -----------------------------AW----------------------------- Ljap   22 -----------------------------AW----------------------------- Gmax  121 MQWYAFHKEPPGGVDEPNGKKERLLRIFEGWCDNAVDLILATEEEAILRRDIYDRIPTLT Mtru  121 MQWYAFHQEPAGGVDTPNGKKERLLKIFEGWCDNAIDLIVATEEEAILRRDIYDRTPTLT Atha  121 MQWYAFHEEPAGGADAPNGMKKRLFEIFDGWCDNVLDLLHATEEEAILRRDIYDRSPGFT   Lfil   23 ---------------------------PLDSYQLASELDNAWEQSIKSGSPIEIDSSLRS Ljap   24 ---------------------------PLDSYQLASELDNAWEQSIRSGSPIEIDSSLRS Gmax  181 WGKGRVTLLGDSVHAMQPNMGQGGCMAIEDSYQLAWELENAWEQSIKSGSPIDIDSSLRS Mtru  181 WGKGRVTLLGDSVHAMQPNMGQGGCMAIEDGYQLAFELDNAWQQSAKSGSTIDIASSLKS Atha  181 WGKGRVTLLGDSIHAMQPNMGQGGCMAIEDSFQLALELDEAWKQSVETTTPVDVVSSLKR   Lfil   56 YERERRLRVAIIHGMARMAALMASTYKAYLGVGLGPLEFLTKFRIPHPGRVG Ljap   57 YERERRLRVAIIHGMARMAALMASTYKAYLGVGLGPLEFLTKFRIPHPGRVG Gmax  241 YERERRLRVAIIHGMARMAALMASTYKAYLGVGLGPLEFLTKFRIPHPGRVG Mtru  241 YERERRLRVTFVHGMARMAALMASTYKAYLGVGLGPFEFLTKFRIPHPGRVG Atha  241 YEESRRLRVAIIHAMARMAAIMASTYKAYLGVGLGPLSFLTKFRVPHPGRVG       187    Figure S17. Amino acid alignment for ZEP-3  Lfil    1 KKNLRILVAGGGIGGLVFALAAKRKGFEVVVFEKDMSAIRGEGKYRGPIQIQSNALAALE Ljap    1 ------------------------------------------------------------ Gmax    1 KKKLRVLVAGGGIGGLVFALAAKRKGFEVVVFEKDMSAIRGEGQYRGPIQIQSNALAALE Mtru    1 KKQLKVLVAGGGIGGLVFALAAKRKGFEVVVFEKDLSAIRGEGQYRGPIQIQSNALAALE Atha    1 KKKSRVLVAGGGIGGLVFALAAKKKGFDVLVFEKDLSAIRGEGKYRGPIQIQSNALAALE   Lfil   61 AIDPDVADEVMRLGCITGDRINGLVDGVSGSWYVKFDTFTPAVERGLPVTRVISRMVLQG Ljap    1 ------------------------------------------------------------ Gmax   61 AIDLEVAEEVLRVGCITGDRINGLVDGISGSWYIKFDTFTPAAERGLPVTRVISRMALQE Mtru   61 AIDMNVADEVMRVGCITGDRINGLVDGVSGSWYIKFDTFTPAAERGLPVTRVISRMALQE Atha   61 AIDIEVAEQVMEAGCITGDRINGLVDGISGTWYVKFDTFTPAASRGLPVTRVISRMTLQQ   Lfil  121 ILARAVGDDIIMNASNVVSFVDDGNKVTVELENGEKYEGDLLVGADGIWSKVRKQLFGLT Ljap    1 ------------------------------------------------------------ Gmax  121 ILAHAVGEDVIMNDSNVVDFVDHGDKVTVELENGQKYDGDLLVGADGIWSKVRKKLFGQT Mtru  121 ILARAVGDDVIMNGSNVVDFIDHETKVTVVLDNGQKYDGDLLVGADGIWSKVRTKLFGST Atha  121 ILARAVGEDVIRNESNVVDFEDSGDKVTVVLENGQRYEGDLLVGADGIWSKVRNNLFGRS   Lfil  181 EAVYSGYTCYTGIADFVPADIESVGYRVFLGHKQYFVSSDVGAGKMQWYAFHQEPPGGVD Ljap    1 ------------------------------------------------------------ Gmax  181 EATYSGYTCYTGIADFVPADIESVGYRVFLGHKQYFVSSDVGAGKMQWYGFHQEPAGGAD Mtru  181 EATYSGYTCYTGIADFVPPDIESVGYRVFLGHKQYFVSSDVGAGKMQWYAFHQEPAGGVD Atha  181 EATYSGYTCYTGIADFIPADIESVGYRVFLGHKQYFVSSDVGGGKMQWYAFHEEPAGGAD   Lfil  241 IPNGRKERVLKIFEGWCDNVIELIVATEEEAILRRDIYDRTPTLTWGKGCVTLLGDSVHA Ljap    1 ------------------------------------------------------------ Gmax  241 IPNGKKERLLKIFKGWCDNVIDLIHATEEEAILRRDIYDRTPTFTWGKGHVTLLGDSIHA Mtru  241 TPNGKKERLLKIFEGWCDNAIDLIVATEEEAILRRDIYDRTPTLTWGKGRVTLLGDSVHA Atha  241 APNGMKKRLFEIFDGWCDNVLDLLHATEEEAILRRDIYDRSPGFTWGKGRVTLLGDSIHA   Lfil  301 MQPNLGQGGCMAIEDGYQLALELDNAWQESIKSGSPIDIDSSLKSYERERRLRVAIIHAM Ljap    1 ------------------------------------------------------------ Gmax  301 MQPNMGQGGCMAIEDSYQLALELDNAWQQSIKSGSPIDIDSSLKSYERERRLRVAIVHGM Mtru  301 MQPNMGQGGCMAIEDGYQLAFELDNAWQQSAKSGSTIDIASSLKSYERERRLRVTFVHGM Atha  301 MQPNMGQGGCMAIEDSFQLALELDEAWKQSVETTTPVDVVSSLKRYEESRRLRVAIIHAM   Lfil  361 ARMAALMASTYKPYLGVGLGPLEFLTKFRVPHPGRVGGRFFIDKMMPLMLNWVLGGNSSK Ljap    1 ------------------------------------------------------------ Gmax  361 ARMAAMMASTYKAYLGVGLGPLEFLTKFRIPHPGRVGGRFFIDKMMPLMLNWVLGGNSSK Mtru  361 ARMAALMASTYKAYLGVGLGPFEFLTKFRIPHPGRVGGRFFIQKSMPLMLNWVLGGNSSK Atha  361 ARMAAIMASTYKAYLGVGLGPLSFLTKFRVPHPGRVGGRFFVDIAMPSMLDWVLGGNSEK   Lfil  421 LEGRPLCCRLSDKANDQLHRWFEDDDALERTITGEWILLPCGDEAGLSKPICLRHDETKP Ljap    1 ------------------------------------------------------------ Gmax  421 LEGRPVCCRLSDKANDQLHRWFEDNDALERAINGEWILLPCGDEAGPTKPICLTQDEMKP Mtru  421 LEGRPLCCRLSDKASDQLHTWFEDDDALERTINGEWILLPCGDVPGHVKPISLNQDDTKP Atha  421 LQGRPPSCRLTDKADDRLREWFEDDDALERTIKGEWYLIPHGDDCCVSETLCLTKDEDQP 188      Lfil  481 CIIGQEDYPGSSITITLPQVSQMHAQINYKDGAFFLTDLQSQHGTWITDNEGRRNRVPPN Ljap    1 ----QEDYPGSSITITLPQVSQMHAQINYKDGAFFLTDLQSQHGTWITDNEGRRNRVPPN Gmax  481 CIIGQKDHPGSSIIIPLPQVSQMHARINYKDGAFFLTDLRSLHGTWITDNEGRRYRVPPN Mtru  481 YIIGQEDYPGSLITIPLPQVSQLHARINFKDGAFFLTDLRSQHGTWITDNEGRRYMVSPN Atha  481 CIVGDQDFPGMRIVIPSSQVYKLYACSCDLQRRSFLLDGSSKRTRNLC------------   Lfil  541 CPARVRPSDLIEFG Ljap   57 CPARVRPSDLIEFG Gmax  541 YPARVRPSDVVEFG Mtru  541 YPARIRPSHVIEFG Atha      --------------    189    Appendix 6 Supplementary Figures S18 – S19 Amino acid alignments for non-differentially expressed flavonoid genes in Lotus filicaulis  Figure S18. Amino acid alignment for F3H-2 Lfil    1 MEVSGKPIRVQSLAHSGLSRVPPE------------------------------YIQPPQ Ljap    1 MEVSGEPIRVQSLAHSGLSRVPPE------------------------------YIQPPQ Gmax    1 MT-----TRVQSLAQSGLSRVPPQ------------------------------YIQPPQ Mtru    1 MELTGEPIRVQSIIQSGLSQVPPE------------------------------YIQPPQ Atha    1 MY----------LMSSFSSSLPPYPFHSKISTIKTSSLGELESKTTSDSHKSGFLLSTSS   Lfil   31 NR---PVHHNQPDPSTNIPTINLSGFDPNHR----ESIRRACREWGAFHVTNHGVPTTLL Ljap   31 NR---PVHHNQPDPSTNIPTINLSGFDPNHRDSTRESIRRACREWGAFHVTNHGVPTTLL Gmax   26 TR---PVRHTAPEP-DSIPVIDLSSFDPTQRASTRDSIARACREWGAFHVTNHGVPPSLL Mtru   31 NR---PIN---TDPTATIPAIDLFNFNTNHRNSTLESISHACRGWGAFHVTNHGIPPSLL Atha   51 HRKLDLISH-YPGDAASIPTVDLSSSD-----SAREAIGDACRDWGAFHVINHGVPIHLL   Lfil   84 DSLRRMGLAFFNECPMPEKLRYACAAGSAASEGYGSRMLVSD--ENNDGAQVLDWRDYFD Ljap   88 DSLRRSGLAFFNECPMPEKLHYACAAGSAASEGYGSRMLVSD--ENNDGAQVLDWRDYFD Gmax   82 ASLRRAGLSFFSDTPIPDKLRYSCSA--AASEGYGSKMLATTTSDQNDAVQVLDWRDYFD Mtru   85 DAVRNSGLTFFNNCPMSEKLKYSCTAGTAASEGYGSRMLVSS--ND---HEVLDWRDYFD Atha  105 DRMRSLGLSFFQDSPMEEKLRYACDSTSAASEGYGSRMLLGA--KD---DVVLDWRDYFD   Lfil  142 HHTLPLSRRNPNRWPEFSSGYRELVARYSDEMKALAQKLLSLISESLGLRPSCIEDVVGE Ljap  146 HHTLPLSRRNPNRWPEFSSGYRELVARYSDEMKALAQKLLSLISESLGLRPSCIEDVVGE Gmax  140 HHTLPLSRRNPNRWPEFPADYRELVATYSDEMKILAQKLLALISESLGLRASCIEDAVGE Mtru  140 HHTFPLSRRNPINWPDFTSDYREIMVNYSDEMKILAQKLLALISESLGLQSSCIEDAVGD Atha  160 HHTFPPSRRNPSHWPIHPSDYRQVVGEYGDEMKKLAQMLLGLISESLGLPCSSIEEAVGE   Lfil  202 FYQNITISYYPPCPQPDLTLGLQSHSDFGAITLLIQDDVGGLQVLKNGDGGGGGDSWVTV Ljap  206 FYQNITISYYPPCPQPDLTLGLQSHSDFGAITLLIQDDVGGLQVLKNGDG--GGDSWITV Gmax  200 FYQNITISYYPPCPEPDLTLGLQSHSDMGAITLLIQDDVGGLQVLKG------GNKWVTV Mtru  200 IYQNITVSYYPPCPQPDLTLGLQSHSDFGAVTLLIQDDVGGLQVLKDGD---GGDKWVTV Atha  220 IYQNITVTYYPPCPQPELTLGLQSHSDFGAITLLIQDDVEGLQLYKDA-------QWLTV   Lfil  262 QPLSDAILVLLADQTEIITNGKYRSCVHRAITNPDRARLSVATFHDPAKTVRISPASELI Ljap  264 QPLSDAILVLLADQTEVNKKT--L------------------------------------ Gmax  254 QPLSDAILVLLADQTEIITNGKYRSCEHRAITNPDRARLSVATFHDPAKTVKISPASELI Mtru  257 KPLSDAILVLLGDQTEIITNGKYRSCVHRAVTNPDRARLSVATFHDPAKTVKIFPVSELI Atha  273 PPISDAILILIADQTEIITNGRYKSAQHRAVTNANRARLSVATFHDPSKTARIAPVSQL-   Lfil  322 DESSPAKFRDVVYGDYVTSWYTKGPEGKRNIDALVLES* Ljap  286 -------------------------------------P* Gmax  314 NDSSLAKYRDVVYGDYVSSWYTKGPGGKRNIDALLLDP* Mtru  317 NDSSPAKYRGVVYGDYVSSWYTKGPDGKRNIDALLLES* Atha  332 ---SPPSYKEVVYGQYVSSWYSKGPEGKRNLDALLY--*  190    Figure S19. Amino acid alignment for F3H  Lfil    1 MGEVDPAFIQDPEHRPKLSI------IKAEGIPVIDLSPIISQTVSDPSAIEGLVKEIGE Ljap    1 MGEVDPAFIQEPEHRPNLSI------IQAEGIPVIDLSPIINKTLSDPSAIEGLVKKVGR Gmax    1 MGEVDTAFIQEPEHRPKLSP------NQAEGIPIIDLSPITNHAVSDPSAIENLVKEIES Mtru    1 MGEVDPAFVQEQEHRPKLSI------IEAKGIPEIDLSPILHHAVPNPSDIENLVKEIGS Atha    1 MGELDEAFIQAPEHRPNTHLTNSGDFIFSDEIPTIDLSSLE-DTHHDKTA---IAKEIAE   Lfil   55 ACKEWGFFQVTNHGVPLSLRQSIDEASRKFFAQSIEEKRKIYRDESTISGYYDTEHTKNV Ljap   55 ACKEWGFFQVTNHGVPLSLRQSIDEASRKFFAQSMEEKRKIYRDESTISGYYDTEHTKNV Gmax   55 ACKEWGFFQVTNHGVPLTLRQNIEKASRLFFEQTQEEKKKVSRDEKSTTGYYDTEHTKNV Mtru   55 ASKEWGFFQVTNHGVPLSLRQRLEEASRLFFAQSLEEKKKVARDEVNPTGYYDTEHTKNV Atha   57 ACKRWGFFQVINHGLPSALRHRVEKTAAEFFNLTTEEKRKVKRDEVNPMGYHDEEHTKNV   Lfil  115 RDWKEVFDFVAREPTVVPLTSDDEDDDRLTQWTNKSPEYPPNFRVILQEYIQELEKLAFK Ljap  115 RDWKEVFDFVATEPTVVPLTSD-EDDDRLTQWTNKSPEYPPNFSVILQEYMQEMEKLAFK Gmax  115 RDWKEVFDFLAKDPTFIPVTSD-EHDDRVTHWTNVSPEYPPNFRDIMEEYIQEVEKLSFK Mtru  115 RDWKEVFDFLSQDPTLVPRSSD-EHDDGVIQWTNQSPQYPPQFRAILEEYIQEVEKLAYR Atha  117 RDWKEIFDFFLQDSTIVPASPE-PEDTELRKLTNQWPQNPSHFREVCQEYAREVEKLAFR   Lfil  175 LLELIALSIGLEAKRFEQFFTKDQTSFIRLNHYPPCPFPHLALGVGRHKDAGGLTILAQD Ljap  174 LLELIALSLGLEAKRFEQFFTKDQTSFIRLNHYPPCPFPHLALGVGRHKDAGGLTILAQD Gmax  174 LLELIALSLGLEAKRFEEFFMKDQTSFIRLNHYPPCPYPHLALGVGRHKDGGALTVLAQD Mtru  174 LLELIALSLGLEAKRFEEFF-KYQTSFIRFNHYPPCPYPHLALGVGRHKDAGALTILAQD Atha  176 LLELVSISLGLPGDRLTGFF-NEQTSFLRFNHYPPCPNPELALGVGRHKDGGALTVLAQD   Lfil  235 QVGGLEVKRKADQQWVRVEPTPDAFIINVCDSIQVWSNDAYESVEHRVMVNSEKERFSIP Ljap  234 EVGGLEVKRKADQQWVRVEPTSDAFIINVCDSIQF------------------------- Gmax  234 EVGGLEVKRKADQEWIRVKPTPDAYIINVGDLIQVWSNDAYESVEHRVMVNSEKERFSIP Mtru  233 EVGGLEVKRKSDQQWVLVKPTPDAYIINVGDIIQVWSNDAYESVEHRVMVNSEKERFSIP Atha  235 SVGGLQVSRRSDGQWIPVKPISDALIINMGNCIQVWTNDEYWSAEHRVVVNTSKERFSIP   Lfil  295 YFFNPAHDCEVKPLDELTNEQNPSKYRPYEWGKFFVNRMGGNLKKKNVENIQIYHY---- Ljap  269 ----------------------------------FVCRFGAM------------------ Gmax  294 FFFNPAHDIEVKPLEELTNEHNPSKYRPYKWGKFLVHRKGSNFKKQNVENIQIYHYKIT* Mtru  293 FFFFPAHDTVVKPLEELTNEENPPKYRPYNWGKFLVNRKSSNFEKKKVENIQIYHYKIA* Atha  295 FFFFPSHEANIEPLEELISEENPPCYKKYNWGKFFVSRNRSDFKKLEVENIQIDHFK-A*       191    Appendix 7  Supplementary Figures S20 – S28 Amino acid alignments for red-differentially expressed flavonoid genes in Lotus filicaulis  Figure S20. Amino acid alignment for ANS  Lfil    1 MAPTVVERVESLSGSGIQSIPKEYVRPKEELANIGDVFEEEKK-VGPQVPTIDLKEIDSP Ljap    1 MAPTVVERVESLSGSGIQSIPKEYVRPKEELANIGDVFEEEKK-VGPQVPTIDLKDIDSP Gmax    1 MG-TVAPRVESLASSGIKCIPKEYVRPQEELKSIGNVFEEEKK-EGLQVPTIDLREIDSE Mtru    1 MG-TVAQRVESLALSGISSIPKEYVRPKEELANIGNIFDEEKK-EGPQVPTIDLKEINSS Atha    1 MV--AVERVESLAKSGIISIPKEYIRPKEELESINDVFLEEKKEDGPQVPTIDLKNIESD   Lfil   60 DEFVRAKCREKLRKAAEEWGVMHLVNHGIPDELLNQLKSAGAEFFSLPVEEKEKYANDQA Ljap   60 DEFVRAKCREKLRKAAEEWGVMHLVNHGIPDELLNQLKSAGAEFFSLPVEEKEKYANDQA Gmax   59 DEVVRGKCREKLKKAAEEWGVMHLVNHGIPDELIERVKKAGETFFGLAVEEKEKYANDLE Mtru   59 DEIVRGKCREKLKKAAEEWGVMHLVNHGISDDLINRLKKAGETFFELPVEEKEKYANDQS Atha   59 DEKIRENCIEELKKASLDWGVMHLINHGIPADLMERVKKAGEEFFSLSVEEKEKYANDQA   Lfil  120 AGNVQGYGSKLANNASGQLEWEDYFFHLIFPEDKRDLSIWPKTPSYYTEVTSDYARRLRV Ljap  120 AGNVQGYGSKLANNASGQLEWEDYFFHLIFPEDKRDLSIWPKTPSYYTEVTSDYARRLRV Gmax  119 SGKIQGYGSKLANNASGQLEWEDYFFHLAFPEDKRDLSFWPKKPADYIEVTSEYAKRLRG Mtru  119 SGKIQGYGSKLANNASGQLEWEDYFFHCIFPEDKRDLSIWPKTPADYTKVTSEYAKELRV Atha  119 TGKIQGYGSKLANNASGQLEWEDYFFHLAYPEEKRDLSIWPKTPSDYIEATSEYAKCLRL   Lfil  180 LASKILEVLSLELGLEEGRLEKEVGGMEELLLQMKINYYPKCPQPELALGVEAHTDISAL Ljap  180 LASKILEVLSLELGLEEGRLEKEVGGMEELLLQMKINYYPKCPQPELALGVEAHTDISAL Gmax  179 LATKILEALSIGLGLEGRRLEKEVGGMEELLLQLKINYYPICPQPELALGVEAHTDVSSL Mtru  179 LASKIMEVLSLELGLEGGRLEKEAGGMEELLLQMKINYYPICPQPELALGVEAHTDVSSL Atha  179 LATKVFKALSVGLGLEPDRLEKEVGGLEELLLQMKINYYPKCPQPELALGVEAHTDVSAL   Lfil  240 TFLLHNMVPGLQLFYEGKWVTAKCVPDSILMHIGDTTEILSNGKFKSILHRGLVNKEKVR Ljap  240 TFLLHNMVPGLQLFYEGKWVTAKCVPDSILMHIGDTTEILSNGKFKSILHRGLVNKEKVR Gmax  239 TFLLHNMVPGLQLFYEGQWVTAKCVPDSILMHIGDTIEILSNGKYKSILHRGLVNKEKVR Mtru  239 TFLLHNMVPGLQLFYEGKWVTAKCVPDSILMHIGDTIEILSNGKYKSILHRGLVNKEKVR Atha  239 TFILHNMVPGLQLFYEGKWVTAKCVPDSIVMHIGDTLEILSNGKYKSILHRGLVNKEKVR   Lfil  300 ISWAVFCEPPKEKIILKPLPELVTETEPARFPPRTFAQHIHHKLFRKDQEASAQ--SK* Ljap  300 ISWAVFCEPPKEKIILKPLPELVTETEPARFPPRTFAQHIHHKLFRKDQEASAQ--SK* Gmax  299 ISWAVFCEPPKEKIILQPLPELVTETEPARFPPRTFAQHIHHKLFRKDQEGLPN----* Mtru  299 ISWAVFCEPPKEKIILKPLPELVTEKEPARFPPRTFAQHIHHKLFRKDEEEKKDDPKK* Atha  299 ISWAVFCEPPKDKIVLKPLPEMVSVESPAKFPPRTFAQHIEHKLFGKEQEELVSEKND*       192    Figure S21. Amino acid alignment for CHS-2  Lfil    1 MV-----SVAEIRKAQRAEGPATILAIGTANPPNCVDQSTYPDFYFKITNSEHMTELKEK Ljap    1 MV-----SVAEIRKAQRAEGPATILAIGTANPPNCVDQSTYPDFYFKITNSEHMTELKEK Gmax    1 MV-----SVAEIRQAQRAEGPATILAIGTANPPNRVDQSTYPDYYFRITNSDHMTELKEK Mtru    1 MV-----SVSEIRKAQRAEGPATILAIGTANPANCVEQSTYPDFYFKITNSEHKTELKEK Atha    1 MVMAGASSLDEIRQAQRADGPAGILAIGTANPENHVLQAEYPDYYFRITNSEHMTDLKEK   Lfil   56 FQRMCDKSMIKKRYMYLNEEILKENPNLCAYMAPSLDARQDMVVVEVPRLGKEAATKAIK Ljap   56 FQRMCDKSMIKKRYMYLNEEILKENPNLCAYMAPSLDARQDMVVVEVPRLGKEAATKAIK Gmax   56 FQRMCDKSMIKTRYMYLNEEILKENPNMCAYMAPSLDARQDMVVVEVPKLGKEAAVKAIK Mtru   56 FQRMCDKSMIKRRYMYLTEEILKENPSVCEYMAPSLDARQDMVVVEVPRLGKEAAVKAIK Atha   61 FKRMCDKSTIRKRHMHLTEEFLKENPHMCAYMAPSLDTRQDIVVVEVPKLGKEAAVKAIK   Lfil  116 EWGQPKSKITHLIFCTTSGVDMPGADYQLTKLLGLRPSVKRYMMYQQGCFAGGTVLRLAK Ljap  116 EWGQPKSKITHLIFCTTSGVDMPGADYQLTKLLGLRPSVKRYMMYQQGCFAGGTVLRLAK Gmax  116 EWGQPKSKITHLIFCTTSGVDMPGADYQLTKQLGLRPYVKRYMMYQQGCFAGGTVLRLAK Mtru  116 EWGQPKSKITHLIVCTTSGVDMPGADYQLTKLLGLRPYVKRYMMYQQGCFAGGTVLRLAK Atha  121 EWGQPKSKITHVVFCTTSGVDMPGADYQLTKLLGLRPSVKRLMMYQQGCFAGGTVLRIAK   Lfil  176 DLAENNKGARVLVVCSEITAVTFRGPNDTHLDSLVGQALFGDGAAAVIVGSDPVPEI-EK Ljap  176 DLAENNKGARVLVVCSEITAVTFRGPNDTHLDSLVGQALFGDGAAAVIVGSDPVPEI-EK Gmax  176 DLAENNKGARVLVVCSEITAVTFRGPSDTHLDSLVGQALFGDGAAAVIVGSDPIPQV-EK Mtru  176 DLAENNKGARVLVVCSEVTAVTFRGPSDTHLDSLVGQALFGDGAAALIVGSDPVPEI-EK Atha  181 DLAENNRGARVLVVCSEITAVTFRGPSDTHLDSLVGQALFSDGAAALIVGSDPDTSVGEK   Lfil  235 PLFELVWTAQTIAPDSDGAIDGHLREVGLTFHLLKDVPGIVSKNIEKALIEAFQPLGISD Ljap  235 PLFELVWTAQTIAPDSDGAIDGHLREVGLTFHLLKDVPGIVSKNIDKALVEAFQPLNISD Gmax  235 PLYELVWTAQTIAPDSEGAIDGHLREVGLTFHLLKDVPGIVSKNIDKALFEAFNPLNISD Mtru  235 PIFEMVWTAQTIAPDSEGAIDGHLREAGLTFHLLKDVPGIVSKNITKALVEAFEPLGISD Atha  241 PIFEMVSAAQTILPDSDGAIDGHLREVGLTFHLLKDVPGLISKNIVKSLDEAFKPLGISD   Lfil  295 YNSIFWIAHPGGPAILDQVEQKLSLKPEKMKATREVLSEYGNMSSACVLFILDEMRKKSA Ljap  295 YNSIFWIAHPGGPAILDQVEQKLGLKPEKMKATRNVLSDYGNMSSACVLFILDEMRKKSA Gmax  295 YNSIFWIAHPGGPAILDQVEQKLGLKPEKMKATRDVLSEYGNMSSACVLFILDEMRRKSA Mtru  295 YNSIFWIAHPGGPAILDQVEQKLALKPEKMNATREVLSEYGNMSSACVLFILDEMRKKST