In order to isolate putative TCP-like homologs in Asparagales, searches were performed using previously reported TCP genes from eudicots, monocots and in particular Orchidaceae as queries (Mondragón-Palomino and Trontin, 2011; Preston and Hileman, 2012; De Paolo et al., 2015; Horn et al., 2015). Searches included homologs from all the three main clades of TCP genes: CYC-like, CIN-like and PCF-like. Searches were done using BLAST tools (Altschul et al., 1990) in the orchid specific available databases including Orchidbase 2.0 (http://orchidbase.itps.ncku.edu.tw/) (Tsai et al., 2013), Orchidstra (http://orchidstra2.abrc.sinica.edu.tw/orchidstra2/index.php) (Su et al., 2013), as well as the more inclusive OneKP database (http://www.bioinfodata.org/Blast4OneKP/). All core eudicot sequences, were isolated from Phytozome (https://phytozome.jgi.doe.gov/pz/portal.html) and genbank (https://www.ncbi.nlm.nih.gov/genbank/).

In addition to the available databases we generated two transcriptomes from C. trianae (Orchidaceae) and H. decumbens (Hypoxidaceae). The transcriptome for each species was generated from mixed material from 3 biological replicates and included vegetative and reproductive meristems, floral buds, young leaves and fruits in as many developmental stages as possible. Total RNA was purified and used for the preparation of one mRNA (polyA) HiSeq library for each species. RNA-seq experiments were conducted using truseq mRNA library construction kit (Illumina, San Diego, California, USA) and sequenced in a HiSeq 2000 instrument reading 100 base paired end reads.

The transcriptome was assembled de novo with Trinity v2 following default settings. Read cleaning was performed with prinseq-lite v0.20.4 with a quality threshold of Q35 and a minimum read length of 50 bases. Contig metrics are as follows: (1) H. decumbens total assembled bases: 73,787,751; total number of contigs (>101 bases): 157,153; average contig length: 469 bp; largest contig: 15,554 bp; contig N50: 1075 bp; contig GC%: 46,42. (2) C. trianae total assembled bases: 63,287,862 bp; total number of contigs (>101 bases): 109,708; average contig length: 576 bp; largest contig: 9321 bp; contig N50: 1401 bp; contig GC%: 42,73. Homologous gene search was performed using BLASTN with the query sequences downloaded from GenBank and other databases (see above). In order to estimate the relative abundance of the assembled contigs, cleaned reads were mapped against the de novo assembled dataset and counted with two different strategies. The first one involved the mapping algorithm of the software Newbler v2.9 where only the unique matching read pairs were accepted as positive counts. The second one involved the mapping algorithm BOWTIE2 and raw reads counts as well as RPKM were calculated to each assembled contig (Table 1).

All sequences isolated were compiled with Bioedit (http://www.mbio.ncsu.edu/bioedit/bioedit.html). Sequences shorter than 200 bp lacking similarity with a region of the putative bHLH motif were discarded. Nucleotide sequences were subsequently aligned using the online version of MAFFT (http://mafft.cbrc.jp/alignment/software/) (Katoh et al., 2002) with a gap open penalty of 3.0, offset value of 1.0 and all other default settings. The alignment was then refined by hand using Bioedit considering as a reference the 60–70 aa reported as conserved in the TCP protein domain (Cubas et al., 1999). To better understand the evolution of the TCP gene lineage, and to integrate previous eudicot and monocot specific phylogenetic analyses (Damerval and Manuel, 2003; Hileman and Baum, 2003; Reeves and Olmstead, 2003; Howarth and Donoghue, 2006; Damerval et al., 2007; Bartlett and Specht, 2011; Mondragón-Palomino and Trontin, 2011; Preston and Hileman, 2012; Horn et al., 2015), we performed Maximum likelihood (ML) phylogenetic analyses using the nucleotide sequences with RaxML-HPC2 BlackBox through the CIPRES Science Gateway (https://www.phylo.org/) (Miller et al., 2010). Bootstrapping (BS) was performed according to the default criteria in RAxML (200–600 replicates). The PCF-like gene from Amborella trichopoda (AtrTCP4) as well as all other PCF-like sequences were used as the outgroup. To find the molecular evolution model that best fit our data, we used the jModelTest package implemented in MEGA6 (Posada and Crandall, 1998). Trees were observed and edited using FigTree v1.4.0 (http://tree.bio.ed.ac.uk/software/figtree/) (Rambaut, 2014). Newly isolated sequences from our own generated transcriptomes from C. trianae (Orchidaceae) and H. decumbens (Hypoxidaceae) can be found under Genbank numbers KY296315–KY296347. All sequences included in the phylogenetic analyses can be found in the Supplementary Table 1.

