Genomic comparison of two independent seagrass lineages reveals habitat-driven convergent evolution

Genomic comparison of two independent seagrass lineages reveals habitat-driven convergent evolution Abstract Seagrasses are marine angiosperms that live fully submerged in the sea. They evolved from land plant ancestors, with multiple species representing at least three independent return-to-the-sea events. This raises the question of whether these marine angiosperms followed the same adaptation pathway to allow them to live and reproduce under the hostile marine conditions. To compare the basis of marine adaptation between seagrass lineages, we generated genomic data for Halophila ovalis and compared this with recently published genomes for two members of Zosteraceae, as well as genomes of five non-marine plant species (Arabidopsis, Oryza sativa, Phoenix dactylifera, Musa acuminata, and Spirodela polyrhiza). Halophila and Zosteraceae represent two independent seagrass lineages separated by around 30 million years. Genes that were lost or conserved in both lineages were identified. All three species lost genes associated with ethylene and terpenoid biosynthesis, and retained genes related to salinity adaptation, such as those for osmoregulation. In contrast, the loss of the NADH dehydrogenase-like complex is unique to H. ovalis. Through comparison of two independent return-to-the-sea events, this study further describes marine adaptation characteristics common to seagrass families, identifies species-specific gene loss, and provides molecular evidence for convergent evolution in seagrass lineages. Gene loss, Halophila ovalis, marine adaptation, NDH complex, osmoregulation, seagrass, Zostera muelleri Introduction Seagrasses are a polyphyletic group of flowering plants that live fully submerged in the marine environment and form monospecific meadows resembling terrestrial grasses. The morphology of seagrasses varies among species, though common features include long, strap-shaped leaves and simple flowers. Seagrasses belong to a basal lineage that diverged around 140 million years ago (Mya), before the divergence of the Poaceae within the monocotyledon clade. Although similar in form, seagrass species represent at least three independent return-to-the-sea events (Les et al., 1997). The convergent evolution of seagrasses is characterized by common physiological and morphological features that possibly represent a collection of marine adaptation traits. For example, seagrass leaves lack stomata, and gas exchange occurs through permeable cuticles, while seagrass roots and rhizomes have aerenchyma to enhance gas transport. Seagrasses have also adapted to variable quality and low levels of light, which attenuates quickly in seawater (Larkum et al., 2006; Strydom et al., 2017) and have effective osmoregulation to survive in the saline aqueous environment (Koch et al., 2007; Touchette, 2007). Seagrasses are adapted to aquatic reproduction, where the transport and capture of pollen grains is carried out on or below the water surface. Current seagrass taxonomy contains around 72 species forming three families, Zosteraceae, Hydrocharitaceae and Cymodoceaceae complex (Les et al., 1997; Short et al., 2011; Nguyen et al., 2015). Recent genome-wide comparative studies of two species in the Zosteraceae provided the first insight into genomic adaptation to the marine environment (Golicz et al., 2015; Lee et al., 2016; Olsen et al., 2016). Genes associated with the synthesis and signalling of volatile substances, including ethylene, methyljasmonate, and terpenoids, were lost in both Zostera muelleri and Z. marina. Genes associated with morphological adaptation, including those for stomatal cell differentiation, flower development and pollen formation, were also absent or greatly reduced in number. An increase in gene families associated with low light harvesting and cell wall modification was observed and postulated to contribute to survival in the light-attenuated and high salinity environment. These gene losses, gene modifications, and gene family expansions in the two Zostera species may not reflect the independent adaptation of other seagrass lineages to the marine environment, and analysis of a second lineage is required to answer the question whether they share a common adaptation pathway to the ocean. Halophila ovalis is a seagrass species in the family Hydrocharitaceae, and is an ideal model for comparison with the Zostera species. The seagrass subclade in Hydrocharitaceae is embedded within branches of largely diverse aquatic angiosperms, including freshwater species (Larkum et al., 2006), indicating the independent rise of marine adaption phenotypes. As the likelihood of convergent evolution is predicted to decrease with phylogenetic distance (Ord and Summers, 2015), the divergence time difference between the seagrass subclade in Hydrocharitaceae (55 Mya; Chen et al., 2012) and Zosteraceae (25 Mya; Coyer et al., 2013) highlights the importance of this study. Moreover, since examples of parallel evolution, where similar phenotypes are generated from a similar genetic process of independent convergent evolution (Ord and Summers, 2015), are not abundant in plants (examples include carnivorous species (Fukushima et al., 2017), recurrence of C4 photosynthesis (reviewed in Washburn et al., 2016) and convergent mutations in loci during domestication (Paterson et al., 1995)), and that habitat is the most common factor associated with reported examples of repeated evolution (Ord and Summers, 2015), independent seagrass lineages are excellent subjects for study. In this work, a genomic comparison between seagrasses of Hydrocharitaceae and Zosteraceae was explored to determine whether the gene loss previously identified in Z. muelleri and Z. marina is also observed in H. ovalis. We also attempt to identify any seagrass-specific genes that are present in one or both lineages. Halophila ovalis genome sequencing data were compared with the annotated genomes of Z. marina and Z. muelleri, together with representative land plants. Our study demonstrates that lost genes associated with the synthesis and signalling of volatile substances, as well as stomatal development, are shared by both seagrass lineages. Genes that are uniquely conserved across the two lineages are enriched in pathways related to cell osmoregulation, and provide molecular evidence for independent marine colonization. Results also revealed the loss of the NADH dehydrogenase-like (NDH) protein complex in H. ovalis, a characteristic that is not shared by the other two seagrass species. This study provides a more complete description of marine adaptation, and suggest a parallel convergent evolution of two independent return-to-the-sea events in seagrasses separated by 30 million years. Materials and methods Genome sequencing of H. ovalis One H. ovalis plant sample was collected at Swan River, Claremont, Perth, Western Australia (coordinates: 32° 0′ 3.98″ S, 115° 45′ 18.31″ E). The growth tips of the seagrass thalli were carefully removed, rinsed in sterile water, and inspected for visible external contamination. Seven hundred milligrams of tissue was placed in 5 ml tubes, flash frozen in liquid nitrogen, and bead-pulverized using a 2010 Geno/Grinder (SPEX SamplePrep, USA). The Qiagen DNeasy Plant Mini Kit was used for the extraction of the DNA. The frozen powdered plant material was suspended in 3 ml of Buffer AP1 and 28 µl of RNAse A was added. After incubating at 65 °C, 910 µl of Buffer AP2 was added. The tubes were incubated on ice for 5 min and centrifuged thereafter to collect plant debris. Lysate (450 µl) was transferred to each of five to six QIAshredder tubes. The remainder of the DNA extraction steps were followed according to the kit protocol. The extracted DNA of each repetition was pooled after elution. DNA concentration was quantified using a Qubit 3.0 Fluorometer (Thermo Fisher Scientific) and visualized using a Labchip GX Touch 24 (PerkinElmer). The extracted DNA was submitted to the Australian Genome Research Facility (AGRF) for library preparation and whole genome sequencing. The libraries for genome sequencing were prepared using the Illumina TruSeq Nano DNA HT Library Preparation Kit, according to the manufacturer’s instructions. Genomic DNA was sequenced using an Illumina HiSeqX sequencer with 150 bp paired-end technology at the Garvan Institute of Medical Research. A total of 510485779 paired-end reads were sequenced. Based on previous flow cytometry analysis of two other Hydrocharitaceae members, Najas minor (2C=7.28) and Eldodea Canadensis (2C=7.54) (Hidalgo et al., 2015), as well as genome size prediction (3628962593 bp, k=45) using the software Kmergenie (Chikhi and Medvedev, 2014) the sequencing coverage was estimated as ~40×. The sequences were deposited in a public repository (NCBI BioProject Accession PRJNA396090). Clones and low quality reads were removed using Sickle (Joshi and Fass, 2011). Pipeline to identify lost and conserved genes The identification of lost and conserved genes was achieved using the mapping of whole genome shotgun sequencing reads against reference genomes based on a previous approach (Golicz et al., 2015). The reads were mapped to coding sequences (CDS) of reference species using dc-megaBLAST (Camacho et al., 2009) with e-value 1e−5. A custom python