Atha  301 WNSLFWIAHPGGPAILDQVEIKLGLKEEKMRATRHVLSEYGNMSSACVLFILDEMRRKSA   Lfil  355 QDGLKTTGEGLEWGVLFGFGPGLTIETVVLRSVA-I* Ljap  355 ENGLKTTGEGLEWGVLFGFGPGLTIETVVLRSVA-I- Gmax  355 ENGHKTTGEGLEWGVLFGFGPGLTIETVVLHSVA-I* Mtru  355 IDGLKTTGEGLEWGVLFGFGPGLTIETVVLRSVA-I* Atha  361 KDGVATTGEGLEWGVLFGFGPGLTVETVVLHSVP-L*       193    Figure S22. Amino acid alignment for CHS-4  Lfil    1 MV-----SVAEIRKAQRAEGPATIFAIGTANPPNCVDQSTYPDFYFRVTNSEHKTELKEK Ljap    1 MV-----SVAEIRKAQRAEGPATIFAIGTANPPNCVDQSTYPDFYFRVTNSEHKTELKEK Gmax    1 MV-----SVAEIRQAQRAEGPATILAIGTANPPNRVDQSTYPDYYFRITNSDHMTELKEK Mtru    1 MV-----SVSEIRNAQRAEGPATILAIGTANPANCVEQSTYPDFYFKITNSEHKTELKEK Atha    1 MVMAGASSLDEIRQAQRADGPAGILAIGTANPENHVLQAEYPDYYFRITNSEHMTDLKEK   Lfil   56 FQRMCDKSMIKKRYMHLTEDLLKENPNMCAYMAPSLDARQDMVVVEVPRLGKEAAVKAI- Ljap   56 FQRMCDKSMIKKRYMHLTEDLLKENPNMCAYMAPSLDARQDMVVVEVPRLGKEAAIKAIK Gmax   56 FQRMCDKSMIKTRYMYLNEEILKENPNMCAYMAPSLDARQDMVVVEVPKLGKEAAVKAIK Mtru   56 FQRMCDKSMIKRRYMYLTEEILKENPSVCEYMAPSLDARQDMVVVEVPRLGKEAAVKAIK Atha   61 FKRMCDKSTIRKRHMHLTEEFLKENPHMCAYMAPSLDTRQDIVVVEVPKLGKEAAVKAIK   Lfil  114 ------------------------------------------------------------ Ljap  116 EWGQPKSKITHLIFCTTSGVDMPGADYQLTKLLGLRPYVKRYMMYQQGCFAGGTVLRLAK Gmax  116 EWGQPKSKITHLIFCTTSGVDMPGADYQLTKQLGLRPYVKRYMMYQQGCFAGGTVLRLAK Mtru  116 EWGQPKSKITHLIVCTTSGVDMPGADYQLTKLLGLRPYVKRYMMYQQGCFAGGTVLRLAK Atha  121 EWGQPKSKITHVVFCTTSGVDMPGADYQLTKLLGLRPSVKRLMMYQQGCFAGGTVLRIAK   Lfil  114 ------------------------------------------------------------ Ljap  176 DLAENNKGARVLVVCSELTAVTFRGPSDTHLDSLVGQALFGDGAAALIVGSDPIPEI-EK Gmax  176 DLAENNKGARVLVVCSEITAVTFRGPSDTHLDSLVGQALFGDGAAAVIVGSDPIPQV-EK Mtru  176 DLAENNKGARVLVVCSEVTAVTFRGPSDTHLDSLVGQALFGDGAAALIVGSDPVPEI-EK Atha  181 DLAENNRGARVLVVCSEITAVTFRGPSDTHLDSLVGQALFSDGAAALIVGSDPDTSVGEK   Lfil  114 ------------------------------------------------------------ Ljap  235 PLFEMVWTAQTIAPDSEGAIDGHLREVGLTFHLLKDVPGIVSKNIEKALIEAFQPLGISD Gmax  235 PLYELVWTAQTIAPDSEGAIDGHLREVGLTFHLLKDVPGIVSKNIDKALFEAFNPLNISD Mtru  235 PIFEMVWTAQTIAPDSEGAIDGHLREAGLTFHLLKDVPGIVSKNINKALVEAFEPLGISD Atha  241 PIFEMVSAAQTILPDSDGAIDGHLREVGLTFHLLKDVPGLISKNIVKSLDEAFKPLGISD   Lfil  114 ------------------------------------------------------------ Ljap  295 YNSIFWIAHPGGPAILDQVEQKLGLKPEKMNATRDVLSNYGNMSSACVLFILDEMRKKSA Gmax  295 YNSIFWIAHPGGPAILDQVEQKLGLKPEKMKATRDVLSEYGNMSSACVLFILDEMRRKSA Mtru  295 YNSIFWIAHPGGPAILDQVEQKLALKPEKMKATREVLSEYGNMSSACVLFILDEMRKKSA Atha  301 WNSLFWIAHPGGPAILDQVEIKLGLKEEKMRATRHVLSEYGNMSSACVLFILDEMRRKSA   Lfil  114 ------------------------------------ Ljap  355 QDGLKTTGEGLKWGVLFGFGPGLTIETVVLRSVAI* Gmax  355 ENGHKTTGEGLEWGVLFGFGPGLTIETVVLHSVAI* Mtru  355 QDGLKTTGEGLEWGVLFGFGPGLTIETVVLRSVAI* Atha  361 KDGVATTGEGLEWGVLFGFGPGLTVETVVLHSVPL*       194    Figure S23. Amino acid alignment for CHS-5  Lfil    1 ------------------------------------------------------------ Ljap    1 MV-----SVAEIRKAQRAEGPATIFAIGTANPPNCVDQSTYPDFYFRVTNSEHKTELKEK Gmax    1 MV-----SVAEIRQAQRAEGPATILAIGTANPPNRVDQSTYPDYYFRITNSDHMTELKEK Mtru    1 MV-----SVSEIRKAQRAEGPATILAIGTANPPNCVEQSTYPDFYFRITNSEHKTELKEK Atha    1 MVMAGASSLDEIRQAQRADGPAGILAIGTANPENHVLQAEYPDYYFRITNSEHMTDLKEK   Lfil    1 ------------------------------------------------------------ Ljap   56 FQRMCDKSMIKKRYMHLTEDLLKENPNMCAYMAPSLDARQDMVVVEVPRLGKEAAVKAIK Gmax   56 FQRMCDKSMIKTRYMYLNEEILKENPNMCAYMAPSLDARQDMVVVEVPKLGKEAAVKAIK Mtru   56 FQRMCDKSMIKRRYMYLTEEILKENPSVCEYMAPSLDARQDMVVVEVPRLGKEAAVKAIK Atha   61 FKRMCDKSTIRKRHMHLTEEFLKENPHMCAYMAPSLDTRQDIVVVEVPKLGKEAAVKAIK   Lfil    1 ------------------------------------------------------------ Ljap  116 EWGQPKSKITHLIFCTTSGVDMPGADYQLTKLLGLRPYVKRYMMYQQGCFAGGTVLRLAK Gmax  116 EWGQPKSKITHLIFCTTSGVDMPGADYQLTKQLGLRPYVKRYMMYQQGCFAGGTVLRLAK Mtru  116 EWGQPKSKITHLIVCTTSGVDMPGADYQLTKLLGLRPYVKRYMMYQQGCFAGGTVLRLAK Atha  121 EWGQPKSKITHVVFCTTSGVDMPGADYQLTKLLGLRPSVKRLMMYQQGCFAGGTVLRIAK   Lfil    1 -MAENNKGARVLVVCSELTAVTFRGPSDTHLDSLVGQALFGDGAAALIVGSDPVPEV-EK Ljap  176 DLAENNKGARVLVVCSELTAVTFRGPSDTHLDSLVGQALFGDGAAALIVGSDPVPEV-EK Gmax  176 DLAENNKGARVLVVCSEITAVTFRGPSDTHLDSLVGQALFGDGAAAVIVGSDPIPQV-EK Mtru  176 DLAENNKGARVLVVCSEVTAVTFRGPSDTHLDSLVGQALFGDGAAALIVGSDPIPEI-EK Atha  181 DLAENNRGARVLVVCSEITAVTFRGPSDTHLDSLVGQALFSDGAAALIVGSDPDTSVGEK   Lfil   59 PLFELVWTAQTIAPDSEGAIDGHLREVGLTFHLLKDVPG--------------------- Ljap  235 PLFELVWTAQTIAPDSEGAIDGHLREVGLTFHLLKDVPGIVSKNIEKALIEAFQPLGISD Gmax  235 PLYELVWTAQTIAPDSEGAIDGHLREVGLTFHLLKDVPGIVSKNIDKALFEAFNPLNISD Mtru  235 PIFEMVWTAQTIAPDSEGAIDGHLREAGLTFHLLKDVPEIVSKNINKALVDAFQPLGVSD Atha  241 PIFEMVSAAQTILPDSDGAIDGHLREVGLTFHLLKDVPGLISKNIVKSLDEAFKPLGISD   Lfil   97 ------------------------------------------------------------ Ljap  295 YNSIFWIAHPGGPAILDQVEQKLSLKPEKMRATREVLSEYGNMSSACVLFILDEMRKKSA Gmax  295 YNSIFWIAHPGGPAILDQVEQKLGLKPEKMKATRDVLSEYGNMSSACVLFILDEMRRKSA Mtru  295 YNSIFWIAHPGGPAILDQVEQKLALKPEKMKATREVLSEYGNMSSACVLFILDEMRKKSA Atha  301 WNSLFWIAHPGGPAILDQVEIKLGLKEEKMRATRHVLSEYGNMSSACVLFILDEMRRKSA   Lfil   97 ------------------------------------  Ljap  355 QDGLKTTGEGLEWGVLFGFGPGLTIETVVLRSVTI* Gmax  355 ENGHKTTGEGLEWGVLFGFGPGLTIETVVLHSVAI* Mtru  355 QDGLKTTGEGHEWGVLFGFGPGLTIETVVLRSVAI* Atha  361 KDGVATTGEGLEWGVLFGFGPGLTVETVVLHSVPL*       195    Figure S24. Amino acid alignment for DFR  Lfil    1 MGSVPETVCVTGAAGFIGSWLVMRLMERGYMVRATVRDPANMKKVKHLLELPEAKTKLTL Ljap    1 MGSVPETVCVTGAAGFIGSWLVMRLMERGYMVRATVRDPANMKKVKHLLELPEAKTKLTL Gmax    1 MGSASESVCVTGASGFIGSWLVMRLIERGYTVRATVRDPVNMKKVKHLVELPGAKSKLSL Mtru    1 MGSMAETVCVTGASGFIGSWLVMRLMERGYMVRATVRDPENLKKVSHLLELPGAKGKLSL Atha    1 MVSQKETVCVTGASGFIGSWLVMRLLERGYFVRATVRDPGNLKKVQHLLDLPNAKTLLTL   Lfil   61 WKADLAEEGSFDEAIKGCTGVFHVATPMDFESKDPENEVIKPTINGVLDIMKASQKAKTV Ljap   61 WKADLAEEGSFDEAIKGCTGVFHVATPMDFESKDPENEVIKPTINGVLDIMKACQKAKTV Gmax   61 WKADLAEEGSFDEAIKGCTGVFHVATPMDFESKDPENEVIKPTINGVLDIMKACLKAKTV Mtru   61 WKADLGEEGSFDEAIKGCTGVFHVATPMDFESKDPENEMIKPTIKGVLDIMKACLKAKTV Atha   61 WKADLSEEGSYDDAINGCDGVFHVATPMDFESKDPENEVIKPTVNGMLGIMKACVKAKTV   Lfil  121 RRLVFTSSAGTLNVIEHQKQMFDESCWSDVEFCRRVKMTGWMYFVSKTLAEQEAWKFAKE Ljap  121 RRLVFTSSAGTLNVIEHQKQMFDESCWSDVEFCRRVKMTGWMYFVSKTLAEQEAWKFAKE Gmax  121 RRLIFTSSAGTLNVIERQKPVFDDTCWSDVEFCRRVKMTGWMYFVSKTLAEKEAWKFAKE Mtru  121 RRFIFTSSAGTLNVTEDQKPLWDESCWSDVEFCRRVKMTGWMYFVSKTLAEQEAWKFAKE Atha  121 RRFVFTSSAGTVNVEEHQKNVYDENDWSDLEFIMSKKMTGWMYFVSKTLAEKAAWDFAEE   Lfil  181 HGIDFITIIPPLVVGSFLMPTMPPSLITALSPITGNEAHYSIIKQGQYVHLDDLCLAHIF Ljap  181 HGIDFITIIPPLVVGSFLMPTMPPSLITALSPITGNEAHYSIIKQGQYVHLDDLCLAHIF Gmax  181 QGLDFITIIPPLVVGPFLMPTMPPSLITALSPITGNEDHYSIIKQGQFVHLDDLCLAHIF Mtru  181 HNMDFITIIPPLVVGPFLIPTMPPSLITALSPITGNEAHYSIIKQGQFVHLDDLCEAHIF Atha  181 KGLDFISIIPTLVVGPFITTSMPPSLITALSPITRNEAHYSIIRQGQYVHLDDLCNAHIF   Lfil  241 LFEHPESEGRYICSASEATIHDIAKLINSKYPEYNIPTKFKNIPDELELVRFSSKKIKDM Ljap  241 LFEHPESEGRYICSASEATIHDIAKLINSKYPEYNIPTKFKNIPDELELVRFSSKKIKDM Gmax  241 LFEEPEVEGRYICSACDATIHDIAKLINQKYPEYKVPTKFKNIPDQLELVRFSSKKITDL Mtru  241 LFEHMEVEGRYLCSACEANIHDIAKLINTKYPEYNIPTKFNNIPDELELVRFSSKKIKDL Atha  241 LYEQAAAKGRYICSSHDATILTISKFLRPKYPEYNVPSTFEGVDENLKSIEFSSKKLTDM   Lfil  301 GFEFKYSLEDMYTGAIDTCKEKGLLPKAAENPSN-------------------------- Ljap  301 GFEFKYSLEDMYTGAIDTCKEKGLLPKAAENPSN-------------------------- Gmax  301 GFKFKYSLEDMYTGAIDTCRDKGLLPKPAEKGLF-------------------------- Mtru  301 GFEFKYSLEDMYTEAIDTCIEKGLLPKFVK--ST-------------------------- Atha  301 GFNFKYSLEEMFIESIETCRQKGFLPVSLSYQSISEIKTKNENIDVKTGDGLTDGMKPCN   Lfil  335 -------G-------------K* Ljap  335 -------G-------------K* Gmax  335 -------TKPAETPVNAI-MHK* Mtru  333 -------N-------------K* Atha  361 KTETGITGERTDAPMLAQQMCA*       196    Figure S25. Amino acid alignment for F3H  Lfil    1 MASFKPKTLTTLAQQNTLESSFVRDEDERPKVAYNNFSNEIPVISLAGIDEVDGRRSEIC Ljap    1 MASFKPKTLTTLAQQNTLESSFVRDEDERPKVAYNNFSNEIPVISLAGIDEVDGRRSEIC Gmax    1 MAPT-AKTLTYLAQEKTLESSFVRDEEERPKVAYNEFSDEIPVISLAGIDEVDGRRREIC Mtru    1 MAP--AQTLTYLAQEKTLESSFVREEDERPKVAYNNFSNEIPIISLDGIDDAGGRRAEIC Atha    1 ------------------------------------------------------------   Lfil   61 NKIVEACENWGIFQVVDHGVDTELVSHMTTLAKEFFALPPEEKLRFDMTGGKKGGFIVSS Ljap   61 NKIVEACENWGIFQVVDHGVDTELVSHMTTLAKEFFALPPEEKLRFDMTGGKKGGFIVSS Gmax   60 EKIVEACENWGIFQVVDHGVDQQLVAEMTRLAKEFFALPPDEKLRFDMSGAKKGGFIVSS Mtru   59 NKIVEACENWGIFQVVDHGVDSKLISEMTRFAKGFFDLPPEEKLRFDMSGGKKGGFIVSS Atha    1 ---------------------------MTRLARDFFALPPEDKLRFDMSGGKKGGFIVSS   Lfil  121 HLQGESVQDWREIVTYFSYPIRNRDYSRWPDTPAGWKAVTEEYSEKLMGLACKLLEVLSE Ljap  121 HLQGESVQDWREIVTYFSYPIRNRDYSRWPDTPAGWKAVTEEYSEKLMGLACKLLEVLSE Gmax  120 HLQGESVQDWREIVTYFSYPKRERDYSRWPDTPEGWRSVTEEYSDKVMGLACKLMEVLSE Mtru  119 HLQGEAVKDWRELVTYFSYPIRQRDYSRWPDKPEGWKEVTEQYSEKLMNLACKLLEVLSE Atha   34 HLQGEAVQDWREIVTYFSYPVRNRDYSRWPDKPEGWVKVTEEYSERLMSLACKLLEVLSE   Lfil  181 AMGLEKEALTKACVDMEQKVVVNYYPKCPQPDLTLGLKRHTDPGTITLLLQDQVGGLQAT Ljap  181 AMGLEKEALTKACVDMDQKVVVNYYPKCPQPDLTLGLKRHTDPGTITLLLQDQVGGLQAT Gmax  180 AMGLEKEGLSKACVDMDQKVVVNYYPKCPQPDLTLGLKRHTDPGTITLLLQDQVGGLQAT Mtru  179 AMGLEKDALTKACVDMDQKVVINYYPKCPQPDLTLGLKRHTDPGTITLLLQDQVGGLQAT Atha   94 AMGLEKESLTNACVDMDQKIVVNYYPKCPQPDLTLGLKRHTDPGTITLLLQDQVGGLQAT   Lfil  241 RDNGKTWITVQPLEGAFVVNLGDHGHYLSNGRFKNADHQAVVNSNSSRLSIATFQNPAPD Ljap  241 RDNGKTWITVQPVEGAFVVNLGDHGHYLSNGRFKNADHQAVVNSNSSRLSIATFQNPAPD Gmax  240 RDNGKTWITVQPVEAAFVVNLGDHAHYLSNGRFKNADHQAVVNSNHSRLSIATFQNPAPN Mtru  239 KDNGKTWITVQPVEGAFVVNLGDHGHYLSNGRFKNADHQAVVNSNYSRLSIATFQNPAPD Atha  154 RDNGKTWITVQPVEGAFVVNLGDHGHFLSNGRFKNADHQAVVNSNSSRLSIATFQNPAPD   Lfil  301 ATVYPLKVREGEKSVMEEPITFAEMYRRKMSKDIELARMKKLAKEK-KLQDLE------- Ljap  301 ATVYPLKVREGEKSVMEEPITFAEMYRRKMSKDIELARMKKLAKEK-KLQDLE------- Gmax  300 ATVYPLKIREGEKPVMEEPITFAEMYRRKMSKDIEIARMKKLAKEK-HLQDLENEKHLQE Mtru  299 ATVYPLKIRDGEKSVMEEPITFAEMYRRKMSKDLEIARMKKLAKEEKELRDLE------- Atha  214 ATVYPLKVREGEKAILEEPITFAEMYKRKMGRDLELARLKKLAKEE-RD-----------   Lfil  353 ---KAKLEPKPMNEIFA* Ljap  353 ---KAKLEPKPMNEIFA* Gmax  359 LDQKAKLEAKPLKEILA* Mtru  352 ---KAKIEAKPLNEILA* Atha  262 ----HKEVDKPVDQIFA*       197    Figure S26. Amino acid alignment for F3’H  Lfil    1 M-FPWMIIGFATITFLIFIHRVVKF-STR-PSLPLPPGPKPWPIIGNFPHMGPVPHHSLA Ljap    1 M-FPWMIIGFATITFLIFIHRVVKF-STR-PSLPLPPGPKPWPIIGNFPHMGPVPHHSLA Gmax    1 M-SP-LIVALATIAAAILIYRIIKF-ITR-PSLPLPPGPKPWPIVGNLPHMGPVPHHSLA Mtru    1 M-SLWFI-AIASFTLCILIYRFMKF-AKRSSSLPLPPGPKPWPIIGNMPHLGPAPHQSIA Atha    1 MATLFLTILLATVLFLIL--RIFSHRRNRSHNNRLPPGPNPWPIIGNLPHMGTKPHRTLS   Lfil   58 ALARAHGPLMHLKLGFVDVVVAASAAVAEQFLKVHDANFSSRPPNAGAKYIAYNYQDLVF Ljap   58 ALARAHGPLMHLKLGFVDVVVAASAAVAEQFLKVHDANFSSRPPNAGAKYIAYNYQDLVF Gmax   57 ALARIHGPLMHLRLGFVDVVVAASASVAEQFLKIHDSNFSSRPPNAGAKYIAYNYQDLVF Mtru   58 ALAKIHGPLMHLKLGFVDVIVAASGSVAEQFLKVHDANFSSRPPNTGAKYIAYNYQDLVF Atha   59 AMVTTYGPILHLRLGFVDVVVAASKSVAEQFLKIHDANFASRPPNSGAKHMAYNYQDLVF   Lfil  118 APYGARWRYLRKITNLHLFSGKALDNFKHLRQEEVSRLTRNISK--SNSKAVNLGQLLNV Ljap  118 APYGARWRYLRKITNLHLFSGKALDNFKHLRQEEVSRLTRNISK--SNSKVVNLGQLLNV Gmax  117 APYGPRWRLLRKLTSVHLFSGKAMNEFRHLRQEEVARLTCNLAS--SDTKAVNLGQLLNV Mtru  118 APYGPRWRMLRKISSVHLFSNKVMEEFKHLRQEEVARLTSNLASNYSDTKAVNLGQLLNV Atha  119 APYGHRWRLLRKISSVHLFSAKALEDFKHVRQEEVGTLTRELVR--VGTKPVNLGQLVNM   Lfil  176 CTTNALSRVMIGRRVFNDGDGGCDPRADEFKAMVVELMVLAGVFNIGDFIPSLEWLDLQG Ljap  176 CTTNALSRVMIGRRVFNDGDGGCDPRADEFKAMVVELMVLAGVFNIGDFIPSLEWLDLQG Gmax  175 CTTNALARAMIGRRVFNDGNGGCDPRADEFKAMVMEVMVLAGVFNIGDFIPSLEWLDLQG Mtru  178 CTTNALARVMLGRRVFNDGNGGCDPKADEFKEMVLELMVLAGVFNISDFIPSLEWLDLQG Atha  177 CVVNALGREMIGRRLFG---ADADHKADEFRSMVTEMMALAGVFNIGDFVPSLDWLDLQG   Lfil  236 VQAKMKKLHNRFDEFLTSIIEEH-NTSSKSENHKDLLSTLLSLKDVP-DDDGNRLNDIEI Ljap  236 VQAKMKKLHNRFDEFLTSIIEEH-NTSSKSENHKDLLSTLLSLKDVP-DDDGNKLNDIEI Gmax  235 VQAKMKKLHKRFDAFLTSIIEEHNNSSSKNENHKNFLSILLSLKDVR-DDHGNHLTDTEI Mtru  238 VQAKMKKLHKKFDAFLTNIIDERENSNFKSEKHKDLLSTLLLLKEET-DVDGNKLTYIEI Atha  234 VAGKMKRLHKRFDAFLSSILKEH-EMNGQDQKHTDMLSTLISLKGTDLDGDGGSLTDTEI   Lfil  294 KALLLNMFTAGTDTSASTTEWAIAELIRSPRILAQVQQELDTVVGRERNVREDDLPHLPY Ljap  294 KALLLNMFTAGTDTSASTTEWVIAELIRSPRILAQVQQELDTVVGRERNVREDDLPHLPY Gmax  294 KALLLNMFTAGTDTSSSTTEWAIAELIKNPQILAKLQQELDTVVGRDRSVKEEDLAHLPY Mtru  297 KALLLNMFAAGTDTSSSTTEWAIAELIRNPRILAQVQQELDNVVGRDRNVKEDDIPNLPY Atha  293 KALLLNMFTAGTDTSASTVDWAIAELIRHPDIMVKAQEELDIVVGRDRPVNESDIAQLPY   Lfil  354 LQAVVKETFRLHPSTPLSLPRVASESCEVLGYHIPKGSTLLVNVWAIARDPKEWAEPLEF Ljap  354 LQAVVKETFRLHPSTPLSLPRVASESCEVLGYHIPKGSTLLVNVWAIARDPKEWAEPLEF Gmax  354 LQAVIKETFRLHPSTPLSVPRAAAESCEIFGYHIPKGATLLVNIWAIARDPKEWNDPLEF Mtru  357 LQAVIKETFRLHPSTPLSLPRIASESCEIFGYHIPKGSTLLVNVWAIARDPKEWVDPLEF Atha  353 LQAVIKENFRLHPPTPLSLPHIASESCEINGYHIPKGSTLLTNIWAIARDPDQWSDPLAF   Lfil  414 KPERF-LEGDKVDVDVKGNDFKVIPFGAGRRICAGMSLGLRMVQLLTATLVHSFNWELEN Ljap  414 KPERF-LEGDKVDVDVKGNDFKVIPFGAGRRICAGMSLGLRMVQLLTATLVHSFNWELEN Gmax  414 RPERFLLGGEKADVDVRGNDFEVIPFGAGRRICAGLSLGLQMVQLLTAALAHSFDWELED Mtru  417 KPERFLPGGEKCDVDVKGNDFEVIPFGAGRRICPGMSLGLRMVQLLTATLAHSFDWELEN Atha  413 KPERFLPGGEKSGVDVKGSDFELIPFGAGRRICAGLSLGLRTIQFLTATLVQGFDWELAG 198      Lfil  473 GLNHEKLNMDEAYGLTLQRAVPLSVYSRPRLSPHVYAASH-* Ljap  473 GLNHEKLNMDEAYGLTLQRAVPLSVYSRPRLSPHVYAASH-* Gmax  474 CMNPEKLNMDEAYGLTLQRAVPLSVHPRPRLAPHVYSMSS-* Mtru  477 GLNAGKMNMDEGYGLTLQRAVPLSVHPKPRLSPHVYSSCF-* Atha  473 GVTPEKLNMEESYGLTLQRAVPLVVHPKPRLAPNVYGLGSG*         199    Figure S27. Amino acid alignment for MAX1  Lfil    1 MVFMDFEWLFQIPS-VPWSSAMFTLLATIG-GFSVYLYGPYWGVRKVPGPPSVPLIGHLP Ljap    1 MVFMDFEWLFQIPS-VPWSSAMFTLLATIG-GFLVYLYGPYWGVRKVPGPPSVPLIGHLP Gmax    1 MVSIVLEWLFPFPC-VAMF---TTLLMLIG-GLLGYLYGPYWGLRKVPGPPTLPLVGHLP Mtru    1 MVFMDLEWLFPIPISVSFA---STILALAG-GWLIYLYEPYWRVRKVPGPPSLPLVGHLH Atha    1 MKTQHQWWEVLDPF-LTQHEALIAFLTFAAVVIVIYLYRPSWSVCNVPGPTAMPLVGHLP   Lfil   59 LLAKYGPDVFSVLAKQYGPIYRFHMGRQPLIIIADAELCKEAGIKKFKDITNRSIPSPIS Ljap   59 LLAKYGPDVFSVLAKQYGPIYRFHMGRQPLIIIADAELCKEAGIKKFKDITNRSIPSPIS Gmax   56 