Plant materials and growth conditions

Male and female accessions of Marchantia polymorpha L., Takaragaike (Tak)-1 and Tak-2, respectively, were used as wild-type plants38. Plants were cultured on half-strength Gamborg’s B5 medium containing 1% sucrose and 1.3% agar under continuous light condition from white fluorescent tubes (50 to 60 µmol m−2 s−1) at 22 °C. To induce the sexual reproduction, thalli developed from gemmae on half-strength Gamborg’s B5 medium were transferred to soil under continuous white light supplemented with far-red light irradiation39. Arabidopsis thaliana plants were grown on soil in growth chambers at 24 °C under continuous illumination. duo1-4/+ has been described previously12. Male plants of Haplomitrium minioides were collected in Higashi-Hiroshima City (Hiroshima Prefecture, Japan) on 24 May, 2012. Plants of Chara braunii, C. australis, and C. leptospora (=C. globularis40,41) that have been maintained in culture in Sakayama lab (Kobe University, Japan)41 were used. The strains of heterothallic Closterium peracerosum-strigosum-littorale complex used in this work were NIES-67 (mt+) and NIES-68 (mt-), which were obtained from the National Institute for Environmental Studies, Ibaraki, Japan. The respective vegetative cells were cultured in nitrogen-supplemented medium (C medium; http://mcc.nies.go.jp/02medium-e.html#c) under a 16 h light/8 h dark cycle.

Plasmid constructions and plant transformation

The genome regions of MpDUO1 and MpMID used for plasmid constructions in this study are shown in Supplementary Figs. 1b and 6b, respectively. To generate the Mpduo1-1ko and Mpmid-1ko, the upper and lower arms were amplified from Tak-1 genomic DNA and were cloned into the PacI and AscI sites, respectively, of the pJHY-TMp1 vector42. To generate the MpDUO1-Citrineki, the upper and lower arms were amplified from Tak-1 genomic DNA and were cloned into the AscI and PacI sites, respectively, of pJHY-TMp1-Cit in which a Citrine ORF was cloned into the HindIII site of pJHY-TMp1 vector. The resultant plasmids were introduced into sporelings derived from crosses between Tak-1 and Tak-2. Screening for gene-targeted lines was performed by genomic PCR42. A single gemma from each T1 line was isolated to establish the line.

To generate Mprkd mutants in MpDUO1-Citrineki background, RKD locus was edited using the CRISPR-Cas9 system43. Candidates were selected on the basis of gemma cup phenotype32,33 and PCR products with genomic DNA as the template were analyzed for each candidate to confirm mutations. Several male mutant lines with phenotypes in antheridia32 were chosen for analysis of MpDUO1-Citrine expression.

To construct proMpDUO1:GUS, the genomic fragment of the upstream region of MpDUO1 (proMpDUO1-1) was amplified from Tak-1 genomic DNA and cloned into the EcoRI site of pENTR1A vector (Life Technologies) and then transferred to pMpGWB30444 through the Gateway technology (Life Technologies). The resultant plasmid was introduced into regenerated thalli of Tak-1 and Tak-2. To construct proMpDUO1:AtDUO1, the 5′-upstream region (proMpDUO1-2) and 3′-downstream region (MpDUO1 3′ region) of MpDUO1 were amplified from Tak-1 genomic DNA. The proMpDUO1-2 was cloned between SalI and BamHI sites and MpDUO1 3′ region was cloned in EcoRV site, respectively, of pENTR1A to generate pENTR1A_proMpDUO1_3′ MpDUO1. AtDUO1 ORF was inserted between BamHI and NotI sites of pENTR1A_proMpDUO1_3′ MpDUO1 and the proMpDUO1:AtDUO1:3′MpDUO1 fragment was transferred into pMpGWB30144 through the Gateway technology. To construct proMpDUO1:MpDUO1 for MpDUO1-1ko complementation, the genomic fragment containing the 5′-upstream region and coding region of MpDUO1 was amplified from Tak-1 genomic DNA. The fragment was cloned into pDONR221 (Life Technologies), then into pMpGWB10144 through the Gateway technology (Life Technologies). The resultant plasmids were introduced into regenerated thalli of Mpduo1-1ko. T1 lines were selected on the half-strength Gamborg’s B5 medium containing 0.5 µM chlorsulfuron and a single gemma from each T1 line was isolated to establish the line.

