Gamete-specific expression of TALE class HD genes activates the diploid sporophyte program in Marchantia polymorpha

Eukaryotic life cycles alternate between haploid and diploid phases and in phylogenetically diverse unicellular eukaryotes, expression of paralogous homeodomain genes in the two gametes directs the haploid-to-diploid transition. In the unicellular Chlorophyte alga Chlamydomonas KNOX and BELL TALE-homeodomain genes mediate the transition. Here we demonstrate that in the liverwort Marchantia polymorpha paternal (sperm) expression three of the five phylogenetically diverse BELL genes, MpBELL234, and maternal (egg) expression of MpKNOX1 mediate the haploid-to-diploid transition. Loss-of-function alleles of either result in zygotic or early embryonic arrest. In land plants both the haploid gametophyte and diploid sporophyte are complex multicellular organisms. Expression of MpKNOX1 and two other paralogs, MpBELL1 and MpKNOX2, during sporophyte development is consistent with a later role in patterning the sporophyte. These results indicate that the ancestral mechanism to activate diploid gene expression was retained in early diverging land plants and subsequently co-opted during evolution of the diploid sporophyte body.


Main text: Introduction
The life cycles of eukaryotes alternate between haploid and diploid phases, initiated by meiosis and gamete fusion, respectively. Expression of paralogous homeodomain genes in the two gametes directs the haploid-to-diploid transition in gene expression in phylogenetically diverse eukaryotes, including the ascomycete fungus Saccharomyces cerevisiae (Goutte andJohnson, 1988, Herskowitz, 1989), the basidiomycete fungi Coprinopsis cinerea and Ustilago maydis (Gillissen et al., 1992, Hull et al., 2005, Kues et al., 1992, Spit et al., 1998, Urban et al., 1996, the Amoebozoa Dictyostelium discoideum (Hedgethorne et al., 2017), the brown alga Ectocarpus (Arun et al., 2019), the red alga Pyropia yezoensis (Mikami et al., 2019), and the unicellular Chlorophyte alga Chlamydomonas reinhardtii (Ferris and Goodenough, 1987, Lee et al., 2008, Nishimura et al., 2012, Zhao et al., 2001. This broad phylogenetic distribution suggest this was an ancestral function of homeodomain genes [reviewed in (Bowman et al., 2016b)]. In the Viridiplantae the paralogous genes are two subclasses, KNOX and BELL, of TALE-class homeodomain (HD) genes. In Chlamydomonas, the minus (-) gamete expresses a KNOX protein (GSM1) and the plus (+) gamete expresses a BELL protein (GSP1), and upon gamete fusion the two proteins heterodimerize and translocate to the nucleus activating zygotic gene expression (Lee et al., 2008). GSM1 and GSP1 are necessary for diploid gene expression, and when ectopically expressed together in vegetative haploid cells are sufficient to induce the diploid genetic program (Ferris and Goodenough, 1987, Lee et al., 2008, Nishimura et al., 2012, Zhao et al., 2001. Biologically, the expression of a unique paralog in each type of gamete, coupled with the requirement for heterodimerization for functionality, is a mechanism to preload gametes such that immediately following gamete fusion/fertilization a distinct diploid genetic program is initiated. Heterodimerization of TALE-HD paralogs is mediated by subclass-specific protein domains N-terminal of the homeodomain that are in some cases conserved in phylogenetically disparate eukaryotes (Bellaoui et al., 2001, Burglin, 1997. For example, Viridiplantae KNOX proteins and metazoan MEIS proteins share a homologous heterodimerization domain thus defining a TALE-HD class, named MEINOX, present in the ancestral eukaryote (Burglin, 1997). In contrast, heterodimerization domains of MEINOX partners are not as well conserved (Joo et al., 2018). Some MEINOX partners in animals have a characteristic PBC-C heterodimerization domain (Burglin, 1997), and PBC-related domains are found in some algal BELL-related TALE-HD proteins, suggesting its presence in the ancestral eukaryote (Joo et al., 2018). In contrast, land plant BELL proteins possess conserved SKY and BELL domains, collectively termed POX (pre-homeobox), that mediate heterodimerization with KNOX partners (Bellaoui et al., 2001, Hackbusch et al., 2005, Smith et al., 2002. POX domains are either highly diverged from, or unrelated to, PBC domains. Finally, in other algal BELL-related TALE-HD proteins neither heterodimerization domain is evident (Joo et al., 2018). Regardless, when tested for dimerization potential, Archaeplastida BELL-related TALE-HD proteins interact only with MEINOX TALE-HD partners and not with other BELL-related TALE-HD proteins (Joo et al., 2018). Land plants are characterized by an alternation of generations whereby complex multicellular bodies develop in both haploid (gametophyte) and diploid (sporophyte) phases of the life cycle (Hofmeister, 1862) and elements of the KNOX/BELL system have been implicated in regulating the land plants haploid to diploid transition. In the moss Physcomitrella patens, one subclass of KNOX genes, KNOX1, is required for proper proliferation and differentiation in the diploid body (Sakakibara et al., 2008, Singer andAshton, 2007), while another subclass, KNOX2, acts to suppress the haploid genetic program during diploid development (Sakakibara et al., 2013). In addition to being expressed during sporophyte development, both classes of KNOX genes are expressed in the egg cell during gamete formation (Sakakibara et al., 2013, Sakakibara et al., 2008. The expression of KNOX genes in egg cells suggests that this gamete may correspond to the (-) gamete in Chlamydomonas, which also expresses a KNOX protein, implying that the male gamete in land plants might express BELL proteins, as does the (+) gamete in Chlamydomonas (Sakakibara et al., 2013, Sakakibara et al., 2008. Consistent with this hypothesis, ectopic expression of a Physcomitrella BELL paralog, PpBELL1, has been noted to induce the diploid genetic program in specific cell types of the gametophyte, but PpBELL1 was paradoxically reported to be expressed in the egg cell rather than the male gamete as might have been expected (Horst et al., 2016). However, expression of PpBELL1 has also been reported to be induced by activation of glutamate channels in the sperm, with PpBELL1 subsequently functionally active in the sporophyte (Ortiz-Ramírez et al., 2017). Finally, in Physcomitrella the KNOX/BELL diploid genetic program is actively suppressed during vegetative haploid development via polycomb-mediated repression (Mosquna et al., 2009, Okano et al., 2009). These observations prompted our investigation of the homologous genetic programs in the liverwort Marchantia polymorpha.