LLAKYGPDVFSLLAKQYGPIYRFHMGRQPLILVADPELCKEVGIKKFKDIPNRSIPSPIS Mtru   57 LLAKHGPDVFSVLAKQYGPIYRFHMGRQPLIIVADAELCKEVGIKKFKDIPNRSTPSPIK Atha   60 LMAKYGPDVFSVLAKQYGPIFRFQMGRQPLIIIAEAELCREVGIKKFKDLPNRSIPSPIS   Lfil  119 ASPLHQKGLFFTKDSQWSTMRNTILSLYQPSHLSRLVPTMQSFIESATQNLDSQKEDFIF Ljap  119 ASPLHQKGLFFTKDSQWSTMRNTILSLYQPSHLSRLVPTMQSFIESATQNLDSQNEDFIF Gmax  116 ASPLHQKGLFFTRDSRWSTMRNTILSVYQPSHLASLVPTMQSFIESATQNLDTPNEDIIF Mtru  117 ASPLHQKGLFFSRDSQWSTMRNTILSVYQPSHLSRLVPTMQSFIESATQNLDSQKEDIFF Atha  120 ASPLHKKGLFFTRDKRWSKMRNTILSLYQPSHLTSLIPTMHSFITSATHNLDSKPRDIVF   Lfil  179 SNLSLSLATDVIGQAAFGVDFGLSKPQPVCDEIKSVN---KEVRDSSTGNEVSDFINQHI Ljap  179 SNLSLSLATDVIGQAAFGVDFGLSRPQSVRDESG--N---KEVRGSGAGNEVSDFINQHI Gmax  176 SNLSLRLATDVIGEAAFGVNFGLSKPHSVCESIKSVSV--NNVR--NDDDEVSDFINQHI Mtru  177 SNLSLKLATDVIGQAAFGVNFGLSQSHSVHNESKNVATDNKDLMNASGSNEVTDFINQHI Atha  180 SNLFLKLTTDIIGQAAFGVDFGLSGKKPIKD------------------VEVTDFINQHV   Lfil  236 YSTTQLKMDLSGSFSIILGLLVPILQEPFRQVLKRIPGTMDWKIERTNRKLSGRLDEIVE Ljap  234 YSTTQLKMDLSGSFSIILGLLVPILQEPFRQILKRIPGTMDWKIECTNRKLSGRLDEIVE Gmax  232 YSTTQLKMDLSGSFSIILGLLAPILQEPFRQILKRIPGTMDSKIESTNEKLSGPLDEIVK Mtru  237 YSTTQLKMDLSGSFSIILGLLVPILQEPFRQILKRIPGTMDWKIERTNEKLGGRLDEIVE Atha  222 YSTTQLKMDLSGSLSIILGLLIPILQEPFRQVLKRIPGTMDWRVEKTNARLSGQLNEIVS   Lfil  296 KRMKDRVRSSKDFLSLILNARESKTVSENVFTPDYISAVTYEHLLAGSATTSFTLSSIVY Ljap  294 KRMKDKVRSSKDFLSLILNARESKTVSENVFTPDYISAVTYEHLLAGSATTSFTLSSIVY Gmax  292 RRMEDKNRTSKNFLSLILNARESKKVSENVFSPDYISAVTYEHLLAGSATTAFTLSSIVY Mtru  297 KRTKDRTRSSKDFLSLILNARESKAVSENVFTPEYISAVTYEHLLAGSATTSFTLSSVVY Atha  282 KRAKEAETDSKDFLSLILKARESDPFAKNIFTSDYISAVTYEHLLAGSATTAFTLSSVLY   Lfil  356 LVAGHPEVEKKMLQEIDGFGPVDQTPTSQDLQEKFPYLDQVIKEAMRYYTVSPLVARETS Ljap  354 LVAGHPEVEKKMLQEIDGFGPVDQTPTSQDLQEKFPYLDQVIKEAMRYYTVSPLVARETS Gmax  352 LVAGHREVEKKLLQEIDGFGPPDRIPTAQDLHDSFPYLDQVIKEAMRFYTVSPLVARETS Mtru  357 LVAAHPEVEKKMLEEIDGYGSLDQIPTSQDLHDKFPYLDQVIKEAMRFYIVSPLVARETS Atha  342 LVSGHLDVEKRLLQEIDGFGNRDLIPTAHDLQHKFPYLDQVIKEAMRFYMVSPLVARETA   Lfil  416 NEVEIGGYLLPKGTWVWLALGVVAKDPRNFPEPEKFKPERFDPKCEEMKRRHPYAFIPFG Ljap  414 NEVEIGGYLLPKGTWVWLALGVVAKDPRNFPEPEKFKPERFDPKCEEMKRRHPYAFIPFG Gmax  412 NEVEIGGYLLPKGTWVWLALGVLAKDPRNFPEPEKFKPERFDPKCEEMKRRHPYAFIPFG Mtru  417 NEVEIGGYLLPKGTWVWLALGVLAKDHKNFPEPEKFKPERFDPNCEEMKQRHPYAFIPFG Atha  402 KEVEIGGYLLPKGTWVWLALGVLAKDPKNFPEPEKFKPERFDPNGEEEKHRHPYAFIPFG 200     Lfil  476 IGPRACIGQKFSLQEIKLSLIHLYRKYLFRHSPNMENPLELEYGIVLNFKHGVKVRAIKR Ljap  474 IGPRACIGQKFSLQEIKLSLIHLYRKYLFRHSPNMENPLELEYGIVLNFKHGVKVRAIKR Gmax  472 IGPRACIGQKFSLQEIKLTLIHLYQKYVFRHSVDMEKPVEMEYGMVLNFKHGIKLRVIRR Mtru  477 IGPRACIGQKFSMQEIKLSLIHLYKKYLFRHSADMESPLELEYGIVLNFKHGVKFSVIKR Atha  462 IGPRACVGQRFALQEIKLTLLHLYRNYIFRHSLEMEIPLQLDYGIILSFKNGVKLRTIKR  Lfil  536 TERSC* Ljap  534 TERSC* Gmax  532 T----* Mtru  537 TEMSC* Atha  522 F----*         201    Figure S28. Amino acid alignment for UFGT73C5  Lfil    1 MVFQEN---QPHFILFPLMAQGHIIPMVDIARLLAQRGVIVTIFTTPKNASRFTSVLSRA Ljap    1 ------------------------------------------------------------ Gmax    1 MVFQTN--NNPHFILFPLMAQGHIIPMMDIARLLAHRGVIVTIFTTPKNASRFNSVLSRA Mtru    1 MVLPANINDVPHFVLFPLIAQGHIIPMIDIAKLLAQRGVIVTIFTTPKNASRFTSVLSRA Atha    1 MVSETTKSSPLHFVLFPFMAQGHMIPMVDIARLLAQRGVIITIVTTPHNAARFKNVLNRA   Lfil   58 ISSGLQIRLVELQFPYKQAGLPEGCENFDMATSKGMFSKLFRGITMLQNSAEELFEKLSP Ljap    1 ---------------------------------------------------EELFEKLSP Gmax   59 ISSGLQIRLVQLHFPSKEAGLPEGCENFDMVTSIDMVYKMFNVINMLHKQAEEFFEALTP Mtru   61 VSSGLQIKIVTLNFPTKQAGLPEGCENFDMVDSIDMRMNLFHAITLLQKPAEELFDALTP Atha   61 IESGLPINLVQVKFPYLEAGLQEGQENIDSLDTMERMIPFFKAVNFLEEPVQKLIEEMNP   Lfil  118 RPSCIISDFCIPWTAQVAAKYNIPRISFHGFSCFCLHCLLKVQT-SKVTESITSETEYFT Ljap   10 RPSCIISDFCIPWTAQVAAKYNIPRISFHGFSCFCLHCLLKVQT-SKVTESITSETEYFT Gmax  119 KPSCIISDFCIPWTAQVAQKHCIPRISFHGFACFCLHCMLMVHT-SNVCESTASESEYFT Mtru  121 KPSCIISDFFIPWTIQIAENHKIPRISFHGFSCFCLHCMLKIQT-SKILERVDSESEYFT Atha  121 RPSCLISDFCLPYTSKIAKKFNIPKILFHGMGCFCLLCMHVLRKNREILDNLKSDKELFT   Lfil  177 VPGIPDQIQVNKSQIPGPM---S---DDLKDYGEQMYEAEKSSYGVIINTFEELENAYVK Ljap   69 VPGIPDQIQVNKSQIPGPM---S---DDLKDYGEQMYEAEKSSYGVIINTFEELENAYVK Gmax  178 IPGIPDQIQVTKEQIPMMI---SNSDEEMKHFREQMRDADIKSYGVIINTFEELEKAYVR Mtru  180 VPGIPDQIQVTKEQIPGIL---K---GELKEFGEKMHDAEMKSYGEIINTFEELEKAYVK Atha  181 VPDFPDRVEFTRTQVPVETYVPA---GDWKDIFDGMVEANETSYGVIVNSFQELEPAYAK   Lfil  231 DYKKERNDKVWCIGPVSLCNKDGLDKAQRGNKASINEHHCLKWLDLQQPKSVVYVCLGSL Ljap  123 DYKKERNDKVWCIGPVSLCNKDGLDKAQRGNKASINEHHCLKWLDLQQPKSVVYVCLGSL Gmax  235 DYKKVRNDKVWCIGPVSLCNQDNLDKVQRGNHASINEHHCLKWLDLQPPKSAVYVCFGSL Mtru  234 DYKKEKNGKVWFVGPVSLCNKDGLDKAQRGIIASISEHHCLKWLDLHQPKSVVYACLGSL Atha  238 DYKEVRSGKAWTIGPVSLCNKVGADKAERGNKSDIDQDECLKWLDSKKHGSVLYVCLGSI   Lfil  291 CNLIPSQLMELALALEATERPFIWVIREGNQF-QELE-KWMSEEGFEERTKGRGLVIRGW Ljap  183 CNLIPSQLMELALALEATERPFIWVIREGNQF-QELE-KWMGEKGFEERTKGRGLVIRGW Gmax  295 CNLIPSQLVELALALEDTKKPFVWVIREGNKF-QELEKKWISEEGFEERTKGRGLIIRGW Mtru  294 CNLIPSQLMELALALEATNRPFIWVIREGNKSSEELE-KWISE----ERNKGRGLIIRGW Atha  298 CNLPLSQLKELGLGLEESQRPFIWVIRGWEKY-KELV-EWFSESGFEDRIQDRGLLIKGW   Lfil  349 APQVLILSHPAIGGFLTHCGWNSTLEGISAGVPMVTWPLFADQFLNEKLVTQVLRIGVSL Ljap  241 APQVLILSHPAIGGFLTHCGWNSTLEGISAGVPMVTWPLFADSF-EENLSRPPGRLPVSX Gmax  354 APQVLILSHPSIGGFLTHCGWNSTLEGISAGVPMITWPLFADQFLNEKLVTQVLKIGVSV Mtru  349 APQVLILSHPSIGGFLTHCGWNSTLEGISAGLPLVTWPLFADQFLNEKLVTQVLRIGVSL Atha  356 SPQMLILSHPSVGGFLTHCGWNSTLEGITAGLPLLTWPLFADQFCNEKLVVEVLKAGVRS   Lfil  409 GVEVPLKWGEEEKTGVLVKKEDIKKGICLLMDDESKESEERRQKARELSEIAKKAVENGG Ljap  300 RHMLPLSIRKIE------------------------------------------------ Gmax  414 GMEVPMKFGEEEKTGVLVKKEDIKRAICIVMDDDGEESKDRRERATKLSEIAKRAVEKEG Mtru  409 GVEVPMRLGVEESLGVLVKKEGIKEAICMVMD-EGEESKERRERASKLSEMAKRAVEKGG Atha  416 GVEQPMKWGEEEKIGVLVDKEGVKKAVEELMG-ESDDAKERRRRAKELGDSAHKAVEEGG 202      Lfil  469 SSYHNMTLLIQDIMQ----------------------  Ljap  311 ------------------------------------- Gmax  474 SSHLDMTLLIQDIMQQSSSKEEVRTSLKESQFSCNA* Mtru  468 SSHLNINLLIQDIMEQ--------------------* Atha  475 SSHSNISFLLQDIMELAEP---------------NN*       203    Appendix 8  Supplementary Figures S29 – S30 Amino acid alignments for yellow-differentially expressed flavonoid genes in Lotus filicaulis  Figure S29. Amino acid alignment for CHS-1 Lfil    1 MV-----TVEEIRNAQRSSGPATILAFGTATPSHCVMQADYPDYYFRITNSEHMTDLKEK Ljap    1 MV-----TVEEIRNAQRSSGPATILAFGTATPSHCVMQADYPDYYFRITNSEHMTDLKEK Gmax    1 MV-----TVEEIRNAQRSHGPATILAFGTATPSNCVSQADYPDYYFRITNSEHMTDLKEK Mtru    1 MV-----TVEEIRKAQRSNGPATILAFGTATPSHCVTQAEYPDYYFRITNSEHMTDLKEK Atha    1 MVMAGASSLDEIRQAQRADGPAGILAIGTANPENHVLQAEYPDYYFRITNSEHMTDLKEK   Lfil   56 FKRMCEKSMIRKRYMHLTEEILKENPAMCAYMAPSLDARQDLVVVEVPKLGKEAAAKAIK Ljap   56 FKRMCEKSMIRKRYMHLTEEILKENPAMCAYMAPSLDARQDLVVVEVPKLGKEAAAKAIK Gmax   56 FKRMCEKSMIKKRYMHLTEEFLKENPNMCEYMAPSLDVRQDVVVMEVPKLGKQAATKAIK Mtru   56 FKRMCEKSMIKKRYMHITEEFLKENPNMCAYMAPSLDARQDLVVVEVPKLGKDAAKKAIA Atha   61 FKRMCDKSTIRKRHMHLTEEFLKENPHMCAYMAPSLDTRQDIVVVEVPKLGKEAAVKAIK   Lfil  116 EWGQPKSKITHLVFCTTSGVDMPGADYQLTKLLGLKPSVKRLMMYQQGCFAGGTVLRLAK Ljap  116 EWGQPKSKITHLVFCTTSGVDMPGADYQLTKLLGLKPSVKRLMMYQQGCFAGGTVLRLAK Gmax  116 EWGQPKSKITHLVFCTTSGVDMPGADYQLTKLLGLRPSVKRLMMYQQGCFAGGTVLRLAK Mtru  116 EWGQPKSKITHVVFCTTSGVDMPGADYQLTKLLGLKPSVKRLMMYQQGCFAGGTVLRLAK Atha  121 EWGQPKSKITHVVFCTTSGVDMPGADYQLTKLLGLRPSVKRLMMYQQGCFAGGTVLRIAK   Lfil  176 DLAENNKGARVLVVCSEITAVTFRGPSDTHLDSLVGQALFGDGAAAIIVGADPDLAV-ER Ljap  176 DLAENNKGARVLVVCSEITAVTFRGPSDTHLDSLVGQALFGDGAAAIIVGADPDLAV-ER Gmax  176 DLAENNKGARVLVVCSEITAVTFRGPSDTHLDSLVGQALFGDGAAALIIGSDPDPAV-ER Mtru  176 DLAENNKNARVLVVCSEITAVTFRGPSDTHLDSLVGQALFGDGAAAMIIGADPDLTV-ER Atha  181 DLAENNRGARVLVVCSEITAVTFRGPSDTHLDSLVGQALFSDGAAALIVGSDPDTSVGEK   Lfil  235 PIFQLVSAGQTILPDSDGAIDGHLREVGLTFHLLKDVPGIISKHIEKSLSEAFAPIGISD Ljap  235 PIFQLVSAGQTILPDSDGAIDGHLREVGLTFHLLKDVPGIISKHIEKSLSEAFAPIGISD Gmax  235 PIFEMISAAQTILPDSDGAIDGHLREVGLTFHLLKDVPGIISKNIEKSLVEAFEPIGISD Mtru  235 PIFEIVSAAQTILPDSDGAIDGHLREVGLTFHLLKDVPGIISKNIEKSLVEAFAPIGISD Atha  241 PIFEMVSAAQTILPDSDGAIDGHLREVGLTFHLLKDVPGLISKNIVKSLDEAFKPLGISD   Lfil  295 WNSLFWIAHPGGPAILDQVEAKLRLKEEKLRSTRHVLGEYGNMSSACVLFILDEVRRRSK Ljap  295 WNSLFWIAHPGGPAILDQVEAKLRLKEEKLRSTRHVLGEYGNMSSACVLFILDEVRRRSK Gmax  295 WNSIFWIAHPGGPAILDQVEEKLRLKPEKLQSTRHVLSEYGNMSSACVLFILDEMRKKSK Mtru  295 WNSIFWVAHPGGPAILDQVEEKLRLKEEKLRSTRHVLSEYGNMSSACVLFILDEMRKRSK Atha  301 WNSLFWIAHPGGPAILDQVEIKLGLKEEKMRATRHVLSEYGNMSSACVLFILDEMRRKSA   Lfil  355 EEGKETTGDGLEWGVLFGFGPGLTVETVVLHSVPLEAX Ljap  355 EEGKETTGDGLEWGVLFGFGPGLTVETVVLHSVPLEAX Gmax  355 EEGKSTTGEGLEWGVLFGFGPGLTVETVVLHSVPLEGX Mtru  355 EEGKITTGEGLEWGVLFGFGPGLTVETVVLHSVPVQGX Atha  361 KDGVATTGEGLEWGVLFGFGPGLTVETVVLHSVPLX-- 204    Figure S30. Amino acid alignment for PAP-1  Lfil    1 MDLETLYSPCFM---SNSNWFVQESAHNTEWSREDNKRFESALAIY-DKDTPDRWLNVAA Ljap    1 MDLETLYSPCFM---PNSNWFVQESAHSTEWSREDNKRFESALAIY-DKDTPDRWLNVAA Gmax    1 MELETIYPPCFM---PNSNWFVQES-HSTEWTREDNKKFESALAIY-DNDTPDRWFKVAA Mtru    1 MDLETLYSPYFM---LDTNWLVEES-YQTEWSREDNKKFESALAIY-DKDTPDRWLKVAE Atha    1 MSSSTMYRGVNMFSPANTNWIFQEV-REATWTAEENKRFEKALAYLDDKDNLESWSKIAD   Lfil   57 MIPGKTVLDVIKQYRELEEDVGEIEAGHVPVPGYHSSS------FTFEVVENQNFD---- Ljap   57 MIPGKTVLDVIKQYRELEEDVGEIEAGHVPVPGYHSSS------FTFEVVENQNFD---- Gmax   56 MIPGKTVFDVIKQYRELEEDVSEIEAGRVPIPGYLASS------FTFELVDNHNYD---- Mtru   56 MIPGKTVFDVIKQYRELVEDVSEIEAGNVPIPGYLASS------FTFEVVEKQNYD---- Atha   60 LIPGKTVADVIKRYKELEDDVSDIEAGLIPIPGYGGDASSAANSDYFFGLENSSYGYDYV   Lfil  107 --GLKRKPGTT--LR--GSDHERKKGVPWTEEEHKRFLMGLLKYGKGDWRNIARNFVMTK Ljap  107 --GLKRKPGTT--LR--GSDHERKKGVPWTEEEHKRFLMGLLKYGKGDWRNIARNFVMTK Gmax  106 --GCRRRL-AP--VR--GSDQERKKGVPWTEDEHRRFLMGLLKYGKGDWRNISRNFVVTK Mtru  106 --GNRRRH-VT--VR--GSDHERKKGVPWTEEEHRRFLMGLLKYGKGDWRNISRNFVVTK Atha  120 VGGKRSSPAMTDCFRSPMPEKERKKGVPWTEDEHLRFLMGLKKYGKGDWRNIAKSFVTTR   Lfil  161 TPTQVASHAQKYYIRQKV-SGGKDKRRPSIHDITTVNLTETSTTSENNKPLSFK------ Ljap  161 TPTQVASHAQKYYIRQKV-SGGKDKRRPSIHDITTVNLTETSITSENNKPLSFK------ Gmax  159 TPTQVASHAQKYYIRQKV-SGGKDKRRPSIHDITTVNLTETSASDKN-KPQLFNASPVLA Mtru  159 TPTQVASHAQKYYIRQKVSSGGKDKRRPSIHDITTVTLTETSSPSEN-KSLLVNVSPMQQ Atha  180 TPTQVASHAQKYFLRQL--TDGKDKRRSSIHDITTVNIPDADASATA-TTADVALSP---   Lfil  214 ---------------STSNYNSGSLMVF-NPNCDDLLMMPSSSDIITSKTLKLQGQDLYD Ljap  214 ---------------STSNYNSGSLMVF-NPNCDDLLMMPSSSDIITSKTLKLQGQDLYD Gmax  217 PQQKLNSISKVQLGWTSSHYNDGSFMVF-NPNSDALFV--SSSPDVTSMALKMQGQDLYD Mtru  218 -----------KMGWSTSHYNDGS----------------------------PQGQDLYD Atha  234 -----------------TPAN--SFDVFLQPN----------------------------   Lfil  258 CSLHEAYAKLKIPSFRAAPRNFNKEAVFGIHVL* Ljap  258 CSLHEAYAKSKIPSFRAAPRNFNKEAVFGIHVL* Gmax  274 CALHEAYAKVKVPGFSMAPRDFNNEAVFGIHAL* Mtru  239 CSFHEAYAKLKVSGFATASRDFNKGAVFGIHAL* Atha  247 --PHYSFASASASSYYNAFPQWS----------*       205    Appendix 9  Supplementary Figures S31 – S34 Amino acid alignments for non-differentially expressed lignin genes in Lotus filicaulis  Figure S31. Amino acid alignment for CCR  Lfil    1 ------------------------------------------------------------ Ljap    1 ------------------------------------------------------------ Gmax    1 MPT--DTSSVSG--EIVCVTGAGGFIASWLVKLLLEKGYTVRGTVRNPDDPKNGHLKELE Mtru    1 MPAYDNTSSVSGGDQTVCVTGAGGFIASWLVKLLLERGYTVRGTVRNPEDPKNGHLKELE Atha    1 MPV--DVASPAG--KTVCVTGAGGYIASWIVKILLERGYTVKGTVRNPDDPKNTHLRELE   Lfil    1 ------------------------------------------------------------ Ljap    1 ------------------------------------------------------------ Gmax   57 GGKERLTLHKVDLFDIASIKAALHGCHGVFHTASPVTDNPEEMVEPAVKGTKNVIIAAAE Mtru   61 GARERLTLHKVDLLDLQSIQSVVHGCHGVFHTASPVTDNPDEMLEPAVNGTKNVIIASAE Atha   57 GGKERLILCKADLQDYEALKAAIDGCDGVFHTASPVTDDPEQMVEPAVNGAKFVINAAAE   Lfil    1 ------------------------------------------------------------ Ljap    1 ------------------------------------------------------------ Gmax  117 AKVRRVVFTSSIGTVYMDPNTSRDALVDESFWSDLEYCKNTKNWYCYGKTVAEQAAWDVA Mtru  121 AKVRRVVFTSSIGTVYMDPNTSRDVVVDESYWSDLEHCKNTKNWYCYGKTVAEQSAWDIA Atha  117 AKVKRVVITSSIGAVYMDPNRDPEAVVDESCWSDLDFCKNTKNWYCYGKMVAEQAAWETA   Lfil    1 ------------------------------------------------------------ Ljap    1 ------------------------------------------------------------ Gmax  177 KERGVDLVVVNPVLVIGPLLQPTINASTIHILKYLTGSAKTYVNATQAYVHVRDVALAHI Mtru  181 KENQVDLVVVNPVVVLGPLLQPTINASTIHILKYLNGAAKTYVNATQSYVHVKDVALAHL Atha  177 KEKGVDLVVLNPVLVLGPPLQPTINASLYHVLKYLTGSAKTYANLTQAYVDVRDVALAHV   Lfil    1 ----------------------------------------------PRVKPYIFSNQKLK Ljap    1 ----------------------------------------CSDEKNPRVKPYIFSNQKLK Gmax  237 LVYETPSASGRFICAESSLHRGELVEILAKFFPEYPIPTKCSDEKNPRVKPYIFSNQKLK Mtru  241 LVYETNSASGRYICCETALHRGEVVEILAKYFPEYPLPTKCSDEKNPRVKPYKFSNQKLK Atha  237 LVYEAPSASGRYLLAESARHRGEVVEILAKLFPEYPLPTKCKDEKNPRAKPYKFTNQKIK   Lfil   15 DLGLEFTPVKQCLYETVKNLQEKGHLPVLIN----QQEL--------I* Ljap   21 DLGLEFTPVKQCLYETVMNLQEKGHLPVLIN----QQEL--------I- Gmax  297 DLGLEFTPVKQCLYDTVKNLQENGHLPVPPK----QKDS--------Y* Mtru  301 DLGLEFTPVKQCLYDTVRSLQEKGHLPIPPM----QEDS--------A* Atha  297 DLGLEFTSTKQSLYDTVKSLQEKGHLAPPPPPPSASQESVENGIKIGS*      206    Figure S32. Amino acid alignment for F5H  Lfil    1 MDSLPTWQTIANLRHEQLPMAFLFMVPMLLLLGLASRIRKRPPYPPGPKGLPIIGNMLMM Ljap    1 MDSLPTWQTIANLRHEQLPMAFLFMVPMLLLLGLASRIRKRPPYPPGPKGLPIIGNMLMM Gmax    1 M---------ANLDLDPFQTSILILVPIALLVALLSRTRRRAPYPPGPKGLPIIGNMLMM Mtru    1 MDSLLKSPIMENLKEEPFLMAIMFIVPLILLLGLVSRILKRPRYPPGPIGLPIIGNMLMM Atha    1 MESSI-SQTLSKLSDP--TTSLVIVVSLFIFISFITR-RRRPPYPPGPRGWPIIGNMLMM   Lfil   61 DQLTHRGLATLAKQYGGIFHLRMGFIHMVAVSDPDAARQVLQVHDNIFSNRPATIAISYL Ljap   61 DQLTHRGLATLAKQYGGIFHLRMGFIHMVAVSDPDAARQVLQVHDNIFSNRPATIAISYL Gmax   52 EQLTHRGLANLAKHYGGIFHLRMGFLHMVAISDPVAARQVLQVQDNIFSNRPATIAISYL Mtru   61 DQLTHRGLANLAKKYGGIFHLRMGFLHMVAISDADAARQVLQVQDNIFSNRPATVAIKYL Atha   57 DQLTHRGLANLAKKYGGLCHLRMGFLHMYAVSSPEVARQVLQVQDSVFSNRPATIAISYL   