script, calculate_blast_coverage.py (downloadable at https://github.com/AppliedBioinformatics/H_ovalis_supplementary.git), was used to calculate the horizontal coverage of each CDS. The average coverage of each CDS across multiple reference species was calculated. If the average coverage was <2%, which means that mapped reads covered less than 2% of the length of a CDS, the orthologue was considered lost. If the average coverage was >50%, the orthologue was conserved. Orthologous gene cluster construction A set of 16007 orthologous gene clusters (OGCs) conserved between seven model species with at least one gene originating from a monocot species, termed OGCsM (as defined in Table S1 in Golicz et al., 2015), was used to represent orthologues highly conserved in plants. Gene clusters unique to Zosteraceae were identified using all-against-all comparison with BLASTP (Camacho et al., 2009) using the following parameters: ‘blastp -evalue 1e-5’, followed by OrthoMCL (Li et al., 2003) between Z. muelleri, Z. marina, one dicot (Arabidopsis), and three other monocots (Oryza sativa, Musa acuminata, and Spirodela polyrhiza) (species selection based on Lee et al., 2016). This group of orthologous genes was termed OGCZ. Lost and conserved H. ovalis, Z. muelleri and Z. marina genes in OGCsM Primary transcript CDSs of five species (four land plants: Arabidopsis, Oryza sativa, Musa acuminata, and Phoenix dactylifera; one floating freshwater plant: Spirodela polyrhiza; versions as listed in Golicz et al., 2015) were used as references for mapping of reads from H. ovalis. Presence and absence results from a previous publication were used for Z. muelleri and Z. marina (Golicz et al., 2015). For each orthologue in OGCsM, lost or conserved status was assigned in each species. Lost and conserved H. ovalis genes in OGCZ Primary transcript CDSs of Z. muelleri (http://www.appliedbioinformatics.com.au/index.php/Seagrass_Zmu_Genome; Lee et al., 2016) and Z. marina (Phytozome 10; Olsen et al., 2016) were used as references for H. ovalis read mapping. For each orthologue in OGCZ, lost or conserved status was assigned in H. ovalis. Gene ontology enrichment and word cloud plotting GO annotation and enrichment were performed using the topGO package (Alexa and Rahnenfuhrer, 2010) based on a previous approach (Golicz et al., 2015). OGCsM was used as background, except for the GO enrichment of OGCZ genes where Arabidopsis whole proteome (TAIR10) was used. A word cloud was generated and coloured to represent the enriched significance of GO terms using the wordcloud package (Fellows, 2014). Inferring gene function through the level of protein domain conservation OGCZ proteins of Z. muelleri, Z. marina and Arabidopsis were compared with TIGRFAM, ProDom, Panther, PfamA and PrositePatterns using InterProScan (version 5.14, Jones et al., 2014) for motif and domain annotation. Domains of each protein were assigned with InterProScan IDs. The InterProScan IDs were compared between Arabidopsis and Zosteraceae genes for each OGCZ cluster. Assembly of H. ovalis protein and multiple sequence alignments with orthologues of other species Halophila ovalis reads aligned to CDS of 50S ribosomal protein L16 were extracted and assembled using Spades v3.10.1 (Bankevich et al., 2012) with the following commands: spades.py, only-assembler, 1 reads_1.fasta, 2 reads_2.fasta. Corresponding protein was aligned to the assembled contigs using Exonerate (Slater and Birney, 2005) with the following parameters: exonerate, model protein2genome, E 1, bestn 1, score 100, softmaskquery no, softmasktarget yes, minintron 20, maxintron 20000, ryo “>HAL_%qi_%qd\n%tas”. The aligned target regions were translated to protein sequences using the translate tool in ExPASy (Gasteiger et al., 2003). Each H. ovalis protein sequence obtained was aligned with orthologues of selected species (Table S1 at JXB online) using MAFFT (Katoh et al., 2002). A phylogenetic tree was plotted with PhyML (Guindon et al., 2009) assuming the JTT model for amino acid substitution and gamma parameter for invariable sites (based on Huang et al., 2016) using the alignments excluding the outgroup (charophyte and chlorophyte). The multiple-sequence alignments were visualized and coloured using Jalview (Waterhouse et al., 2009). Results Read alignment of H. ovalis to reference species CDS A total of 112202319 H. ovalis reads (10.9%) were discarded in the process of clonal removal and quality-based filtering (Table S2). Out of the remaining 908769239 H. ovalis reads, 2.7% (24495631) aligned to Arabidopsis CDS, 5.6% (50565060) aligned to Oryza sativa CDS, 1.3% (11617255) aligned to Musa acuminata CDS, 0.8% (7367361) aligned to Phoenix dactylifera CDS and 1.8% (16600802) aligned to Spirodela polyrhiza CDS. For the seagrass reference species, 1.8% (16727940) and 0.5% (5005993) of H. ovalis reads aligned to Z. muelleri and Z. marina CDSs, respectively. Conservation of core biological processes A total of 4367 OGCsM genes, out of 16007, were conserved in H. ovalis. When compared with conserved genes previously described in Z. muelleri and Z. marina (Golicz et al., 2015; Lee et al., 2016; Olsen et al., 2016), 3335 (76.4%) genes were conserved in all three seagrass species, 377 genes were shared with either Z. muelleri or Z. marina, and 655 genes were only conserved in H. ovalis. A total of 508 genes were only conserved in the Zosteraceae species. A full list of genes conserved in H. ovalis and their presence in other seagrass species is presented in Table S3. The GO terms enriched in these 4367 OGCsM genes conserved in H. ovalis involved core biological pathways such as photosynthesis, chlorophyll biosynthesis, and glycolytic processes, as well as response to stresses such as cadmium (Table 1). Table 1. Significantly enriched biological process GO terms in the genes conserved in H. ovalis compared with five other plant species (Arabidopsis, Oryza sativa, Musa acuminata, Phoenix dactylifera, and Spirodela polyrhiza) GO ID Term P value GO:0046686 Response to cadmium ion 3.0 × 10−30 GO:0006412 Translation 4.1 × 10−28 GO:0046496 Nicotinamide nucleotide metabolic process 3.9 × 10−16 GO:0006099 Tricarboxylic acid cycle 1.7 × 10−13 GO:0015991 ATP hydrolysis-coupled proton transport 1.0 × 10−12 GO:1901566 Organonitrogen compound biosynthetic process 7.9 × 10−11 GO:0043039 tRNA aminoacylation 1.2 × 10−10 GO:0006090 Pyruvate metabolic process 1.9 × 10−10 GO:1901293 Nucleoside phosphate biosynthetic process 2.3 × 10−10 GO:0009156 Ribonucleoside monophosphate biosynthetic process 2.7 × 10−10 GO:0009225 Nucleotide-sugar metabolic process 5.4 × 10−10 GO:0007264 Small GTPase-mediated signal transduction 8.2 × 10−9 GO:0046034 ATP metabolic process 8.4 × 10−9 GO:0006108 Malate metabolic process 1.4 × 10−8 GO:0006006 Glucose metabolic process 1.8 × 10−8 GO:0034622 Cellular macromolecular complex assembly 2.3 × 10−8 GO:0071702 Organic substance transport 2.4 × 10−8 GO:0018105 Peptidyl-serine phosphorylation 3.2 × 10−8 GO:0009250 Glucan biosynthetic process 2.0 × 10−7 GO:0016192 Vesicle-mediated transport 2.0 × 10−7 GO:0010499 Proteasomal ubiquitin-independent protein catabolic process 3.4 × 10−7 GO:0043094 Cellular metabolic compound salvage 6.7 × 10−7 GO:0015994 Chlorophyll metabolic process 1.2 × 10−6 GO:0034613 Cellular protein localization 1.4 × 10−6 GO:0006536 Glutamate metabolic process 1.9 × 10−6 GO:0005985 Sucrose metabolic process 5.3 × 10−6 GO:0098656 Anion transmembrane transport 5.7 × 10−6 GO:0015672 Monovalent inorganic cation transport 7.5 × 10−6 GO:0009932 Cell tip growth 9.9 × 10−6 GO:0006081 Cellular aldehyde metabolic process 1.0 × 10−5 GO:0018298 Protein–chromophore linkage 1.0 × 10−5 GO:0030163 Protein catabolic process 1.3 × 10−5 GO:0048588 Developmental cell growth 1.3 × 10−5 GO:0006102 Isocitrate metabolic process 2.4 × 10−5 GO:0006607 NLS-bearing protein import into nucleus 2.5 × 10−5 GO:0015977 Carbon fixation 5.1 × 10−5 GO:0015979 Photosynthesis 5.4 × 10−5 GO:0006563 L-Serine metabolic process 6.0 × 10−5 GO:0006268 DNA unwinding involved in DNA replication 6.9 × 10−5 GO:0007035 Vacuolar acidification 6.9 × 10−5 GO:0009768 Photosynthesis, light harvesting in photosystem I 7.2 × 10−5 GO:0016197 Endosomal transport 8.1 × 10−5 GO:0030048 Actin filament-based movement 0.00012 GO:0009651 Response to salt stress 0.00012 GO:0006206 Pyrimidine nucleobase metabolic process 0.00013 GO:0030243 Cellulose metabolic process 0.00014 GO:0097164 Ammonium ion metabolic process 0.00015 GO:0010315 Auxin efflux 0.00015 GO:0006551 Leucine metabolic process 0.00017 GO:0006085 Acetyl-CoA biosynthetic process 0.00018 GO:0045899 Positive regulation of RNA polymerase II transcriptional preinitiation complex assembly 0.00020 GO:0032012 Regulation of ARF protein signal transduction 0.00020 GO:0009735 Response to cytokinin 0.00027 GO:0006782 Protoporphyrinogen IX biosynthetic process 0.00030 GO:0009846 Pollen germination 0.00032 GO:1901679 Nucleotide transmembrane transport 0.00041 GO:0030042 Actin filament depolymerization 0.00048 GO:0006558 L-Phenylalanine metabolic process 0.00050 GO:0006544 Glycine metabolic process 0.00057 GO:0035999 Tetrahydrofolate interconversion 0.00096 GO:0009066 Aspartate family amino acid metabolic process 0.00103 GO:0006222 UMP biosynthetic process 0.00113 GO:0046500 S-Adenosylmethionine metabolic process 0.00125 GO:0015804 Neutral amino acid transport 0.00125 GO:0006097 