For Arabidopsis complementation assay in Atduo1-4/+, proAtDUO1:AtDUO1-Clover, proAtDUO1:MpDUO1-Clover and proAtDUO1:Chimera1 were inserted into the multisite Gateway T-DNA destination vector pAlligatorR4345 through the Gateway technology (Life Technologies). AtDUO1 cDNA synthesized from Col-0 pollen RNA as well as MpDUO1 cDNA synthesized from Tak-1 young antheridiophore RNA were cloned into pDONR221 with attB1 and attB2 sites. The additional Gateway technology vectors used in this study are pENTR_2-r3_Clover45, pENTR_4-1r_proAtDUO19,10, and pAlligatorG43_proHTR10:HTR10-mCherry45.

For promoter conservation assay, proMpDUO1:H2B-Clover and proAtDUO1:H2B-Clover were inserted into the multisite Gateway T-DNA destination vector pAlligatorR43 and pAlligatorG4345, respectively. Promoters of MpDUO1 (proMpDUO1-2) and AtDUO1 were cloned into pDONR-P4P1r (Life Technologies). pENTR221-H2B vector was previously described45. The mRuby2 clone (Addgene) was introduced into pDONR-P2rP3 (Life Technologies).

For transient luciferase assay, the firefly and Renilla luciferase constructs built with pK7m24GW346 and pB2GW747, respectively, were used9. Chimera 1 to 7 DNA fragments with attB1 and attB2 sites were generated by fusion PCR method48, and then cloned into pDONR221 vector (Life Technologies) through the Gateway technology. These entry clones as well as At DUO1 and Mp DUO1 cDNA entry clones were then recombined into pB2GW7 to generate Pro35S constructs for the transient luciferase assay.

For the protein-binding DNA microarray assay, synthesized Escherichia coli-codon-optimized fragments (ThermoFisher Scientific) encoding MYB domains of AtDUO1, MpDUO1, Chimera 4, 5, 6 as well as MpR2R3-MYB21 and KflMYB were cloned into pDONR221 and then transferred to the destination vector pMAL-C2 vector (New England Biolabs) through Gateway technology, generating the in-frame fusion of Maltose Binding Protein (MBP) and MYB DNA-binding domain.

Transgenic Arabidopsis plants were generated using the floral dip method49 and T1 transgenic plants were screened based on each selection marker of the construct. All oligonucleotides and synthesized fragment sequences used for this study are listed in Supplementary Table 3.

Histochemical GUS staining

Histochemical staining for GUS activity was performed by a common procedure50 with some modifications. The thalli, antheridiophores, archegoniophores, and sporophytes of proMpDUO1:GUS-expressing plants were fixed in 90% (v/v) acetone, vacuum-infiltrated and incubated at 37 °C overnight in the GUS assay solution containing 100 mM sodium phosphate buffer (pH 7.2), 5 mM potassium-ferrocyanide, 5 mM potassium-ferricyanide, 0.1% (v/v) Triton X-100 and 0.5 mg ml−1 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (X-Gluc). Chlorophyll in the tissue were removed by incubation in 70% (v/v) ethanol.