Lfil  121 TYDRADMAFAHYGPFWRQMRKLCVMKLFSRKRAESWQSVRDEVDAAVRDVDQNTGTPVNI Ljap  121 TYDRADMAFAHYGPFWRQMRKLCVMKLFSRKRAESWQSVRDEVDAAVRDVDQNTGTPVNI Gmax  112 TYDRADMAFAHYGPFWRQMRKLCVMKLFSRKRAESWQSVRDEVDAAVRAVASSVGKPVNI Mtru  121 TYDRADMAFAHYGPFWRQMRKLCVMKLFSRKHAESWQSVRDEVDYAVRTVSDNIGNPVNI Atha  117 TYDRADMAFAHYGPFWRQMRKVCVMKVFSRKRAESWASVRDEVDKMVRSVSCNVGKPINV   Lfil  181 GEVVFNLTKNIIYRAAFGSSSKEGQDEFIGILQEFSKLFGAFNIADFIPYIGAIDPQGLN Ljap  181 GEVVFNLTKNIIYRAAFGSSSKEGQDEFIGILQEFSKHFGAFNIADFIPYIGAIDPQGLN Gmax  172 GELVFNLTKNIIYRAAFGSSSQEGQDE-------------------------------LN Mtru  181 GELVFNLTKNIIYRAAFGSSSREGQDEFIGILQEFSKLFGAFNISDFVPCFGAIDPQGLN Atha  177 GEQIFALTRNITYRAAFGSACEKGQDEFIRILQEFSKLFGAFNVADFIPYFGWIDPQGIN   Lfil  241 ARLVKARAALDSFIGKIIDEHVQKKRGGGGGVDGD-GETDMVDELLAFYTEEEVAGSKGS Ljap  241 ARLVKARAALDSFIGKIIDEHVQKKRGGGGGVDGD-GETDMVDELLAFYTEEEVAGSKGS Gmax  201 SRLARARGALDSFSDKIIDEHVHKMKNDKSSEIVD-GETDMVDELLAFYSEEAK-----L Mtru  241 ARLVKARKELDSFIDKIIDEHVQKKKS-----VVD-EETDMVDELLAFYSEEAK-----L Atha  237 KRLVKARNDLDGFIDDIIDEHMKKKENQNAVDDGDVVDTDMVDDLLAFYSEEAK-----L   Lfil  300 SNESEDLQNSIKLTKDNIKAIIMDVMFGGTETVASAIEWAISELMKTPEELKRVQQELAD Ljap  300 SNESEDLQNSIKLTKDNIKAIIMDVMFGGTETVASAIEWAMSELMKTPEELKRVQQELAD Gmax  255 NNESDDLQNSIRLTKDNIKAIIMDVMFGGTETVASAIEWAMAELMRSPEDQKRVQQELAD Mtru  290 NNESDDLNNSIKLTKDNIKAIIMDVMFGGTETVASAIEWAMAELMKSPEDLKKVQQELAE Atha  292 VSETADLQNSIKLTRDNIKAIIMDVMFGGTETVASAIEWALTELLRSPEDLKRVQQELAE   Lfil  360 VVGLDRVVEESDFEKLTHLKCALKETLRLHPPIPLLLHESAEDVTVGGYFIPKKARVMIN Ljap  360 VVGLDRVVEESDFEKLTHLKCALKETLRLHPPIPLLLHETAEDVTIGGYFIPKKARVMIN Gmax  315 VVGLDRRAEESDFEKLTYLKCALKETLRLHPPIPLLLHETAEDATVGGYLVPKKARVMIN Mtru  350 VVGLSRQVEESDFEKLTYLKCALKETLRLHPPIPLLLHETAEEATVNGYFIPKQARVMIN Atha  352 VVGLDRRVEESDIEKLTYLKCTLKETLRMHPPIPLLLHETAEDTSIDGFFIPKKSRVMIN   Lfil  420 VWAIGRDKNCWEEPDSFKPSRFLKPGVPDFKGSNFEFIPFGSGRRSCPGMQLGLYALDLA Ljap  420 VWAIGRDKNCWEEPNSFKPSRFLKPGVPNFKGSNFEFIPFGSGRRSCPGMQLGLYALDLA Gmax  375 AWAIGRDKNSWEEPESFKPARFLKPGVPDFKGSNFEFIPFGSGRRSCPGMVLGLYALELT Mtru  410 AWAIGRDANCWEEPQSFKPSRFLKPGVPDFKGSNFEFIPFGSGRRSCPGMQLGLYALDLA Atha  412 AFAIGRDPTSWTDPDTFRPSRFLEPGVPDFKGSNFEFIPFGSGRRSCPGMQLGLYALDLA 207      Lfil  480 VAHLLHCFTWELPDGMKPSEMDMSDVFGLTAPRASRLVAIPTKRVICPLS* Ljap  480 VAHLLHCFTWELPDGMKPSEMDMSDVFGLTAPRASRLVAIPTKRVVCPLS* Gmax  435 VAHLLHCFTWELPDGMKPSEMDMGDVFGLTAPRSTRLIAVPTKRVVCPLF* Mtru  470 VAHLLHCFTWELPDGMKPSEMDMSDVFGLTAPRASRLVAIPTKRVLCPLD* Atha  472 VAHILHCFTWKLPDGMKPSELDMNDVFGLTAPKATRLFAVPTTRLICAL-*       208    Figure S33. Amino acid alignment for POD-2  Lfil    1 ------------------------------------------------------------ Ljap    1 MANSMSLLM-LLSLLAFAPLCLCHKK--MGGYLYPQFYDYSCPQAKEIVKSIVANAVARE Gmax    1 MANSMSFFL-LLSLLAFAPLCLCHYN--QEGYLYPQFYDYSCPQVQHIVKSVLAKYVAEQ Mtru    1 MANSLSFLM-LLSLLAFAPFCLCHKK--MGSYLYPQFYDYSCPQAQNIVKSILANAVAKE Atha    1 MAKSLNILIAALSLIAFSPFCLCSKAYGSGGYLFPQFYDQSCPKAQEIVQSIVAKAFEHD   Lfil    1 -----------------------------------------------------------K Ljap   58 TRTAASILRLHFHDCFVKGCDASLLLDGSGSFTSEKVSNPNRNSARGFEVIDEIKQALEK Gmax   58 PRLAASILRLHFHDCFVKGCDASLLLDSSVNIISEKGSNPNRNSARGFEVVDAIKAELER Mtru   58 PRIAASLLRLHFHDCFVKGCDASILLDNSGSIISEKGSNPNRNSARGFEVIDEIKYALEK Atha   61 PRMPASLLRLHFHDCFVKGCDASILLDSSGTIISEKRSNPNRNSARGFELIEEIKHALEQ   Lfil    2 ECPQVVSCADILALAARDSTVLTGGPSWEVPLGRRDSRSASLSGSNNNIPAPNNTFQTIL Ljap  118 ECPQVVSCADILALAARDSTVLTGGPSWEVPLGRRDSRSASLSGSNNNIPAPNNTFQTIL Gmax  118 KCPSTVSCADILTLAARDSVVLTGGPSWEVPLGRRDSLGASISGSNNNIPAPNNTFQTIL Mtru  118 ECPHTVSCADILAIAARDSTVLAGGPNWEVPLGRRDSLGASLSGSNNNIPAPNNTFQTIL Atha  121 ECPETVSCADILALAARDSTVITGGPSWEVPLGRRDARGASLSGSNNDIPAPNNTFQTIL   Lfil   62 TKFKLQGLNIVDLVALSGSHT--------------------------------------- Ljap  178 TKFKLQGLNIVDLVALSGSHTIGNSRCASFRQRLYNQTGNGKADFTLDPNYAAELRTQCP Gmax  178 TKFNLQGLDLVDLVALSGGHTIGNARCTTFKQRLYNQSGNGEPDSTLDQYYAATLRNRCP Mtru  178 TKFKLQGLDIVDLVALSGSHTIGKSRCTSFRQRLYNQTGNGKQDFTLDQYYAAELRTQCP Atha  181 TKFKRQGLDLVDLVSLSGSHTIGNSRCTSFRQRLYNQSGNGKPDMTLSQYYATLLRQRCP   Lfil   82 ------------------------------------------------------------ Ljap  238 KSGGDQNLVFLDPVTPTKFDNTYFKNLLAYKGLLSSDEILLTANQESAQLVKLYAERNDL Gmax  238 SSGGDQNLFFLDYATPYKFDNSYFTNLLAYKGLLSSDQVLFTMNQESAELVKLYAERNDI Mtru  238 RSGGDQNLFFLDYVTPTKFDNNYFKNLLAYKGLLSSDEILLTKNQESAELVKLYAERNDL Atha  241 RSGGDQTLFFLDFATPFKFDNHYFKNLIMYKGLLSSDEILFTKNKQSKELVELYAENQEA   Lfil   82 ------------------------------------- Ljap  298 FFEQFAKSMIMMGNISPLTGSKGEIRKNCRVIN---* Gmax  298 FFEQFAKSMIKMGNISPLTNSKGEIRENCRRINA--* Mtru  298 FFEQFAKSMIKMGNISPLTGSRGNIRTNCRVINTW-* Atha  301 FFEQFAKSMVKMGNISPLTGAKGEIRRICRRVNHAY*       209    Figure S34. Amino acid alignment for POD-1  Lfil    1 MVNSIKCFL-LLS-LAFVPFCHCKKEQ--GVNLYPQFYDQSCPKAQEIVKSIVAKAFAKD Ljap    1 MVNSIKCLL-LLS-LAFVPFCHCKKEQ--GVNLYPQFYDQSCPKAQEIVKSIVAKAFAKD Gmax    1 MVKSINFLL-LLSLLAFVPSCHCKKKI--GGYLYPQFYDGSCPRAQEIVQSIVAKAVAKE Mtru    1 MVNSINFLL-LFSLLVFAPCCHCKTKL--GDYLYPQFYDESCPKVEEIVKSVVAKAVTKE Atha    1 MAKSLNILIAALSLIAFSPFCLCSKAYGSGGYLFPQFYDQSCPKAQEIVQSIVAKAFEHD   Lfil   57 PRMAASLLRLHFHDCFVKGCDGSVLLDSSGTIISEKRSNPNRNSARGFEVIDEIKSAIEK Ljap   57 PRMAASLLRLHFHDCFVKGCDGSVLLDSSGTIISEKRSNPNRNSARGFEVIDEIKSAIEK Gmax   58 PRMAASLLRLHFHDCFVKGCDASVLLDSSGTIISEKRSNPNRDSARGFEVIDEIKSALEK Mtru   58 PRMAASLLRLHFHDCFVKGCDASVLLDSSGTIISEKRSNPNRNSARGFEVIEEIKSAVEK Atha   61 PRMPASLLRLHFHDCFVKGCDASILLDSSGTIISEKRSNPNRNSARGFELIEEIKHALEQ   Lfil  117 ECPHTVSCADILAIAARDSTVLTGGPSWGVPLGRRDSLGASLSGSNQNIPAPNNTFQTIL Ljap  117 ECPHTVSCADILAIAARDSTVLTGGPSWGVPLGRRDSLGASLSGSNQNIPAPNNTFQTIL Gmax  118 ECPHTVSCADILALAARDSTVLTGGPSWGVPLGRRDSLGASISGSNNNIPAPNNTFQTIL Mtru  118 ECPQTVSCADILTLAARDSTVLTGGPSWDVPLGRRDSLGASISGSNNNIPAPNNTFQTIL Atha  121 ECPETVSCADILALAARDSTVITGGPSWEVPLGRRDARGASLSGSNNDIPAPNNTFQTIL   Lfil  177 TKFKLKGLDIVDLVALSGSHTIGDSRCTSFRQRLYNQTGNGKADFTLEQSYAAKLRTQCP Ljap  177 TKFKLKGLDIVDLVALSGSHTIGDSRCTSFRQRLYNQTGNGKADFTLDQSYAAKLRTQCP Gmax  178 TKFKLKGLDIVDLVALSGSHTIGNSRCTSFRQRLYNQTGNGKADFTLDQVYAAELRTRCP Mtru  178 TKFKLKGLNIVDLVALSGSHTIGDSRCTSFRQRLYNQTGNGKSDFTLDQNYAAQLRTRCP Atha  181 TKFKRQGLDLVDLVSLSGSHTIGNSRCTSFRQRLYNQSGNGKPDMTLSQYYATLLRQRCP   Lfil  237 RSGGDQNLFVLDFVTPVKFDNNYFKNLLAFKGLLSSDEILLTDNKESADLVKKYAERNDL Ljap  237 RSGGDQNLFVLDFVTPVKFDNNYFKNLLAFKGLLSSDEILLTDNKESADLVKKYAERNDL Gmax  238 RSGGDQNLFVLDFVTPIKFDNFYYKNLLANKGLLSSDEILLTKNQVSADLVKQYAENNDL Mtru  238 RSGGDQNLFVLDFVTPVKFDNNYYKNLLANKGLLSSDEILLTKNQVSADLVKKYAESNDL Atha  241 RSGGDQTLFFLDFATPFKFDNHYFKNLIMYKGLLSSDEILFTKNKQSKELVELYAENQEA   Lfil  297 FFEQFAKSMVKMGNITPLTGSRGEIRRICRRINN--* Ljap  297 FFEQFAKSMVKMGNITPLTGSRGEIRRICRRINN--* Gmax  298 FFEQFAKSMVKMGNITPLTGSRGEIRKNCRGINK--* Mtru  298 FFEQFAKSMVKMGNITPLTGSRGEIRKRCRKINN--* Atha  301 FFEQFAKSMVKMGNISPLTGAKGEIRRICRRVNHAY*       210    Appendix 10  Supplementary Figures S35 Amino acid alignments for red-differentially expressed lignin genes in Lotus filicaulis  Figure S35. Amino acid alignment for CAD  Lfil    1 MSNE-EGKLVCVTGASGYIASWIVKFLLEHGYTVRATVRDLSNPNKVEHLVKLDVAKERL Ljap    1 MSNE-EGKLVCVTGASGYIASWIVKFLLEHGYTVRATVRDLSNPNKVEHLVKLDVAKERL Gmax    1 MSNNNAGKVVCVTGASGFIASWIVKFLLQRGYTVRATVRYPSNLKKVDHLVKLEGAKERL Mtru    1 MMSG-EGKVVCVTGANGFIASWIVKFLLQCGYTVRATVRHPSNSEKVDHLVKLDGAKERL Atha    1 MSSE-EEKTVCVTGASGYIASWIVKLLLLRGYTVKASVRDPNDPRKTEHLLALEGAEERL   Lfil   60 QLFKADLLEEGSFDSVIQGCHGVFHVASPVLMFVEDPQAELIDPAVKGTLNVLKSCAKSP Ljap   60 QLFKADLLEEGSFDSVIQGCHGVFHVASPVLMFVEDPQAELIDPAVKGTLNVLKSCAKSP Gmax   61 QLFKADLLEEGSFDSVVEGCHGVFHTASPVRFVVNDPQAELLDPAVKGTLNVLKSCAKSP Mtru   60 QLFKADLLEEGSFDSVVEGCDGVFHTASPVRFVVNDPQAELIDPALKGTLNVLQSCAKSP Atha   60 KLFKANLLEEGSFDSAIDGCEGVFHTASPFYHDVKDPQAELLDPAVKGTINVLSSCLKTS   Lfil  120 SVKRVVLTSSTSAVLFNGRPKSPEVVVDETWFSDPDFLRESKFFHKWY------------ Ljap  120 SVKRVVLTSSTSAVLFNGRPKSPEVVVDETWFSDPDFLRESKV---CV------------ Gmax  121 SVKRVVLTSSISAVAFNRRPKTPQVVVDETWFSDPDVCRELEL---WYTLSKTLAEDAAW Mtru  120 SVKRVILTSSISAVVFDTRPKNPGVIVDETWFSNPDLCRESKL---WYTLSKTLAEAAAW Atha  120 SVKRVVLTSSIAAVAFNGMPRTPETIVDETWFADPDYCRASKL---WYVLSKTLAENAAW   Lfil  168 ---------------------------------------------TLSKTMA--EDAA-- Ljap  165 ------------------------------------------PFPNLSIGWVDVKDVAKA Gmax  178 KFVNENSIDMISINPTMVAGPLLQPEINESVEPILNLINGK-PFPNKSFGWVDVKDVANA Mtru  177 KFVNENSIDMVAINPTMVAGPLLQPELNGSVEPILNLISGI-PFPNKAYGWCNVKDVANA Atha  177 KFAKENNLQLVSINPAMVIGPLLQPTLNTSAAAVLSLIKGAQTFPNATFGWVNVKDVANA   Lfil  179 ------------------------------------------------------WKFAK- Ljap  183 HIQAYEITSASGRYCLVERVAHFSELLEFYMICIQHCKFQISVWTMNHMSQH--FNFHKK Gmax  237 HILAYEIASASGRYCLVERVIHYSELATILR--GLYPTLQIPDKCEVDEPYIPTYQISTE Mtru  236 HILAYETASASGRYCLAERVVHYSELAMILR--DLYPTLQISDKCEDDGPYMPTYQISKE Atha  237 HIQAFENPDADGRYCLVERVAHYSEVVNILH--DLYPDFQLPEKCADEKIYIPTYKVSKE   Lfil  184 -EN-KIDMVVILPSMVVGPLLRPEVNFS-VEPVLNIINGVPF-P- Ljap  241 EPR-AXESSLLLXKXVF----RKPXKAS-EKRR-------LST--  Gmax  295 KAKKDLGIEFTPLEVSL----RETVESFREKKI------VNFNP* Mtru  294 KAK-SLGIEFTSLEVTL----KETVESFREKKI------VNF--* Atha  295 KAE-SLGVEFVPLEVSI----KETVESLRDKGF------IRF--*       211    Appendix 11  Supplementary Figures S36 – S40 Amino acid alignments for yellow-differentially expressed lignin genes in Lotus filicaulis  Figure S36. Amino acid alignment for C3’H  Lfil    1 MSLLLAIPISLITLLLSYTLYQ---RLKFKLPPGPRPWPVVGNLYDIKPVRFRCFAEWAQ Ljap    1 MSLLLAIPISLITLLLSYTLYQ---RLKFKLPPGPRPWPVVGNLYDIKPVRFRCFAEWAQ Gmax    1 MALLLIVPISLVTLWLGYTLYQ---RLRFKLPPGPRPWPVVGNLYDIKPVRFRCFAEWAQ Mtru    1 MSPLIIVFATIAAAILIYRLLNLITKPSLPLPPGPSPWPIVGNLPHMGPVPHHALAVLAL Atha    1 MSWFLIA-VATIAAVVSYKLIQ---RLRYKFPPGPSPKPIVGNLYDIKPVRFRCYYEWAQ   Lfil   58 SYGPIISVWFGSTLNVVVSNSELAKEVLKEKDQQLADRHRSRSAAKFSRDGQDLIWADYG Ljap   58 SYGPIISVWFGSTLNVVVSNSELAKEVLKEKDQQLADRHRSRSAAKFSRDGQDLIWADYG Gmax   58 SYGPIISVWFGSTLNVIVSNSELAKEVLKEHDQQLADRHRSRSAAKFSRDGKDLIWADYG Mtru   61 KHGPLMHLRLGYVDVIVAASASVAEQFLKVHDTNFSSRPTNSGAKYLAYNYQDLVFAPYG Atha   57 SYGPIISVWIGSILNVVVSSAELAKEVLKEHDQKLADRHRNRSTEAFSRNGQDLIWADYG   Lfil  118 PHYVKVRKVCTLELFSPKRLEALRPIREDEVTAMVESVFHDCTNSENLGKGLLVKKYLGT Ljap  118 PHYVKVRKVCTLELFSPKRLEALRPIREDEVTAMVESVFHDCTNSENLGKGLLVKKYLGT Gmax  118 PHYVKVRKVCTLELFTPKRLESLRPIREDEVTTMVESVYNHCTTTGNLGKAILVRKHLGS Mtru  121 PRWRLLRKISYVHMFSSKALDEFRHIRQEEVARLIRNLASSGSKAVNLGQ------MLNV Atha  117 PHYVKVRKVCTLELFTPKRLESLRPIREDEVTAMVESVFRDCNLPENRAKGLQLRKYLGA   Lfil  178 VAFNNITRLAFGKRFVNSE---GVIDEQGVEFKAIVANGLKLGASLAMAEHIPWLRWMFP Ljap  178 VAFNNITRQAFGKRFVNSE---GVIDEQGVEFKAIVANGLKLGASLAMAEHIPWLRWMFP Gmax  178 VAFNNITRLAFGKRFVNSE---GVMDEQGVEFKAIVENGLKLGASLAMAEHIPWLRWMFP Mtru  175 CTTNALARVMIGRRVFNEGNGGCECDPRADEFKSMVVELMVLAGVFNIGDFLPAFEWLDL Atha  177 VAFNNITRLAFGKRFMNAE---GVVDEQGLEFKAIVSNGLKLGASLSIAEHIPWLRWMFP   Lfil  235 L-EEEAFAKHGARRDRLTRAIMEEHTQARQKSGGAKQHFVDALLTLQDKYDLSEDT---- Ljap  235 L-EEEAFAKHGARRDRLTRAIMEEHTQARQKSGGAKQHFVDALLTLQDKYDLSEDT---- Gmax  235 L-EEGAFAKHGARRDRLTRAIMTEHTEARKKSGGAKQHFVDALLTLQDKYDLSEDT---- Mtru  235 QGVQGKMKKLHKRFDTFLTSIIEDHRISKSE---KHNDLLSTLLSLKEKLPEDEDTLNDT Atha  234 A-DEKAFAEHGARRDRLTRAIMEEHTLARQKSSGAKQHFVDALLTLKDQYDLSEDT----   Lfil  290 -IIGLLWDMITAGMDTTAISVEWAMAELIKNPRVQQKAQEELDRVIGFERVLTETDFSNL Ljap  290 -IIGLLWDMITAGMDTTAISVEWAMAELIKNPRVQQKAQEELDRVIGFERVLTETDFSNL Gmax  290 -IIGLLWDMITAGMDTTAISVEWAMAELIRNPRVQQKVQEELDRVIGLERVMTEADFSSL Mtru  292 EIKALLLNLFTAGTDTTASTTEWAISELIKNPKLMACIQNELDTVVGRDRLVTEQDLTNL Atha  289 -IIGLLWDMITAGMDTTAITAEWAMAEMIKNPRVQQKVQEEFDRVVGLDRILTEADFSRL   Lfil  349 PYLQCVAKEAMRLHPPTPLMLPHRSNANVKIGGYDIPKGSNVHVNVWAVARDPAVWKNPL Ljap  349 PYLQCVAKEAMRLHPPTPLMLPHRSNANVKIGGYDIPKGSNVHVNVWAVARDPAVWKNPL Gmax  349 PYLQCVIKEAMRLHPPTPLMLPHRANANVKVGGYDIPKGSNVHVNVWAVARDPAVWKDPL Mtru  352 PYLEAVVKETLRLHPSTPLSLPRVATQSCEIFNYHIPKGATLLVNVWAISRDPKEWSNPL 212    Atha  348 PYLQCVVKESFRLHPPTPLMLPHRSNADVKIGGYDIPKGSNVHVNVWAVARDPAVWKNPF   Lfil  409 EFRPERFLE----EDVDMKGHDFRLLPFGAGRRVCPGAQLGINLVTSMLGHLLHHFCWAP Ljap  409 EFRPERFLE----EDVDMKGHDFRLLPFGAGRRVCPGAQLGINLVTSMLGHLLHHFCWAP Gmax  409 EFRPERFLE----EDVDMKGHDFRLLPFGAGRRVCPGAQLGINLVTSMLGHLLHHFCWTP Mtru  412 EFKPERFLPGGEKFDVDVRGNDFEVIPFGAGRRICAGMSLGLRMVQLLTATLAHAYDWEL Atha  408 EFRPERFLE----EDVDMKGHDFRLLPFGAGRRVCPGAQLGINLVTSMMSHLLHHFVWTP   Lfil  465 PEGVKPEEIDMVENPGLVTYMRTPVQAVATPRLPSHLYKRVPADI* Ljap  465 PEGVKPEEIDMVENPGLVTYMRTPVQAVATPRLPSHLYKRVPADI* Gmax  465 PEGMKPEEIDMGENPGLVTYMRTPIQALASPRLPSHLYKRVPAEI* Mtru  472 ENGVSPEKLNMDEAYGLTLQRAVPILAHPRPRLSPHLYL------* Atha  464 PQGTKPEEIDMSENPGLVTYMRTPVQAVATPRLPSDLYKRVPYDM*        213    Figure S37. Amino acid alignment for CAD  Lfil    1 MGSLE-AERTTVGWAARDPSGILSPYTFTLRNTGPDDVYIKVHYCGVCHTDVHQVKNDLG Ljap    1 MGSLE-AERTTVGWAARDPSGILSPYTFTLRNTGPDDVYIKVHYCGVCHTDVHQVKNDLG Gmax    1 MGSLE-AERTTVGLAARDPSGILSPYTYNLRNTGPDDVYIKVHYCGICHSDLHQIKNDLG Mtru    1 MGSIEVAERTTVGLAAKDPSGILTPYTYTLRNTGPDDVYIKIHYCGVCHSDLHQIKNDLG Atha    1 MGIME-AERKTTGWAARDPSGILSPYTYTLRETGPEDVNIRIICCGICHTDLHQTKNDLG   Lfil   60 MSNYPMVPGHEVVGEVLEVGSDVTRFTVGEIVGAGLLVGCCKKCTACQSDIEQYCKKKIW Ljap   60 MSNYPMVPGHEVVGEVLEVGSDVTRFTVGEIVGAGLLVGCCKKCTACQSDIEQYCKKKIW Gmax   60 MSNYPMVPGHEVVGEVLEVGSDVSRFRVGELVGVGLLVGCCKNCQPCQQDIENYCSKKIW Mtru   61 MSNYPMVPGHEVVGEVLEVGSNVTRFKVGEIVGVGLLVGCCKSCRACDSEIEQYCNKKIW Atha   60 MSNYPMVPGHEVVGEVVEVGSDVSKFTVGDIVGVGCLVGCCGGCSPCERDLEQYCPKKIW   Lfil  120 NYNDVYVDGKPTQGGFAETIVVEQKFVVKIPEGMAPEQVAPLLCAAVTVYSPLSHFGLKE Ljap  120 NYNDVYVDGKPTQGGFAETIIVEQKFVVKIPEGMAPEQVAPLLCAAVTVYSPLSHFGLKE Gmax  120 SYNDVYVDGKPTQGGFAETMVVEQKFVVKIPEGLAPEQVAPLLCAGVTVYSPLVHFGLKE Mtru  121 SYNDVYTDGKITQGGFAESTVVEQKFVVKIPEGLAPEQVAPLLCAGVTVYSPLSHFGLKT Atha  120 SYNDVYINGQPTQGGFAKATVVHQKFVVKIPEGMAVEQAAPLLCAGVTVYSPLSHFGLKQ   Lfil  180 SGLRGGILGLGGVGHMGVIIAKAMGHHVTVISSSDRKKKEAIEDLGADAYLVSSDTTSMQ Ljap  180 SGLRGGILGLGGVGHMGVIIAKAMGHHVTVISSSDRKKKEAIEDLGADAYLVSSDTTSMQ Gmax  180 SGLRGGILGLGGVGHMGVKIAKALGHHVTVISSSDKKKQEALEHLGADQYLVSSDVTAMQ Mtru  181 PGLRGGILGLGGVGHMGVKVAKAFGHHVTVISSSDKKKKEALEDLGADSYLVSSDTVGMQ Atha  180 