Glyoxylate cycle 0.00125 GO:0030433 Endoplasmic reticulum-associated ubiquitin- dependent protein degradation 0.00132 GO:0019627 Urea metabolic process 0.00169 GO:0015800 Acidic amino acid transport 0.00169 GO:0000398 mRNA splicing, via spliceosome 0.00170 GO:0010540 Basipetal auxin transport 0.00235 GO:0035435 Phosphate ion transmembrane transport 0.00258 GO:0006457 Protein folding 0.00262 GO:0051259 Protein oligomerization 0.00265 GO:0006525 Arginine metabolic process 0.00265 GO:0016482 Cytoplasmic transport 0.00284 GO:0016036 Cellular response to phosphate starvation 0.00288 GO:0043604 Amide biosynthetic process 0.00295 GO:0019395 Fatty acid oxidation 0.00317 GO:0006570 Tyrosine metabolic process 0.00326 GO:0052646 Alditol phosphate metabolic process 0.00326 GO:0010043 Response to zinc ion 0.00346 GO:0055085 Transmembrane transport 0.00377 GO:0000338 Protein deneddylation 0.00436 GO:0006002 Fructose 6-phosphate metabolic process 0.00436 GO:0043650 Dicarboxylic acid biosynthetic process 0.00439 GO:0010501 RNA secondary structure unwinding 0.00439 GO:0009629 Response to gravity 0.00447 GO:0006103 2-Oxoglutarate metabolic process 0.00490 GO:0009833 Plant-type primary cell wall biogenesis 0.00490 GO:0006610 Ribosomal protein import into nucleus 0.00490 GO:0010541 Acropetal auxin transport 0.00540 GO:0006012 Galactose metabolic process 0.00540 GO:0006511 Ubiquitin-dependent protein catabolic process 0.00630 GO:0051275 β-Glucan catabolic process 0.00735 GO:0009141 Nucleoside triphosphate metabolic process 0.00770 GO:0018208 Peptidyl-proline modification 0.00803 GO:0006486 Protein glycosylation 0.00804 GO:0009624 Response to nematode 0.00847 GO:1904659 Glucose transmembrane transport 0.00872 GO:0046323 Glucose import 0.00872 GO:0006631 Fatty acid metabolic process 0.00878 GO:0044282 Small molecule catabolic process 0.00886 GO ID Term P value GO:0046686 Response to cadmium ion 3.0 × 10−30 GO:0006412 Translation 4.1 × 10−28 GO:0046496 Nicotinamide nucleotide metabolic process 3.9 × 10−16 GO:0006099 Tricarboxylic acid cycle 1.7 × 10−13 GO:0015991 ATP hydrolysis-coupled proton transport 1.0 × 10−12 GO:1901566 Organonitrogen compound biosynthetic process 7.9 × 10−11 GO:0043039 tRNA aminoacylation 1.2 × 10−10 GO:0006090 Pyruvate metabolic process 1.9 × 10−10 GO:1901293 Nucleoside phosphate biosynthetic process 2.3 × 10−10 GO:0009156 Ribonucleoside monophosphate biosynthetic process 2.7 × 10−10 GO:0009225 Nucleotide-sugar metabolic process 5.4 × 10−10 GO:0007264 Small GTPase-mediated signal transduction 8.2 × 10−9 GO:0046034 ATP metabolic process 8.4 × 10−9 GO:0006108 Malate metabolic process 1.4 × 10−8 GO:0006006 Glucose metabolic process 1.8 × 10−8 GO:0034622 Cellular macromolecular complex assembly 2.3 × 10−8 GO:0071702 Organic substance transport 2.4 × 10−8 GO:0018105 Peptidyl-serine phosphorylation 3.2 × 10−8 GO:0009250 Glucan biosynthetic process 2.0 × 10−7 GO:0016192 Vesicle-mediated transport 2.0 × 10−7 GO:0010499 Proteasomal ubiquitin-independent protein catabolic process 3.4 × 10−7 GO:0043094 Cellular metabolic compound salvage 6.7 × 10−7 GO:0015994 Chlorophyll metabolic process 1.2 × 10−6 GO:0034613 Cellular protein localization 1.4 × 10−6 GO:0006536 Glutamate metabolic process 1.9 × 10−6 GO:0005985 Sucrose metabolic process 5.3 × 10−6 GO:0098656 Anion transmembrane transport 5.7 × 10−6 GO:0015672 Monovalent inorganic cation transport 7.5 × 10−6 GO:0009932 Cell tip growth 9.9 × 10−6 GO:0006081 Cellular aldehyde metabolic process 1.0 × 10−5 GO:0018298 Protein–chromophore linkage 1.0 × 10−5 GO:0030163 Protein catabolic process 1.3 × 10−5 GO:0048588 Developmental cell growth 1.3 × 10−5 GO:0006102 Isocitrate metabolic process 2.4 × 10−5 GO:0006607 NLS-bearing protein import into nucleus 2.5 × 10−5 GO:0015977 Carbon fixation 5.1 × 10−5 GO:0015979 Photosynthesis 5.4 × 10−5 GO:0006563 L-Serine metabolic process 6.0 × 10−5 GO:0006268 DNA unwinding involved in DNA replication 6.9 × 10−5 GO:0007035 Vacuolar acidification 6.9 × 10−5 GO:0009768 Photosynthesis, light harvesting in photosystem I 7.2 × 10−5 GO:0016197 Endosomal transport 8.1 × 10−5 GO:0030048 Actin filament-based movement 0.00012 GO:0009651 Response to salt stress 0.00012 GO:0006206 Pyrimidine nucleobase metabolic process 0.00013 GO:0030243 Cellulose metabolic process 0.00014 GO:0097164 Ammonium ion metabolic process 0.00015 GO:0010315 Auxin efflux 0.00015 GO:0006551 Leucine metabolic process 0.00017 GO:0006085 Acetyl-CoA biosynthetic process 0.00018 GO:0045899 Positive regulation of RNA polymerase II transcriptional preinitiation complex assembly 0.00020 GO:0032012 Regulation of ARF protein signal transduction 0.00020 GO:0009735 Response to cytokinin 0.00027 GO:0006782 Protoporphyrinogen IX biosynthetic process 0.00030 GO:0009846 Pollen germination 0.00032 GO:1901679 Nucleotide transmembrane transport 0.00041 GO:0030042 Actin filament depolymerization 0.00048 GO:0006558 L-Phenylalanine metabolic process 0.00050 GO:0006544 Glycine metabolic process 0.00057 GO:0035999 Tetrahydrofolate interconversion 0.00096 GO:0009066 Aspartate family amino acid metabolic process 0.00103 GO:0006222 UMP biosynthetic process 0.00113 GO:0046500 S-Adenosylmethionine metabolic process 0.00125 GO:0015804 Neutral amino acid transport 0.00125 GO:0006097 Glyoxylate cycle 0.00125 GO:0030433 Endoplasmic reticulum-associated ubiquitin- dependent protein degradation 0.00132 GO:0019627 Urea metabolic process 0.00169 GO:0015800 Acidic amino acid transport 0.00169 GO:0000398 mRNA splicing, via spliceosome 0.00170 GO:0010540 Basipetal auxin transport 0.00235 GO:0035435 Phosphate ion transmembrane transport 0.00258 GO:0006457 Protein folding 0.00262 GO:0051259 Protein oligomerization 0.00265 GO:0006525 Arginine metabolic process 0.00265 GO:0016482 Cytoplasmic transport 0.00284 GO:0016036 Cellular response to phosphate starvation 0.00288 GO:0043604 Amide biosynthetic process 0.00295 GO:0019395 Fatty acid oxidation 0.00317 GO:0006570 Tyrosine metabolic process 0.00326 GO:0052646 Alditol phosphate metabolic process 0.00326 GO:0010043 Response to zinc ion 0.00346 GO:0055085 Transmembrane transport 0.00377 GO:0000338 Protein deneddylation 0.00436 GO:0006002 Fructose 6-phosphate metabolic process 0.00436 GO:0043650 Dicarboxylic acid biosynthetic process 0.00439 GO:0010501 RNA secondary structure unwinding 0.00439 GO:0009629 Response to gravity 0.00447 GO:0006103 2-Oxoglutarate metabolic process 0.00490 GO:0009833 Plant-type primary cell wall biogenesis 0.00490 GO:0006610 Ribosomal protein import into nucleus 0.00490 GO:0010541 Acropetal auxin transport 0.00540 GO:0006012 Galactose metabolic process 0.00540 GO:0006511 Ubiquitin-dependent protein catabolic process 0.00630 GO:0051275 β-Glucan catabolic process 0.00735 GO:0009141 Nucleoside triphosphate metabolic process 0.00770 GO:0018208 Peptidyl-proline modification 0.00803 GO:0006486 Protein glycosylation 0.00804 GO:0009624 Response to nematode 0.00847 GO:1904659 Glucose transmembrane transport 0.00872 GO:0046323 Glucose import 0.00872 GO:0006631 Fatty acid metabolic process 0.00878 GO:0044282 Small molecule catabolic process 0.00886 View Large Table 1. Significantly enriched biological process GO terms in the genes conserved in H. ovalis compared with five other plant species (Arabidopsis, Oryza sativa, Musa acuminata, Phoenix dactylifera, and Spirodela polyrhiza) GO ID Term P value GO:0046686 Response to cadmium ion 3.0 × 10−30 GO:0006412 Translation 4.1 × 10−28 GO:0046496 Nicotinamide nucleotide metabolic process 3.9 × 10−16 GO:0006099 Tricarboxylic acid cycle 1.7 × 10−13 GO:0015991 ATP hydrolysis-coupled proton transport 1.0 × 10−12 GO:1901566 Organonitrogen compound biosynthetic process 7.9 × 10−11 GO:0043039 tRNA aminoacylation 1.2 × 10−10 GO:0006090 Pyruvate metabolic process 1.9 × 10−10 GO:1901293 Nucleoside phosphate biosynthetic process 2.3 × 10−10 GO:0009156 Ribonucleoside monophosphate biosynthetic process 2.7 × 10−10 GO:0009225 Nucleotide-sugar metabolic process 5.4 × 10−10 GO:0007264 Small GTPase-mediated signal transduction 8.2 × 10−9 GO:0046034 ATP metabolic process 8.4 × 10−9 GO:0006108 Malate metabolic process 1.4 × 10−8 GO:0006006 Glucose metabolic process 1.8 × 10−8 GO:0034622 Cellular macromolecular complex assembly 2.3 × 10−8 GO:0071702 Organic substance transport 2.4 × 10−8 GO:0018105 Peptidyl-serine phosphorylation 3.2 × 10−8 GO:0009250 Glucan biosynthetic process 2.0 × 10−7 GO:0016192 Vesicle-mediated transport 2.0 × 10−7 GO:0010499 Proteasomal ubiquitin-independent protein catabolic process 3.4 × 10−7 GO:0043094 Cellular metabolic compound salvage 6.7 × 10−7 GO:0015994 Chlorophyll metabolic process 1.2 × 10−6 GO:0034613 Cellular protein localization 1.4 × 10−6 GO:0006536 Glutamate metabolic process 1.9 × 10−6 GO:0005985 Sucrose metabolic process 5.3 × 10−6 GO:0098656 Anion transmembrane transport 5.7 × 10−6 GO:0015672 Monovalent inorganic cation transport 7.5 × 10−6 GO:0009932 Cell tip growth 9.9 × 10−6 GO:0006081 Cellular aldehyde metabolic process 1.0 × 10−5 GO:0018298 Protein–chromophore linkage 1.0 × 10−5 GO:0030163 Protein catabolic process 1.3 × 10−5 GO:0048588 Developmental cell growth 1.3 × 10−5 GO:0006102 Isocitrate metabolic process 2.4 × 10−5 GO:0006607 NLS-bearing protein import into nucleus 2.5 × 10−5 GO:0015977 Carbon fixation 5.1 × 10−5 GO:0015979 Photosynthesis 5.4 × 10−5 GO:0006563 L-Serine metabolic process 6.0 × 10−5 GO:0006268 DNA unwinding involved