RNA in situ hybridization

Probe and tissue preparation and hybridization were performed as described14. A probe fragment for each gene was amplified from Tak-1 cDNA with a set of gene-specific primers (Supplementary Table 3) and was cloned into the pCR-BluntII-TOPO vector (Life Technologies). DIG-labeled anti-sense and sense RNA probes were synthesized with a DIG RNA Labeling kit (SP6/T7) (Roche) according to the manufacturer’s instructions. Antheridiophore receptacles of Tak-1 at stage 3 to 414 were fixed in a solution containing 3% (w/v) paraformaldehyde and 0.25% glutaraldehyde in 0.1 M phosphate buffer, pH 7.0, and 0.05% Triton X-100, dehydrated and embedded in paraffin. Eight-µm sections were made with a microtome, applied to an APS-coated glass slide, and then deparaffinized and rehydrated. They were treated with 1 µg ml−1 of proteinase K (ThermoFisher Scientific) in 100 mM Tris-HCl, pH 8.0, 50 mM EDTA, pH 8.0 at 37 °C for 30 min, subsequently fixed in 4% (w/v) paraformaldehyde in PBS (7 mM Na 2 HPO 4 , 3 mM NaH 2 PO 4 , 130 mM NaCl) for 10 min, and treated with 0.5% (v/v) acetic anhydride in 100 mM triethanolamine for 10 min. Sections were incubated at 55 °C for 2 h in pre-hybridization buffer [50% formamide (w/v), 5 × SSC, 40 µg ml−1 salmon sperm DNA] and then in the same buffer with 100 ng of probes for more than 16 h. After treatment with 50 µg ml−1 of RNase A (SIGMA) in RNase buffer (10 mM Tris-HCl, pH 8.0, 500 mM NaCl, 1 mM EDTA) at 37 °C for 30 min, sections were washed in 0.2 × SSC (30 mM NaCl, 3 mM sodium citrate, pH 7.0) at 55 °C for 1 h, incubated with 1% (w/v) Blocking Reagent (Roche) in buffer 1 (100 mM Tris-HCl, pH 7.5, 150 mM NaCl) for 1 h and then incubated with 1/1000 diluted Anti-Digoxigenin-AP, Fab fragments (Roche) with buffer 1 containing 1% (w/v) blocking reagent for 1 h. The slides were subsequently washed three times with buffer 1 for 10 min each, rinsed with buffer 2 (100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 50 mM MgCl 2 ) for 5 min and then covered with NBT/BCIP (Roche) diluted 1/125 in buffer 2. After incubation at 22 °C for more than 16 h in the dark, the reaction was stopped by immersing the slides in TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).

Confocal microscopy

The accumulation of MpDUO1-Citrine was observed in isolated antheridia and sperm of MpDUO1-Citrineki plant. The Citrine fluorescence was detected in a range from 525 to 565 nm with confocal laser scanning microscopy (FV-1000; Olympus and LSM780; Zeiss) after excitation at 515 nm. An antheridiophore of the MpDUO1-Citrineki plant was hand-sectioned with a blade and immersed in 1 μg ml−1 4′,6-diamidino-2-phenylindole (DAPI) solution. Antheridia were isolated from the sections and observed immediately under a confocal microscope (FV-1000, Olympus) with the following setting: MpDUO1-Citrine was excited by 488-nm laser and detected by a GaAsP detector with 535−565-nm window; DAPI was excited by 405-nm laser and detected by a photomultiplier tube with 425−475-nm window.

Clover and mRuby2 in the transgenic Arabidopsis pollens were excited at 488 and 561 nm, respectively and detected in a range from 495 to 540 and from 566 to 600 nm, respectively, with confocal laser scanning microscopy (LSM780; Zeiss). In brief, 3-4 open flowers were collected in a microfuge tube containing 300 μl of the solution [0.1 M sodium phosphate (pH 7.0), 1 mM EDTA, 0.1% Triton X-100]. After brief vortexing and centrifugation, 15 μl of the pollen pellet was transferred to a microscope slide and imaged12.

Feulgen staining of antheridia

Feulgen staining51 was performed with some modifications. The antheridiophores receptacles of Tak-1 at stage 514 were fixed overnight in 3:1 mixture of ethanol and glacial acetic acid at 4 °C overnight. After fixation, the equal volume of ethanol was added and the samples were incubated for 1 h. Then the samples were hydrated in graded ethanol solutions. The samples were rinsed three times with distilled water for 15 min each and hydrolyzed in 5 N HCl for 1 h. After hydrolysis, the samples were rinsed three times with distilled water for 5 min each and stained with Schiff’s reagent (Sigma-Aldrich) for 2 h. The samples were rinsed twice for 15 min each with distilled water and were dehydrated in graded ethanol solutions and 100% ethanol. The 100% ethanol was exchanged with the fresh one at hourly intervals until it remained colorless after the exchange. Then LR White resin (Sigma-Aldrich) was added to make a 1:1 mixture of 100% ethanol and LR White and the samples were left for 1 h at room temperature. Then the mixture was replaced with pure LR White and left at room temperature overnight. The antheridia were manually dissected from antheridiophores and placed in fresh LR White on a standard glass microscope slide and a cover glass was gently lowered over the antheridia in LR White. The samples were incubated at 60 °C overnight. The cover glass can be carefully removed from the polymerized LR White. The fluorescence was detected at 535 nm and longer with LSM510 META Confocal Imaging System (Zeiss) after excitation at 488 nm by an argon laser.