PGLRGGILGLGGVGHMGVKIAKAMGHHVTVISSSNKKREEALQDLGADDYVIGSDQAKMS   Lfil  240 EAADSLDYIIDTVPVGHPLEPYLSLLKLDGKLILMGVINTPLQFITPMVMLGRRSITGSF Ljap  240 EAADSLDYIIDTVPVGHPLEPYLSLLKLDGKLILMGVINTPLQFITPMVMLGRRSITGSF Gmax  240 EAADSLDYIIDTVPVGHPLEPYLSLLKLDGKLILMGVINTPLQFVSPMVMLGRRSITGSF Mtru  241 EAADSLDYIIDTVPVGHPLEPYLSLLKIDGKLILMGVINTPLQFVTPMVMLGRKSITGSF Atha  240 ELADSLDYVIDTVPVHHALEPYLSLLKLDGKLILMGVINNPLQFLTPLLMLGEKVITGSF   Lfil  300 IGSIKETEEMLEFWKEKGLTSMIEIVNMDYINKAFERLEKNDVRYRFVVDVKGSKLIDQ* Ljap  300 IGSIKETEEMLEFWKEKGLTSMIEIVNMDYINKAFERLEKNDVRYRFVVDVKGSKLIDQ* Gmax  300 IGSMKETEEMLEFWKEKGLSSMIEVVNMDYINKAFERLEKNDVRYRFVVDVKGSKLVD-* Mtru  301 VGSVKETEEMLEFWKEKGLSSMIEIVTMDYINKAFERLEKNDVRYRFVVDVKGSKFED-* Atha  300 IGSMKETEEMLEFCKEKGLSSIIEVVKMDYVNTAFERLEKNDVRYRFVVDVEGSNLDA-*        214    Figure S38. Amino acid alignment for CCoA-OMT  Lfil    1 ------------------------------------------------------------ Ljap    1 ------------------------------------------------------------ Gmax    1 MTL----------IKELEQQPNQIAGHKELAHKSLLQSDALYQYILETSVYPREHESLKE Mtru    1 MAI----------NNG-EEQQNQLIGDADLAHKTLLQSDALYQYILDTSVFPREHPCLKE Atha    1 MATTTTEATKTSSTNGEDQKQSQNLRHQEVGHKSLLQSDDLYQYILETSVYPREPESMKE   Lfil    1 ------------------------------------------------------------ Ljap    1 ------------------------------------------------------------ Gmax   51 LRELTEKHPWNLMATPPDEGQLLGMLLKLINAKNTMEIGVFTGYSLLSTALALPSDG--- Mtru   50 LREMTEKHPRNHMAIPGDEGQLISMLVKLINAKNTLEIGVYTGYSLLSTALALPSDGKVL Atha   61 LREVTAKHPWNIMTTSADEGQFLNMLIKLVNAKNTMEIGVYTGYSLLATALALPEDGKIL   Lfil    1 ---------ILGLPIIEKAGVAHKIDFREGPALPVLDELLEDENNKGTFDFVFVDADKDN Ljap    1 -----------------------------------------------SFLWML------I Gmax  108 -----------------KAGVAHKIDFREGPALPLLDQLIKDEKNKGAFDFIYVDADKDN Mtru  110 ALDKSREYYELGLPMIEKAGVAHKIDFRQGLALPLLDELLQDENNKGTFDFVFVDADKNN Atha  121 AMDVNRENYELGLPIIEKAGVAHKIDFREGPALPVLDEIVADEKNHGTYDFIFVDADKDN   Lfil   52 YLNYHKRVIELVKVGGLIGFDNTLWNGSVVSAPDAPMMDYLKHYRGYVIELNKYLAQDSR Ljap    8 RMNYHKRVIELVKVGGLIGFDNTLWNGSVVSAPDAPMMDYLKHYRAYVIELNKYLAQDSR Gmax  151 YLNYHKRVIELVKVGGLVGYDNTLWNGSVVAPPDAPLMDYVKYYRDFVMELNKALALDSR Mtru  170 YLNYHVRMIDLVKVGGLIGYDNTLWSGSVAAPPDAPMMDFIRNLRGYVIEFNKHLAQDSR Atha  181 YINYHKRLIDLVKIGGVIGYDNTLWNGSVVAPPDAPMRKYVRYYRDFVLELNKALAADPR   Lfil  112 IEICQLPVGDGITLCRRII* Ljap   68 IEICQLPVGDGITLCRRII- Gmax  211 VEICQLPVGDGITLCRRII* Mtru  230 IEICQLSIADGITLCRRIK* Atha  241 IEICMLPVGDGITICRRIS*        215    Figure S39. Amino acid alignment for CCR-2  Lfil    1 MPAAAAES---PSSPTGETICVTGAGGFIASWMVKLLLQKGYTVRGTVRNPDDPKNGHLR Ljap    1 MPAA-----------AGETICVTGAGGFIASWMVKLLLQKGYTVRGTVRNPDDPKNGHLR Gmax    1 MPSAEA---------SSETICVTGAGGFIASWMVKLLLEKGYTVRGTLRNPDDPKNGHLK Mtru    1 MPAATAAAAAESSSVSGETICVTGAGGFIASWMVKLLLEKGYTVRGTLRNPDDPKNGHLK Atha    1 MPVDVA-------SPAGKTVCVTGAGGYIASWIVKILLERGYTVKGTVRNPDDPKNTHLR   Lfil   58 ELEGASDRLTLIKVDLLDLNSVRAAVHGSHGVFHTASPVTDNPEEMVEPAVNGAKNVIIA Ljap   50 ELEGASDRLTLIKVDLLELNSVRAAVHGSHGVFHTASPVTDNPEEMVEPAVNGAKNVIIA Gmax   52 EFEGASERLTLHKVDLLHLDSVRSVINGCHGVFHTASPVTDNPEEMVEPAVSGAKNVIIA Mtru   61 KLEGAKERLTLVKVDLLDLNSVKEAVNGCHGVFHTASPVTDNPEEMVEPAVNGAKNVIIA Atha   54 ELEGGKERLILCKADLQDYEALKAAIDGCDGVFHTASPVTDDPEQMVEPAVNGAKFVINA   Lfil  118 AAEAKVRRVVFTSSIGAVYMDPKRSVDSVVDESCWSDLEFCKDTKNWYCYGKAVAEQAAW Ljap  110 AAEAKVRRVVFTSSIGAVYMDPKRSVDSVVDESCWSDLEFCKDTK--------------- Gmax  112 AAEAKVRRVVFTSSIGAVYMDPSRSIDLVVDESCWSDLEYCKNTKNWYCYGKAVAEQAAW Mtru  121 GAEAKVRRVVFTSSIGAVYMDPNRSVDVEVDESCWSDLEFCKKTKNWYCYGKAVAEAAAW Atha  114 AAEAKVKRVVITSSIGAVYMDPNRDPEAVVDESCWSDLDFCKNTKNWYCYGKMVAEQAAW   Lfil  178 DTAKEKGVDLVVVNPVLVLGPLLQPTINASTVHILKYLTGSAKTYANATQAYADVRDVAL Ljap  155 ---------------------------------------------ANATQAYADVRDVAL Gmax  172 DTAKENGVDLVVVNPVLVLGPLLQPTINASTIHILKYLTGSAKTYANATQAYVHVRDVAL Mtru  181 DVAKEKGVDLVVVNPVLVLGPLLQPTINASTIHILKYLTGSAKTYANATQAYVHVRDVAL Atha  174 ETAKEKGVDLVVLNPVLVLGPPLQPTINASLYHVLKYLTGSAKTYANLTQAYVDVRDVAL   Lfil  238 AHILVYEKPEASGRYICSESSLHRGELVEILAKHFPEYPMPTKCKDEKNPRAKPYIFSNQ Ljap  170 AHILVYERPEASGRYICSESSLHRGELVEILAKHFPEYPMPTKCKDEKNPRAKPYIFSNQ Gmax  232 AHILVYEKPSASGRYLCAESSLHRGELVEILAKYFPEYPVPTKCSDEKNPRAKPYTFSNQ Mtru  241 AHILVYEKPSASGRYLCAETSLHRGELVEILAKYFPEYPIPTKCSDEKNPRVKPHIFSNK Atha  234 AHVLVYEAPSASGRYLLAESARHRGEVVEILAKLFPEYPLPTKCKDEKNPRAKPYKFTNQ   Lfil  298 KLKDLGLEFTPVSHSLYETVKSLQEKGHLT--IPK---QEDSIAVKS* Ljap  230 KLKDLGLEFTPVSHSLYETVKSLQEKGHLT--IPK---QEDSIAVKS* Gmax  292 KLKDLGLEFTPVSQCLYETVKSLQEKGHLP--VPAKQQEEDSTTVKS* Mtru  301 KLKDLGLEFTPVSECLYETVKSLQDQGHLS--IPN---KEDSLAVKS* Atha  294 KIKDLGLEFTSTKQSLYDTHRKNPWKMALRSGLEK---LINSLKYPP*        216    Figure S40. Amino acid alignment for CCR-1  Lfil    1 ------------------------------------------------------------ Ljap    1 ------------------------------------------------------------ Gmax    1 MPT--DTSSVSG--EIVCVTGAGGFIASWLVKLLLEKGYTVRGTVRNPDDPKNGHLKELE Mtru    1 MPAYDNTSSVSGGDQTVCVTGAGGFIASWLVKLLLERGYTVRGTVRNPEDPKNGHLKELE Atha    1 MPV--DVASPAG--KTVCVTGAGGYIASWIVKILLERGYTVKGTVRNPDDPKNTHLRELE   Lfil    1 ------------------------------------------------------------ Ljap    1 ------------------------------------------MLEPAVNGARNVVMAAAE Gmax   57 GGKERLTLHKVDLFDIASIKAALHGCHGVFHTASPVTDNPEEMVEPAVKGTKNVIIAAAE Mtru   61 GARERLTLHKVDLLDLQSIQSVVHGCHGVFHTASPVTDNPDEMLEPAVNGTKNVIIASAE Atha   57 GGKERLILCKADLQDYEALKAAIDGCDGVFHTASPVTDDPEQMVEPAVNGAKFVINAAAE   Lfil    1 ------------------------------------------------------------ Ljap   19 TKVQRVVFTSSIGTVYMDPNRSRDEVVDESCWSDLEYCKNTKNWYCYGKTVAEQVAWEAA Gmax  117 AKVRRVVFTSSIGTVYMDPNTSRDALVDESFWSDLEYCKNTKNWYCYGKTVAEQAAWDVA Mtru  121 AKVRRVVFTSSIGTVYMDPNTSRDVVVDESYWSDLEHCKNTKNWYCYGKTVAEQSAWDIA Atha  117 AKVKRVVITSSIGAVYMDPNRDPEAVVDESCWSDLDFCKNTKNWYCYGKMVAEQAAWETA   Lfil    1 -------------------------------------------------VDVKDAALAHV Ljap   79 KEKGVDLVVVNPVLVIGPLLQPTINASTIHILKYLTGSAKTYVNATQAYVDVKDAALAHV Gmax  177 KERGVDLVVVNPVLVIGPLLQPTINASTIHILKYLTGSAKTYVNATQAYVHVRDVALAHI Mtru  181 KENQVDLVVVNPVVVLGPLLQPTINASTIHILKYLNGAAKTYVNATQSYVHVKDVALAHL Atha  177 KEKGVDLVVLNPVLVLGPPLQPTINASLYHVLKYLTGSAKTYANLTQAYVDVRDVALAHV   Lfil   12 LVYETPSASGRYICAESSLHRGELVEILAKYFPEYPIPTKCSDEKNPRVKPYIFSNQKLK Ljap  139 LVYETPSASGRYICAESSLHRGELVEILAKYFPEYPIPT--------------------- Gmax  237 LVYETPSASGRFICAESSLHRGELVEILAKFFPEYPIPTKCSDEKNPRVKPYIFSNQKLK Mtru  241 LVYETNSASGRYICCETALHRGEVVEILAKYFPEYPLPTKCSDEKNPRVKPYKFSNQKLK Atha  237 LVYEAPSASGRYLLAESARHRGEVVEILAKLFPEYPLPTKCKDEKNPRAKPYKFTNQKIK   Lfil   72 DLGLEFTPV---------------------------------------- Ljap      ------------------------------------------------- Gmax  297 DLGLEFTPVKQCLYDTVKNLQENGHLPVPPKQKDS------------Y* Mtru  301 DLGLEFTPVKQCLYDTVRSLQEKGHLPIPPMQEDS------------A* Atha  297 DLGLEFTSTKQSLYDTVKSLQEKGHLAPPPPPPSASQESVENGIKIGS*       217    Appendix 12  Supplementary Figures S41 – S47 Amino acid alignments for yellow-differentially expressed carotenoid genes in Lotus sessilifolius  Figure S41. Amino acid alignment for CRTISO  Lses    1 VKGARVLVLEKYVIPGGSSGFYQRDGYTFDVGSSVMFGFSDKGNLNLITQALEAVGCQMQ Ljap    1 VKGARVLVLEKYVIPGGSSGFYQRDGYTFDVGSSVMFGFSDKGNLNLITQALEAVGCKMQ Gmax    1 VKGARVLVLEKYVIPGGSSGFYQRDGYTFDVGSSVMFGFSDKGNLNLITQALEAVGCRMQ Mtru    1 VKGAKVLVLEKYVIPGGSSGFYHREGYTFDVGSSVMFGFSDKGNLNLITQALEAVGCRMQ Atha    1 VKEARVLVLEKYLIPGGSSGFYERDGYTFDVGSSVMFGFSDKGNLNLITQALKAVGRKME   Lses   61 VVPDPTTVHFHLPNNLSVRVHREYDKFIEELTCYFPHEKEGILKFYGECWKIFNALNSLE Ljap   61 VVPDPTTVHFHLPNNLSVRVHREYDKFIEELTCYFPHEKEGILKFYGECWKIFNALNLLE Gmax   61 VVPDPTTVHFHLPNNLSVRVHKEYDKFIEELTSYFPHEKEGILKFYGECWKIFNALNSLE Mtru   61 VIPDPTTVHFHLPNHLSVRVHREYDKFIEELTSYFPHEKDGILKFYGECWKIFNALNSLE Atha   61 VIPDPTTVHFHLPNNLSVRIHREYDDFIAELTSKFPHEKEGILGFYGDCWKIFNSLNSLE   Lses  121 LKSLEEPLYLFGQFFQKPLECLTLAYYLPQNAGAIARKYIQDPQLLSFIDAECFIVSTVN Ljap  121 LKSLEEPLYLFGQFFQKPLECLTLAYYLPQNAGAIARKYIQDPQLLSFIDAECFIVSTVN Gmax  121 LKSLEEPLYLFGQFFRRPLECLTLAYYLPQNAGAIARKYIQDPQLLSFIDAECFIVSTVN Mtru  121 LKSLEEPLYLFGQFFQKPLECLTLAYYLPQNAGAIARKYIQDPQLLSFIDAECFIVSTVN Atha  121 LKSLEEPIYLFGQFFQKPLECLTLAYYLPQNAGAIARKYIKDPQLLSFIDAECFIVSTVN   Lses  181 ALQTPMINAAMVLCDRHFGGINYPLGGVGGIAKSLAKGLADQGSEILYKANVTSVIIEQG Ljap  181 ALQTPMINAAMVLCDRHFGGINYPLGGVGGIAKSLAKGLADQGSEILYKANVTSVIIEQG Gmax  181 ALQTPMINAAMVLCDRHFGGINYPLGGVGGIAKSLAKGLVDQGSEIVYKANVTSIIIEQG Mtru  181 ALQTPMINASMVLCDRHFGGINYPLGGVGGIAKSLAKGLVDQGSQILYKANVTSIITEQG Atha  181 ALQTPMINASMVLCDRHYGGINYPVGGVGGIAKSLAEGLVDQGSEIQYKANVKSIILDHG   Lses  241 KAVGVRLSDGREFFAKTIISNATRWDT--------------------------------- Ljap  241 KAVGVRLSDGREFFAKTIISNATRWDTFG------------------------------- Gmax  241 KAVGVRLSNGREFFAKTIISNATRWDTFGKLLKGVPLPKEEENFQKVYVKAPSFLSIHMG Mtru  241 KAVGVRLSDGREFFAKTIISNATRWDTFGKLVKGESLPKEEENFQKVYVKAPSFLSIHMG Atha  241 KAVGVRLADGREFFAKTIISNATRWDTFGKLLKGEKLPKEEENFQKVYVKAPSFLSIHMG   Lses      ------------------------------------------------------------ Ljap  270 ------------------------------------------------------------ Gmax  301 VKAEVLPPDTDCHHFVLESNWSKLEEPYGSIFLSIPTVLDSSLAPEGRHILHIFTTSSME Mtru  301 VKAEVLPPDTDCHHFVLENNWTSLEEPYGSIFLSIPTILDSSLAPEGRHILHIFTTSSMD Atha  301 VKAEVLPPDTDCHHFVLEDDWKNLEEPYGSIFLSIPTILDSSLAPDGRHILHIFTTSSIE   Lses      ------------------------------------------------------------ Ljap  270 ------------------------------------------------------------ Gmax  361 DWEGLSRVEYEAKKQLVADEITSRLENKLFPGLRSSIDFMEVGTPKTHRRYLARDEGTYG Mtru  361 DWQGLSRIDYEAKKQVVADEIISRLEKKLFPGLRSSIDFIEVGTPKTHRRYLARDEGTYG 218    Atha  361 DWEGLPPKEYEAKKEDVAARIIQRLEKKLFPGLSSSITFKEVGTPRTHRRFLARDKGTYG   Lses      ------------------------------------------------------------ Ljap  270 ------------------------------------------------------------ Gmax  421 PMPRRIPKGLLGMPFNTTGIDGLYCVGDSCFPGQGVIAVAFSGVMCAHRVAADIGLEKKS Mtru  421 PMPRSIPKGLLGMPFNTTSIDGLYCVGDSCFPGQGVIAVAFSGVMCAHRVAADIGLEKKS Atha  421 PMPRGTPKGLLGMPFNTTAIDGLYCVGDSCFPGQGVIAVAFSGVMCAHRVAADIGLEKKS   Lses      --------------- Ljap  270 ------------WFR Gmax  481 PVLDSMLLRLLGWLR Mtru  481 PVLDGMLLGLLGWLR Atha  481 RVLDVGLLGLLGWLR       219    Figure S42. Amino acid alignment for CYB  Lses    1 RASSSALLELVPEFKKENLDFELPLYDSTKGAVVDLAVVGGGPAGLAVAQQVSEAGLSVC Ljap    1 RASSSALLELVPEVKKENLDFELPLYDSMKGAVVDLAVVGGGPAGLAVAQQVSEAGLSVC Gmax    1 RASSSALLELVPEFKKENLDFELPLYDSSKGAMVDLAVVGGGPAGLAVAQQVSEAGLSVC Mtru    1 RASSSALLELVPEFKKENLDFELPLYDSTKGTVVDLVVVGGGPAGLAVAQQVSEAGLSVC Atha    1 VSGSAALLDLVPETKKENLDFELPLYDTSKSQVVDLAIVGGGPAGLAVAQQVSEAGLSVC   Lses   61 AIDPNPRLIWPNNYGVWVDEFEAMDLLDCLDTTWSGAVVYIDDKTKKYLDRPYGRVNRKL Ljap   61 AIDPNPRLIWPNNYGVWVDEFEAMDLLDCLDTTWSGAVVYIDDKTKKYLDRPYGRVNRKL Gmax   61 AIDPNPRLIWPNNYGVWVDEFEAMDLLDCLDTIWSGAVVFIDDKTKKDLDRPYGRVNRKL Mtru   61 AIDPNPRLIWPNNYGVWVDEFEAMDLLDCLDKTWSGAVVYIDDKTKKDLDRPYGRVNRKL Atha   61 SIDPSPKLIWPNNYGVWVDEFEAMDLLDCLDTTWSGAVVYVDEGVKKDLSRPYGRVNRKQ   Lses  121 LKSKMLQKCISNGVKFHQAKVIKVIHEESKSMLICNDGVTIQATVVLDATGFSRSLVQYD Ljap  121 LKSKMLQKCISNGVKFHQAKVIKVIHEESKSMLICNDGVTIQATVVLDATGFSRSLVQYD Gmax  121 LKSKMLQKCISNGVKFHQAKVIKVIHEEAKSLLICNDGVTVHATVVLDATGFSRCLVQYD Mtru  121 LKSKMLQKCIANGVKFHQAKVIKVIHEESKSMLICNDGVTVQATVVLDATGFSRCLVQYD Atha  121 LKSKMLQKCITNGVKFHQSKVTNVVHEEANSTVVCSDGVKIQASVVLDATGFSRCLVQYD   Lses  181 KPYNPGYQVAYGILAEVEEHPFDVDKMLFMDWRDSHLDNDMELKERNSKIPTFLYAMPFS Ljap  181 KPYNPGYQVAYGILAEVEEHPFDVDKMLFMDWRDSHLDNNMELKEKNSRIPTFLYAMPFS Gmax  181 KPYNPGYQVAYGILAEVDEHPFDVDKMLFMDWRDSHLDNDMELKQRNSRIPTFLYAMPFS Mtru  181 KPYNPGYQVAYGILAEVEEHPFDVDKMLFMDWRDSHLDNDLKLKERNSKIPTFLYAMPFS Atha  181 KPYNPGYQVAYGIVAEVDGHPFDVDKMVFMDWRDKHLDSYPELKERNSKIPTFLYAMPFS   Lses  241 STKIFLEETSLVARPGLRMDDIQDRMVARLKHLGIKVKSIEEDEHCVIPMGGPLPVLPQR Ljap  241 STKIFLEETSLVARPGLRMDDIQDRMVARLKHLGIKVKSIEEDEHCVIPMGGPLPVLPQR Gmax  241 STKIFLEETSLVARPGLRMDDIQERMAARLKHLGIRVKSIEEDEHCVIPMGGPLPVLPQR Mtru  241 STKIFLEETSLVARPGLRMDDIQDRMVARLKHLGINVKSIEEDEQCVIPMGGPLPVLPQR Atha  241 SNRIFLEETSLVARPGLRMEDIQERMAARLKHLGINVKRIEEDERCVIPMGGPLPVLPQR   Lses  301 VVGIGGTAGMVHPSTGYMVARTLAAAPIVANSIVQYLGSDRGISGDHLSAQVWKDLWPIE Ljap  301 VIGIGGTAGMVHPSTGYMVARTLAAAPIVANAIVQYLGSDRGISGDGLSSLVWKDLWPIE Gmax  301 VVGIGGTAGMVHPSTGYMVARTLAAAPIVANSIVQCLGSDRGFSGDEISAQVWKDLWPIQ Mtru  301 VVGIGGTAGMVHPSTGYMVARTLAAAPIVANAIVQYLGSDRGLLGDEVSARVWKDLWPIE Atha  301 VVGIGGTAGMVHPSTGYMVARTLAAAPIVANAIVRYLGSPNSLRGDQLSAEVWRDLWPIE   Lses  361 RRRQREFFCFGMDILLKLDLAGTKRFFDAFFDLEPRYWHGFLSSRLFLPELLFFGLSLFS Ljap  361 RRRQREFFCFGMDILLKLDLPGTRRFFDAFFDLEPHYWHGFLSSRLFLPELLVFGLSLFS Gmax  361 RRRQREFFCFGMDILLKLDLPGTKRFFDAFFDLEPHYWHGFLSSRLFLHELLFFGLSLFS Mtru  361 RRRQREFFCFGMDVLLKLDLPGTRRFFDAFFNLEPHYWHGFLSSRLYLPELFTFGLSLFS Atha  361 RRRQREFFCFGMDILLKLDLDATRRFFDAFFDLQPHYWHGFLSSRLFLPELLVFGLSLFS   Lses  421 YASNTSRIEIMAKGTVP Ljap  421 YASNTSRIEIMAKGTVP Gmax  421 YASNTSRVEIMAKGTLP Mtru  421 YASNTSRLEIMAKGTLP Atha  421 HASNTSRLEIMTKGTVP 220    Figure S43. Amino acid alignment for E-CHY  Lses    1 ------------------------------------------------------------ Ljap    1 SLSLSPLSKSSINKTKPSSWVSPDWLTSLSRSLTAGKNDDSGIPIASAKLDDVSDLLGGA Gmax    1 SLSLTPLSKSSINKTKPSSWLSPDWLTSLSRSLTAG-NDVSGIPVASAKLDDVSDLLGGA Mtru    1 SLPLSHLSKSSTNKTKSTSWVSPDWLTSLSKSLTTSKNDDSNIPIASAKLDDVSDLLGGA   Lses    1 ------------------------------------------------------------ Ljap   61 LFLPLFKWMKEYGPIYRLAAGPRNFVVVSDPAIAKHVLKNYGKYGKGLVAEVSEFLFGSG Gmax   60 LFLPLFKWMQDYGPIYRLAAGPRNFVVVSDPAIAKHVLRNYGKYAKGLVAEVSEFLFGSG Mtru   61 LFLPLFKWMNEYGPIYRLAAGPRNFVVVSDPAIAKHVLKNYGKYGKGLVAEVSEFLFGDG   Lses    1 ------------------------------------------KLQPAALNGTAVNMEDKF Ljap  121 FAIAEGPLWTARRRAVVPSLHKRYLSVIVDRVFCRCAERLVEKLQPDALNGTAVNMEDKF Gmax  120 FAIAEGPLWTARRRAVVPSLHKRYLSVIVDRVFCRCAERLVEKLQPDALNGTAVNMEAKF Mtru  121 FAIAEGPLWTARRRAVVPSLHKRYLSIMVDRVFCKCAERLVEKLQADAVNGTAVNMEDKF   Lses   19 SQLTLDVIGLSVFNYDFDSLNADSPVIGAVYTALKEAEARSTDLLPYWQGEFLRKINPRQ Ljap  181 SQLTLDVIGLSVFNYDFDSLNADSPVIGAVYTALKEAEARSTDLLPYWQVEFLRKIIPRQ Gmax  180 