in DNA replication 6.9 × 10−5 GO:0007035 Vacuolar acidification 6.9 × 10−5 GO:0009768 Photosynthesis, light harvesting in photosystem I 7.2 × 10−5 GO:0016197 Endosomal transport 8.1 × 10−5 GO:0030048 Actin filament-based movement 0.00012 GO:0009651 Response to salt stress 0.00012 GO:0006206 Pyrimidine nucleobase metabolic process 0.00013 GO:0030243 Cellulose metabolic process 0.00014 GO:0097164 Ammonium ion metabolic process 0.00015 GO:0010315 Auxin efflux 0.00015 GO:0006551 Leucine metabolic process 0.00017 GO:0006085 Acetyl-CoA biosynthetic process 0.00018 GO:0045899 Positive regulation of RNA polymerase II transcriptional preinitiation complex assembly 0.00020 GO:0032012 Regulation of ARF protein signal transduction 0.00020 GO:0009735 Response to cytokinin 0.00027 GO:0006782 Protoporphyrinogen IX biosynthetic process 0.00030 GO:0009846 Pollen germination 0.00032 GO:1901679 Nucleotide transmembrane transport 0.00041 GO:0030042 Actin filament depolymerization 0.00048 GO:0006558 L-Phenylalanine metabolic process 0.00050 GO:0006544 Glycine metabolic process 0.00057 GO:0035999 Tetrahydrofolate interconversion 0.00096 GO:0009066 Aspartate family amino acid metabolic process 0.00103 GO:0006222 UMP biosynthetic process 0.00113 GO:0046500 S-Adenosylmethionine metabolic process 0.00125 GO:0015804 Neutral amino acid transport 0.00125 GO:0006097 Glyoxylate cycle 0.00125 GO:0030433 Endoplasmic reticulum-associated ubiquitin- dependent protein degradation 0.00132 GO:0019627 Urea metabolic process 0.00169 GO:0015800 Acidic amino acid transport 0.00169 GO:0000398 mRNA splicing, via spliceosome 0.00170 GO:0010540 Basipetal auxin transport 0.00235 GO:0035435 Phosphate ion transmembrane transport 0.00258 GO:0006457 Protein folding 0.00262 GO:0051259 Protein oligomerization 0.00265 GO:0006525 Arginine metabolic process 0.00265 GO:0016482 Cytoplasmic transport 0.00284 GO:0016036 Cellular response to phosphate starvation 0.00288 GO:0043604 Amide biosynthetic process 0.00295 GO:0019395 Fatty acid oxidation 0.00317 GO:0006570 Tyrosine metabolic process 0.00326 GO:0052646 Alditol phosphate metabolic process 0.00326 GO:0010043 Response to zinc ion 0.00346 GO:0055085 Transmembrane transport 0.00377 GO:0000338 Protein deneddylation 0.00436 GO:0006002 Fructose 6-phosphate metabolic process 0.00436 GO:0043650 Dicarboxylic acid biosynthetic process 0.00439 GO:0010501 RNA secondary structure unwinding 0.00439 GO:0009629 Response to gravity 0.00447 GO:0006103 2-Oxoglutarate metabolic process 0.00490 GO:0009833 Plant-type primary cell wall biogenesis 0.00490 GO:0006610 Ribosomal protein import into nucleus 0.00490 GO:0010541 Acropetal auxin transport 0.00540 GO:0006012 Galactose metabolic process 0.00540 GO:0006511 Ubiquitin-dependent protein catabolic process 0.00630 GO:0051275 β-Glucan catabolic process 0.00735 GO:0009141 Nucleoside triphosphate metabolic process 0.00770 GO:0018208 Peptidyl-proline modification 0.00803 GO:0006486 Protein glycosylation 0.00804 GO:0009624 Response to nematode 0.00847 GO:1904659 Glucose transmembrane transport 0.00872 GO:0046323 Glucose import 0.00872 GO:0006631 Fatty acid metabolic process 0.00878 GO:0044282 Small molecule catabolic process 0.00886 GO ID Term P value GO:0046686 Response to cadmium ion 3.0 × 10−30 GO:0006412 Translation 4.1 × 10−28 GO:0046496 Nicotinamide nucleotide metabolic process 3.9 × 10−16 GO:0006099 Tricarboxylic acid cycle 1.7 × 10−13 GO:0015991 ATP hydrolysis-coupled proton transport 1.0 × 10−12 GO:1901566 Organonitrogen compound biosynthetic process 7.9 × 10−11 GO:0043039 tRNA aminoacylation 1.2 × 10−10 GO:0006090 Pyruvate metabolic process 1.9 × 10−10 GO:1901293 Nucleoside phosphate biosynthetic process 2.3 × 10−10 GO:0009156 Ribonucleoside monophosphate biosynthetic process 2.7 × 10−10 GO:0009225 Nucleotide-sugar metabolic process 5.4 × 10−10 GO:0007264 Small GTPase-mediated signal transduction 8.2 × 10−9 GO:0046034 ATP metabolic process 8.4 × 10−9 GO:0006108 Malate metabolic process 1.4 × 10−8 GO:0006006 Glucose metabolic process 1.8 × 10−8 GO:0034622 Cellular macromolecular complex assembly 2.3 × 10−8 GO:0071702 Organic substance transport 2.4 × 10−8 GO:0018105 Peptidyl-serine phosphorylation 3.2 × 10−8 GO:0009250 Glucan biosynthetic process 2.0 × 10−7 GO:0016192 Vesicle-mediated transport 2.0 × 10−7 GO:0010499 Proteasomal ubiquitin-independent protein catabolic process 3.4 × 10−7 GO:0043094 Cellular metabolic compound salvage 6.7 × 10−7 GO:0015994 Chlorophyll metabolic process 1.2 × 10−6 GO:0034613 Cellular protein localization 1.4 × 10−6 GO:0006536 Glutamate metabolic process 1.9 × 10−6 GO:0005985 Sucrose metabolic process 5.3 × 10−6 GO:0098656 Anion transmembrane transport 5.7 × 10−6 GO:0015672 Monovalent inorganic cation transport 7.5 × 10−6 GO:0009932 Cell tip growth 9.9 × 10−6 GO:0006081 Cellular aldehyde metabolic process 1.0 × 10−5 GO:0018298 Protein–chromophore linkage 1.0 × 10−5 GO:0030163 Protein catabolic process 1.3 × 10−5 GO:0048588 Developmental cell growth 1.3 × 10−5 GO:0006102 Isocitrate metabolic process 2.4 × 10−5 GO:0006607 NLS-bearing protein import into nucleus 2.5 × 10−5 GO:0015977 Carbon fixation 5.1 × 10−5 GO:0015979 Photosynthesis 5.4 × 10−5 GO:0006563 L-Serine metabolic process 6.0 × 10−5 GO:0006268 DNA unwinding involved in DNA replication 6.9 × 10−5 GO:0007035 Vacuolar acidification 6.9 × 10−5 GO:0009768 Photosynthesis, light harvesting in photosystem I 7.2 × 10−5 GO:0016197 Endosomal transport 8.1 × 10−5 GO:0030048 Actin filament-based movement 0.00012 GO:0009651 Response to salt stress 0.00012 GO:0006206 Pyrimidine nucleobase metabolic process 0.00013 GO:0030243 Cellulose metabolic process 0.00014 GO:0097164 Ammonium ion metabolic process 0.00015 GO:0010315 Auxin efflux 0.00015 GO:0006551 Leucine metabolic process 0.00017 GO:0006085 Acetyl-CoA biosynthetic process 0.00018 GO:0045899 Positive regulation of RNA polymerase II transcriptional preinitiation complex assembly 0.00020 GO:0032012 Regulation of ARF protein signal transduction 0.00020 GO:0009735 Response to cytokinin 0.00027 GO:0006782 Protoporphyrinogen IX biosynthetic process 0.00030 GO:0009846 Pollen germination 0.00032 GO:1901679 Nucleotide transmembrane transport 0.00041 GO:0030042 Actin filament depolymerization 0.00048 GO:0006558 L-Phenylalanine metabolic process 0.00050 GO:0006544 Glycine metabolic process 0.00057 GO:0035999 Tetrahydrofolate interconversion 0.00096 GO:0009066 Aspartate family amino acid metabolic process 0.00103 GO:0006222 UMP biosynthetic process 0.00113 GO:0046500 S-Adenosylmethionine metabolic process 0.00125 GO:0015804 Neutral amino acid transport 0.00125 GO:0006097 Glyoxylate cycle 0.00125 GO:0030433 Endoplasmic reticulum-associated ubiquitin- dependent protein degradation 0.00132 GO:0019627 Urea metabolic process 0.00169 GO:0015800 Acidic amino acid transport 0.00169 GO:0000398 mRNA splicing, via spliceosome 0.00170 GO:0010540 Basipetal auxin transport 0.00235 GO:0035435 Phosphate ion transmembrane transport 0.00258 GO:0006457 Protein folding 0.00262 GO:0051259 Protein oligomerization 0.00265 GO:0006525 Arginine metabolic process 0.00265 GO:0016482 Cytoplasmic transport 0.00284 GO:0016036 Cellular response to phosphate starvation 0.00288 GO:0043604 Amide biosynthetic process 0.00295 GO:0019395 Fatty acid oxidation 0.00317 GO:0006570 Tyrosine metabolic process 0.00326 GO:0052646 Alditol phosphate metabolic process 0.00326 GO:0010043 Response to zinc ion 0.00346 GO:0055085 Transmembrane transport 0.00377 GO:0000338 Protein deneddylation 0.00436 GO:0006002 Fructose 6-phosphate metabolic process 0.00436 GO:0043650 Dicarboxylic acid biosynthetic process 0.00439 GO:0010501 RNA secondary structure unwinding 0.00439 GO:0009629 Response to gravity 0.00447 GO:0006103 2-Oxoglutarate metabolic process 0.00490 GO:0009833 Plant-type primary cell wall biogenesis 0.00490 GO:0006610 Ribosomal protein import into nucleus 0.00490 GO:0010541 Acropetal auxin transport 0.00540 GO:0006012 Galactose metabolic process 0.00540 GO:0006511 Ubiquitin-dependent protein catabolic process 0.00630 GO:0051275 β-Glucan catabolic process 0.00735 GO:0009141 Nucleoside triphosphate metabolic process 0.00770 GO:0018208 Peptidyl-proline modification 0.00803 GO:0006486 Protein glycosylation 0.00804 GO:0009624 Response to nematode 0.00847 GO:1904659 Glucose transmembrane transport 0.00872 GO:0046323 Glucose import 0.00872 GO:0006631 Fatty acid metabolic process 0.00878 GO:0044282 Small molecule catabolic process 0.00886 View Large Gene loss in H. ovalis and comparison of lost genes between the three seagrass species A total of 1822 OGCsM genes were lost in H. ovalis, and these were compared with those previously reported as lost in both Z. muelleri and Z. marina (Golicz et al., 2015; Lee et al., 2016; Olsen et al., 2016) (Table S4). A total of 1197 (65.6%) lost genes were shared between all three seagrass species, 187 were shared with either Z. muelleri or Z. marina, and 412 were only lost in H. ovalis. In comparison, 743 genes were only lost in the Zosteraceae lineage. Enriched GO terms for the 1822 OGCsM genes highlighted the loss of genes associated with ethylene synthesis and perception, and stomatal development (Table 2). The presence or absence of genes involved in stomatal development, ethylene synthesis and signalling, and terpenoid biosynthesis in H. ovalis, Z. marina, and Z. muelleri are listed in Table 3. Table 2. Significantly enriched