In order to detect previously reported, as well as to identify new, conserved motifs, 77 TCP-like genes were selected representing major model eudicot and monocot groups of this study (i.e., A. majus, O. sativa, Aloe vera, P. equestris, C. trianae and H. decumbens). Sequences were permanently translated and uploaded as amino acids to the online MEME server (http://meme-suite.org/tools/meme) and run with all the default options (Bailey et al., 2006). Specific analyses for each of the TCP clades (i.e., CYC-like, CIN-like, PCF-like) were also performed.

To examine and compare the expression patterns of TCP-like genes we used floral buds, dissected floral organs, leaves, and fruits of C. trianae and H. decumbens. Preanthetic floral buds of H. decumbens were dissected into sepals, petals, stamens and carpels. In addition whole floral buds, inmature fruits (F1, right after tepals shed off), mature fruits (F2, before lignification), and young leaves were also collected. Preanthetic floral buds of C. trianae were dissected into sepals, lateral petals, labellum (or lip), gynostemium, and ovary. Young leaves were also collected. Total RNA was isolated from each organ collected using the SV Total RNA Isolation System kit (Promega, Madison, WI, USA), and resuspended in 20 μl of DEPC water. RNA was treated with DNAseI (Roche, Basel, Switzerland) and quantified with a NanoDrop 2000 (Thermo Scientific, Waltham, MA) (Wilfinger et al., 1997). Three Micrograms of RNA were used as a template for cDNA synthesis (SuperScriptIII RT, Invitrogen) using OligodT primers. The cDNA was diluted 1:4 for amplification reactions by RT-PCR. Primers were designed in specific regions like portions flanking the conserved domains for each copy found in C. trianae and H. decumbens (Supplementary Table 2). Each amplification reaction incorporated 9 μl of EconoTaq (Lucigen, Middleton, WI), 6 μl of nuclease free water, 1 μl of BSA (5 μg/ml), 1 μl of Q solution (5 μg/μl) 1 μl fwd primer (10 mM), 1 μl rev primer (10 mM), and 1 μl of template cDNA for a total of 20 μl. Thermal cycling profiles followed an initial denaturation step (94°C for 30 s), an annealing step (50–59°C for 30 s) and an extension step with polymerase (72°C for 10 min), all by 30–38 amplification cycles. ACTIN2 was used as a load control. PCR products were run on a 1.0% agarose gel stained with ethidium bromide and digitally photographed using a Whatman Biometra® BioDoc Analyzer.

We were able to isolate 168 sequences belonging to the CYC/TB1-like clade (Supplementary Table 1). Our sampling includes 15 sequences from four species of Poales, 10 sequences from seven species of Asparagales (incl. Orchidaceae), eight from five species of Commelinales (Commelina, Alstroemeria, Tradescantia), 14 from nine species of basal angiosperms and 121 from 46 species of eudicots. Only one homolog from Curculigo spp. (CurTB1) and one homolog from H. decumbens (HydTB1) were recovered from our blast searches. Moreover, an exhaustive search in Orchidaceae specific databases resulted in eight additional copies: three from P. equestris (PETCP06750, PETCP06749, PETCP11715), two from P. aphrodite (PaTB1, PaTCP06749) and one from C. trianae (CtrTB1) (Epidendroideae); one homolog from O. italica (OitaTB1) (Orchidoideae); and one homolog from Vanilla shenzhenica (VaTCP06749) (Vanilloideae). CYC/TB1-like homologs seem to have undergone size reduction in Asparagales, and only the searches made with monocot TB1 genes yielded positive hits. Interestingly, in the two transcriptomes newly obtained in the present research, the CYC/TB1-like contigs were supported by fewer reads (when compared to MADS-box APETALA3 floral organ identity genes and other TCP-like genes; Table 1) suggesting low expression of these transcripts.