Nuclear shape quantification

Fiji package52 was used to quantify shapes [circularity (4π*area/perimeter2), aspect ratio (major_axis/minor_axis), and solidity (area/convex_area)] of Feulgen-stained Marchantia antheridia nuclei obtained from Z-projected confocal images. In brief, nuclear images were smoothened through Gaussian blur function, followed by threshold adjustment to capture the shape of nuclei. Nuclear images which are either partial or overlapping with others were excluded from quantification.

TEM analysis of antheridia

The antheridiophore receptacles of Tak-1 at stage 414 were fixed with 2% each of paraformaldehyde and glutaraldehyde in 0.05 M cacodylate buffer pH 7.4 at 4 °C overnight. After fixation, the samples were washed three times with 0.05 M cacodylate buffer for 30 min each, and were post-fixed with 2% osmium tetroxide in 0.05 M cacodylate buffer at 4 °C for 3 h. The samples were dehydrated in graded ethanol solutions then in 100% ethanol. The samples were infiltrated with propylene oxide twice for 30 min each and were put into a 7:3 mixture of propylene oxide and resin, Quetol-651 (Nissin EM Co.) for 1 h, then the mixture was kept in a tube without cap overnight to volatilize propylene oxide. The samples were transferred to a fresh resin and were polymerized at 60 °C for 48 h. The polymerized resins were ultra-thin sectioned at 80 nm with a diamond knife using Ultracut UCT (Leica) and the sections were mounted on copper grids. They were stained with 2% uranyl acetate at room temperature for 15 min, and then they were washed with distilled water followed by being secondary-stained with Lead stain solution (Sigma-Aldrich) at room temperature for 3 min. The grids were observed by JEM-1400Plus (JEOL Ltd.) at an acceleration voltage of 80 kV. Digital images were taken with a CCD camera, VELETA (Olympus).

Immunofluorescence staining

The fixation and permeabilization of antheridia were carried out essentially according to previous report53. Isolated spermatid cells were incubated with a polyclonal anti-centrin antibody against a recombinant protein corresponding to a full-length centrin from a brown alga Scytosiphon lomentaria (a gift from Dr. Taizo Motomura, Hokkaido University, Japan) diluted 1:500 with PBS [137 mM NaCl, 2.68 mM KCl, 8.1 mM Na 2 HPO 4 , and 1.47 mM KH 2 PO 4 , pH 7.4] in a moist chamber for 90 min at 37 °C. After a PBS wash, incubation with a monoclonal anti α-tubulin (T5168, Sigma) diluted 1:500 with PBS was performed for 60 min at 37 °C. After a wash again with PBS, the samples were incubated for 60 min at 37 °C with an equal mixture of Alexa 488-conjugated goat anti-rabbit IgG (H + L) (A11034, Invitrogen) diluted 1:200 with PBS and Alexa 568-conjugated goat anti-mouse IgG (H + L) (A11031, Invitrogen) diluted 1:200 with PBS. After washing with PBS for 10 min, the nuclei were stained with 1 μg ml−1 DAPI in PBS. Slides were mounted using Vectashield (Vector Laboratory) and observed using a confocal laser scanning microscope (FV-1000, Olympus).

Quantitative RT-PCR analysis

Total RNA was extracted with an RNeasy Plant Mini Kit (QIAGEN) according to the manufacturer’s protocol and the quality and quantity of the resultant RNA were evaluated using a NanoDrop 2000c spectrophotometer (ThermoFisher Scientific). One μg of total RNA was reverse-transcribed in a 20 μl reaction mixture using Transcriptor (Roche). After the reaction, the mixture was diluted with 180 μl of distilled water and 2 μl aliquots were used for PCR in a 10 μl PCR reaction mixture containing 1 μl of 10× Ex Taq buffer, 1 μl of 2 mM dNTPs, 0.4 μl of 10 μM each of primers, and 0.05 μl of Ex Taq DNA polymerase (Takara) for semi-quantitative RT-PCR analysis. The PCR products were separated on a 2.5% (w/v) agarose gel, stained with ethidium bromide, and visualized under UV light. The primers used in these experiments are listed in Supplementary Table 3. For quantitative RT-PCR analysis, the cDNA samples were diluted with 220 µl of distilled water and 2 μl aliquots were amplified with the CFX96 Real-time PCR Detection System (Bio-Rad) using SYBR Premix Ex Taq (Tli RNaseH Plus) (Takara). The two-step PCR cycling program was performed according to manufacturer’s protocol. The primers used in these experiments are listed in Supplementary Table 3. MpACT1 was used as an internal control.