biological process GO terms in the genes conserved in five other plant species (Arabidopsis, Oryza sativa, Musa acuminata, Phoenix dactylifera and Spirodela polyrhiza) but absent in H. ovalis Function GO ID Term P value Ethylene synthesis and signalling GO:0009835 Fruit ripening 4.1 × 10−10 GO:0042218 1-Aminocyclopropane-1-carboxylate biosynthetic process 6.3 × 10−10 GO:0009693 Ethylene biosynthetic process 1.9 × 10−8 GO:0010105 Negative regulation of ethylene-activated signalling pathway 1.2 × 10−6 Stomata development GO:0010375 Stomatal complex patterning 0.00012 GO:2000038 Regulation of stomatal complex development 0.00608 Others GO:0045168 Cell–cell signalling involved in cell fate commitment 3.1 × 10−6 GO:0006952 Defence response 9.4 × 10−6 GO:0009626 Plant-type hypersensitive response 1.0 × 10−5 GO:0031640 Killing of cells of other organism 8.4 × 10−5 GO:0010039 Response to iron ion 0.00011 GO:0034644 Cellular response to UV 0.00014 GO:0071484 Cellular response to light intensity 0.00018 GO:0009773 Photosynthetic electron transport in photosystem I 0.00023 GO:0080027 Response to herbivore 0.00034 GO:0033473 Indoleacetic acid conjugate metabolic process 0.00034 GO:0009696 Salicylic acid metabolic process 0.00059 GO:0033609 Oxalate metabolic process 0.00093 GO:0050832 Defence response to fungus 0.00113 GO:0071423 Malate transmembrane transport 0.00209 GO:0042542 Response to hydrogen peroxide 0.00234 GO:1900426 Positive regulation of defence response to bacterium 0.00239 GO:0010876 Lipid localization 0.00313 GO:0018106 Peptidyl-histidine phosphorylation 0.00404 GO:0046688 Response to copper ion 0.00558 GO:0010257 NADH dehydrogenase complex assembly 0.00608 GO:0009838 Abscission 0.00704 GO:0071732 Cellular response to nitric oxide 0.00791 Function GO ID Term P value Ethylene synthesis and signalling GO:0009835 Fruit ripening 4.1 × 10−10 GO:0042218 1-Aminocyclopropane-1-carboxylate biosynthetic process 6.3 × 10−10 GO:0009693 Ethylene biosynthetic process 1.9 × 10−8 GO:0010105 Negative regulation of ethylene-activated signalling pathway 1.2 × 10−6 Stomata development GO:0010375 Stomatal complex patterning 0.00012 GO:2000038 Regulation of stomatal complex development 0.00608 Others GO:0045168 Cell–cell signalling involved in cell fate commitment 3.1 × 10−6 GO:0006952 Defence response 9.4 × 10−6 GO:0009626 Plant-type hypersensitive response 1.0 × 10−5 GO:0031640 Killing of cells of other organism 8.4 × 10−5 GO:0010039 Response to iron ion 0.00011 GO:0034644 Cellular response to UV 0.00014 GO:0071484 Cellular response to light intensity 0.00018 GO:0009773 Photosynthetic electron transport in photosystem I 0.00023 GO:0080027 Response to herbivore 0.00034 GO:0033473 Indoleacetic acid conjugate metabolic process 0.00034 GO:0009696 Salicylic acid metabolic process 0.00059 GO:0033609 Oxalate metabolic process 0.00093 GO:0050832 Defence response to fungus 0.00113 GO:0071423 Malate transmembrane transport 0.00209 GO:0042542 Response to hydrogen peroxide 0.00234 GO:1900426 Positive regulation of defence response to bacterium 0.00239 GO:0010876 Lipid localization 0.00313 GO:0018106 Peptidyl-histidine phosphorylation 0.00404 GO:0046688 Response to copper ion 0.00558 GO:0010257 NADH dehydrogenase complex assembly 0.00608 GO:0009838 Abscission 0.00704 GO:0071732 Cellular response to nitric oxide 0.00791 View Large Table 2. Significantly enriched biological process GO terms in the genes conserved in five other plant species (Arabidopsis, Oryza sativa, Musa acuminata, Phoenix dactylifera and Spirodela polyrhiza) but absent in H. ovalis Function GO ID Term P value Ethylene synthesis and signalling GO:0009835 Fruit ripening 4.1 × 10−10 GO:0042218 1-Aminocyclopropane-1-carboxylate biosynthetic process 6.3 × 10−10 GO:0009693 Ethylene biosynthetic process 1.9 × 10−8 GO:0010105 Negative regulation of ethylene-activated signalling pathway 1.2 × 10−6 Stomata development GO:0010375 Stomatal complex patterning 0.00012 GO:2000038 Regulation of stomatal complex development 0.00608 Others GO:0045168 Cell–cell signalling involved in cell fate commitment 3.1 × 10−6 GO:0006952 Defence response 9.4 × 10−6 GO:0009626 Plant-type hypersensitive response 1.0 × 10−5 GO:0031640 Killing of cells of other organism 8.4 × 10−5 GO:0010039 Response to iron ion 0.00011 GO:0034644 Cellular response to UV 0.00014 GO:0071484 Cellular response to light intensity 0.00018 GO:0009773 Photosynthetic electron transport in photosystem I 0.00023 GO:0080027 Response to herbivore 0.00034 GO:0033473 Indoleacetic acid conjugate metabolic process 0.00034 GO:0009696 Salicylic acid metabolic process 0.00059 GO:0033609 Oxalate metabolic process 0.00093 GO:0050832 Defence response to fungus 0.00113 GO:0071423 Malate transmembrane transport 0.00209 GO:0042542 Response to hydrogen peroxide 0.00234 GO:1900426 Positive regulation of defence response to bacterium 0.00239 GO:0010876 Lipid localization 0.00313 GO:0018106 Peptidyl-histidine phosphorylation 0.00404 GO:0046688 Response to copper ion 0.00558 GO:0010257 NADH dehydrogenase complex assembly 0.00608 GO:0009838 Abscission 0.00704 GO:0071732 Cellular response to nitric oxide 0.00791 Function GO ID Term P value Ethylene synthesis and signalling GO:0009835 Fruit ripening 4.1 × 10−10 GO:0042218 1-Aminocyclopropane-1-carboxylate biosynthetic process 6.3 × 10−10 GO:0009693 Ethylene biosynthetic process 1.9 × 10−8 GO:0010105 Negative regulation of ethylene-activated signalling pathway 1.2 × 10−6 Stomata development GO:0010375 Stomatal complex patterning 0.00012 GO:2000038 Regulation of stomatal complex development 0.00608 Others GO:0045168 Cell–cell signalling involved in cell fate commitment 3.1 × 10−6 GO:0006952 Defence response 9.4 × 10−6 GO:0009626 Plant-type hypersensitive response 1.0 × 10−5 GO:0031640 Killing of cells of other organism 8.4 × 10−5 GO:0010039 Response to iron ion 0.00011 GO:0034644 Cellular response to UV 0.00014 GO:0071484 Cellular response to light intensity 0.00018 GO:0009773 Photosynthetic electron transport in photosystem I 0.00023 GO:0080027 Response to herbivore 0.00034 GO:0033473 Indoleacetic acid conjugate metabolic process 0.00034 GO:0009696 Salicylic acid metabolic process 0.00059 GO:0033609 Oxalate metabolic process 0.00093 GO:0050832 Defence response to fungus 0.00113 GO:0071423 Malate transmembrane transport 0.00209 GO:0042542 Response to hydrogen peroxide 0.00234 GO:1900426 Positive regulation of defence response to bacterium 0.00239 GO:0010876 Lipid localization 0.00313 GO:0018106 Peptidyl-histidine phosphorylation 0.00404 GO:0046688 Response to copper ion 0.00558 GO:0010257 NADH dehydrogenase complex assembly 0.00608 GO:0009838 Abscission 0.00704 GO:0071732 Cellular response to nitric oxide 0.00791 View Large Table 3. Presence and absence of genes involved in stomatal development, ethylene synthesis and signalling, and terpenoid biosynthesis in OGCsM, H. ovalis, Z. marina, and Z. muelleri Gene ID Protein name Function Conserved in OGCsM Presence in H. ovalis Presence in Z. muelleri Presence in Z. marina Stomata development AT1G04110 SBT1.2 Spacing and patterning + NA − − AT4G12970 EPFL9 Spacing and patterning + − − − AT2G20875 EPF1 Spacing and patterning + − − − AT1G80080 TMM Spacing and patterning + − − − AT1G34245 EPF2 Spacing and patterning + − − − AT2G02820 MYB88 Differentiation − NA NA − AT3G06120 MUTE Differentiation + − − − AT5G53210 SPCH Differentiation + − − − AT3G24140 FAMA Differentiation + NA NA − AT1G12860 SCRM2 Differentiation − NA NA − AT1G14350 FLP Differentiation + − − − Ethylene synthesis and signalling AT2G19590 ACO1 ACC oxidase + − − − AT1G62380 ACO2 ACC oxidase + − − − AT1G05010 ACO4 ACC oxidase + − − − AT1G77330 ACO5 ACC oxidase + − − − AT3G61510 ACS1 ACC synthase + − − − AT1G01480 ACS2 ACC synthase + − − − AT2G22810 ACS4 ACC synthase + − − − AT5G65800 ACS5 ACC synthase + − − − AT4G11280 ACS6 ACC synthase + − − − AT4G26200 ACS7 ACC synthase + − − − AT4G37770 ACS8 ACC synthase + − − − AT3G49700 ACS9 ACC synthase + − − − AT4G08040 ACS11 ACC synthase + − − − AT2G40940 ERS1 Ethylene receptor + − − − AT1G66340 ETR1 Ethylene receptor + − − − AT3G23150 ETR2 Ethylene receptor + − − − AT3G04580 EIN4 Ethylene receptor + − − − AT5G03730 CTR1 Raf-like kinase + NA NA − AT5G03280 EIN2 Signal transducer + NA − − AT2G25490 EBF1 EIN2 degradation + − − − AT5G25350 EBF2 EIN2 degradation + − − − Terpenoid biosynthesis AT3G25820 TPS-CIN Terpene synthase + − − − AT3G25830 TPS23 Terpene synthase + − − − AT4G16740 TPS03 Terpene synthase − − − − AT2G24210 TPS10 Terpene synthase + − − − AT3G25810 TPS24 Terpene synthase − − − − Gene ID Protein name Function Conserved in OGCsM Presence in H. ovalis Presence in Z. muelleri Presence in Z. marina Stomata development AT1G04110 SBT1.2 Spacing and patterning + NA − − AT4G12970 EPFL9 Spacing and patterning + − − − AT2G20875 EPF1 Spacing and patterning + − − − AT1G80080 TMM Spacing and patterning + − − − AT1G34245 EPF2 Spacing and patterning + − − − AT2G02820 MYB88 Differentiation − NA NA − AT3G06120 MUTE Differentiation + − − − AT5G53210 SPCH Differentiation + − − − AT3G24140 FAMA Differentiation + NA NA − AT1G12860 SCRM2 Differentiation − NA NA − AT1G14350 FLP Differentiation + − − − Ethylene synthesis and signalling AT2G19590 ACO1 ACC oxidase + − − − AT1G62380 ACO2 ACC oxidase + − − − AT1G05010 ACO4 ACC oxidase + − − − AT1G77330 ACO5 ACC oxidase + − − − AT3G61510 ACS1 ACC synthase + − − − AT1G01480 