M. polymorpha possesses phylogenetically diverse TALE homeodomain proteins
The M. polymorpha genome encodes nine TALE-HD-related family members: four KNOX genes and five BELL genes [ (Bowman et al., 2017); Figure 1], all of which are expressed in the haploid sexual organs or diploid sporophyte, with minimal or no expression detected by rtPCR in the haploid vegetative thallus (Figure 1-figure supplement 1). Of the four KNOX genes, three are KNOX1 subclass, with only one encoding a HD, and one KNOX2 subclass (Figure 1-figure supplement 1). The two subclasses arose via gene duplication in an ancestral charophycean alga (Sakakibara, 2016, Joo et al., 2018, Frangedakis et al., 2017. Expression of MpKNOX1 (Mp5g01600/Mapoly0175s0020), which encodes a HD, is predominantly detected in archegoniophores and young sporophytes. In contrast, the KNOX1 genes lacking an HD (MpKNOX1A, Mp4g12450/Mapoly0174s0007; MpKNOX1B, Mp2g11140/Mapoly0023s0081) are predominantly expressed in antheridiophores. MpKNOX2 (Mp7g05320/Mapoly0194s0001) is expressed primarily during sporophyte development.
In contrast to the M. polymorpha KNOX genes, only one BELL gene (MpBELL1, Mp8g18310/Mapoly0213s0014) is phylogenetically related to previously described land plant BELL genes, whereas the other four are more closely related to algal TALE-HD genes ( Figure   2). MpBELL1 harbors a conserved canonical land plant BELL homeodomain sequence and a discernible, albeit divergent, BELL domain characteristic of other land plant BELL genes. to other algal BELL classes is enigmatic (Figure 2). The fourth algal-like sequence, MpBELL5 (Mp5g11060/Mapoly0093s0028), is phylogenetically distinct, and resides in the previously defined GLX-basal clade (Joo et al., 2018). Unlike some algal BELL-related proteins (Joo et al., 2018), the presence of a PBC domain is not easily discernible in any of the predicted M. polymorpha BELL protein sequences. Thus, a diversity of BELL-related paralogs arose in an algal ancestor and has persisted in M. polymorpha. The presence of an algal-related Metzgeria BELL sequence implies that BELL diversity may exist throughout liverworts.  Table 1. All protein annotation models are based on the Marchantia genome assembly of v5.1 except for MpKNOX2. The MpKNOX2 model is based on sequences derived from RT-PCR. In genes with multiple homeodomains, they are denoted a, b and c. Gene models were assembled using wormweb (http://wormweb.org/exonintron). anneal; 1 minute extension, except MpKNOX1, MpKNOX2, MpBELL1 at 2 minutes). The expected size band from cDNA is indicated by a red arrow at left of each panel. We were unable to amplify a product from MpBELL2.  (Sakakibara, 2016, Joo et al., 2018, Frangedakis et al., 2017. The KNOX2 genes, including charophyte sequences, form a well-supported monophyletic clade (posterior probability = 1). The KNOX1 clade is less well-supported with regard to the early diverging charophyte sequences; however, land plant KNOX1 genes, including MpKNOX1, share a conserved MEINOX domain (see also Figure 3-supplemental figure 1)   values within the polytomy in the canonical land plant clade were omitted for clarity. highlighted by blue arrows) in the homeodomain Lee et al. (Lee et al., 2008) suggested that C.
reinhardtii HDG1 was orthologous to canonical land plant BELL genes (characterized by I and V residues at the positions); this is supported by phylogenetic analysis (Figure 2). The second (b) and third (c) homeodomains of MpBELL2, MpBELL3, and MpBELL4 are progressively more divergent that the carboxyl-most (a) homeodomain