The resulting ML topology recovered the three previously established core eudicot subclades (Howarth and Donoghue, 2006) with very low support (BS < 50), namely CYC1/TCP18, CYC2/TCP1, and CYC3/TCP12 (Figure 1). Our analysis also recovers the previously identified duplication of CYC-like genes in basal eudicots (Citerne et al., 2013), and another in Poales, the latter resulting in the REP1/TB1 clades (Mondragón-Palomino and Trontin, 2011). Most basal angiosperms and many monocots outside Poales have single copy CYC-like genes that predate the independent duplications in eudicots and Poales (Figure 1). Intraspecific duplications in monocots have occurred in Zea mays, as well as in the orchids P. equestris and P. aphrodite (Figure 1; BS = 100). Outside of the monocots, local specific duplications have also occurred in the basal angiosperms Aristolochia ringens and Persea americana, in the basal eudicots Circaeaster and Nelumbo, and in core eudicots such as Gerbera, Antirrhinum, Citrus, Glycine, Populus, and Gossypium (Figure 1).

Members of the CYC/TB1-like clade show very little variation in the ca. 60 amino acid TCP domain (sensu Cubas et al., 1999) consisting of a putative basic-Helix-Loop-Helix (bHLH) domain (Figure 2). We were able to identify the highly conserved residues previously reported for the putative bipartite Nuclear Localization Signal (NLS) at the N-terminus flanking the bHLH, which provide hydrophobicity in the α-helices and in the loop region between the two. The loop itself is highly conserved in all CYC/TB1-like sequences except for AmDICH and AmCYC that have an A > P change at position 42. The second helix contains the LxxLL motif in all CYC2 proteins; this motif is modified into a VxWLx motif in other CYC-like proteins.

Our MEME analysis identified motifs 1 and 2 corresponding to the TCP domain (Supplementary Figure 1). At the start of Helix I, between positions 24–29 we found specific amino acids exclusive to CYC protein homologs. Outside the TCP domain, motifs 7 and 10 (reported also by Bartlett and Specht, 2011) and motifs 36–40, 42, and 43 (reported also by De Paolo et al., 2015) were recovered in our analysis as conserved in all CYC proteins. In addition, the protein interaction R domain (motif 11) putatively involved in hydrophilic α-helix formation in TCP Class II genes (shared between CYC and CIN proteins) was also identified (Supplementary Figure 1; Cubas et al., 1999). However, this motif is absent from CtrTB1 and REP-1 (Supplementary Figure 1; Yuan et al., 2009). Previously unidentified motifs include motif 4, exclusive to Epidendroideae and motif 5, exclusive to Phalaenopsis species. Whereas, most orchid CYC-like proteins (including O. italica OitaTB1 and C. trianae CtrTB1) do not share any common motifs with the canonical A. majus paralogs outside the TCP domain, the Phalaenopsis CYC-like homolog (PETCP11715) shares motifs 6, 9, and 12 with AmDICH or AmCYC.

A total of 155 CIN-like homologs were recovered and unlike the CYC-like sampling, most CIN-like sequences belong to the Asparagales (Supplementary Table 1). Our sampling contains 57 sequences from 35 non-Orchidaceae Asparagales species, including four paralogs from H. decumbens labeled HydCIN1-HydCIN4. A total of 78 CIN-like homologs were isolated, including four homologs from two Apostasioideae species, nine homologs from three Vanilloideae species, five homologs from three Cypripedioideae species, 22 homologs from seven Orchidoideae species and 38 homologs from 11 Epidendroideae species. Furthermore, six paralogs were identified in C. trianae labeled CtrCIN1-CtrCIN6. Searches outside Asparagales were restricted to six homologs from O. sativa, two from A. trichopoda and 12 homologs from five eudicots species, including the canonical A. majus CINCINNATA (AmCIN).