ACS2 ACC synthase + − − − AT2G22810 ACS4 ACC synthase + − − − AT5G65800 ACS5 ACC synthase + − − − AT4G11280 ACS6 ACC synthase + − − − AT4G26200 ACS7 ACC synthase + − − − AT4G37770 ACS8 ACC synthase + − − − AT3G49700 ACS9 ACC synthase + − − − AT4G08040 ACS11 ACC synthase + − − − AT2G40940 ERS1 Ethylene receptor + − − − AT1G66340 ETR1 Ethylene receptor + − − − AT3G23150 ETR2 Ethylene receptor + − − − AT3G04580 EIN4 Ethylene receptor + − − − AT5G03730 CTR1 Raf-like kinase + NA NA − AT5G03280 EIN2 Signal transducer + NA − − AT2G25490 EBF1 EIN2 degradation + − − − AT5G25350 EBF2 EIN2 degradation + − − − Terpenoid biosynthesis AT3G25820 TPS-CIN Terpene synthase + − − − AT3G25830 TPS23 Terpene synthase + − − − AT4G16740 TPS03 Terpene synthase − − − − AT2G24210 TPS10 Terpene synthase + − − − AT3G25810 TPS24 Terpene synthase − − − − Categories are: gene present (+), gene absent (−), and information not available (NA). View Large Table 3. Presence and absence of genes involved in stomatal development, ethylene synthesis and signalling, and terpenoid biosynthesis in OGCsM, H. ovalis, Z. marina, and Z. muelleri Gene ID Protein name Function Conserved in OGCsM Presence in H. ovalis Presence in Z. muelleri Presence in Z. marina Stomata development AT1G04110 SBT1.2 Spacing and patterning + NA − − AT4G12970 EPFL9 Spacing and patterning + − − − AT2G20875 EPF1 Spacing and patterning + − − − AT1G80080 TMM Spacing and patterning + − − − AT1G34245 EPF2 Spacing and patterning + − − − AT2G02820 MYB88 Differentiation − NA NA − AT3G06120 MUTE Differentiation + − − − AT5G53210 SPCH Differentiation + − − − AT3G24140 FAMA Differentiation + NA NA − AT1G12860 SCRM2 Differentiation − NA NA − AT1G14350 FLP Differentiation + − − − Ethylene synthesis and signalling AT2G19590 ACO1 ACC oxidase + − − − AT1G62380 ACO2 ACC oxidase + − − − AT1G05010 ACO4 ACC oxidase + − − − AT1G77330 ACO5 ACC oxidase + − − − AT3G61510 ACS1 ACC synthase + − − − AT1G01480 ACS2 ACC synthase + − − − AT2G22810 ACS4 ACC synthase + − − − AT5G65800 ACS5 ACC synthase + − − − AT4G11280 ACS6 ACC synthase + − − − AT4G26200 ACS7 ACC synthase + − − − AT4G37770 ACS8 ACC synthase + − − − AT3G49700 ACS9 ACC synthase + − − − AT4G08040 ACS11 ACC synthase + − − − AT2G40940 ERS1 Ethylene receptor + − − − AT1G66340 ETR1 Ethylene receptor + − − − AT3G23150 ETR2 Ethylene receptor + − − − AT3G04580 EIN4 Ethylene receptor + − − − AT5G03730 CTR1 Raf-like kinase + NA NA − AT5G03280 EIN2 Signal transducer + NA − − AT2G25490 EBF1 EIN2 degradation + − − − AT5G25350 EBF2 EIN2 degradation + − − − Terpenoid biosynthesis AT3G25820 TPS-CIN Terpene synthase + − − − AT3G25830 TPS23 Terpene synthase + − − − AT4G16740 TPS03 Terpene synthase − − − − AT2G24210 TPS10 Terpene synthase + − − − AT3G25810 TPS24 Terpene synthase − − − − Gene ID Protein name Function Conserved in OGCsM Presence in H. ovalis Presence in Z. muelleri Presence in Z. marina Stomata development AT1G04110 SBT1.2 Spacing and patterning + NA − − AT4G12970 EPFL9 Spacing and patterning + − − − AT2G20875 EPF1 Spacing and patterning + − − − AT1G80080 TMM Spacing and patterning + − − − AT1G34245 EPF2 Spacing and patterning + − − − AT2G02820 MYB88 Differentiation − NA NA − AT3G06120 MUTE Differentiation + − − − AT5G53210 SPCH Differentiation + − − − AT3G24140 FAMA Differentiation + NA NA − AT1G12860 SCRM2 Differentiation − NA NA − AT1G14350 FLP Differentiation + − − − Ethylene synthesis and signalling AT2G19590 ACO1 ACC oxidase + − − − AT1G62380 ACO2 ACC oxidase + − − − AT1G05010 ACO4 ACC oxidase + − − − AT1G77330 ACO5 ACC oxidase + − − − AT3G61510 ACS1 ACC synthase + − − − AT1G01480 ACS2 ACC synthase + − − − AT2G22810 ACS4 ACC synthase + − − − AT5G65800 ACS5 ACC synthase + − − − AT4G11280 ACS6 ACC synthase + − − − AT4G26200 ACS7 ACC synthase + − − − AT4G37770 ACS8 ACC synthase + − − − AT3G49700 ACS9 ACC synthase + − − − AT4G08040 ACS11 ACC synthase + − − − AT2G40940 ERS1 Ethylene receptor + − − − AT1G66340 ETR1 Ethylene receptor + − − − AT3G23150 ETR2 Ethylene receptor + − − − AT3G04580 EIN4 Ethylene receptor + − − − AT5G03730 CTR1 Raf-like kinase + NA NA − AT5G03280 EIN2 Signal transducer + NA − − AT2G25490 EBF1 EIN2 degradation + − − − AT5G25350 EBF2 EIN2 degradation + − − − Terpenoid biosynthesis AT3G25820 TPS-CIN Terpene synthase + − − − AT3G25830 TPS23 Terpene synthase + − − − AT4G16740 TPS03 Terpene synthase − − − − AT2G24210 TPS10 Terpene synthase + − − − AT3G25810 TPS24 Terpene synthase − − − − Categories are: gene present (+), gene absent (−), and information not available (NA). View Large Halophila ovalis lost genes encoding NADH dehydrogenase-like complex assembly The five most significantly enriched GO terms in the 412 genes that were only lost in H. ovalis were cellular response to light intensity (GO:0071484), cellular response to UV (GO:0034644), photosynthetic electron transport in photosystem I (GO:0009773), NADH dehydrogenase complex assembly (GO:0010257), and cellular response to salt stress (GO:0071472). A complete list of all significantly enriched terms is given in Table S5. Closer examination revealed the loss of 23 (15 nuclear and 8 chloroplast) genes that encode the five subcomplexes in the NDH complex (Table 4). In addition, 17 genes required for the supercomplex formation, including tethering of NDH to photosystem I, assembly of subunits, accessory proteins, and transcription factors, were absent in H. ovalis. Two proteins required for nitrate uptake and assimilation, nitrogen reductase 1 (NR1) and nitrate transporter (NRT3.1), were also lost in H. ovalis. Table 4. Presence and absence of 40 nuclear and chloroplast-encoded genes involved in formation of the NDH complex Gene ID Protein name Function Presence in OGCsM Presence in H. ovalis Presence in Z. muelleri Presence in Z. marina Nuclear encoded AT1G70760 NDHL Subunit A + NA + + AT4G37925 NDHM Subunit A + − + + AT5G58260 NDHN Subunit A + NA + + AT1G74880 NDHO Subunit A + − + + AT4G23890 NDHS Subunit ED + − NA + AT4G09350 NDHT Subunit ED + − + + AT5G21430 NDHU Subunit ED + − + + AT1G15980 PNSB1 Subunit B + − NA + AT1G64770 PNSB2 Subunit B + − + + AT3G16250 PNSB3 Subunit B + − + + AT1G18730 PNSB4 Subunit B + − + + AT2G39470 PNSL1 Subunit B + − + + AT1G14150 PNSL2 Subunit L + − + + AT3G01440 PNSL3 Subunit L + NA + + AT4G39710 PNSL4 Subunit L + − + + AT5G13120 PNSL5 Subunit L + + + + AT2G47910 CRR6 Complex formation + − + + AT5G39210 CRR7 Complex formation + − + + AT1G45474 Lhca5 Complex formation + − + + AT1G19150 Lhca6 Complex formation + NA + + AT1G26230 CRR27 Complex formation + NA + + AT1G51100 CRR41 Complex formation + NA + + AT2G05620 PGR5 Proton gradient regulation + NA + + AT4G22890 PGRL1A Proton gradient regulation + + + + AT3G46790 CRR2 Unknown + NA + + AT2G01590 CRR3 Unknown + − − + AT5G20935 CRR42 Unknown + − + + AT2G01918 PQL3 Unknown + − − + AT1G55370 NDF5 Unknown + − + + Chloroplast encoded ATCG00890 ATCG01250 NDHB Subunit M + NA + + ATCG01010 NDHF Subunit M + NA + + ATCG00440 NDHC Subunit M + − + + ATCG01050 NDHD Subunit M + − + + ATCG01070 NDHE Subunit M + − + + ATCG01100 NDHA Subunit M + − + + ATCG01080 NDHG Subunit M + NA + + ATCG01110 NDHH Subunit A + + + + ATCG00420 NDHJ Subunit A + − + + ATCG00430 NDHK Subunit A + − + + ATCG01090 NDHI Subunit A + − + + Gene ID Protein name Function Presence in OGCsM Presence in H. ovalis Presence in Z. muelleri Presence in Z. marina Nuclear encoded AT1G70760 NDHL Subunit A + NA + + AT4G37925 NDHM Subunit A + − + + AT5G58260 NDHN Subunit A + NA + + AT1G74880 NDHO Subunit A + − + + AT4G23890 NDHS Subunit ED + − NA + AT4G09350 NDHT Subunit ED + − + + AT5G21430 NDHU Subunit ED + − + + AT1G15980 PNSB1 Subunit B + − NA + AT1G64770 PNSB2 Subunit B + − + + AT3G16250 PNSB3 Subunit B + − + + AT1G18730 PNSB4 Subunit B + − + + AT2G39470 PNSL1 Subunit B + − + + AT1G14150 PNSL2 Subunit L + − + + AT3G01440 PNSL3 Subunit L + NA + + AT4G39710 PNSL4 Subunit L + − + + AT5G13120 PNSL5 Subunit L + + + + AT2G47910 CRR6 Complex formation + − + + AT5G39210 CRR7 Complex formation + − + + AT1G45474 Lhca5 Complex formation + − + + AT1G19150 Lhca6 Complex formation + NA + + AT1G26230 CRR27 Complex formation + NA + + AT1G51100 CRR41 Complex formation + NA + + AT2G05620 PGR5 Proton gradient regulation + NA + + AT4G22890 PGRL1A Proton gradient regulation + + + + AT3G46790 CRR2 Unknown + NA + + AT2G01590 CRR3 Unknown + − − + AT5G20935 CRR42 Unknown + − + + AT2G01918 PQL3 Unknown + − − + AT1G55370 NDF5 Unknown + − + + Chloroplast encoded ATCG00890 ATCG01250 NDHB Subunit M + NA + + ATCG01010 NDHF Subunit M + NA + + ATCG00440 NDHC Subunit M + − + + ATCG01050 NDHD Subunit M + − + + ATCG01070 NDHE Subunit M + − + + ATCG01100 NDHA Subunit M + − + + ATCG01080 NDHG Subunit M + NA + + ATCG01110 NDHH Subunit A + + + + ATCG00420 NDHJ Subunit A + − + + ATCG00430 NDHK Subunit A + − + + ATCG01090 NDHI Subunit A + − + + Categories are: gene present (+), gene absent (−), and information not available (NA). View Large Table 4. Presence and absence of 40 nuclear and chloroplast-encoded genes involved in formation of the NDH complex Gene ID Protein name Function Presence in OGCsM Presence in H. ovalis Presence in Z. muelleri Presence in Z. marina Nuclear encoded AT1G70760 NDHL Subunit A + NA + + AT4G37925 NDHM Subunit A + − + + AT5G58260 NDHN Subunit A + NA + + AT1G74880 NDHO Subunit A + − + + AT4G23890 NDHS Subunit ED + − NA + AT4G09350 NDHT Subunit ED + − + + AT5G21430 NDHU Subunit ED + − + + AT1G15980 