Vegetative gametophytic knock-down of MpE(z) de-represses MpKNOX2 and MpBELL1 expression
In the moss P. patens, genome wide polycomb repressive complex 2 (PRC2)-mediated repression of gene expression is observed in the shift from gametophyte to sporophyte and at least a subset of KNOX and BELL TALE-HD genes exhibit polycomb-mediated repression in the P. patens vegetative gametophyte (Mosquna et al., 2009, Okano et al., 2009. Thus, we first examined whether any Marchantia TALE-HD genes are repressed in the vegetative gametophyte in a PRC2-dependent manner.
PRC2, found in both animals and plants (Goodrich et al., 1997, Jones andGelbart, 1993), acts as an epigenetic regulator of genetic programs, and thus provides a 'cellular memory' (Cao et al., 2002, Czermin et al., 2002, Saurin et al., 2001. Each PRC2 includes a member of the SET domain family, which has methyltransferase activity that enzymatically methylates histones (Fischle et al., 2003), with trimethylation of lysine 27 at histone 3 (H3K27me3) being a genomic imprint of PRC2 that is inherited throughout a cell lineage (Cao et al., 2002, Czermin et al., 2002, Muller et al., 2002 We specifically examined the H3K27me3 pattern relative to whether gene expression

Maternal MpKNOX1 is required for post-zygotic embryo development
During the gametophyte generation MpKNOX1 is expressed specifically in the egg cell, but is not detected at the stage prior when the venter canal cell is present (Figure 4A      All scales (FPKM), except those in the strand-specific expression panels, are set to a standard within each outlined box; all H3K27me3 panels set to 50; for RNA-seq data the FPKM values (if less than 10, rounded to .1) are presented. Scales in the strand-specific expression panels vary according to tissue to allow potential weakly-expressed transcripts to be visualized.