SQLTLDVIGLSVFNYNFDSLNTDSPVIEAVYTALKEAEARSTDLLPYWKFKFLCKIIPRQ Mtru  181 SQLTLDVIGLSVFNYNFDALNSDSPVIEAVYTALKEAEARSTDLLPYWKIDFLCKIIPRQ   Lses   79 ------------------------------------------------------------ Ljap  241 IKAENAVTIIRKTVEDLIEKCKEIVESEGERIDGDEYVNDSDPSILRFLLASREEVSSDQ Gmax  240 IKAEEAVSVIRKTVEDLIEKCREIVESEGERIDVEEYVNDSDPSILRFLLASREEVSSVQ Mtru  241 IKAENAVTVIRKTVEDLIEQCKEIVESEGERIDADEYVNDADPSILRFLLASREEVSSVQ   Lses      ------------------------------------------------------------ Ljap  301 LRDDLLSMLVAGHETTGSVLTWTLYLLSKDSSSLAKVQEEVDRVLQGRRPTFEDMKNLKF Gmax  300 LRDDLLSLLVAGHETTGSVLTWTLYLLSKDSSSLAKAQEEVDRVLQGRRPTYEDIKDLKF Mtru  301 LRDDLLSMLVAGHETTGSVLTWTLYLLSKDSSSLAKAQEEVDRVLQGRRPTYEDMKDLKF   Lses      ------------------------------------------------------------ Ljap  361 LTRCITESLRLYPHPPVLIRRAQVPDELPGAYKVNAGQDIMISVYNIHHSSEVWDRAEEF Gmax  360 LTRCIIESLRLYPHPPVLIRRAQVPDELPGGYKLDAGQDIMISVYNIHRSSEVWDRAEEF Mtru  361 LNRCIIESLRLYPHPPVLIRRSQIPDELPGDYKIDAGQDIMISVYNIHHSSKVWDRAEEF   Lses      ------------------------------------------------------------ Ljap  421 LPERFDLDGPMPNETNTDFRFIPFSGGPRKCVGDQFALLEATVSLAIFLQHMNFELVPDQ Gmax  420 VPERFDLDGPVPNETNTDFRFIPFSGGPRKCVGDQFALMEAIVALAIFLQHMNFELVPDQ Mtru  421 LPERFDLDGPVPNETNTDFRFIPFSGGPRKCVGDQFALLEATVALAVFLQHMNFELVPDQ   Lses      ------------------------- Ljap  481 NISMTTGATIHTTNGLYMKLSQRVK Gmax  480 NISMTTGATIHTTNGLYMKLSRRLK Mtru  481 NIGMTTGATIHTTNGLYMKLSQRLK   221    Figure S44. Amino acid alignment for PSY  Lses    1 VLLWVNCGKGNSISLLGLGGRGSRSQRRFRLCSGISFASFSSAVAEPSRSSEERVYEVVL Ljap    1 -----------------LGGRGSRSQRRYRLCSGISFASFSSAVAEPSRSSEERVYEVVL Gmax    1 VLLWVSCGKENPISIVGLGGRGGRSQRRFGLCNGISFASFSPAVADPSRSSEERVYEVVL Mtru    1 VLLWVNCGKENAISLMGLVGRDVRTQRRFRHCSGISFACFSSAVVEPTRSSEERVYEVVL Atha    1 AVLWVATSNPDPMNNCGLV-RVLESSRLFSPCQNQRLSLVASPSGEIALSSEEKVYNVVL   Lses   61 KQAALVKEQKRNTKRDLNLDIEDDFTNKDLLNAAYDRCGEVCAEYAKTFYLGTQLMTEER Ljap   44 KQAALVKEQKRDTKRKLNLDIEDDFTNKDLLSGAYDRCGEVCAEYAKTFYLGTQLMTEER Gmax   61 KQAALVKEQDKGTKRALNLDIEGDLTNGDLLSDAYDRCGEVCAEYAKTFYLGTQLMTQER Mtru   61 KQAALVKEQRKDIKRGLNLDIEGDFTNGELLSSAYDRCGDVCAEYAKTFYLGTQLMTQER Atha   60 KQAALVNKQLRSSSYDLDVKQDVVLPSLSLLGEAYDRCGEVCAEYAKTFYLGTLLMTPER   Lses  121 RKAIWAIYVWCRRTDELVDGPNSPHITPKALDRWEQRLSDVFEGRPYDMYDAALSDTVTK Ljap  104 RKAIWAIYVWCRRTDELVDGPNSPHITPKALDRWEQRLSDVFEGRPYDMYDAALSDTVTK Gmax  121 RKAIWAIYVWCRRTDELVDGPNASHITPKALDRWEQRLSDVFEGRPYDMYDAALSDTVSK Mtru  121 RKAIWAIYVWCRRTDELVDGPNASHITPKALDRWEQRLTDVFEGRPYDMYDAALSDTVTK Atha  120 RKAIWAIYVWCRRTDELVDGPNASHITPMALDRWEARLEDLFRGRPFDMLDAALADTVAR   Lses  181 YPVDIQPFKDMIEGMRLDLRKSRYDNFDELYLYCYYVAGTVGLMSVPVMGIAPESKASTE Ljap  164 YPVDIQVNLMLLTG---------------------------------------------- Gmax  181 YPVDIQPFKDMIEGMRLDLRKSRYNSFDELYLYCYYVAGTVGLMSVPVMGIAPESKATTE Mtru  181 YPVDIQPFKDMIEGMRLDLRKSRYNNFDELYLYCYYVAGTVGLMSVPVMGIEPQSKASTE Atha  180 YPVDIQPFRDMIEGMRMDLKKSRYQNFDDLYLYCYYVAGTVGLMSVPVMGIDPKSKATTE   Lses  241 SVYNAALALGIANQLTNILRDVGEDARRGRVYLPQDELAKAGLSDEDIFSGRVTDKWRNF Ljap      ------------------------------------------------------------ Gmax  241 SVYNAALALGIANQLTNILRDVGEDARRGRVYLPQDELAQAGLTDDDIFRGKVTDKWRNF Mtru  241 SIYNAALALGIANQLTNILRDVGEDARRGRVYLPQDELAQAGLSDDDIFRGRVTDKWRNF Atha  240 SVYNAALALGIANQLTNILRDVGEDARRGRVYLPQDELAQAGLSDEDIFAGKVTDKWRNF   Lses  301 MKGQIRRARMFFDEAEKGVSELSSASRWPVWASLLLYRQILDSIEANDYNNFTKRAYVGK Ljap      ------------------------------------------------------------ Gmax  301 MKGQIQRARMFFDEAEKGVSELNSASRWPVWASLLLYGQILDSIEANDYNNFTKRAYVGK Mtru  301 MKGQIKRARMFFDEAEKGVSELSSASRWPVWASLLLYRQILDSIEANDYNNFTKRAYVGK Atha  300 MKMQLKRARMFFDEAEKGVTELSAASRWPVWASLLLYRRILDEIEANDYNNFTKRAYVGK   Lses  361 AKKLLSLPAA Ljap      ---------- Gmax  361 VKKLLSLPAA Mtru  361 AKKLLSLPVA Atha  360 VKKIAALPLA      222    Figure S45. Amino acid alignment for ZEP-1 Lses    1 ------------------------------------------------------------ Gmax    1 MATTLCYNSLNPSTTVFSRTHFSVPGCRTRKQRKKVMHVKTPSKKQLRILVAGGGIGGLV Mtru    1 MVSTLSHKCLSPSMTTFSRTHFSNPGNRTTKQRRKLMQVKTPQKKQLKVLVAGGGIGGLV Atha    1 MGSTPFCYSINPSKLDFTRTHVFSPGVSGFRSRRALLGVKTEKKKKSRVLVAGGGIGGLV   Lses    1 ----------------------------------------------------------IT Gmax   61 FALAAKRKGFEVMVFEKDLSAIRGEGQYRGPIQIQSNALAALEAIDSEVADEVMRVGCIT Mtru   61 FALAAKRKGFEVVVFEKDLSAIRGEGQYRGPIQIQSNALAALEAIDMNVADEVMRVGCIT Atha   61 FALAAKKKGFDVLVFEKDLSAIRGEGKYRGPIQIQSNALAALEAIDIEVAEQVMEAGCIT   Lses    3 GDRINGLVDGVSGSWYVKFDTFTPAVERGLPVTRVISRMVLQGILARAVGDDIIMNASNV Gmax  121 GDRINGLVDGVSGSWYVKFDTFTPAVERGLPVTRVISRMVLQEILARAVGEDIIMNASNV Mtru  121 GDRINGLVDGVSGSWYIKFDTFTPAAERGLPVTRVISRMALQEILARAVGDDVIMNGSNV Atha  121 GDRINGLVDGISGTWYVKFDTFTPAASRGLPVTRVISRMTLQQILARAVGEDVIRNESNV   Lses   63 VNFVDDGNKVTVELENGEKYEGDLLVGADGIWSKVRKQLFGLTEAVYSGYTC-------- Gmax  181 VNFVDDGNKVTVELENGQKYEGDVLVGADGIWSKVRKQLFGLTEAVYSGYTCYTGIADFV Mtru  181 VDFIDHETKVTVVLDNGQKYDGDLLVGADGIWSKVRTKLFGSTEATYSGYTCYTGIADFV Atha  181 VDFEDSGDKVTVVLENGQRYEGDLLVGADGIWSKVRNNLFGRSEATYSGYTCYTGIADFI   Lses      ------------------------------------------------------------ Gmax  241 PADIETVGYRVFLGHKQYFVSSDVGAGKMQWYAFHKEPPGGVDEPNGKKERLLRIFEGWC Mtru  241 PPDIESVGYRVFLGHKQYFVSSDVGAGKMQWYAFHQEPAGGVDTPNGKKERLLKIFEGWC Atha  241 PADIESVGYRVFLGHKQYFVSSDVGGGKMQWYAFHEEPAGGADAPNGMKKRLFEIFDGWC   Lses      ------------------------------------------------------------ Gmax  301 DNAVDLILATEEEAILRRDIYDRIPTLTWGKGRVTLLGDSVHAMQPNMGQGGCMAIEDSY Mtru  301 DNAIDLIVATEEEAILRRDIYDRTPTLTWGKGRVTLLGDSVHAMQPNMGQGGCMAIEDGY Atha  301 DNVLDLLHATEEEAILRRDIYDRSPGFTWGKGRVTLLGDSIHAMQPNMGQGGCMAIEDSF   Lses      ------------------------------------------------------------ Gmax  361 QLAWELENAWEQSIKSGSPIDIDSSLRSYERERRLRVAIIHGMARMAALMASTYKAYLGV Mtru  361 QLAFELDNAWQQSAKSGSTIDIASSLKSYERERRLRVTFVHGMARMAALMASTYKAYLGV Atha  361 QLALELDEAWKQSVETTTPVDVVSSLKRYEESRRLRVAIIHAMARMAAIMASTYKAYLGV   Lses      -------------------------------------------------------- Gmax  421 GLGPLEFLTKFRIPHPGRVGGRFFVDIMMPSMLSWVLGGNSDKLEGRPLSCRLTDK Mtru  421 GLGPFEFLTKFRIPHPGRVGGRFFIQKSMPLMLNWVLGGNSSKLEGRPLCCRLSDK Atha  421 GLGPLSFLTKFRVPHPGRVGGRFFVDIAMPSMLDWVLGGNSEKLQGRPPSCRLTDK        223    Figure S46. Amino acid alignment for ZEP-2 Lses    1 CRLSDKANDQLQRWFEDDDALERAVNGEWILLPCGDEAGLSKPICLSQDEMKPCMIGQED Ljap    1 ---------------------------------------------------------QED Gmax    1 CRLSDKANDQLHRWFEDNDALERAINGEWILLPCGDEAGPTKPICLTQDEMKPCIIGQKD Mtru    1 CRLSDKASDQLHTWFEDDDALERTINGEWILLPCGDVPGHVKPISLNQDDTKPYIIGQED   Lses   61 YPGCSITIPLPQVSQMHARINYKDGAFFLTDLQSQHGTWITDNGGRRYRVPPNDPARVRP Ljap    4 YPGSSITITLPQVSQMHAQINYKDGAFFLTDLQSQHGTWITDNEGRRNRVPPNCPARVRP Gmax   61 HPGSSIIIPLPQVSQMHARINYKDGAFFLTDLRSLHGTWITDNEGRRYRVPPNYPARVRP Mtru   61 YPGSLITIPLPQVSQLHARINFKDGAFFLTDLRSQHGTWITDNEGRRYMVSPNYPARIRP   Lses  121 SDLIEFGSNKASYRVKVTR Ljap   64 SDLIEFGSNKVIDDP---- Gmax  121 SDVVEFGSDKASYRVKVTR Mtru  121 SHVIEFGCNQASYRVKVTR           224    Figure S47. Amino acid alignment for ZEP-3  Lses    1 KKKLRILVAGGGIGGLVFALAAKRKGFEVVVFEKDMSAIRGEGKYRGPIQIQSNALAALE Ljap    1 -----LIIAG-------------------------------------------------- Gmax    1 KKKLRVLVAGGGIGGLVFALAAKRKGFEVVVFEKDMSAIRGEGQYRGPIQIQSNALAALE Mtru    1 KKQLKVLVAGGGIGGLVFALAAKRKGFEVVVFEKDLSAIRGEGQYRGPIQIQSNALAALE Atha    1 KKKSRVLVAGGGIGGLVFALAAKKKGFDVLVFEKDLSAIRGEGKYRGPIQIQSNALAALE   Lses   61 AIDLKVADEVMRIGCITGDRINGLVDGISGSWYIKFDTFTPAAERGLPVTRVISRMALQE Ljap    6 ------------------------------------------------------------ Gmax   61 AIDLEVAEEVLRVGCITGDRINGLVDGISGSWYIKFDTFTPAAERGLPVTRVISRMALQE Mtru   61 AIDMNVADEVMRVGCITGDRINGLVDGVSGSWYIKFDTFTPAAERGLPVTRVISRMALQE Atha   61 AIDIEVAEQVMEAGCITGDRINGLVDGISGTWYVKFDTFTPAASRGLPVTRVISRMTLQQ   Lses  121 ILACAVGEDVILNGSNVVDFIDHGNKVTVELENGQKYDGDLLVGADGIWSKVRKKLFGES Ljap    6 ------------------------------------------------------------ Gmax  121 ILAHAVGEDVIMNDSNVVDFVDHGDKVTVELENGQKYDGDLLVGADGIWSKVRKKLFGQT Mtru  121 ILARAVGDDVIMNGSNVVDFIDHETKVTVVLDNGQKYDGDLLVGADGIWSKVRTKLFGST Atha  121 ILARAVGEDVIRNESNVVDFEDSGDKVTVVLENGQRYEGDLLVGADGIWSKVRNNLFGRS   Lses  181 EAIYSGYTCYTGIADFVPPDIESVGYRVFLGHKQYFVSSDVGAGKMQWYAFHQEPPGGVD Ljap    6 ------VSCK-------------------------------------------------- Gmax  181 EATYSGYTCYTGIADFVPADIESVGYRVFLGHKQYFVSSDVGAGKMQWYGFHQEPAGGAD Mtru  181 EATYSGYTCYTGIADFVPPDIESVGYRVFLGHKQYFVSSDVGAGKMQWYAFHQEPAGGVD Atha  181 EATYSGYTCYTGIADFIPADIESVGYRVFLGHKQYFVSSDVGGGKMQWYAFHEEPAGGAD   Lses  241 IPNGRKKRVLKIFKGWCDNVTELIVATEEAAILRRDIYDRTPTLTWGKGRVTLLGDSVHA Ljap   10 ------------------------------------------------------------ Gmax  241 IPNGKKERLLKIFKGWCDNVIDLIHATEEEAILRRDIYDRTPTFTWGKGHVTLLGDSIHA Mtru  241 TPNGKKERLLKIFEGWCDNAIDLIVATEEEAILRRDIYDRTPTLTWGKGRVTLLGDSVHA Atha  241 APNGMKKRLFEIFDGWCDNVLDLLHATEEEAILRRDIYDRSPGFTWGKGRVTLLGDSIHA   Lses  301 MQPNLGQGGGMAIEDGFQLALELDTAWQQSMKSGSSIDIDSSLKSYERERRLRVAVIHAM Ljap   10 ------------------------------------------------------------ Gmax  301 MQPNMGQGGCMAIEDSYQLALELDNAWQQSIKSGSPIDIDSSLKSYERERRLRVAIVHGM Mtru  301 MQPNMGQGGCMAIEDGYQLAFELDNAWQQSAKSGSTIDIASSLKSYERERRLRVTFVHGM Atha  301 MQPNMGQGGCMAIEDSFQLALELDEAWKQSVETTTPVDVVSSLKRYEESRRLRVAIIHAM   Lses  361 ARMAALMASTYKAYLGVGLGPLEFLTMFRVPHPGRVGGRYFIDKMMPLMLNWVLGGNSSK Ljap   10 ------------------------------------------------------------ Gmax  361 ARMAAMMASTYKAYLGVGLGPLEFLTKFRIPHPGRVGGRFFIDKMMPLMLNWVLGGNSSK Mtru  361 ARMAALMASTYKAYLGVGLGPFEFLTKFRIPHPGRVGGRFFIQKSMPLMLNWVLGGNSSK Atha  361 ARMAAIMASTYKAYLGVGLGPLSFLTKFRVPHPGRVGGRFFVDIAMPSMLDWVLGGNSEK   Lses  421 LEGRPLCCRLSDKANDQLRTWFEDDDALESAINGEWTLLPCGDETGHSEPIRISQNEMKP Ljap   10 ------------------------------------------------------------ Gmax  421 LEGRPVCCRLSDKANDQLHRWFEDNDALERAINGEWILLPCGDEAGPTKPICLTQDEMKP Mtru  421 LEGRPLCCRLSDKASDQLHTWFEDDDALERTINGEWILLPCGDVPGHVKPISLNQDDTKP 225    Atha  421 LQGRPPSCRLTDKADDRLREWFEDDDALERTIKGEWYLIPHGDDCCVSETLCLTKDEDQP   Lses  481 CIIGSGTTVTIPSPQVSPMHARINYKDGGFFLIDLRSEHGTWISDIEGKRYRVPPNYPAR Ljap   10 -----ALQLQYLCPRVSPMHARINYKDGGFFLIDLRSEHGTWISDIEGKRYRVPPNYPAR Gmax  481 CIIGSGSSIIIPLPQVSQMHARINYKDGAFFLTDLRSLHGTWITDNEGRRYRVPPNYPAR Mtru  481 YIIGN------------------------------------------------------- Atha  481 CIVGSGMRIVIPSSQVYKLYACSCDLQRRSFLLD--------------------------   Lses  541 IHPSDVLEFGSKK Ljap   65 IHPSDVLEFGSRK Gmax  541 VRPSDVVEFGSDK Mtru      ------------- Atha  515 ---------GSSK    226    Appendix 13  Supplementary Figures S48 – S54 Amino acid alignments for red-differentially expressed flavonoid genes in Lotus sessilifolius  Figure S48. Amino acid alignment for ANS  Lses    1 MAPTVVERVESLSTSGIQSIPKEYVRPKEELENIGNVFEEEKK-DGPQVPTIDLKEIDSP Ljap    1 MAPTVVERVESLSGSGIQSIPKEYVRPKEELANIGDVFEEEKK-VGPQVPTIDLKDIDSP Gmax    1 MG-TVAPRVESLASSGIKCIPKEYVRPEKELKSIGNVFEEEKK-EGPEVPTIDLREIDSE Mtru    1 ---------------------KEYVRPKEELANIGNIFDEEKK-EGPQVPTIDLKEINSS Atha    1 MV--AVERVESLAKSGIISIPKEYIRPKEELESINDVFLEEKKEDGPQVPTIDLKNIESD   Lses   60 DEFVRANCREKLRKAAEEWGVMHLVNHGIPDELLNQLKTAGAEFFSLPVEEKEKYANDQT Ljap   60 DEFVRAKCREKLRKAAEEWGVMHLVNHGIPDELLNQLKSAGAEFFSLPVEEKEKYANDQA Gmax   59 DEVVRGKCRQKLKKAAEEWGVMNLVNHGIQDELIERVKKAGEEFFGLAVEEKEKYANDQE Mtru   39 DEIVRGKCREKLKKAAEEWGVMHLVNHGISDDLINRLKKAGETFFELPVEEKEKYANDQS Atha   59 DEKIRENCIEELKKASLDWGVMHLINHGIPADLMERVKKAGEEFFSLSVEEKEKYANDQA   Lses  120 TGNVQGYGSKLANNASGQLEWEDYFFHLIFPEDKRDLSIWPKTPSYYTEVTSDYARRLRV Ljap  120 AGNVQGYGSKLANNASGQLEWEDYFFHLIFPEDKRDLSIWPKTPSYYTEVTSDYARRLRV Gmax  119 SGKIQGYGSKLANNASGQLEWEDYFFHLVFPEDKRDLSIWPKKPDDYIEVTSEYAKRLRG Mtru   99 SGKIQGYGSKLANNASGQLEWEDYFFHCIFPEDKRDLSIWPKTPADYTKVTSEYAKELRV Atha  119 TGKIQGYGSKLANNASGQLEWEDYFFHLAYPEEKRDLSIWPKTPSDYIEATSEYAKCLRL   Lses  180 LASKVLEVLSLELGLEEGRLEKEVGGMEELLLQMKINYYPKCPQPELALGVEAHTDVSSL Ljap  180 LASKILEVLSLELGLEEGRLEKEVGGMEELLLQMKINYYPKCPQPELALGVEAHTDISAL Gmax  179 LATKMLEALSIGLGLEGGRLEKEVGGMEELLLQLKINYYPICPQPELALGVEAHTDVSSL Mtru  159 LASKIMEVLSLELGLEGGRLEKEAGGMEELLLQMKINYYPICPQPELALGVEAHTDVSSL Atha  179 LATKVFKALSVGLGLEPDRLEKEVGGLEELLLQMKINYYPKCPQPELALGVEAHTDVSAL   Lses  240 TFLVHNMVPGLQLFYQDKWITAKCVPDSILMHIGDTIEILSNGKFKSILHRGLVNKEKVR Ljap  240 TFLLHNMVPGLQLFYEGKWVTAKCVPDSILMHIGDTTEILSNGKFKSILHRGLVNKEKVR Gmax  239 TFLLHNMVPGLQLFYQGQWFTAKCVPNSILMHIGDTIEILSNGKYKSILHRGLVNKEKVR Mtru  219 TFLLHNMVPGLQLFYEGKWVTAKCVPDSILMHIGDTIEILSNGKYKSILHRGLVNKEKVR Atha  239 TFILHNMVPGLQLFYEGKWVTAKCVPDSIVMHIGDTLEILSNGKYKSILHRGLVNKEKVR   Lses  300 ISWAVFCEPPKEKIILKPLPELVTETEPALFPPRTFAQHLHHKLFRKDQEAAAQ--SKX Ljap  300 ISWAVFCEPPKEKIILKPLPELVTETEPARFPPRTFAQHIHHKLFRKDQEASAQ--SKX Gmax  299 ISWAMFCEPPKEKIILQPLPELVTETEPARFPPRTFAQHIHHKLFRKDQEGLPN----X Mtru  279 ISWAVFCEPPKEKIILKPLPELVTEKEPARFPPRTFAQHIHHKLFRKDEEEKKDDPKKX Atha  299 ISWAVFCEPPKDKIVLKPLPEMVSVESPAKFPPRTFAQHIEHKLFGKEQEELVSEKNDX     227    Figure S49. Amino acid alignment for CHS-1  Lses    1 -------TVEEIRNAQRSHGPATILAFGTATPSHCVMQADYPDYYFRITNSEHMTDLKEK Ljap    1 MV-----TVEEIRNAQRSSGPATILAFGTATPSHCVMQADYPDYYFRITNSEHMTDLKEK Gmax    1 MV-----TVEEIRNAQRSHGPATILAFGTATPSNCVSQADYPDYYFRITNSEHMTDLKEK Mtru    1 MV-----TVEEIRKAQRSNGPATILAFGTATPSHCVTQAEYPDYYFRITNSEHMTDLKEK Atha    1 MVMAGASSLDEIRQAQRADGPAGILAIGTANPENHVLQAEYPDYYFRITNSEHMTDLKEK   Lses   54 FKRMCEKSMIRKRYMHLTEEILKENPNMCAYMAPSLDARQDLVVVEVPKLGKDAAAKAIK Ljap   56 FKRMCEKSMIRKRYMHLTEEILKENPAMCAYMAPSLDARQDLVVVEVPKLGKEAAAKAIK Gmax   56 FKRMCEKSMIKKRYMHLTEEFLKENPNMCEYMAPSLDVRQDVVVMEVPKLGKQAATKAIK Mtru   56 FKRMCEKSMIKKRYMHITEEFLKENPNMCAYMAPSLDARQDLVVVEVPKLGKDAAKKAIA Atha   61 FKRMCDKSTIRKRHMHLTEEFLKENPHMCAYMAPSLDTRQDIVVVEVPKLGKEAAVKAIK   Lses  114 EWGQPKSKITHLVFCTTSGVDMPGADYQLTKLLGLKPSVKRLMMYQQGCFAGGTVLRLAK Ljap  116 EWGQPKSKITHLVFCTTSGVDMPGADYQLTKLLGLKPSVKRLMMYQQGCFAGGTVLRLAK Gmax  116 EWGQPKSKITHLVFCTTSGVDMPGADYQLTKLLGLRPSVKRLMMYQQGCFAGGTVLRLAK Mtru  116 EWGQPKSKITHVVFCTTSGVDMPGADYQLTKLLGLKPSVKRLMMYQQGCFAGGTVLRLAK Atha  121 EWGQPKSKITHVVFCTTSGVDMPGADYQLTKLLGLRPSVKRLMMYQQGCFAGGTVLRIAK   