The CIN-like ML analysis shows a duplication (BS = 75) that predates the diversification of angiosperms resulting in the CIN1 (BS = 79) and CIN2 clades (BS = 99) (Figure 3). This is confirmed by the position of the two A. trichopoda CIN paralogs, AtrPCF and AtrTCP2, each in its own clade (Figure 3). Additional support for this early duplication is found in the topology yielded by a second complementary analysis that includes 11 Solanaceae CIN homologs, where CIN1 and CIN2 clades have monocot and core eudicot representatives and, at least CIN2 is well supported (BS = 93) (Supplementary Figure 2). The CIN1 clade has undergone at least two additional duplications resulting in the CIN1a-c clades. It is likely that the duplication resulting in CIN1a and CIN1b/c occurred exclusively in monocots, although the exact timing is unclear. The other duplication resulting in CIN1b and CIN1c appears to be Orchidaceae-specific, prior to the diversification of Orchidoideae and Epidendroideae (Figure 3). On the other hand, the CIN2 clade underwent an independent duplication predating the diversification of Asparagales, resulting in CIN2a and CIN2b subclades. Intraspecific duplications were identified in Hesperaloe, Disporopsis, Maianthemum, Rhodophiaia, Sansevieria and Yucca, (Figure 3). Poales CIN homologs form a clade, with a low BS, in the first analysis, with the exception of OsPCF5 clustered with AmCIN (Figure 3). However, our second analysis shows two Poales clades nested in each angiosperm paralogous CIN1 and CIN2 clades (with low BS), suggesting that the two Poales clades likely resulted from the angiosperm CIN1/2 duplication (Supplementary Figure 2).

CIN-like sequences show high degree of conservation at the N-flank of the TCP domain (Figure 2). The only changes with respect to the key aminoacids in the bHLH domain in CYC proteins are at the second helix where the LxxLL motif shifts to V/IxxLL (Figure 2). Toward the 3′ end of the TCP domain proteins are highly variable, except for motifs 13, 14, 16–18 and 21, reported also by De Paolo et al. (2015) (Supplementary Figure 1). The R domain (motif 11) in CIN proteins is only present in the CIN1a clade and O. sativa homologs OsTCP21, OSTCP8 and OsTCP10. Motif 14, which corresponds to the miR319 binding site is present in most CIN-like sequences. The miR319 binding motif is lacking in HydCIN2, HydCIN3, OsTCP21, OsTCP27, and OsTCP10. All CIN1 subclade sequences share motifs 13, 17, 19, 20 21, and 25; the CIN1a subclade shares motifs 11, 28, 29, 34 and 35, while the CIN1b subclade shares motifs 15, 23, 26, and 30. Synapomorphies for the CIN2 subclade include motifs 16, 18, 22, 24 and only motif 27 is exclusive to Orchidaceae. Finally, CIN2b homologs share motifs 32 and 33. The most divergent C. trianae sequence is CtrCIN6, which only has the motifs 1, 2, 3, 11, 13, 14, 17, and 21.

Our analysis recovered 129 PCF-like homologs (Supplementary Table 1). Similarly to CIN-like genes most sampling is concentrated in Asparagales, thus 53 homologs belong to 35 species of non-Orchidaceae Asparagales and 52 sequences correspond to Orchidaceae. H. decumbens has 10 PCF-like copies (HydPCF1–HydPCF10). Sampling in Orchidaceae includes one homolog from one Vanilloideae species, 14 homologs from seven Orchidoideae species and 37 homologs from 11 Epidendroideae species. We recovered 11 PCF-like homologs from C. trianae (CtrPCF1–CtrPCF11). Sampling in monocots outside Asparagales include 10 homologs from O. sativa (Poales) and sequences that are not monocots are restricted to one homolog from A. trichopoda and 13 homologs from A. thaliana.

Our analysis detected at least five duplication events of PCF-like prior to the diversification of Asparagales, however support is low for all clades (BS = < 50) (Figure 4). In addition, our complementary analysis including 18 Solanaceae PCF-like genes also shows support for at least two rounds of core eudicot specific PCF-like duplications (Supplementary Figure 2).

PCF-like sequences exhibit the shortest basic motif in TCP proteins, with a deletion in the bipartite NLS between positions 10 and 13 (Figure 2; Cubas et al., 1999). The TCP domain shows little conservation in comparison to the TCP II class (CYC and CIN) proteins. For instance, there is a four amino acid deletion in the middle of the basic motif, and only 12 out of the 23 amino acids characterized in both helices and the loop are conserved (Figure 2). Additionally our MEME analysis show that PCF-like proteins do not have an R domain (motif 11), nor a target sequence for miR319 (motif 14) (Supplementary Figure 1).