Paternal MpBELL is required for proper embryo development
To examine whether these MpBELL genes could provide the male counterpart to the female MpKNOX1, we created loss-of-function alleles for each gene ( in sporophyte development that progressed to a globular multicellular stage characteristic of the first week of wild-type development, followed by sporophyte arrest (Figure 6). Only occasionally did sporophytes fail to progress beyond zygote formation, resembling the phenotype observed in female Mpknox1-6 ge mutants. In crosses in which mature sporophytes form, the corresponding archegoniophores remain viable until after the sporophytes have matured, while unfertilized archegoniophores undergo senescence. The archegoniophores with arrested sporophytes senesced in manner similar to unfertilized archegoniophores. In contrast, in the reciprocal cross between Mpbell34 females and wild-type males normal sporophyte development was observed (Figure 6-figure supplement 1).
We next examined whether egg-expressed MpKNOX1 and antheridial-expressed MpBELL proteins could interact. We chose MpBELL4 for analysis due to the truncated nature of MpBELL2 and the extreme length of MpBELL3. In a split YFP BiFC assay, MpKNOX1 on its own is cytoplasmically localized, but when co-expressed with nuclear localized MpBELL4, MpKNOX1 signal becomes nuclear (Figure 6). A similar interaction was observed with MpKNOX2 and MpBELL1 (Figure 6-figure supplement 2). These interactions are selective, since neither interaction between MpKNOX1 and MpBELL1 nor between MpKNOX2 and MpBELL4 were observed (Figure 6-figure supplement 2).
Expression of the fifth M. polymorpha MpBELL gene, MpBELL5, may be restricted to the archegoniophore among tissues examined (Figure 3-figure supplement 3). As with MpKNOX1 ( Figure 4) and MpFGMYB (Hisanaga et al., 2019), the MpBELL5 transcript may also be opposed by a convergent antheridial-expressed transcript (Figure 3-figure   supplement 2).    Figure 6B and C for reference.

Activation of sporophyte gene expression in the vegetative gametophyte
The expression patterns of MpBELL2/3/4 and MpKNOX1 genes are consistent with a role in activating diploid gene expression, and thus we examined whether ectopic co-expression of these genes in vegetative gametophyte is sufficient to activate diploid gene expression ( Figure 6-figure supplement 3A). Co-expression MpKNOX1 and MpBELL3 in the vegetative gametophyte is sufficient to activate MpKNOX2, whose expression is normally limited to the sporophyte, and MpBELL1, whose expression is normally limited to the

An ancestral function for TALE-HD genes in the Viridiplantae
The eukaryotic life cycle alternates between haploid and diploid phases, initiated by meiosis and gamete fusion, respectively. Organisms spanning the phylogenetic diversity of eukaryotes, including ascomycete and basiomycete fungi, Amoebozoa, brown algae, and Chlorophyte algae, have also been shown utilize paralogous homeodomain proteins that heterodimerize following gamete fusion to initiate the diploid genetic program, lending support the idea that this may have been the ancestral function of homeodomain proteins. Our observations in M.
polymorpha indicate that TALE-HD proteins, specifically MpKNOX1 and MpBELL2/3/4, are initially supplied in gametes (egg and sperm, respectively), and that this gametic expression is required for diploid sporophyte development (Figure 7). These data are consistent with a general model for the Viridiplantae whereby KNOX is supplied via the egg (equivalent to the

(-) gamete in Chlamydomonas) and BELL via the sperm (equivalent to the (+) gamete in
Chlamydomonas), and that once together in the zygote, the diploid genetic program can be activated. Thus, the basic tenants of the genetic regulation of the haploid to diploid transition elucidated in another lineage of Viridiplantae also apply to a basal lineage of land plants, consistent with the notion that such a system was present in the common ancestor of extant Viridiplantae.
While crosses with Mpknox1 females always resulted in zygotic arrest, this phenotype was seldom seen in crosses involving Mpbell234 males, but rather, in the latter case a limited amount of embryo development ensued before developmental arrest. Note that this is the opposite of what might have been expected based on ectopic expression of these genes in the vegetative gametophyte where MpBELL3 alone was sufficient to activate some diploid gene expression whereas MpKNOX1 was not (Figure 6-figure supplement 3). There exist several possible, not necessarily mutually exclusive, explanations for the discordance between these two reciprocal crosses. First, the Mpbell alleles may not be null -despite large internal deletions each still encodes an intact conserved carboxyl homeodomain (Figure 1), and perhaps a truncated MpBELL protein is sufficient for limited activity. A corollary of this possibility is that maternal transcripts at the MpBELL3 locus (Figure 5-figure supplement 1 (Marchant, 1713, Mirbel, 1835, Thuret, 1851, Unger, 1837.