Lses  174 DLAENNKGARVLVVCSEITAVTFRGPSDTHLDSLVGQALFGDGAAAMIVGSDPDLAV-ER Ljap  176 DLAENNKGARVLVVCSEITAVTFRGPSDTHLDSLVGQALFGDGAAAIIVGADPDLAV-ER Gmax  176 DLAENNKGARVLVVCSEITAVTFRGPSDTHLDSLVGQALFGDGAAALIIGSDPDPAV-ER Mtru  176 DLAENNKNARVLVVCSEITAVTFRGPSDTHLDSLVGQALFGDGAAAMIIGADPDLTV-ER Atha  181 DLAENNRGARVLVVCSEITAVTFRGPSDTHLDSLVGQALFSDGAAALIVGSDPDTSVGEK   Lses  233 PIFELVSAAQTILPDSDGAIDGHLREVGLTFHLLKDVPGIISKHIEKSLTEAFAPIGVSD Ljap  235 PIFQLVSAGQTILPDSDGAIDGHLREVGLTFHLLKDVPGIISKHIEKSLSEAFAPIGISD Gmax  235 PIFEMISAAQTILPDSDGAIDGHLREVGLTFHLLKDVPGIISKNIEKSLVEAFEPIGISD Mtru  235 PIFEIVSAAQTILPDSDGAIDGHLREVGLTFHLLKDVPGIISKNIEKSLVEAFAPIGISD Atha  241 PIFEMVSAAQTILPDSDGAIDGHLREVGLTFHLLKDVPGLISKNIVKSLDEAFKPLGISD   Lses  293 WNSLFWIAHPGGPAILDQVEVKLRLKEEKLRSTRHVLSEYGNMSSACVLFILDEVRRRSK Ljap  295 WNSLFWIAHPGGPAILDQVEAKLRLKEEKLRSTRHVLGEYGNMSSACVLFILDEVRRRSK Gmax  295 WNSIFWIAHPGGPAILDQVEEKLRLKPEKLQSTRHVLSEYGNMSSACVLFILDEMRKKSK Mtru  295 WNSIFWVAHPGGPAILDQVEEKLRLKEEKLRSTRHVLSEYGNMSSACVLFILDEMRKRSK Atha  301 WNSLFWIAHPGGPAILDQVEIKLGLKEEKMRATRHVLSEYGNMSSACVLFILDEMRRKSA   Lses  353 EEGKETTGEGLEWGVLFGFGPGLTVETVVLHSVP---- Ljap  355 EEGKETTGDGLEWGVLFGFGPGLTVETVVLHSVPLEAX Gmax  355 EEGKSTTGEGLEWGVLFGFGPGLTVETVVLHSVPLEGX Mtru  355 EEGKITTGEGLEWGVLFGFGPGLTVETVVLHSVPVQGX Atha  361 KDGVATTGEGLEWGVLFGFGPGLTVETVVLHSVPL--X     228    Figure S50. Amino acid alignment for CHS-6 Lses    1 MVSVAEIRKAQRAEGPATILAIGTANPPNCVDQSTYPDFYFRITNSEHMTELKEKFQRMC Ljap    1 MVSVAEIRKAQRAEGPATIFAIGTANPPNCVDQSTYPDFYFRVTNSQHKTELKEKFQRMC Gmax    1 MVSVAEIRQAQRAEGPATILAIGTANPPNCVDQSTYPDYYFRITNSEHMTELKEKFQRMC Mtru    1 MVSVSEIRKAQRAEGPATILAIGTANPANCVEQSTYPDFYFKITNSEHKTELKEKFQRMC Atha    1 MVMAGASSLDEIRQAQRADGPAGILAIGTANPENHVLQAEYPDYYFRITNSEHMTDLKEK   Lses   61 DKSMIKKRYMHLTEDLLKENPNMCA----------------------------------- Ljap   61 DKSMIKKRYMHLTEDLLKENPNMCAYMAPSLDARQDMVVVEVPRLGKEAAVKAIKEWGQP Gmax   61 DKSMIKRRYMYLNEEILKENPNMCAYMAPSLDARQDMVVVEVPKLGKEAAVKAIKEWGQP Mtru   61 DKSMIKRRYMYLTEEILKKNPSVCEYMAPSLDARQDMVVVEVPRLGKEAAVKAIKEWGQP Atha   61 FKRMCDKSTIRKRHMHLTEEFLKENPHMCAYMAPSLDTRQDIVVVEVPKLGKEAAVKAIK   Lses      ------------------------------------------------------------ Ljap  121 KSKITHLIFCTTSGVDMPGADYQLTKLLGLRPYVKRNKGALQVARCFVWPKTWLRTTKVL Gmax  121 KSKITHLIFCTTSGVDMPGADYQLTKQLGLRPYVKRYMMYQQGCFAGGTVLRLAKDLAEN Mtru  121 KSKITHLIVCTTSGVDMPGADYQLTKLLGLRPYVKRYMMYQQGCFAGGTVLRLAKDLAEN Atha  121 EWGQPKSKITHVVFCTTSGVDMPGADYQLTKLLGLRPSVKRLMMYQQGCFAGGTVLRIAK   Lses      ------------------------------------------------------------ Ljap  181 VCXMFVLNSLQLPFVALVTLTXTALLGKHCLEMEQLHSLLVPIQCLKLRNLCSSXYGLRK Gmax  181 NKGARVLVVCSEITAVTFRGPSDTHLDSLVGQALFGDGAAAVIVGSDPIPQVEKPLYELV Mtru  181 NKGARVLVVCSEVTAVTFRGPSDTHLDSLVGQALFGDGAAALIVGSDPIPEIERPIFEMV Atha  181 DLAENNRGARVLVVCSEITAVTFRGPSDTHLDSLVGQALFSDGAAALIVGSDPDTSVGEK   Lses      ------------------------------------------------------------ Ljap  241 LLLQIVKEPLMVISVKLDXPFISLKMFPGLFQRTLRKHXSRPSNRXAYLITTQYFGLHTQ Gmax  241 WTAQTIAPDSEGAIDGHLREVGLTFHLLKDVPGIVSKNIDKALFEAFNPLNISDYNSIFW Mtru  241 WTAQTIAPDSEGAIDGHLREAGLTFHLLKDVPGIVSKNITKALVEAFEPLGISDYNSIFW Atha  241 PIFEMVSAAQTILPDSDGAIDGHLREVGLTFHLLKDVPGLISKNIVKSLDEAFKPLGISD   Lses      ------------------------------------------------------------ Ljap  301 AAQQFLIRLSRTNSXSGQA----------------------------------------- Gmax  301 IAHPGGPAILDQVEQKLGLKPEKMKATRDVLSEYGNMSSACVLFILDEMRRKSAENGLKT Mtru  301 IAHPGGPAILDQVEQKLALKPEKMNATREVLSEYGNMSSACVLFILDEMRKKSTQNGLKT Atha  301 WNSLFWIAHPGGPAILDQVEIKLGLKEEKMRATRHVLSEYGNMSSACVLFILDEMRRKSA   Lses      ------------------------------------ Ljap      ------------------------------------ Gmax  361 TGEGLEWGVLFGFGPGLTIETVVLRSVAI------X Mtru  361 TGEGLEWGVLFGFGPGLTIETVVLRSVAI------X Atha  361 KDGVATTGEGLEWGVLFGFGPGLTVETVVLHSVPLX       229    Figure S51. Amino acid alignment for CHS-7 Lses    1 ------------------------------------------------------------ Ljap    1 MVSVAEIRKAQRAEGPATIFAIGTANPANCVDQSTYPDFYFRVTNSEHKTELKEKFQRMC Gmax    1 MVSVAEIRQAQRAEGPATILAIGTANPPNRVDQSTYPDYYFRITNSDHMTELKEKFQRMC Mtru    1 MVSVSEIRKAQRAEGPATIMAIGTANPANCVEQSTYPDFYFKITNSEHKVELKEKFQRMC   Lses    1 ------------------------------------------------------------ Ljap   61 DKSMIKKRYMHLTEDILKENPNMCAYMAPSLDARQDMVVVEVPRLGKEAAVKAIKEWGQP Gmax   61 DKSMIKTRYMYLNEEILKENPNMCAYMAPSLDARQDMVVVEVPKLGKEAAVKAIKEWGQP Mtru   61 DKSMIKRRYMYLTEEILKENPSVCEYMAPSLDARQDMVVVEVPRLGKEAAVKAIKEWGQP   Lses    1 ------------------------------------------------------------ Ljap  121 KSKITHLIFCTTSGVDMPGADYQLTKLLGLRPYVKRYMMYQQGCFAGGTVLRMAKDLAEN Gmax  121 KSKITHLIFCTTSGVDMPGADYQLTKQLGLRPYVKRYMMYQQGCFAGGTVLRLAKDLAEN Mtru  121 KSKITHLIFCTTSGVDMPGADYQLTKLLGLRPYVKRYMMYQQGCFAGGTVLRLAKDLAEN   Lses    1 ------------------------------------------------------------ Ljap  181 NKGARVLVVCSELTAVTFRGPSDTHLDSLVGQALFGDGAAALIVGSDPVPEIEKPLFELV Gmax  181 NKGARVLVVCSEITAVTFRGPSDTHLDSLVGQALFGDGAAAVIVGSDPIPQVEKPLYELV Mtru  181 NKGARVLVVCSEVTAVTFRGPSDTHLDSLVGQALFGDGAAALIVGSDPIPEIEKPIFEMV   Lses    1 -------------------------HLLKDVPGIVSKNIDKALVEAFQPLNISDYNSIFW Ljap  241 WTAQTIAPDSEGAIDGHLREVGLTFHLLKDVPGIVSKNIEKALIEAFQPLGISDYNSIFW Gmax  241 WTAQTIAPDSEGAIDGHLREVGLTFHLLKDVPGIVSKNIDKALFEAFNPLNISDYNSIFW Mtru  241 WTAQTIAPDSEGAIDGHLREAGLTFHLLKDVPGIVSKNIDKALVEAFQPLNISDYNSIFW   Lses   36 IAHPGGPAILDQVEQKLSLKPEKMRATREVLSEYGNMSSACVLFILDEMRKKSAQDGLKT Ljap  301 IAHPGGPAILDQVEQKLSLKPEKMRATREVLSEYGNMSSACVLFILDEMRKKSAQDGLKT Gmax  301 IAHPGGPAILDQVEQKLGLKPEKMKATRDVLSEYGNMSSACVLFILDEMRRKSAENGHKT Mtru  301 IAHPGGPAILDQVEEKLGLKPEKMKATREVLSEYGNMSSACVLFILDEMRKKSAQEGLKT   Lses   96 TGEGLEWGVLFGFGPGLTIETVVLRSVAIX Ljap  361 TGEGLEWGVLFGFGPGLTIETVVLRSVAIX Gmax  361 TGEGLEWGVLFGFGPGLTIETVVLHSVAIX Mtru  361 TGEGLDWGVLFGFGPGLTIETVVLHSVAIX        230    Figure S52. Amino acid alignment for DFR-2  Lses    1 -------ETVCVTGAAGFIGSWLVMRLIERGYTVRATVRDPANMKKVKHLLELPNAKTKL Ljap    1 MSS--ESETVCVTGAAGFIGSWLVMRLIERGYTVRATVRDPANMKKVKHLLELPDAKTKL Gmax    1 MGSSSASESVCVTGASGFIGSWLVMRLIERGYTVRATVRDPANMKKVKHLVELPGAKTKL Mtru    1 MGS--VSETVCVTGASGFIGSWLVMRLMERGYTVRATVRDPDNMKKVKHLLELPGANSKL Atha    1 MVS--QKETVCVTGASGFIGSWLVMRLLERGYFVRATVRDPGNLKKVQHLLDLPNAKTLL   Lses   54 TLWKADLAEEGSFDEAIKGCTGVFHVATPMDFESKDPENEVIKPTINGLLDILKACEKAK Ljap   59 SLWKADLAEEGSFDEAIRGCTGVFHVATPMDFESKDPENEVIKPTINGLLDILKACEKAK Gmax   61 SLWKADLAQEGSFDEAIKGCTGVFHVATPMDFDSKDPENEVIKPTINGLLDIMKACVKAK Mtru   59 SLWKADLGEEGSFDEAIKGCTGVFHVATPMDFESKDPEKEVINPTINGLLDIMKACKKAK Atha   59 TLWKADLSEEGSYDDAINGCDGVFHVATPMDFESKDPENEVIKPTVNGMLGIMKACVKAK   Lses  114 SVRRLVFTSSAGTVDVTEQQKPVIDETCWSDIEFCLRVKMTGWMYFVSKTRAEQEAWKYA Ljap  119 TVRRLVFTSSAGTVDVTEHPKPVIDETCWSDIEFCLRVKMTGWMYFVSKTRAEQEAWKYA Gmax  121 TVRRLVFTSSAGTVDVTEHPNPVIDENCWSDVDFCTRVKMTGWMYFVSKTLAEQEAWKYA Mtru  119 TVRRLVFTSSAGTLDVTEQQNSVIDETCWSDVEFCRRVKMTGWMYFVSKTLAEQEAWKFS Atha  119 TVRRFVFTSSAGTVNVEEHQKNVYDENDWSDLEFIMSKKMTGWMYFVSKTLAEKAAWDFA   Lses  174 KEHNIDFVSVIPPLVVGPFLMPTMPPSLITALSLITGNEAHYSIIKQGQYVHLDDLCLAH Ljap  179 KEHNIDFVSVIPPLVVGPFLMPTMPPSLITALSLITGNEAHYSIIKQGQYVHLDDLCLAH Gmax  181 KEHNIDFISVIPPLVVGPFLMPTMPPSLITALSLITGNESHYHIIKQGQFVHLDDLCLGH Mtru  179 KEHNIDFVSIIPPLVVGPFIMPSMPPSLITALSLITGYEAHYSIIKQGQYIHLDDLCLAH Atha  179 EEKGLDFISIIPTLVVGPFITTSMPPSLITALSPITRNEAHYSIIRQGQYVHLDDLCNAH   Lses  234 IFLFENPKAQGRYMCSAYEATIHEVARMINKKYPEFNVPTKFKDIPDELDIIKFSSKKIT Ljap  239 IFLFENPKAQGRYMCSAYEATIHEVARMINKKYPEFNVPTKFKDIPDELDIIKFSSKKIT Gmax  241 IFVFENPKAEGRYICCSHEATIHDIAKLLNQKYPEYNVLTKFKNIPDELDIIKFSSKKIT Mtru  239 IFLFENPKAHGRYICCSHEATIHEVAKLINKKYPEFNVPTKFKDIPDDLEIIKFSSKKIT Atha  239 IFLYEQAAAKGRYICSSHDATILTISKFLRPKYPEYNVPSTFEGVDENLKSIEFSSKKLT   Lses  294 DLGFKFKYSLEDMYTGAIETCREKGLLPKTS----------------------------- Ljap  299 DLGFKFKYSLEDMYTGAVETCREKGLLPKTA----------------------------- Gmax  301 DLGFKFKYSLEDMFTGAVETCREKGLLPKPE----------------------------- Mtru  299 DLGFIFKYSLEDMFTGAIETCREKGLLPKVT----------------------------- Atha  299 DMGFNFKYSLEEMFIESIETCRQKGFLPVSLSYQSISEIKTKNENIDVKTGDGLTDGMKP   Lses  325 ----ETPVTN------------GTTQKX Ljap  330 ----ETPATN------------GTTQKX Gmax  332 ----ETTVNNELLPKPAETTVNDTMQKX Mtru  330 ----ETPV-N------------DTMKKX Atha  359 CNKTETGITGERTDAP---MLAQQMCAX     231    Figure S53. Amino acid alignment for F3H  Lses    1 --------LTTLAQQNTLESSFVRDEDERPKVAYNNFSNEIPVISLAGIDEVDGRRSEIC Ljap    1 MASFKPKTLTTLAQQNTLESSFVRDEDERPKVAYNNFSNEIPVISLAGIDEVDGRRSEIC Gmax    1 MAPT-AKTLTYLAQEKTLESSFVRDEEERPKVAYNEFSDEIPVISLAGIDEVDGRRREIC Mtru    1 MAP--AQTLTYLAQEKTLESSFVREEDERPKVAYNNFSNEIPIISLDGIDDAGGRRAEIC Atha    1 MAP---GTLTELAGESKLNSKFVRDEDERPKVAYNVFSDEIPVISLAGIDDVDGKRGEIC   Lses   53 NKIVEACENWGIFQVVNHGVDTELVSHMTTLAKEFFALPPEEKLRFDMSGGKKGGFIVSS Ljap   61 NKIVEACENWGIFQVVDHGVDTELVSHMTTLAKEFFALPPEEKLRFDMTGGKKGGFIVSS Gmax   60 EKIVEACENWGIFQVVDHGVDQQLVAEMTRLAKEFFALPPDEKLRFDMSGAKKGGFIVSS Mtru   59 NKIVEACENWGIFQVVDHGVDSKLISEMTRFAKGFFDLPPEEKLRFDMSGGKKGGFIVSS Atha   58 RQIVEACENWGIFQVVDHGVDTNLVADMTRLARDFFALPPEDKLRFDMSGGKKGGFIVSS   Lses  113 HLQGESVQDWREIVTYFSYPIRQRDYSRWPDTPAGWKAVTEEYSEKLMGLACKLLEVLSE Ljap  121 HLQGESVQDWREIVTYFSYPIRNRDYSRWPDTPAGWKAVTEEYSEKLMGLACKLLEVLSE Gmax  120 HLQGESVQDWREIVTYFSYPKRERDYSRWPDTPEGWRSVTEEYSDKVMGLACKLMEVLSE Mtru  119 HLQGEAVKDWRELVTYFSYPIRQRDYSRWPDKPEGWKEVTEQYSEKLMNLACKLLEVLSE Atha  118 HLQGEAVQDWREIVTYFSYPVRNRDYSRWPDKPEGWVKVTEEYSERLMSLACKLLEVLSE   Lses  173 AMGLEKEALTKACVDMDQKVVVNYYPKCPQPDLTLGLKRHTDPGTITLLLQDQVGGLQAT Ljap  181 AMGLEKEALTKACVDMDQKVVVNYYPKCPQPDLTLGLKRHTDPGTITLLLQDQVGGLQAT Gmax  180 AMGLEKEGLSKACVDMDQKVVVNYYPKCPQPDLTLGLKRHTDPGTITLLLQDQVGGLQAT Mtru  179 AMGLEKDALTKACVDMDQKVVINYYPKCPQPDLTLGLKRHTDPGTITLLLQDQVGGLQAT Atha  178 AMGLEKESLTNACVDMDQKIVVNYYPKCPQPDLTLGLKRHTDPGTITLLLQDQVGGLQAT   Lses  233 RDNGKTWITVQPVEGAFVVNLGDHGHYLSNGRFKNADHQAVVNSNSSRLSIATFQNPAPD Ljap  241 RDNGKTWITVQPVEGAFVVNLGDHGHYLSNGRFKNADHQAVVNSNSSRLSIATFQNPAPD Gmax  240 RDNGKTWITVQPVEAAFVVNLGDHAHYLSNGRFKNADHQAVVNSNHSRLSIATFQNPAPN Mtru  239 KDNGKTWITVQPVEGAFVVNLGDHGHYLSNGRFKNADHQAVVNSNYSRLSIATFQNPAPD Atha  238 RDNGKTWITVQPVEGAFVVNLGDHGHFLSNGRFKNADHQAVVNSNSSRLSIATFQNPAPD   Lses  293 ATVYPLKVREGEKSVLDEPITFAEMYRRKMSKDIELARMKKLAKEK-KLQDLE------- Ljap  301 ATVYPLKVREGEKSVMEEPITFAEMYRRKMSKDIELARMKKLAKEK-KLQDLE------- Gmax  300 ATVYPLKIREGEKPVMEEPITFAEMYRRKMSKDIEIARMKKLAKEK-HLQDLENEKHLQE Mtru  299 ATVYPLKIRDGEKSVMEEPITFAEMYRRKMSKDLEIARMKKLAKEEKELRDLE------- Atha  298 ATVYPLKVREGEKAILEEPITFAEMYKRKMGRDLELARLKKLAKEE-RD-----------   Lses  345 ---KAKLEPKPMNEIFA- Ljap  353 ---KAKLEPKPMNEIFAX Gmax  359 LDQKAKLEAKPLKEILAX Mtru  352 ---KAKIEAKPLNEILAX Atha  346 ----HKEVDKPVDQIFAX     232    Figure S54. Amino acid alignment for F3’H  Lses    1 MFQWMIIGFATITVIIFIHRVVKFAT-RP-SLPLPPGPKPWPIIGNLPHMGPVPHHSLAA Ljap    1 MFPWMIIGFATITFLIFIHRVVKFST-RP-SLPLPPGPKPWPIIGNFPHMGPVPHHSLAA Gmax    1 MSPLI-VALATIAAAILIYRIIKFIT-RP-SLPLPPGPKPWPIVGNLPHMGPVPHHSLAA Mtru    1 MSLWF-IAIASFTLCILIYRFMKFAK-RSSSLPLPPGPKPWPIIGNMPHLGPAPHQSIAA Atha    1 MATLF-LTILLATVLFLILRIFSHRRNRSHNNRLPPGPNPWPIIGNLPHMGTKPHRTLSA   Lses   59 LAQTHGPLMHLKLGFVDVVVAASATVAEQFLKVHDANFSSRPPNAGAKYIAYNYQDLVFA Ljap   59 LARAHGPLMHLKLGFVDVVVAASAAVAEQFLKVHDANFSSRPPNAGAKYIAYNYQDLVFA Gmax   58 LARIHGPLMHLRLGFVDVVVAASASVAEQFLKIHDSNFSSRPPNAGAKYIAYNYQDLVFA Mtru   59 LAKIHGPLMHLKLGFVDVIVAASGSVAEQFLKVHDANFSSRPPNTGAKYIAYNYQDLVFA Atha   60 MVTTYGPILHLRLGFVDVVVAASKSVAEQFLKIHDANFASRPPNSGAKHMAYNYQDLVFA   Lses  119 PYGARWRYLRKITSLHLFSGKALDNFKHLRQEEVSRLTRNISK--SNSKVVNLGQLLNVC Ljap  119 PYGARWRYLRKITNLHLFSGKALDNFKHLRQEEVSRLTRNISK--SNSKVVNLGQLLNVC Gmax  118 PYGPRWRLLRKLTSVHLFSGKAMNEFRHLRQEEVARLTCNLAS--SDTKAVNLGQLLNVC Mtru  119 PYGPRWRMLRKISSVHLFSNKVMEEFKHLRQEEVARLTSNLASNYSDTKAVNLGQLLNVC Atha  120 PYGHRWRLLRKISSVHLFSAKALEDFKHVRQEEVGTLTRELVR--VGTKPVNLGQLVNMC   Lses  177 TTNALSRVMIGRRVFNDGDGGCDPRADEFKAMVVELMVLAGVFNIGDFIPSLEWLDLQGV Ljap  177 TTNALSRVMIGRRVFNDGDGGCDPRADEFKAMVVELMVLAGVFNIGDFIPSLEWLDLQGV Gmax  176 TTNALARAMIGRRVFNDGNGGCDPRADEFKAMVMEVMVLAGVFNIGDFIPSLEWLDLQGV Mtru  179 TTNALARVMLGRRVFNDGNGGCDPKADEFKEMVLELMVLAGVFNISDFIPSLEWLDLQGV Atha  178 VVNALGREMIGRRLFGA---DADHKADEFRSMVTEMMALAGVFNIGDFVPSLDWLDLQGV   Lses  237 QAKMKNLHNRFDEFLTSIIEEHDT-SSKSENHKDLLSTLLSLKEV-ADDDGNKLNDIEIK Ljap  237 QAKMKKLHNRFDEFLTSIIEEHNT-SSKSENHKDLLSTLLSLKDV-PDDDGNKLNDIEIK Gmax  236 QAKMKKLHKRFDAFLTSIIEEHNNSSSKNENHKNFLSILLSLKDV-RDDHGNHLTDTEIK Mtru  239 QAKMKKLHKKFDAFLTNIIDERENSNFKSEKHKDLLSTLLLLKEE-TDVDGNKLTYIEIK Atha  235 AGKMKRLHKRFDAFLSSILKEHEM-NGQDQKHTDMLSTLISLKGTDLDGDGGSLTDTEIK   Lses  295 ALLLNMFTAGTDTSASTTEWAIAELIRNPKILAQVQQELDTVVGRDRNVREDDLPHLPYL Ljap  295 ALLLNMFTAGTDTSASTTEWVIAELIRSPRILAQVQQELDTVVGRERNVREDDLPHLPYL Gmax  295 ALLLNMFTAGTDTSSSTTEWAIAELIKNPQILAKLQQELDTVVGRDRSVKEEDLAHLPYL Mtru  298 ALLLNMFAAGTDTSSSTTEWAIAELIRNPRILAQVQQELDNVVGRDRNVKEDDIPNLPYL Atha  294 ALLLNMFTAGTDTSASTVDWAIAELIRHPDIMVKAQEELDIVVGRDRPVNESDIAQLPYL   Lses  355 QAVVKETFRLHPSTPLSLPRVASESCEVLGYHIPKGSTLLVNVWAIARDPKEWADPLEFK Ljap  355 QAVVKETFRLHPSTPLSLPRVASESCEVLGYHIPKGSTLLVNVWAIARDPKEWAEPLEFK Gmax  355 QAVIKETFRLHPSTPLSVPRAAAESCEIFGYHIPKGATLLVNIWAIARDPKEWNDPLEFR Mtru  358 QAVIKETFRLHPSTPLSLPRIASESCEIFGYHIPKGSTLLVNVWAIARDPKEWVDPLEFK Atha  354 QAVIKENFRLHPPTPLSLPHIASESCEINGYHIPKGSTLLTNIWAIARDPDQWSDPLAFK   Lses  415 PERFLQGGGDKVHVDVKGNDFEVIPFGAGRRICAGMSLGLRMVQLLTATLAHSFNWELEN Ljap  415 PERFLE--GDKVDVDVKGNDFKVIPFGAGRRICAGMSLGLRMVQLLTATLVHSFNWELEN Gmax  415 PERFLLG-GEKADVDVRGNDFEVIPFGAGRRICAGLSLGLQMVQLLTAALAHSFDWELED Mtru  418 PERFLPG-GEKCDVDVKGNDFEVIPFGAGRRICPGMSLGLRMVQLLTATLAHSFDWELEN 233    Atha  414 PERFLPG-GEKSGVDVKGSDFELIPFGAGRRICAGLSLGLRTIQFLTATLVQGFDWELAG   Lses  475 GLNHEKLNMDEAYGLTLQRAVPLSVYSRPRLSPHV------- Ljap  473 GLNHEKLNMDEAYGLTLQRAVPLSVYSRPRLSPHVYAAS-HX Gmax  474 CMNPEKLNMDEAYGLTLQRAVPLSVHPRPRLAPHVYSMS-SX Mtru  477 GLNAGKMNMDEGYGLTLQRAVPLSVHPKPRLSPHVYSSC-FX Atha  473 GVTPEKLNMEESYGLTLQRAVPLVVHPKPRLAPNVYGLGSGX     234    Appendix 14  Supplementary