PNSB1 Subunit B + − NA + AT1G64770 PNSB2 Subunit B + − + + AT3G16250 PNSB3 Subunit B + − + + AT1G18730 PNSB4 Subunit B + − + + AT2G39470 PNSL1 Subunit B + − + + AT1G14150 PNSL2 Subunit L + − + + AT3G01440 PNSL3 Subunit L + NA + + AT4G39710 PNSL4 Subunit L + − + + AT5G13120 PNSL5 Subunit L + + + + AT2G47910 CRR6 Complex formation + − + + AT5G39210 CRR7 Complex formation + − + + AT1G45474 Lhca5 Complex formation + − + + AT1G19150 Lhca6 Complex formation + NA + + AT1G26230 CRR27 Complex formation + NA + + AT1G51100 CRR41 Complex formation + NA + + AT2G05620 PGR5 Proton gradient regulation + NA + + AT4G22890 PGRL1A Proton gradient regulation + + + + AT3G46790 CRR2 Unknown + NA + + AT2G01590 CRR3 Unknown + − − + AT5G20935 CRR42 Unknown + − + + AT2G01918 PQL3 Unknown + − − + AT1G55370 NDF5 Unknown + − + + Chloroplast encoded ATCG00890 ATCG01250 NDHB Subunit M + NA + + ATCG01010 NDHF Subunit M + NA + + ATCG00440 NDHC Subunit M + − + + ATCG01050 NDHD Subunit M + − + + ATCG01070 NDHE Subunit M + − + + ATCG01100 NDHA Subunit M + − + + ATCG01080 NDHG Subunit M + NA + + ATCG01110 NDHH Subunit A + + + + ATCG00420 NDHJ Subunit A + − + + ATCG00430 NDHK Subunit A + − + + ATCG01090 NDHI Subunit A + − + + Gene ID Protein name Function Presence in OGCsM Presence in H. ovalis Presence in Z. muelleri Presence in Z. marina Nuclear encoded AT1G70760 NDHL Subunit A + NA + + AT4G37925 NDHM Subunit A + − + + AT5G58260 NDHN Subunit A + NA + + AT1G74880 NDHO Subunit A + − + + AT4G23890 NDHS Subunit ED + − NA + AT4G09350 NDHT Subunit ED + − + + AT5G21430 NDHU Subunit ED + − + + AT1G15980 PNSB1 Subunit B + − NA + AT1G64770 PNSB2 Subunit B + − + + AT3G16250 PNSB3 Subunit B + − + + AT1G18730 PNSB4 Subunit B + − + + AT2G39470 PNSL1 Subunit B + − + + AT1G14150 PNSL2 Subunit L + − + + AT3G01440 PNSL3 Subunit L + NA + + AT4G39710 PNSL4 Subunit L + − + + AT5G13120 PNSL5 Subunit L + + + + AT2G47910 CRR6 Complex formation + − + + AT5G39210 CRR7 Complex formation + − + + AT1G45474 Lhca5 Complex formation + − + + AT1G19150 Lhca6 Complex formation + NA + + AT1G26230 CRR27 Complex formation + NA + + AT1G51100 CRR41 Complex formation + NA + + AT2G05620 PGR5 Proton gradient regulation + NA + + AT4G22890 PGRL1A Proton gradient regulation + + + + AT3G46790 CRR2 Unknown + NA + + AT2G01590 CRR3 Unknown + − − + AT5G20935 CRR42 Unknown + − + + AT2G01918 PQL3 Unknown + − − + AT1G55370 NDF5 Unknown + − + + Chloroplast encoded ATCG00890 ATCG01250 NDHB Subunit M + NA + + ATCG01010 NDHF Subunit M + NA + + ATCG00440 NDHC Subunit M + − + + ATCG01050 NDHD Subunit M + − + + ATCG01070 NDHE Subunit M + − + + ATCG01100 NDHA Subunit M + − + + ATCG01080 NDHG Subunit M + NA + + ATCG01110 NDHH Subunit A + + + + ATCG00420 NDHJ Subunit A + − + + ATCG00430 NDHK Subunit A + − + + ATCG01090 NDHI Subunit A + − + + Categories are: gene present (+), gene absent (−), and information not available (NA). View Large Co-evolution of genes for intracellular transport, cell wall, and ion transport-related genes in H. ovalis and Zostera We identified a set of 1748 genes that are unique to Z. muelleri and Z. marina, termed OGCZ (Fig. S1; Table S6), with 57 also found to be conserved in H. ovalis (Table S7). Putative functions of these 57 genes were inferred by matching their protein domains to the corresponding best aligned Arabidopsis gene. Out of 57 OGCZ groups, 45 have identical domains (indicated as InterProScan IDs) to the corresponding Arabidopsis genes. A total of eight of them have fewer or different domains from Arabidopsis, and four of them have no domains assigned (Table S7). The majority of the 57 OGCZ genes conserved in H. ovalis are predicted to be involved in protein secretion and intracellular transport, with significantly enriched terms annotated with cellular component ontology, including organelles of the intracellular transport pathways, namely Golgi apparatus, trans-Golgi network, and endosome, and nearly half of the remaining terms intracellular transport-related (Fig. 1). A total of 13 genes are predicted to function in protein secretion and intracellular transport, mainly as transport proteins or transport regulators. Nine genes are associated with cell wall construction, organization, and modification, while other predicted functions include ion or proton transport, lipid catabolism, transcription and translation-related, protein ubiquitination, and histone assembly (Table 5). Table 5. Fifty-seven orthologous groups of seagrass-specific genes shared in two Zosteraceae species (Z. muelleri and Z. marina) and Halophila categorized by predicted function Category of related function Name of best TAIR10 hit corresponding to Zostera orthologue ID of best TAIR10 hit corresponding to Zostera orthologue Putative gene function Protein secretion and intracellular transport Endoplasmic reticulum retention defective 2B AT3G25040.1 Retention mechanism Endoplasmic reticulum-type calcium-transporting ATPase 3 AT1G10130.1 Calcium and manganese ion transport RAB GTPase homologue A1F AT5G60860.1 GTPase activity RAB GTPase homologue A2B AT1G07410.1 GTPase activity Secretory carrier 3 AT1G61250.1 Integral membrane protein NOD26-like intrinsic protein 1;2 AT4G18910.1 Aquaporin Mitochondrial substrate carrier family protein AT3G53940.1 Substrate transport Mitochondrial import inner membrane translocase subunit Tim17/Tim22/Tim23 family protein AT5G63000.1 Protein transport Transducin/WD40 repeat-like superfamily protein AT3G01340.1 Protein transport Protein of unknown function AT1G09330.1 — Cell wall Expansin A16 AT3G55500.1 Cell wall loosening Expansin A1 AT1G69530.2 Cell wall loosening Galacturonosyltransferase-like 2 AT3G50760.1 Cell wall organization Xyloglucan endotransglucosylase/hydrolase 5 AT5G13870.1 Cell wall organization Glucan synthase-like 8 AT2G36850.1 Callose synthesis S-Adenosyl-L-methionine-dependent methyltransferases superfamily protein AT4G34050.1 Lignin biosynthesis Peroxidase superfamily protein AT5G05340.1 Lignin biosynthesis Cotton Golgi-related 2 (pectin methyltransferase) AT3G49720.1 Cell wall modification Vascular related NAC-domain protein 1 AT2G18060.1 Xylem secondary cell wall formation Ion flux and sequestering ATP synthase epsilon chain, mitochondrial AT1G51650.1 Proton-transporting ATPase activity Vacuolar proton ATPase A1 AT2G28520.1 Proton-transporting ATPase activity Calmodulin 4 AT1G66410.1 Calcium ion binding Lipid catabolism Trigalactosyldiacylglycerol 5 AT1G27695.1 Lipid transport GDSL-like lipase/acylhydrolase superfamily protein AT1G29670.1 AT5G45670.1 Lipid catabolic process Peroxin 6 AT1G03000.1 Peroxisomal matrix protein import Alkaline phytoceramidase AT4G22330.1 Ceramide synthase involved in sphingolipid metabolism Transcription-related RNA polymerase subunit beta ATCG00190.1 Constituent of RNA polymerase B Pre-mRNA-splicing factor SPF27 homologue AT3G18165.1 mRNA splicing of resistance genes Ribosome/ translation-related Ribosomal protein L16 ATCG00790.1 Structural constituent of ribosome Ribosomal protein S26e family protein AT2G40510.1 Structural constituent of ribosome Ribosomal protein S8e family protein AT5G59240.1 Structural constituent of ribosome Ribosomal protein S2 ATCG00160.1 Structural constituent of ribosome Eukaryotic translation initiation factor 3A AT4G11420.1 Constituent of eukaryotic initiation factor 3 Protein ubiquitination F-box protein PP2-A13 AT3G61060.1 Protein ubiquitination BTB/POZ domain-containing protein AT1G63850.1 Protein ubiquitination Ubiquitin-conjugating enzyme 28 AT1G64230.1 Protein ubiquitination Ubiquitin-like protein 5 AT5G42300.1 Ubiquitin-like modification Histone Histone H2A.2 AT3G20670.1 Histones/DNA binding/nucleosome assembly Histone H3.3 AT4G40030.2 Histones/DNA binding/nucleosome assembly Others Photosystem II light harvesting complex gene 2.1 AT2G05100.1 Constituent of light harvesting complex II Alternative oxidase 1A AT3G22370.1 Alternative oxidase activity Tubulin folding cofactor D AT3G60740.1 Microtubule stability Asparagine synthetase 2 AT5G65010.2 Asparagine biosynthesis Glutamate-1-semialdehyde 2,1-aminomutase 2 AT3G48730.1 Porphyrin-containing compound metabolism Membrane-associated progesterone binding protein 3 AT3G48890.1 Porphyrin binding Thioredoxin superfamily protein AT3G62950.1 Electron carrier activity DNA polymerase epsilon catalytic subunit AT1G08260.1 DNA replication proofreading NAC domain containing protein 32 AT1G77450.1 Transcription factor DNA-binding protein phosphatase 1 AT2G25620.1 Protein phosphatase activity Protein kinase 1B AT2G28930.1 Serine/threonine kinase activity UDP-glycosyltransferase superfamily protein AT5G04480.1 — Adenine nucleotide alpha hydrolases-like superfamily protein AT1G11360.4 — Protein of unknown function (DUF300) AT1G11200.1 — Protein of unknown function (DUF803) AT1G34470.1 — Category of related function Name of best TAIR10 hit corresponding to Zostera orthologue ID of best TAIR10 hit corresponding to Zostera orthologue Putative gene function Protein secretion and intracellular transport Endoplasmic reticulum retention defective 2B AT3G25040.1 Retention mechanism Endoplasmic reticulum-type calcium-transporting ATPase 3 AT1G10130.1 Calcium and manganese ion transport RAB GTPase homologue A1F AT5G60860.1 GTPase activity RAB GTPase homologue A2B AT1G07410.1 GTPase activity Secretory carrier 3 AT1G61250.1 Integral membrane protein NOD26-like intrinsic protein 1;2 AT4G18910.1 Aquaporin