In order to hypothesize functional roles for the Asparagales TCP-like homologs, the expression patterns of all homologs isolated from transcriptomic analysis in H. decumbens and C. trianae were evaluated (Figure 5). Although in both species we were able to dissect floral organs in preanthesis and young leaves, we were not able to find fruits of C. trianae, thus only young and old fruits of H. decumbens were included in the expression study.

The CYC/TB1-like homologs have very different expression patterns in C. trianae and H. decumbens. HydTB1 is expressed in the floral bud, sepals, carpels, young and mature fruits and leaves, whereas CtrTB1 is expressed in all floral whorls and is not expressed in leaves (Figure 5). CIN-like homologs also exhibit different expression patterns in C. trianae and H. decumbens. HydCIN1, HydCIN2 and HydCIN3 are expressed in the floral bud, carpels, fruits and leaves. Only HydCIN2 expression is extended to sepals, petals and stamens at very low levels. Interestingly both HydCIN2 and HydCIN3 lack the miR319 binding site. HydCIN4 expression is restricted to fruits and leaves. CtrCIN1, CtrCIN2, CtrCIN5, and CtrCIN6 are expressed in sepals, petals, lip, gynostemium, and ovary, although, CtrCIN2 expression in sepals and gynostemium occurs at low levels. CtrCIN3 and CtrCIN4 have similar expression patterns to CtrCIN1, however, they are poorly expressed in the ovary and are the only CIN-like homologs that extend their expression to leaves.

Similar to CYC/TB1-like and CIN-like genes, the expression of PCF-like homologs varies dramatically between C. trianae and H. decumbens (Figure 5). CtrPCF1, CtrPCF2, CtrPCF3, CtrPCF4, CtrPCF5, CtrPCF6, CtrPCF9, and CtrPCF11 are expressed in all floral organs and the only copy with expression in leaves is CtrPCF4. CtrPCF3 has low expression in sepals and petals. CtrPCF7 is only expressed in petals and ovary. Finally, CtrPCF10 has expression in all floral whorls except in petals. In H. decumbens only HydPCF3, HydPCF4, HydPCF5, and HydPCF6 are expressed in the floral buds, carpels, fruits and leaves, although HydPCF3 and HydPCF4 have low expression in carpels. HydPCF5 is also expressed in the perianth with higher expression in sepals than in petals. HydPCF7 has restricted expression to the floral buds and the young fruits. HydPCF9 and HydPCF10 are only expressed in young fruits and the remaining copies (HydPCF1, HydPCF2, and HydPCF8) are only expressed at very low levels in the floral buds.

Our study detected a single copy CYC/TB1 gene in each of the species investigated in the Asparagales. The Asparagales CYC/TB1 homologs fall outside of the Poales (TB1/REP clades) or Commelinales identified local duplications, described before and recovered here (Doebley et al., 1995, 1997; Yuan et al., 2009; Mondragón-Palomino and Trontin, 2011). The tree topology suggests that independent duplications have occurred in CYC-like genes in the monocots (Yuan et al., 2009; Mondragón-Palomino and Trontin, 2011; Hileman, 2014). Species-specific duplications in Asparagales were found only in Phalaenopsis (Orchidaceae). Moreover, expression data of CYC/TB1 genes in Asparagales show remarkable differences between H. decumbens and C. trianae, even though sampled organs correspond to fairly well developed tissues. CtrTB1 is expressed homogeneously in all floral whorls while HydTB1 is expressed predominantly in carpels, fruits and leaves (Figure 5). Homogeneous expression of CYC-like genes in dorsal and ventral floral organs in C. trianae suggest that CtrTB1 is likely not playing important roles in maintenance of bilateral symmetry in orchid flowers (see also Horn et al., 2015). However, only pre-anthethic floral buds were sampled and earlier stages are needed to test whether CtrTB1 can be contributing to the establishment of bilateral symmetry in the flower primordia. Comparative expression studies of CYC/TB1-like genes in other Orchidaceae point to significant variations. For instance, while the O. italica homolog OitaTB1 is only expressed in leaves (De Paolo et al., 2015), two CYC/TB1 genes in P. equestris do exhibit differential dorsiventral expression in the floral bud suggesting species specific roles in bilateral symmetry establishment (Lin et al., 2016).