Roles of MpKNOX1 in the Marchantia sporophyte
One of the key innovations of land plants was the evolution of a mutlicellular diploid generation, the embryo, via mitoses interpolated between gamete fusion and meiosis (Bower, 1908). In seed plants, ferns, and mosses KNOX1 activity is associated with continued cell proliferation, including sporophyte apical meristem activity (Hay and Tsiantis, 2010, Sakakibara et al., 2008, Sano et al., 2005. In eukaryotic lineages in which multicellularity evolved in the diploid phase of the life cycle, it is perhaps not surprising that genes involved in the haploid to diploid transition, or paralogs thereof, have been co-opted into roles directing development of the diploid generation. In the basidiomycete fungus Coprinopsis, the same HD heterodimer that initiates the diploid genetic program also directs early developmental stages in the multicellular diploid (Kamada, 2002). Most conspicuously, despite the loss of their role in zygotic gene activation, both non-TALE (e.g. Hox) and TALE HD genes act to pattern the metazoan body (Merabet and Mann, 2016, Pearson et al., 2005, Lewis, 1978. In the syncytial Chlorophyte alga Caulerpa lentillifera, differential expression of TALE-HD genes in the diploid body was speculated to influence differentiation of fronds and stolons, but functional data are lacking (Arimoto et al., 2019). Finally, in land plants, as the multicellular sporophyte evolved increasing complexity, KNOX/BELL genetic modules were co-opted to direct development of novel organs and tissues via regulation of the extent of proliferation (KNOX1) or promotion of differentiation (KNOX2) in conjunction with other gene regulatory networks (Hay andTsiantis, 2010, Furumizu et al., 2015).

Marchantia gametophyte and sporophyte TALE-HD expression is distinctly regulated
The two TALE-HD genes expressed predominantly in during sporophyte development, Of the three BELL genes (MpBELL2/3/4) expressed specifically in the antheridia during sperm differentiation, MpBELL3 and MpBELL4 are marked with H3K27me3 in the vegetative gametophyte, but MpBELL2 is not. However, none of these three genes are de-repressed in amiR-MpE(z)1 lines (Figure 5-figure supplement 1). Likewise, the three KNOX1-related genes whose expression is predominantly gametophytic are neither marked with H3K27me3 in the vegetative gametophyte nor de-repressed in amiR-MpE(z)1 lines (Figure 4, Figure 4-figure supplement 2). In contrast to MpKNOX1, MpSUK1 appears to be marked by H3K27me3 in the vegetative gametophyte, but whether the mark is sex-specific is unknown.
Collectively, these data indicate that TALE-HD genes expressed during later sporophyte development are regulated in a fundamentally different manner than the gamete-expressed TALE-HD genes. Further, de-repression of MpKNOX2 and MpBELL1 in amiR-MpE(z)1 lines implies that their activators are present in the vegetative gametophyte, while activators of the gamete-expressed TALE-HD genes are not present in this tissue.

TALE-HD genes with converse expression patterns
The two KNOX1-related sequences lacking homeodomains are reminiscent of similar, albeit independently evolved, proteins in angiosperms that have been demonstrated to act as inhibitors of KNOX/BELL function by forming inactive heterodimers primarily with BELL partners (Magnani andHake, 2008, Kimura et al., 2008).  (Figure 5-figure supplement 1), suggesting MpBELL5 may be regulated by an antisense transcript in a manner similar to MpKNOX1 (Figure 4) and MpFGMYB (Hisanaga et al., 2019). Thus, male-expressed antisense transcript-mediated repression could provide a general mechanism for female-specific expression of autosomal genes whose regulation may be linked to the feminizing-locus on the female sex chromosome (Bowman, 2016, Knapp, 1935, Lorbeer, 1936.

Marchantia BELL gene diversity resembles that of Viridiplantae algae
The phylogenetic diversity of BELL genes in M. polymorpha more closely resembles the diversity observed in charophyte and Chlorophyte algae than that of other land plants (Joo et al., 2018, Lee et al., 2008. All previously described land plant BELL genes form a single clade that evolved within the charophycean algae (Figure 2). suggesting a role in reproduction, but the function of which is unknown. Likewise, the expression pattern of MpBELL5, a member of the GLX-basal clade that evolved early in the charophytes, also suggests an as yet unresolved function in reproduction. Finally, the structural (two or three homeodomains) and extensive sequence diversity of the MpBELL2/3/4 paralogs is consistent with the rapid evolution of genes involved reproductive processes (Swanson and Vacquier, 2002).