Figures S55 Amino acid alignments for yellow-differentially expressed flavonoid genes in Lotus sessilifolius  Figure S55. Amino acid alignment for FLS  Lses    1 -EVLRVQTVATQSKD-A--SIPSMFVRPKTEQPGLTTVRGVELEVPIIDLSGSDEEKVLR Ljap    1 MEVLRVQSVAAQSKD-A--SIPAMFVRSETEQPGITTVRGVELEVPIIDLNGTDEVKVLS Gmax    1 MEVLRVQTIASKSKD-A--AIPAMFVRAETEQPGITTVQGVNLEVPIIDFSDPDEGKVVH Mtru    1 MEVLRVQTIAHQSKD-ATSTIPSMFLRSETESPGTTTVQGVKLEVPIIDFNNPDEGKIQN Atha    1 MEVERVQDISSSSLLTE--AIPLEFIRSEKEQPAITTFRGPTPAIPVVDLSDPDEESVRR   Lses   57 EIVEASKEWGMFQVVNHEIPVEVIAKLQSVGKHFFELPQGEKEVCGKIDGSDSVEGYGTK Ljap   58 EIVEASKEWGMFQVVNHEIPSEVIAKLQAVGKEFFELPQEEKEVYGKIEGSDSLEGYGTK Gmax   58 EILEASRDWGMFQIVNHDIPSDVIRKLQSVGKMFFELPQEEKELIAKPAGSDSIEGYGTK Mtru   60 EIMEASTKWGMFQIVNHDIPNEVIKKLQSVGKEFFELPQEEKEVIAKPIGSDSLEGYGTK Atha   59 AVVKASEEWGLFQVVNHGIPTELIRRLQDVGRKFFELPSSEKESVAKPEDSKDIEGYGTK   Lses  117 LQKEVNGKKGWVDHLFHIIWPTSAINYRFWPKNPPSYREVNEEYGKYLRSVGDKLFKSLS Ljap  118 LQKEVNGKKGWVDHLFHIIWPTSSINYRFWPKNPASYREVNEEYGKYLRSVADKLFKSMS Gmax  118 LQKEVNGKKGWVDHLFHIVWPPSSINYSFWPQNPPSYREVNEEYCKHLRGVVDKLFKSMS Mtru  120 LEKEVNGKKGWVDHLFHIIWPPSSINYRFWPNNPASYREVNEEYGKYLREVADKLFKSLS Atha  119 LQKDPEGKKAWVDHLFHRIWPPSCVNYRFWPKNPPEYREVNEEYAVHVKKLSETLLGILS   Lses  177 IGLGLEENELKEAAGGDDMIHLLKINYYPPCPCPELVLGVPPHTDMSFMTILVPNEVQGL Ljap  178 IGLGLEENELKEAAGGDDMIHLLKINYYPPCPCPDLVLGVPPHTDMSFVTILVPNEVQGL Gmax  178 VGLGLEENELKEGANEDDMHYLLKINYYPPCPCPDLVLGVPPHTDMSYLTILVPNEVQGL Mtru  180 IGLELEEHELKKAAGGDELIHMLKINYYPPCPVPDLVLGVPPHTDMSFLTILVPNEVQGL Atha  179 DGLGLKRDALKEGLGGEMAEYMMKINYYPPCPRPDLALGVPAHTDLSGITLLVPNEVPGL   Lses  237 QAFRDGHWYDVKYVPNALVIHIGDQMEIVSNGKYKAVLHRTTVNKEETRMSWPVFIEPQG Ljap  238 QAFRDGHWYDVKYVPNALVIHIGDQMEILSNGKYKAVLHRTTVNKEETRMSWPVFIEPQG Gmax  238 QACRDGHWYDVKYVPNALVIHIGDQMEILSNGKYKAVFHRTTVNKDETRMSWPVFIEPKK Mtru  240 QASRDGQWYDVKYVPNALVIHIGDQMEILSNGKYKAVLHRTTVNKDETRMSWPVFIEPKP Atha  239 QVFKDDHWFDAEYIPSAVIVHIGDQILRLSNGRYKNVLHRTTVDKEKTRMSWPVFLEPPR   Lses  297 HHEVGPHSKLVNHDNPPKYKTKKYKDYAYCKLNKIPQ-X Ljap  298 DHEVGPHSKLVNQDNPPKYKTKKYKDYAYCKLNKIPQ-X Gmax  298 EQEVGPHPKLVNQDNPPKYKTKKYKDYAYCKLNKIPQ-X Mtru  300 EHEIGPHPKLVNQENPPKYKTKKFEDYLYCKLNKIPQ-X Atha  299 EKIVGPLPELTGDDNPPKFKPFAFKDYSYRKLNKLPLDX     235    Appendix 15  Supplementary Figures S56 – S57 Amino acid alignments for non-differentially expressed lignin genes in Lotus sessilifolius  Figure S56. Amino acid alignment for CAD  Lses    1 MGSLE-AERTTVGWAARDPSGILSPYTFTLRNTGPDDVYIKVHYCGICHSDIHQVKNDLG Ljap    1 MGSLE-AERTTVGWAARDPSGILSPYTFTLRNTGPDDVYIKVHYCGVCHTDVHQVKNDLG Gmax    1 MGSLE-AERTTVGLAARDPSGILSPYTYNLRNTGPDDVYIKVHYCGICHSDLHQIKNDLG Mtru    1 MGSIEVAERTTVGLAAKDPSGILTPYTYTLRNTGPDDVYIKIHYCGVCHSDLHQIKNDLG Atha    1 MGIME-AERKTTGWAARDPSGILSPYTYTLRETGPEDVNIRIICCGICHTDLHQTKNDLG   Lses   60 MSNYPMVPGHEVVGEVLEVGSDVTRFRAGEIVGAGLLVGCCKNCTACQSDIEQYCSKKIW Ljap   60 MSNYPMVPGHEVVGEVLEVGSDVTRFTVGEIVGAGLLVGCCKKCTACQSDIEQYCKKKIW Gmax   60 MSNYPMVPGHEVVGEVLEVGSDVSRFRVGELVGVGLLVGCCKNCQPCQQDIENYCSKKIW Mtru   61 MSNYPMVPGHEVVGEVLEVGSNVTRFKVGEIVGVGLLVGCCKSCRACDSEIEQYCNKKIW Atha   60 MSNYPMVPGHEVVGEVVEVGSDVSKFTVGDIVGVGCLVGCCGGCSPCERDLEQYCPKKIW   Lses  120 NYNDVYVDGKPTQGGFAETIIVEQKFVVKIPEGMAPEQVAPLLCAGVTVYSPLAHFGLKE Ljap  120 NYNDVYVDGKPTQGGFAETIIVEQKFVVKIPEGMAPEQVAPLLCAAVTVYSPLSHFGLKE Gmax  120 SYNDVYVDGKPTQGGFAETMVVEQKFVVKIPEGLAPEQVAPLLCAGVTVYSPLVHFGLKE Mtru  121 SYNDVYTDGKITQGGFAESTVVEQKFVVKIPEGLAPEQVAPLLCAGVTVYSPLSHFGLKT Atha  120 SYNDVYINGQPTQGGFAKATVVHQKFVVKIPEGMAVEQAAPLLCAGVTVYSPLSHFGLKQ   Lses  180 SGLRGGILGLGGVGHMGVKIAKALGHHVTVISSSDRKKKEAIEDLGVDAYLVSSDTTSMQ Ljap  180 SGLRGGILGLGGVGHMGVIIAKAMGHHVTVISSSDRKKKEAIEDLGADAYLVSSDTTSMQ Gmax  180 SGLRGGILGLGGVGHMGVKIAKALGHHVTVISSSDKKKQEALEHLGADQYLVSSDVTAMQ Mtru  181 PGLRGGILGLGGVGHMGVKVAKAFGHHVTVISSSDKKKKEALEDLGADSYLVSSDTVGMQ Atha  180 PGLRGGILGLGGVGHMGVKIAKAMGHHVTVISSSNKKREEALQDLGADDYVIGSDQAKMS   Lses  240 GAADSLDYIIDTVPVGHPLEPYLSLLKLDGKLILMGVINTPLQFITPMVMLGRRSITGSF Ljap  240 EAADSLDYIIDTVPVGHPLEPYLSLLKLDGKLILMGVINTPLQFITPMVMLGRRSITGSF Gmax  240 EAADSLDYIIDTVPVGHPLEPYLSLLKLDGKLILMGVINTPLQFVSPMVMLGRRSITGSF Mtru  241 EAADSLDYIIDTVPVGHPLEPYLSLLKIDGKLILMGVINTPLQFVTPMVMLGRKSITGSF Atha  240 ELADSLDYVIDTVPVHHALEPYLSLLKLDGKLILMGVINNPLQFLTPLLMLGEKVITGSF   Lses  300 IGSIKETEEMLEFWKEKGLSSMIEVVNMDYINKAFERLEKNDVRYRFVVDVKGS------ Ljap  300 IGSIKETEEMLEFWKEKGLTSMIEIVNMDYINKAFERLEKNDVRYRFVVDVKGSKLIDQX Gmax  300 IGSMKETEEMLEFWKEKGLSSMIEVVNMDYINKAFERLEKNDVRYRFVVDVKGSKLVD-X Mtru  301 VGSVKETEEMLEFWKEKGLSSMIEIVTMDYINKAFERLEKNDVRYRFVVDVKGSKFED-X Atha  300 IGSMKETEEMLEFCKEKGLSSIIEVVKMDYVNTAFERLEKNDVRYRFVVDVEGSNLDA-X     236    Figure S57. Amino acid alignment for CCR-3 Lses    1 -----------KVCVTGAGGFVASWLVKLLLSKGYTVHGTVRQP--GSPKYEHLLKLEKA Ljap    1 ------------------------------------------------------------ Gmax    1 MAA-------KKVCVTGAGGFVASWLVKLLLSKGYIVHGTVRDPEPATQKYEHLLKLHGA Mtru    1 MEASGGVNNNKKVCVTGAGGFVASWLVKLLLSKGYFVHGTVREP--GSPKYEHLLKLEKA Atha    1 MAD----VHKGKVCVTGAGGFLGSWVVDLLLSKDYFVHGTVRDP--DNEKYAHLKKLEKA   Lses   48 SENLTLFKADLLNYESVHSAILGCTAVFHVASPVPSTISSNPQVEVIEPAVKGTANVLEA Ljap    1 -------------------------NVFHVF----------------------------- Gmax   54 SENLTLFKADLLNYESLRSAISGCTAVFHLACPVPSISVPNPQVEMIEPAVKGTTNVLEA Mtru   59 SENLTLFKADILDYESVYSAIVGCSAVFHVASPVPSTVVPNPEVEVIEPAVKGTANVLEA Atha   55 GDKLKLFKADLLDYGSLQSAIAGCSGVFHVACPVPPASVPNPEVELIAPAVDGTLNVLKA   Lses  108 SLQAKVERVVFVSSEAAICMNPNLPKDKLIDESYWSDKDYCKKTQNWYCYSKTEAEEQAL Ljap    7 ------------------------------------------------------------ Gmax  114 SLEAKVQRLVFVSSLAAISNSPNLPKDKVIDESYWSDKDYCKTTQNWYCFSKTEAEEQAL Mtru  119 CLKANVERVVFVSSVAAVAINPNLPKDKAIDESCWSDKDYCKNTKNWYCYAKTEAEEQAL Atha  115 CIEANVKRVVYVSSVAAAFMNPMWSKNQVLDEACWSDQEYCKKTENWYCLAKTRAESEAF   Lses  168 DFAKRTGLSVVSICPTTVLGPILQS-TVNASSLILLKLLKAEGCDSMENKLNWIVDVRDL Ljap    7 -------------------------------GLVFLDCLKLKVLYSMENRLSWIVDVGDL Gmax  174 DFAKRTGLDVVSICPSLVLGPILQSTTVNASSLALLKLLK--GVNSMENKIRWIVDVRDV Mtru  179 HFAKRTGLNVVTICPTLVLGPILQS-TTNASSLVLVKLLK-EGCDSVENKLRWIVDVRDV Atha  175 EFAKRTGLHLVSVCPTLVLGPILQQNTVNASSLVLLKLLK-EGFETRDNQERHLVDVRDV   Lses  227 VDAVLLAYEKLEAEGRYICTSHPIKKRDLVEKLKSIYPNYKYPTKFTEVDGYRRLSSEKL Ljap   36 VDAVLLAYEKLEAEGRYICTSHPIKKRDLVEKLKSIYPYYKYPTKFTEVDSYRRLTSEKL Gmax  232 ADAILLAYEKLEAEGRYICHSHTIKTRDMLEKLKSIYPNYKYPAKYTEVDDYISFSSEKL Mtru  237 VNAILLAYENHEADGRYICTSHAIVTRDLVERLKGIYPNYKYPTNYIEMDDYKMLSSEKL Atha  234 AQALLLVYEKAEAEGRYICTSHTVKEEIVVEKLKSFYPHYNYPKKYIDAEDRVKVSSEKL   Lses  287 QRLGWKSRPLEETLIDSVESYREAGLLQSEX Ljap   96 QRLGWKSRPLEETLIDSVESYREAGLLQSE- Gmax  292 QRLGWKYRSLEETLVDSVESYREAGHLQSEX Mtru  297 QSLGWKLRPLEETLIDSVESYKEAGLLQSQX Atha  294 QKLGWTYRPLEETLVDSVESYRKAKLVD--X         237    Appendix 16  Supplementary Figures S58 – S60 Amino acid alignments for yellow-differentially expressed lignin genes in Lotus sessilifolius  Figure S58. Amino acid alignment for CAD  Lses    1 RRKRMS-NNAGKLVCVTGGSGYIASWIVKFLLEHGYTVRATVRDPSNPKKVEHLLKLDGA Ljap    1 ----MS-NGAGKLVCVTGGSGYIASWIVKFLLEHGYTVRATVRDPNNPNKVEHLLKLDGA Gmax    1 ----MSNNNAGKVVCVTGASGFIASWIVKFLLQRGYTVRATVRYPSNLKKVDHLVKLEGA Mtru    1 ----MS--GEGKVVCVTGASGFIASWIVKFLLQRGYTVRATVRDPSNPKKVDHLLKLDGA   Lses   60 KERLHLFKADLLEEGSFDSVVDGCDGVFHTASPVGLIVKDPQAELMDPAVKGTLNV---- Ljap   56 KERLHLFKADLLEEGSFDSAI---------------------AELIDPAVKGTLNVLKSC Gmax   57 KERLQLFKADLLEEGSFDSVVEGCHGVFHTASPVRFVVNDPQAELLDPAVKGTLNVLKSC Mtru   55 KERLQLFKADLLEEGSFDSVVEGCDGVFHTASPVRFVVNDPQVELIDPALKGTLNVLKSC   Lses      ------------------------------------------------------------ Ljap   95 AKSPSVRRVVLTSSVGAVQYNERPKTPEVLIDETWFSDPDFCRKSKVCASFSXPLFRNXS Gmax  117 AKSPSVKRVVLTSSISAVAFNRRPKTPQVVVDETWFSDPDVCRELELWYTLSKTLAEDAA Mtru  115 AKSTSVKRVVLTSSNAAVSFNTRPKNPEVVVDETWFSNPDFCRESKLWYVLSKTLAEAAA   Lses      ------------------------------------------------------------ Ljap  155 -------------------------------LE--------------------------- Gmax  177 WKFVNENSIDMISINPTMVAGPLLQPEINESVEPILNLINGKPFPNKSFGWVDVKDVANA Mtru  175 WKFVNENNIDMVSLNPTMVAGPLLQPEVNESVEPILNLINGIPFPNKAIGWVNVKDVANA   Lses      ------------------------------------------------------------ Ljap  157 -------AHADGRC---------------------------------------------- Gmax  237 HILAYEIASASGRYCLVERVIHYSELATILRGLYPTLQIPDKCEVDEPYIPTYQISTEKA Mtru  235 HIHAYEIASASGRCLLAERVVHYSELAMILRDLYPTLPISDKQSTSSSKFVIL-LAYTTL   Lses      ---------------------------------- Ljap  164 -------------------------------DSD Gmax  297 KKDLGIEFTPLEVSLRETVESFREKKIVNF-NPX Mtru  294 FHQIGAEPTCIQGMQLNPLNPLIYSYYFGFLPCX        238    Figure S59. Amino acid alignment for CCoA-OMT  Lses    1 MATN------------EDQKQTEAGRHQEVGHKSLLQSDALYQYILETSVYPREHEAMKE Ljap    1 MATN------------EDQKQTEAGRHQEVGHKSLLQSDALYQYILETSVYPREHEAMKE Gmax    1 MAEQ------------NQNQTTEAGRHQEVGHKSLLQSDALYQYILETSVYPREPESMKE Mtru    1 MATN------------EDQNQTESGRHQEVGHKSLLQSDALYQYILETSVFPREHEAMKE Atha    1 MATTTTEATKTSSTNGEDQKQSQNLRHQEVGHKSLLQSDDLYQYILETSVYPREPESMKE   Lses   49 LRDLTAKHPWNIMTTSADEGQFLNMLLKLINAKNTMEIGVYTGYSLLATALAIPDDGKIL Ljap   49 LRDLTAKHPWNIMTTSADEGQFLNMLLKLINAKNTMEIGVYTGYSLLATALAIPDDGKIL Gmax   49 LRELTAKHPWNIMTTSADEGQFLNMLLKLINAKNTMEIGVYTGYSLLATALALPEDGKIL Mtru   49 LREVTAKHPWNIMTTSADEGQFLSMLLKLINAKNTMEIGVYTGYSLLATALAIPEDGKIL Atha   61 LREVTAKHPWNIMTTSADEGQFLNMLIKLVNAKNTMEIGVYTGYSLLATALALPEDGKIL   Lses  109 AMDVNKENYELGLPVIKKAGVAHKIDFREGPALPVLDEMVKDEKNHASYDFIFVDADKDN Ljap  109 AMDVNKENYELGLPVIKKAGVAHKIDFREGPALPVLDEMVKDEKNHGSYDFIFVDADKDN Gmax  109 AMDINRENYELGLPVIKKAGVDHKIEFREGPALPVLDEMIKDEKNHGSYDFIFVDADKDN Mtru  109 AMDINKENYELGLPVIKKAGVDHKIDFREGPALPVLDEMIKDEKNHGSYDFIFVDADKDN Atha  121 AMDVNRENYELGLPIIEKAGVAHKIDFREGPALPVLDEIVADEKNHGTYDFIFVDADKDN   Lses  169 YLNYHKRLIELVKVGGVIGYDNTLWNGSVVAPPDAPLRKYVRYYRDFVLELNKALAADPR Ljap  169 YLNYHKRLIELVKVGGVIGYDNTLWNGSVVAPPDAPLRKYVRYYRDFVLELNKALAVDPR Gmax  169 YLNYHKRLIELVKVGGVIGYDNTLWNGSVVAPPDAPLRKYVRYYRDFVLELNKALAVDPR Mtru  169 YLNYHKRLIDLVKVGGVIGYDNTLWNGSVVAPPDAPLRKYVRYYRDFVLELNKALAVDPR Atha  181 YINYHKRLIDLVKIGGVIGYDNTLWNGSVVAPPDAPMRKYVRYYRDFVLELNKALAADPR   Lses  229 IEICMLPVGDGITICRRIKX Ljap  229 IEICMLPVGDGITICRRIKX Gmax  229 IEICMLPVGDGITICRRIKX Mtru  229 IEICMLPVGDGITICRRIKX Atha  241 IEICMLPVGDGITICRRISX    239    Figure S60. Amino acid alignment for OMT Lses    1 MGSTGETQITPTHVTDEEANLFAMQLASASVLPMVLKSAIELDLLEIIAKAGTGAQISPA Ljap    1 MGSTGETQITPTHVSDEEANLFAMQLASASVLPMVLKSAIELDLLEFIAKAGTGAQLSPA Gmax    1 MGSTGETQITPTHVSDEEANLFAMQLASASVLPMVLKSALELDLLEIIAKAGPGVHLSPS Mtru    1 MGSTGETQITPTHISDEEANLFAMQLASASVLPMVLKSALELDLLEIIAKAGPGAQISPI Atha    1 MGSTAETQLTPVQVTDDEAALFAMQLASASVLPMALKSALELDLLEIMAKNG--SPMSPT   Lses   61 EIASQLPTTNPDAPTVLDRILRLLACYNILTCSVQTQQDGKVQRLYGLAPVAKYLVKNED Ljap   61 EIASQLPTTNPDAPTVLDRMLRLLACYNILTCSVRTEQDGKVQRLYGLAPVAKYLVKNED Gmax   61 DIASRLPTHNPDAPVMLDRILRLLACYNILSFSLRTLPHGKVERLYGLAPVAKYLVRNED Mtru   61 EIASQLPTTNPEAPVMLDRILRLLACYNILTCSVRTQQDGKVQRLYGLATVAKYLVKNED Atha   59 EIASKLPTKNPEAPVMLDRILRLLTSYSVLTCSNRKLSGDGVERIYGLGPVCKYLTKNED   Lses  121 GVSISALNLMNQDKVLMESWYHLKDAVLEGGIPFNKAYGMTAFEYHGTDPRFNKVFNKGM Ljap  121 GVSISALNLMNQDKVLMESWYHLKDAVLEGGIPFNKAYGMTAFEYHGTDPRFNKVFNKGM Gmax  121 GVSIAALNLMNQDKILMESWYYLKDAVLEGGIPFNKAYGMTAFEYHGTDPRFNKVFNKGM Mtru  121 GVSISALNLMNQDKVLMESWYHLKDAVLDGGIPFNKAYGMTAFEYHGTDPRFNKVFNKGM Atha  119 GVSIAALCLMNQDKVLMESWYHLKDAILDGGIPFNKAYGMSAFEYHGTDPRFNKVFNNGM   Lses  181 SDHSTITMKKILETYTGFEGLKSLVDVGGGTGAVINMIVSKYPTIQGINFDLPHVIEDAP Ljap  181 SDHSTITMKKILETYTGFEGLKSLVDVGGGTGAVINMIVSKYPTIKGINFDLPHVIEDAP Gmax  181 ADHSTITMKKILETYTGFEGLKSLVDVGGGTGAVVNMIVSKYPTIKGINFDLPHVIEDAP Mtru  181 SDHSTITMKKILETYTGFEGLKSLVDVGGGTGAVINTIVSKYPTIKGINFDLPHVIEDAP Atha  179 SNHSTITMKKILETYKGFEGLTSLVDVGGGIGATLKMIVSKYPNLKGINFDLPHVIEDAP   Lses  241 SYPGVEHVGGDMFVSVPKADAVFMKWICHDWSDEHCLKFLKNCYEALPDNGKVIVAECIL Ljap  241 SYPGVEHVGGDMFVSVPKADAVFMKWICHDWSDEHCLKFLKNCYEALPDNGKVIVAECIL Gmax  241 SYPGVEHVGGDMFVSVPKADAIFMKWICHDWSDEHCLKFLKNCYEALPDNGKVIVAECIL Mtru  241 SYPGVEHVGGDMFVSIPKADAVFMKWICHDWSDEHCLKFLKNCYEALPDNGKVIVAECIL Atha  239 SHPGIEHVGGDMFVSVPKGDAIFMKWICHDWSDEHCVKFLKNCYESLPEDGKVILAECIL   Lses  301 PVAPDSSLATKGVVHIDVIMLAHNPGGKERTEKEFEALAKGAGFQGFKVMCCAFNSYVME Ljap  301 PVAPDSSLATKGVVHIDVIMLAHNPGGKERTEKEFEALAKGAGFQGFQVLCCAFNSYVME Gmax  301 PVAPDSSLATKGVVHIDVIMLAHNPGGKERTEKEFEALAKGSGFQGFQVLCCAFNTYVME Mtru  301 PVAPDSSLATKGVVHIDAIMLAHNPGGKERTQKEFEDLAKGAGFQGFKVHCNAFNTYIME Atha  299 PETPDSSLSTKQVVHVDCIMLAHNPGGKERTEKEFEALAKASGFKGIKVVCDAFGVNLIE   Lses  361 FLKKVX Ljap  361 FLKKVX Gmax  361 FLKKVX Mtru  361 FLKKVX Atha  359 LLKKLX       240    Appendix 17   Figure S61. Expanded phylogeny of Lotus spp. exhibiting PACC, including those studied in this thesis. Figure and caption are reproduced from Figure 2 in Ojeda et al. 2013:   Molecular tree based on one nuclear (ITS) and three plastid regions (CYB6, trnH-psbA and matK). The tree was randomly selected form a maximum parsimony (MP) analysis from Ojeda et al., 2012a and Ojeda et al., 2012b. Character mapping of the trait flower color change after anthesis in Lotus sections Pedrosia and the “rhyncholotus group”. Red branches show clades where this trait has evolved and the numbers on the tree the times this trait evolved within this groups (1–3). Arrows indicate the numbers of reversals, one of which occurred in three species of the “rhyncholotus group”. The species from the outgroup belong to the Lotus section are not endemic of the Macaronesian region. Species with (*) are distributed in mainland Africa and/or in Europe (a) represents flower color at anthesis (pre-change) and (b) indicates flower color after change (post-change). (A) L. jacobaeus, (B) L. purpureus, (C) L. eriosolen, (D) L. sessilifolius subsp. sessilifolius, (E) L. pyranthus, (F) L. emeroides, (G) L. argyrodes, (H) L. glaucus, (I). L. campylocladus, and (J) L. lancerottensis.   241     

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