Expression of CYC/TB1 genes in Orchidaceae contrasts with studies in Commelinales, Zingiberales, and Alstroemeriaceae that show differential dorsiventral expression of CYC/TB1 genes and hence support convergent recruitment of CYC homologs in the acquisition of bilateral symmetry in different monocots (Bartlett and Specht, 2011; Preston and Hileman, 2012; Hoshino et al., 2014). Within core eudicots, only the CYC2/TCP1 clade members have been linked to shifts toward bilateral floral symmetry. This has been extensively documented for CYC and DICH in A. majus (recent paralogs within the CYC2/TCP1 clade) which are expressed in the dorsal regions of the floral meristems and negatively regulate cell proliferation (Luo et al., 1996, 1999). However, many other CYC2 orthologs have been shown to control bilateral symmetry in Asterales, Brassicales, Dipsacales, Fabales, Lamiales, and Malpighiales (Busch and Zachgo, 2007; Gao et al., 2008; Preston et al., 2009; Wang et al., 2010; Zhang et al., 2010, 2013; Howarth et al., 2011; Tähtiharju et al., 2012; Yang et al., 2012; Ma et al., 2016).

Recruitment of CYC2 homologs in bilateral symmetry is likely facilitated by conserved protein-protein interactions mediated by the LxxLL motif (Heery et al., 1997; Damerval and Manuel, 2003; Reeves and Olmstead, 2003; Howarth and Donoghue, 2006; Li et al., 2009; Preston et al., 2009; Tähtiharju et al., 2012; Parapunova et al., 2014; Ma et al., 2016). If so, it is possible that different CYC-like proteins can form specific homo- and heterodimers, or even have unique partners, and thus protein motifs can provide clues to protein affinity and functional specificity (Kosugi and Ohashi, 2002). Our MEME analysis rescues motifs 6, 9 and 12, shared only between P. equestris PETCP11715 and the A. majus AmCYC and AmDICH, which are not present in other orchid CYC/TB1 proteins (i.e., C. trianae and O. italica) and hence putatively involved in establishing early bilateral floral symmetry in some Orchidaceae species (Supplementary Figure 1). When full length CtrTB1 is compared to the three P. equestris CYC/TB1 proteins very little similarity is detected (PETCP11715–0.38; PETCP06750–0.22; PETCP06750–0.24); for instance, CtrTB1 lacks motif 11 (R-domain). Nevertheless, the TCP domain is extremely conserved (0.91, 0.80, and 0.84 respectively). These results suggest that besides the key bHLH amino acids that target conserved genes, there are likely important motifs in the flanking regions allowing unique interactions and downstream partners in each species.

Conversely, the expression of HydTB1 in H. decumbens is indicative of exclusive roles in carpels, fruits and leaves. This data suggests that HydTB1 may have similar roles to other CYC genes that do not participate in establishing floral bilateral symmetry. Such is the case of the A. thaliana, AtTCP1, which promotes shoot growth and regulates leaf lamina size (Costa et al., 2005; Guo et al., 2010; Koyama et al., 2010b). To date less attention has been given to the putative role of CYC genes in the development of leaves, carpels and fruits, despite the fact that expression has been detected in these organs in other core eudicots. This is the case for CYC homologs in Citrullus, Gerbera, Gossypium, Lotus, Solanum, and some Papaveraceae that have been detected in seedlings, young leaves, and immature fruits (Damerval et al., 2007; Wang et al., 2010; Parapunova et al., 2014; Ma et al., 2016; Shi et al., 2016).