Genetic complementation assay on Atduo1 and Mpduo1-1 ko

For Arabidopsis complementation assay, mature pollen grains from WT and T3 stable homozygous complemented lines (AtComp, MpComp #3 and #4, and Chimera 1 #1 and #2) were examined by fluorescence microscopy. The frequency of bicellular and tricellular pollen grains was determined by scoring the number of respective pollen grains by DAPI staining. The ability of H3.10 activation was assessed by counting the frequency of pollen grains expressing mRuby2 fluorescence marker in sperm (proAtHTR10:HTR10-mRuby2).

For Marchantia complementation assay, the discharged sperm was directly observed without fixation on a Miniscope TM3000 (HITACHI, Japan) to obtain scanning electron microscopic images. To observe the nuclear morphology of sperm, the discharged sperm were stained with DAPI and observed under a confocal microscope (FV-1000, Olympus) with the following setting: DAPI was excited by 405-nm laser and detected by a photomultiplier tube with 425−475-nm window. Movies of discharged sperm were taken under a microscope BX43 (Olympus) with a dry dark-field condenser U-DCD (Olympus). For quantitative motility analysis, sperm discharged in water was observed under a phase-contrast/DIC microscope and video-recorded at the resolution of 1216 × 960 pixels and at the rate of 7 frames per second (fps) for 10 s by a microscope camera DP26 (Olympus). To track individual sperm, an open-source software ImageJ (ver. 1.51n, the Fiji distribution)52 was used. Movies of sperm were first converted into a sequence of individual frames. Their green-channel images were extracted and converted into 8-bit gray-scale, and their black and white values were inverted. To reduce stationary objects in the background, each image was subtracted with a Z projection of the entire frames. Moving paths of sperm were detected by TrackMate ver. 3.4.254, a plugin bundled with the Fiji distribution of ImageJ, with the following modified parameters: initial threshold, auto; linking max distance, 50 pixel; gap-closing max distance, 50 pixel. Tracks with duration of at least 3 s, or 21 frames, were selected and the length of each track was measured to calculate average speed in µm s-1. Three movies were analyzed to obtain sperm swimming velocity for each genotype, and the total numbers of sperm observed are: n = 132 (WT), n = 193 (MpComp #5), n = 14 (AtComp #9), n = 12 (AtChimera #13), n = 22 (CbrChimera #1) for analysis of fraction of motile sperm; n = 117 (WT), n = 67 (MpComp #5), n = 10 (AtComp #9), n = 8 (AtChimera #13), n = 12 (CbrChimera #1) for analysis of swimming velocity.

To test the fertilization ability, mature antheridiophores were immersed in water and aliquots of sperm suspension were observed for discharged sperm and deposited onto archegoniophore receptacles of Tak-2. Sporophyte and sporangium development was observed about a month after crossing.

Protein-binding DNA microarray assay

Recombinant plasmids harboring MBP-MYB fusions were introduced into the BL21 strain of E. coli, and the expression of recombinant proteins was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 6 h at 25 °C. Pellets corresponding to 25 ml of induced E. coli culture for each construct were stored at −80 °C and resuspended in 1 ml 1× binding buffer prior to DNA-binding assay20. Bacterial lysates were sonicated twice for 30 s, and centrifuged twice at 20,000×g to obtain cleared extracts of soluble proteins.