An ancestral function for TALE-HD genes in eukaryotes
In the Viridiplantae (Chlamydomonas and Marchantia) the haploid to diploid transition is mediated by two TALE-HD genes. In the red alga Pyropia yezoensis KNOX gene expression is detected in the diploid conchosporangium, but not in haploid thalli (Mikami et al., 2019).
Since Pyropia, Chlamydomonas and Marchantia span much of the phylogenetic diversity of the Archaeplastida, the common ancestor of this group likely utilized TALE-HD proteins of the KNOX and BELL subfamilies mediate the haploid to diploid transition. The phylogenetic distribution of KNOX and BELL subfamilies and their heterodimerization affinities (Joo et al., 2018), suggests this can likely be extended to all Archaeplastida. In contrast, in both ascomycete and basidiomycete fungi the haploid to diploid transition is mediated by heterodimerzation of a TALE-HD and a non-TALE HD protein (Gillissen et al., 1992, Goutte and Johnson, 1988, Herskowitz, 1989, Hull et al., 2005, Kues et al., 1992, Spit et al., 1998, Urban et al., 1996. In the Amoebozoa Dictyostelium, the two homeodomain-like proteins controlling the haploid to diploid transition are highly divergent, rendering the phylogenetic affinities enigmatic (Hedgethorne et al., 2017). Finally, in the brown alga Ectocarpus two TALE-HD proteins, OUROBOROS and SAMSARA, mediate the transition, however, their lack of gamete specific expression raises questions concerning the precise mechanism (Arun et al., 2019). As these taxa span eukaryotic phylogenetic diversity, one role of homeodomain genes in the ancestral eukaryote was to regulate the haploid to diploid transition -the evolution of the homeodomain in the ancestral eukaryote was associated with evolution of a novel life cycle (Figure 7-figure   supplement 1). In arguably the most intensively studied taxon, the Metazoa, zygotic gene activation has been replaced by maternally derived pluripotency factors (Schulz and Harrison, 2019), but homeodomain genes have been retained. Given the ancestral eukaryote possessed a minimum of two HD genes, including one TALE-HD and one non-TALE-HD (Bharathan et al., 1997, Burglin, 1997, Derelle et al., 2007, it is an intriguing question as to whether, ancestrally, both TALE and non-TALE paralogs acted in the diploid to haploid transition. The two possible ancestral scenarios (TALE + non-TALE or TALE + TALE), require either a co-option of a pre-existing paralog (e.g. non-TALE), or alternatively, a TALE gene duplication and transference of function to the new paralog, respectively. To resolve the ancestral condition broader phylogenetic sampling and functional analyses across additional unicellular Bikont eukaryotic lineages, particularly in the Excavata, and the paraphyletic sister groups of the Metazoa (Choanoflagellates, Filasterea, and Ichthyosporea) might be informative (Figure 7-figure supplement 1). Resolution of the ancestral condition could inform whether primary ancestral function of both TALE-HD and non-TALE-HD was regulating the haploid to diploid transition or whether the non-TALE-HD genes had another fundamental function in ancestral eukaryote.

Artificial microRNA design
We used the same amiR as described in Flores-Sandoval et al. (Flores-Sandoval et al., 2016).