Here we show the first comprehensive phylogenetic analysis of CIN-like genes. Our results point to the occurrence of at least one duplication event predating angiosperm diversification, at least one duplication occurring prior to the origin of the Asparagales and one specific duplication prior to the diversification of Orchidoideae and Epidendroideae (Figure 3, Supplementary Figure 2; Floyd and Bowman, 2007; Martín-Trillo and Cubas, 2010). By comparison to CYC genes, CIN genes functional characterization is restricted to model species only. The canonical CINCINNATA in A. majus has dual roles in limiting the growth of leaf margins while promoting epidermal cell differentiation in petals (Nath et al., 2003; Crawford et al., 2004). The cin mutant in A. majus, as well as the tcp4 mutant in A. thaliana, exhibit curly leaves as a result of excessive growth in leaf margins (Crawford et al., 2004; Koyama et al., 2010a). CIN regulates leaf shape through direct or indirect negative regulation of the boundary CUP-SHAPED COTYLEDON (CUC) genes, likely via the activation of ASSYMETRIC LEAVES 1 (AS1), miR164, INDOLE-3-ACETIC ACID3/SHORT HYPOCOTYL2 (IAA3/SHY2), and SMALL AUXIN UP RNA (SAUR) (Koyama et al., 2007, 2010a). CIN also controls leaf development through the regulation of cell proliferation by activating miR396, CYCLIN-DEPENDENT KINASE INHIBITOR/KIP RELATED PROTEIN 1 (ICK1/KRP1) and jasmonate biosynthesis (Schommer et al., 2014). Similarly, AtTCP4 regulates the transition between cell proliferation and differentiation, controlling cytokinin and auxin receptors (Efroni et al., 2013; Das Gupta et al., 2014). AtTCP4 also controls leaf senescence, maintains petal growth, and regulates early embryo development and seed viability, and finally it regulates jasmonic acid (JA) biosynthesis by the activation of LIPOXIGENASE2 (LOX2) (Schommer et al., 2008; Nag et al., 2009; Sarvepalli and Nath, 2011; Danisman et al., 2012). Leaf patterning is also controlled by CIN-like homologs in tomato and rice (Ori et al., 2007; Yang et al., 2013; Zhou et al., 2013; Ballester et al., 2015).

In addition to the roles of CIN genes in leaf patterning, other functions in carpel and fruit development have been identified. AtTCP2 and AtTCP3 are known to activate NGATHA genes that regulate carpel apical patterning. NGA orthologs from A. thaliana, rice, tomato and bean have conserved putative TCP binding site suggesting that this regulation is conserved in monocots and dicots (Ballester et al., 2015). Moreover, the tcp3 mutant has shorter siliques with a crinkled surface (Koyama et al., 2007; Ballester et al., 2015). Additionally, expression analyses in different Solanaceae species suggest an important contribution of these genes to fruit development and maturation, as they are downstream targets of key ripening regulators including RIPENING INHIBITOR (RIN), COLORLESS NON-RIPENING (CNR) and SlAP2a (Supplementary Figure 3; Crawford et al., 2004; Parapunova et al., 2014). More recently PeCIN8, a P. equestris CIN-like homolog, was shown to be broadly expressed and to have roles in leaf cell proliferation, determining the fruit final size and controlling proper embryo and ovule development (Lin et al., 2016). In summary, CIN-like genes are pleiotropic regulators of cell division and differentiation in leaves, petals, carpels, ovules, fruits and seeds across angiosperms.

This study identified significant changes in the helix I residues and the loop between Orchidaceae sequences and other Asparagales CIN-like proteins (Figure 2). For instance, a number of motifs involved in protein interaction, including the R domain characteristic of class II TCP proteins were only identified in the CIN1a clade, but are absent in all other paralogous clades (Supplementary Figure 1, Cubas et al., 1999; Damerval and Manuel, 2003). Here we have also identified conserved motifs like the miR319 binding site, previously reported in CIN-like homologs from O. italica (Nag et al., 2009; De Paolo et al., 2015), for all CIN-like sequences in Asparagales. This indicates, that Asparagales homologs are also regulated by miR319, similar to AtTCP4, and other CIN-like Arabidopsis paralogs, including AtTCP2/3/10 and 24 (Palatnik et al., 2003; Koyama et al., 2007; Schommer et al., 2008, 2012; Koyama et al., 2010a; Danisman et al., 2012).