Second strand of DNA was synthesized in a primer extension reaction with 32 U Thermo Sequenase Polymerase (USB), 163 μM dNTPs, 1.63 μM Cy5-dUTP (GE Healthcare) and 1.17 μM oligonucleotide primer (5′-CAGCACGGACAACGGAACACAGAC-3′) in a 900 μl total volume reaction. DNA microarray was incubated with the mixture in a hybridization oven for 10 min at 85 °C, the temperature gradually reduced up to 60 °C during 30 min and hold at this temperature for 90 min. The slide was then rapidly transferred to wash solution (1× PBS, 0.01% Triton X-100), incubated at 37 °C for 10 min with agitation and rinsed in 1× Phosphate Buffered Saline (PBS) for 3 min at room temperature. The slide was spun dry by centrifugation (1 min at 500 rpm) and scanned at 2 μm resolution in a Agilent’s DNA Microarray Scanner for monitoring the amount of dsDNA. The binding mixture obtained from cleared bacterial lysates was adjusted to 175 μl and to contain 2% milk and 0.89 μg of denatured salmon sperm DNA (ssDNA). Double stranded DNA microarray was incubated with the binding mixture in a humid chamber for 2.5 h at room temperature. Slides were then washed three times PBS-1% Tween 20 (5 min), three times in PBS-0.01% Triton X-100 (5 min) and spun dry by centrifugation. DNA–protein complexes were incubated with 16 μg of Rabbit polyclonal to Maltose Binding Protein (Abcam) in PBS-2% milk for 16 h at room temperature. Slides were washed 3× in PBS-0.05% Tween 20, 3× in PBS-0.01% Triton X-100 (5 min each wash) and dried. Labeling of DNA–protein complexes were performed by incubating the microarrays with 0.4 μg of goat anti-rabbit IgG DyLight 549 conjugated (Pierce) in PBS-2% milk for 3 h at room temperature, followed by the same washes as before and the slides dried for scanning.

We used the nPBM11 design containing 167,773 different oligonucleotide probes19 synthesized in an Agilent’s SurePrint G3 4 × 180k format (Agilent Technologies). DNA microarrays were scanned in a DNA Microarray Scanner at 2-μm resolution and quantified with Feature Extraction 9.0 software (Agilent Technologies). Normalization of probe intensities and calculation of E- and Z-scores of all the possible 8-mers were carried out with the PBM Analysis Suite55. Perl scripts were modified to adapt them to nPBM11 microarray dimensions and to input files generated by Feature Extraction software.

Transient luciferase assay

Agrobacterium-mediated transient transformation of Nicotiana tabacum leaf was carried out as described by Sparkes et al.56 modified as detailed below. Agrobacterium strains were combined as required at an OD600 of 0.1 for reporter and effector vectors and an OD600 of 0.02 for the Renilla luciferase control vector in infiltration media (280 mM d-glucose, 50 mM MES, 2 mM Na 3 PO 4 ·12H 2 O, 0.1 mM acetosyringone). 4–6-week-old Nicotiana tabacum plants were grown in greenhouse conditions. Agrobacterium suspensions were taken up in 1 ml syringes and the underside of leaves gently rubbed to remove a small region of the cuticle. The syringe tip was placed at these regions and Agrobacterium suspensions were gently infiltrated. Plants were placed in a growth chamber under normal growth conditions and left for 2 days. Each leaf disc from an infiltrated region was excised using a 9 mm cork-borer and ground to homogeneity in 300 μl of 1× Passive Lysis Buffer (Promega) in a chilled mortar and pestle. Leaf extracts were centrifuged at 16,000×g for 5 minutes at 4 °C to pellet cell debris. Two separate 25 μl aliquots were assayed separately for firefly and Renilla luciferase activities in 100 μl of the respective assay buffer. The firefly luciferase assay buffer (25 mM glycylglycine, 15 mM KPO 4 pH 8.0, 4 mM EGTA, 2 mM ATP, 1 mM DTT, 15 mM MgSO 4 , 0.1 mM CoA, 75 μM luciferin with final pH adjusted to 8.0) and Renilla luciferase assay buffer (1.1 M NaCl, 2.2 mM Na 2 EDTA, 0.22 M KPO 4 pH 5.1, 0.44 mg/ml BSA, 1.43 μM coelenterazine with final pH adjusted to 5.0) were prepared immediately before measurement57. Luminescence was measured in white 96-well plates with a FLUOstar Omega (BMG LABTECH Ltd) microplate reader as relative luminescence units (RLUs) integrated over 10 seconds. Extracts of discs taken from non-infiltrated leaves were assayed to determine mean background RLU values, which were subtracted from those for extracts of infiltrated leaves. Normalized luciferase activity (FLuc/RLuc) was calculated for each extract from the ratio of background subtracted RLUs obtained in firefly luciferase (FLuc) and Renilla (RLuc) luciferase assays.