Ecotypes, plant growth, transformation and induction
M. polymorpha ssp ruderalis, ecotype BoGa obtained from the Botanical Garden of Osnabrueck, Germany was used in SZ lab. Plant cultivation, transformation and induction was according to Ishizaki et al. (Ishizaki et al., 2008) and Althoff et al. (Althoff et al., 2014). M.
polymorpha ssp ruderalis, ecotype MEL was used in JLB lab and grown, transformed and induced according to Ishizaki et al. (Ishizaki et al., 2008) and Flores-Sandoval et al. (Flores-Sandoval et al., 2015). In case of double transformations, sporelings were co-transformed using two constructs featuring different selectable markers. Spores were drained and plated on selection media for approximately 2 weeks and then subjected to a second round of selection before being transferred to ½ B5 plates. For induction of reproductive organs, plants were transferred to white light supplemented with far red light (735 nm; 45 lmol/m2/s) on ½ B5 media supplemented with 1 % glucose.
Genotyping, RNA extraction, cDNA synthesis and sqRT-PCR DNA was extracted using a modified protocol from Edwards et al. (Edwards et al., 1991).
Instead of vacuum drying, the pelleted DNA was air-dried. Amplicons were directly sequenced.
Complete or partial coding nucleotide sequences were manually aligned as amino acid translations using Se-Al v2.0a11 for Macintosh (http://tree.bio.ed.ac.uk/software/seal/). We excluded ambiguously aligned sequence to produce an alignment of 228 nucleotides (76 amino acids) for 82 BELL sequences (see Figure 2-figure supplement 1). Alignments of KNOX genes included the homeodomain, MEINOX (KNOX) and ELK domains (Joo et al., 2018), comprising 606 nucleotides (202 amino acids) for 103 KNOX sequences. Alignments of nucleotides and amino acids were employed in subsequent Bayesian analysis. Bayesian phylogenetic analysis was performed using Mr. Bayes 3.2.1 . Analyses were performed on both the nucleotide and amino acid alignments. The fixed rate model option JTT + I was used based on analysis of the alignments with ProTest 2.4 (Abascal et al., 2005). The Bayesian analyses for the nucleotide data sets were run for 5,000,000 (BELL) or 1,000,000 (KNOX) generations, which was sufficient for convergence of the two simultaneous runs (BELL, 0.0418; KNOX, 0.0416). Analyses for the amino acid data sets were run for 4,000,000 (BELL) or 2,000,000 (KNOX) generations, which was sufficient for convergence of the two simultaneous runs (BELL, 0.037; KNOX, 0.039). In both cases, to allow for the burn-in phase, 50% of the total number of saved trees was discarded.
The graphic representation of the trees was generated using the FigTree (version 1.4.0) software (http://tree.bio.ed.ac.uk/software/figtree/). Sequence alignments and command files used to run the Bayesian phylogenetic analyses provided upon request.

RNA-seq
G2 gemmae were grown under normal growth conditions (23°C, 24h white light) on ½ B5 Gamborg's media for 10 days, and then transferred via forceps to plates containing 5μM 17-βestradiol. Plants were sampled at specific time points and immediately frozen in liquid nitrogen, then ground up using mortal and pestle. RNA was extracted from samples at time point 0 (not induced), plus 5 time points (

ChIP-seq
Wild-type and proEF1:XVE>>amiR-MpE(z) SkmiR166 G2 were grown on ½ B5 media for 2 weeks. a vacuum for 10 minutes. After addition of 1.6mL 2.5M Glycine, sample was mixed for an additional 5 minutes in a vacuum. Following centrifugation (10 minutes at 2500rpm, 4°C) the pellet was resuspended in ice-cold diH2O, and centrifugation was repeated with an ice-cold diH2O wash. Pellet was freeze dried for 30 minutes and stored at -80˚C

Induction of
Chromatin was prepped by resuspending freeze dried pellet in 5mL nuclei isolation buffer, 5µL protease inhibitor cocktail (PIC) and 50µL 0.1M PMSF. Samples were incubated on ice for 10 minutes and subsequently homogenized by 20 strokes of douncing. Following centrifugation (10 minutes at 2500rpm, 4°C) the pellet was resuspended in 500µL DOC sonication buffer, 5µL PIC and 5µL 0.1M PMSF, and incubated on ice for 10 minutes. Samples were sonicated for 10 minutes in 30 second pulses in the presence of glass beads. Following centrifugation, the supernatant was aliquoted and stored at -80˚C.
Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, proteinase K and heat for de-crosslinking, followed by ethanol precipitation. Pellets were resuspended and the resulting DNA was quantified on a NanoDrop spectrophotometer.
Extrapolation to the original chromatin volume allowed quantitation of the total chromatin yield. An aliquot of chromatin (6 ug) was precleared with protein A agarose beads (Invitrogen).
Genomic DNA regions of interest were isolated using 12 ug of antibody against H3K27me3 (Millipore, 07-449, Lot 2653203). Complexes were washed, eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65˚C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation.
Illumina sequencing libraries were prepared from the ChIP and Input DNAs by the standard consecutive enzymatic steps of end-polishing, dA-addition, and adaptor ligation. After a final PCR amplification step, the resulting DNA libraries were quantified and sequenced on Illumina's NextSeq 500 (75 nt reads, single end). Reads were aligned to the M. polymorpha v3.1 genome using the BWA algorithm (v0.6.1-r104; default settings; (Li and Durbin, 2009)).

Data repository
The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus (Edgar et al., 2002) and are accessible through GEO Series accession number GSE147756 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE147756).