In comparison with reported functional data from model eudicots and monocots, the CIN-like differential expression observed in C. trianae and H. decumbens homologs points to three testable hypotheses. (1) While in H. decumbens all CIN-like genes are expressed in young leaves and may play roles in leaf development, only two of the six paralogs identified in C. trianae (CtrCIN3/4) are likely involved in cell division and differentiation during leaf development, similar to the P. equestris PeCIN8 (Lin et al., 2016). (2) While CIN-like genes are likely playing key roles in both perianth and fertile organs development and growth in C. trianae, their contribution to perianth development and growth is less clear in H. decumbens, perhaps only with HydCIN2 involved in cell proliferation in the perianth. (3) In both species CIN-like genes are strongly expressed in carpels and fruits, suggesting that their role in carpel patterning, ovule development as well as fruit maturation is likely conserved in Asparagales. Nevertheless, mRNA expression data for all CIN-like genes must be interpreted with caution given the putative conserved miR139 postranscriptional regulation.

Our results on the evolution of PCF-like genes points to numerous duplication events within Asparagales in comparison to the CYC-like and the CIN-like clades. In addition, the topology recovered suggests that PCF-like gene duplications in monocots are independent from the one that occurred in core eudicots (Figure 4, Supplementary Figure 2). Functional data available for PCF-like genes suggest redundancy with CIN-like genes (Aguilar-Martínez and Sinha, 2013; Danisman et al., 2013). For instance, both PCF-like and CIN-like genes control leaf development through regulation of LOX2. However, while AtTCP20 (PCF-like) inhibits, AtTCP4 (CIN-like) induces the expression of LOX2 (Danisman et al., 2012). PCF-like genes are also involved in the regulation of circadian clock genes, that is the case of CCA1 Hiking Expedition CHE (AtTCP21) (Pruneda-Paz et al., 2009; Giraud et al., 2010). In addition dimers formed between AtTCP15 and other TCP-like proteins (AtTCP2, AtTCP3, AtTCP11) are known to regulate circadian cycles, cell proliferation in floral organs and leaves, and to promote seed germination (Koyama et al., 2007; Kieffer et al., 2011; Resentini et al., 2015). PCF-like genes are also expressed in ovule, seed and fruit development in Solanum lycopersicum, Solanum tuberosum, O. sativa and in P. equestris, suggesting that they mediate cell proliferation in the carpel to fruit transition in both eudicots and monocots (Supplementary Figure 3; Kosugi and Ohashi, 1997; Yao et al., 2007; Parapunova et al., 2014; Lin et al., 2016).

Expression detected here of PCF-like homologs in C. trianae and H. decumbens show broad expression of most paralogs in C. trianae (except CtrPCF7) contrasting with a restricted expression of most paralogs in H. decumbens (except HydPCF5; Figure 5). Such expression patterns are in accordance with all putative functions identified in other monocots and in core eudicots including, but not restricted to, cell proliferation control in leaves, carpels, ovules, fruits and seeds (Lin et al., 2016). All data available point to pleiotropic roles of TCP-like genes with a high degree of redundancy among paralogs. Interestingly, when compared to CYC-like and CIN-like genes, the PCF-like gene HydPCF5 in H. decumbens is more likely to be playing cell proliferation roles in the perianth.

In conclusion, the Asparagales, unlike core eudicot model plants, have a reduction in the number of CYC-like homologs and an increase of CIN-like and PCF-like copies. Characteristic key amino acids in the bHLH domain, flanking motifs and binding sites are for the most part conserved in Asparagales CIN-like and PCF like proteins suggesting similar conserved mechanisms of post-transcriptional regulation and interacting partners. The most notorious exception to this is the lack of a miR319 binding site in HydCIN2/3. Nevertheless, the CYC-like proteins in Asparagales seem to be poorly expressed and have undergone important shifts in protein domains, suggesting changes in regulation as well as in protein-protein interactions. The orchid included in this study has similar numbers of TCP copies when compared to Asparagales with radially symmetrical flowers, and only the Epidendrioideae and Orchidoideae seem to have an additional CIN paralog lacking in other Orchidaceae and Asparagales. Expression data suggests different roles of TCP-like genes in C. trianae and H. decumbens, pointing to: (1) a possible decoupling of TB1 homologs from bilateral symmetry in C. trianae; (2) conserved roles of CIN-like and PCF-like genes in the control of cell proliferation in carpels, ovules and fruits in both species; and (3) preferential leaf expression of CIN-like and PCF-like genes in H. decumbens and perianth expression in C. trianae. Here we have performed the first large scale analysis of TCP genes in Asparagales which will provide a platform for in depth comparative expression analyses as well as much needed functional studies of these genes in emerging model orchids.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.