Sequences, alignment, and phylogenetic tree construction

Sequences of Klebsormidium MYB TFs and DUO1 MYB TFs of conjugating green algae were obtained from published data58,59,60. The 5′ and 3′ parts of CpeDUO1 cDNA fragment were amplified separately by PCR from cDNA from conjugation induced early stage. Sequences of land plant DUO1, DAZ1, GCS1/HAP2, and GEX2 were obtained from www.phytozome.jgi.doe.gov. Amino acid sequence alignment illustrated in Supplementary Figure 1a was generated by CLC Workbench 7 package (QIAGEN).

RNA preparation and RNA-seq analysis

For the preparation of total RNA from vegetative cultures of Closterium peracerosum-strigosum-littorale complex, mating-type (mt)+ and mt− cells were harvested at 0 (start time of light illumination), 6, 12, and 18 h, respectively. For the preparation of total RNA from mating cultures, vegetative growing cells of the mt+ and mt− were collected, washed three times with nitrogen-depleted medium (MI medium)61, and incubated separately in MI medium (3.6 × 105 cells/72 ml in 300-ml Erlenmeyer flasks) under continuous light for 2, 8, and 24 h (gamete induced mt+ and mt-, respectively). The cells of both mating types, which had been separately cultured in MI medium (at 3.6×105/72 ml in 300-ml Erlenmeyer flasks) for 24 h, were mixed and co-incubated (at 3.6 × 105 each/72 ml in 300-ml Erlenmeyer flasks) for 1, 2, 4, 6, 8, 12, 16, 20, and 24 h (conjugation induced early) and for 48, 72, 96 h (conjugation induced late). For the preparation of total RNA from germinated zygote, zygotes were dried once and incubated in C medium for 12, 24, 48, 72 h under a 16 h light/8 h dark cycle (germinated). The harvested cells were frozen in liquid nitrogen and total RNA was isolated using TRIZOL Plus Kit (Invitrogen, Carlsbad, CA, USA), in accordance with supplier instructions. Paired-end libraries were generated with TruSeq RNA Sample Preparation Kit (Illumina, www.illumina.com), according to manufacturer’s instructions. Sequencing was carried out 76 bps with a Genome Analyzer IIx using standard reagents. All high-quality sequences were de novo assembled with Trinity62. Expression frequency of the contigs was calculated by RSEM63, using all high-quality sequences.

DUO1 DNA-binding motif search

The DUO1 consensus DNA-binding motif (5′-RRCSGTT-3′) generated in this study was used to search the upstream regions of AtDAZ1, AtGCS1/HAP2, and AtGEX2 homologs of some angiosperms, a lycophyte, and some bryophytes by using the dna-pattern tool from Regulatory Sequence Analysis Tools (RSAT) web server (http://www.rsat.eu/)64 with default parameters. Up to 2-kb upstream regions between the putative transcriptional start site and the upstream neighboring gene were collected from Phytozome ver.11 (https://phytozome.jgi.doe.gov/pz/portal.html). The genes used in this experiment are listed in Supplementary Table 4.

Selection pressure analyses

Branch model tests were performed using codeML in PAML65 on DUO1 and S18 MYB domain sequences from genes in Supplementary Table 415,58,59. Default parameters were used, to the exception of cleandata = 0 to prevent removal of codon information around region B. Likelihood ratio tests were performed between models and chi-squared tests were performed to assess statistical significance. Maximum-likelihood tree shown in Supplementary Figure 4 was generated in PhyML 3.166 using the LG + I + G protein substitution model, selected according to ProtTest 367. Full results are listed in Supplementary Table 2.

RT-PCR and Southern hybridization analysis

Gene expression analysis of three Chara species was performed using standard procedures40 with the following modifications. A probe fragment for each gene was amplified from cDNA of each species with a set of gene-specific primers (Supplementary Table 3) and was cloned into the pCR-BluntII-TOPO vector (Life Technologies), which contains SP6 and T7 polymerase-binding sites. RT-PCR was performed with a set of gene-specific primers (Supplementary Table 3). Probe synthesis, hybridization, washing, and detection were performed by using DIG High Prime DNA Labeling and Detection Starter Kit II (Roche) according to the manufacturer’s protocol. Hybridization was performed at 55 °C.