Mitochondrial substrate carrier family protein AT3G53940.1 Substrate transport Mitochondrial import inner membrane translocase subunit Tim17/Tim22/Tim23 family protein AT5G63000.1 Protein transport Transducin/WD40 repeat-like superfamily protein AT3G01340.1 Protein transport Protein of unknown function AT1G09330.1 — Cell wall Expansin A16 AT3G55500.1 Cell wall loosening Expansin A1 AT1G69530.2 Cell wall loosening Galacturonosyltransferase-like 2 AT3G50760.1 Cell wall organization Xyloglucan endotransglucosylase/hydrolase 5 AT5G13870.1 Cell wall organization Glucan synthase-like 8 AT2G36850.1 Callose synthesis S-Adenosyl-L-methionine-dependent methyltransferases superfamily protein AT4G34050.1 Lignin biosynthesis Peroxidase superfamily protein AT5G05340.1 Lignin biosynthesis Cotton Golgi-related 2 (pectin methyltransferase) AT3G49720.1 Cell wall modification Vascular related NAC-domain protein 1 AT2G18060.1 Xylem secondary cell wall formation Ion flux and sequestering ATP synthase epsilon chain, mitochondrial AT1G51650.1 Proton-transporting ATPase activity Vacuolar proton ATPase A1 AT2G28520.1 Proton-transporting ATPase activity Calmodulin 4 AT1G66410.1 Calcium ion binding Lipid catabolism Trigalactosyldiacylglycerol 5 AT1G27695.1 Lipid transport GDSL-like lipase/acylhydrolase superfamily protein AT1G29670.1 AT5G45670.1 Lipid catabolic process Peroxin 6 AT1G03000.1 Peroxisomal matrix protein import Alkaline phytoceramidase AT4G22330.1 Ceramide synthase involved in sphingolipid metabolism Transcription-related RNA polymerase subunit beta ATCG00190.1 Constituent of RNA polymerase B Pre-mRNA-splicing factor SPF27 homologue AT3G18165.1 mRNA splicing of resistance genes Ribosome/ translation-related Ribosomal protein L16 ATCG00790.1 Structural constituent of ribosome Ribosomal protein S26e family protein AT2G40510.1 Structural constituent of ribosome Ribosomal protein S8e family protein AT5G59240.1 Structural constituent of ribosome Ribosomal protein S2 ATCG00160.1 Structural constituent of ribosome Eukaryotic translation initiation factor 3A AT4G11420.1 Constituent of eukaryotic initiation factor 3 Protein ubiquitination F-box protein PP2-A13 AT3G61060.1 Protein ubiquitination BTB/POZ domain-containing protein AT1G63850.1 Protein ubiquitination Ubiquitin-conjugating enzyme 28 AT1G64230.1 Protein ubiquitination Ubiquitin-like protein 5 AT5G42300.1 Ubiquitin-like modification Histone Histone H2A.2 AT3G20670.1 Histones/DNA binding/nucleosome assembly Histone H3.3 AT4G40030.2 Histones/DNA binding/nucleosome assembly Others Photosystem II light harvesting complex gene 2.1 AT2G05100.1 Constituent of light harvesting complex II Alternative oxidase 1A AT3G22370.1 Alternative oxidase activity Tubulin folding cofactor D AT3G60740.1 Microtubule stability Asparagine synthetase 2 AT5G65010.2 Asparagine biosynthesis Glutamate-1-semialdehyde 2,1-aminomutase 2 AT3G48730.1 Porphyrin-containing compound metabolism Membrane-associated progesterone binding protein 3 AT3G48890.1 Porphyrin binding Thioredoxin superfamily protein AT3G62950.1 Electron carrier activity DNA polymerase epsilon catalytic subunit AT1G08260.1 DNA replication proofreading NAC domain containing protein 32 AT1G77450.1 Transcription factor DNA-binding protein phosphatase 1 AT2G25620.1 Protein phosphatase activity Protein kinase 1B AT2G28930.1 Serine/threonine kinase activity UDP-glycosyltransferase superfamily protein AT5G04480.1 — Adenine nucleotide alpha hydrolases-like superfamily protein AT1G11360.4 — Protein of unknown function (DUF300) AT1G11200.1 — Protein of unknown function (DUF803) AT1G34470.1 — Gene functions were predicted with corresponding Arabidopsis gene of highest sequence similarity. View Large Table 5. Fifty-seven orthologous groups of seagrass-specific genes shared in two Zosteraceae species (Z. muelleri and Z. marina) and Halophila categorized by predicted function Category of related function Name of best TAIR10 hit corresponding to Zostera orthologue ID of best TAIR10 hit corresponding to Zostera orthologue Putative gene function Protein secretion and intracellular transport Endoplasmic reticulum retention defective 2B AT3G25040.1 Retention mechanism Endoplasmic reticulum-type calcium-transporting ATPase 3 AT1G10130.1 Calcium and manganese ion transport RAB GTPase homologue A1F AT5G60860.1 GTPase activity RAB GTPase homologue A2B AT1G07410.1 GTPase activity Secretory carrier 3 AT1G61250.1 Integral membrane protein NOD26-like intrinsic protein 1;2 AT4G18910.1 Aquaporin Mitochondrial substrate carrier family protein AT3G53940.1 Substrate transport Mitochondrial import inner membrane translocase subunit Tim17/Tim22/Tim23 family protein AT5G63000.1 Protein transport Transducin/WD40 repeat-like superfamily protein AT3G01340.1 Protein transport Protein of unknown function AT1G09330.1 — Cell wall Expansin A16 AT3G55500.1 Cell wall loosening Expansin A1 AT1G69530.2 Cell wall loosening Galacturonosyltransferase-like 2 AT3G50760.1 Cell wall organization Xyloglucan endotransglucosylase/hydrolase 5 AT5G13870.1 Cell wall organization Glucan synthase-like 8 AT2G36850.1 Callose synthesis S-Adenosyl-L-methionine-dependent methyltransferases superfamily protein AT4G34050.1 Lignin biosynthesis Peroxidase superfamily protein AT5G05340.1 Lignin biosynthesis Cotton Golgi-related 2 (pectin methyltransferase) AT3G49720.1 Cell wall modification Vascular related NAC-domain protein 1 AT2G18060.1 Xylem secondary cell wall formation Ion flux and sequestering ATP synthase epsilon chain, mitochondrial AT1G51650.1 Proton-transporting ATPase activity Vacuolar proton ATPase A1 AT2G28520.1 Proton-transporting ATPase activity Calmodulin 4 AT1G66410.1 Calcium ion binding Lipid catabolism Trigalactosyldiacylglycerol 5 AT1G27695.1 Lipid transport GDSL-like lipase/acylhydrolase superfamily protein AT1G29670.1 AT5G45670.1 Lipid catabolic process Peroxin 6 AT1G03000.1 Peroxisomal matrix protein import Alkaline phytoceramidase AT4G22330.1 Ceramide synthase involved in sphingolipid metabolism Transcription-related RNA polymerase subunit beta ATCG00190.1 Constituent of RNA polymerase B Pre-mRNA-splicing factor SPF27 homologue AT3G18165.1 mRNA splicing of resistance genes Ribosome/ translation-related Ribosomal protein L16 ATCG00790.1 Structural constituent of ribosome Ribosomal protein S26e family protein AT2G40510.1 Structural constituent of ribosome Ribosomal protein S8e family protein AT5G59240.1 Structural constituent of ribosome Ribosomal protein S2 ATCG00160.1 Structural constituent of ribosome Eukaryotic translation initiation factor 3A AT4G11420.1 Constituent of eukaryotic initiation factor 3 Protein ubiquitination F-box protein PP2-A13 AT3G61060.1 Protein ubiquitination BTB/POZ domain-containing protein AT1G63850.1 Protein ubiquitination Ubiquitin-conjugating enzyme 28 AT1G64230.1 Protein ubiquitination Ubiquitin-like protein 5 AT5G42300.1 Ubiquitin-like modification Histone Histone H2A.2 AT3G20670.1 Histones/DNA binding/nucleosome assembly Histone H3.3 AT4G40030.2 Histones/DNA binding/nucleosome assembly Others Photosystem II light harvesting complex gene 2.1 AT2G05100.1 Constituent of light harvesting complex II Alternative oxidase 1A AT3G22370.1 Alternative oxidase activity Tubulin folding cofactor D AT3G60740.1 Microtubule stability Asparagine synthetase 2 AT5G65010.2 Asparagine biosynthesis Glutamate-1-semialdehyde 2,1-aminomutase 2 AT3G48730.1 Porphyrin-containing compound metabolism Membrane-associated progesterone binding protein 3 AT3G48890.1 Porphyrin binding Thioredoxin superfamily protein AT3G62950.1 Electron carrier activity DNA polymerase epsilon catalytic subunit AT1G08260.1 DNA replication proofreading NAC domain containing protein 32 AT1G77450.1 Transcription factor DNA-binding protein phosphatase 1 AT2G25620.1 Protein phosphatase activity Protein kinase 1B AT2G28930.1 Serine/threonine kinase activity UDP-glycosyltransferase superfamily protein AT5G04480.1 — Adenine nucleotide alpha hydrolases-like superfamily protein AT1G11360.4 — Protein of unknown function (DUF300) AT1G11200.1 — Protein of unknown function (DUF803) AT1G34470.1 — Category of related function Name of best TAIR10 hit corresponding to Zostera orthologue ID of best TAIR10 hit corresponding to Zostera orthologue Putative gene function Protein secretion and intracellular transport Endoplasmic reticulum retention defective 2B AT3G25040.1 Retention mechanism Endoplasmic reticulum-type calcium-transporting ATPase 3 AT1G10130.1 Calcium and manganese ion transport RAB GTPase homologue A1F AT5G60860.1 GTPase activity RAB GTPase homologue A2B AT1G07410.1 GTPase activity Secretory carrier 3 AT1G61250.1 Integral membrane protein NOD26-like intrinsic protein 1;2 AT4G18910.1 Aquaporin Mitochondrial substrate carrier family protein AT3G53940.1 Substrate transport Mitochondrial import inner membrane translocase subunit Tim17/Tim22/Tim23 family protein AT5G63000.1 Protein transport Transducin/WD40 repeat-like superfamily protein AT3G01340.1 Protein transport Protein of unknown function AT1G09330.1 — Cell wall Expansin A16 AT3G55500.1 Cell wall loosening Expansin A1 AT1G69530.2 Cell wall loosening Galacturonosyltransferase-like 2 AT3G50760.1 Cell wall organization Xyloglucan endotransglucosylase/hydrolase 5 AT5G13870.1 Cell wall organization Glucan synthase-like 8 AT2G36850.1 Callose synthesis S-Adenosyl-L-methionine-dependent methyltransferases superfamily protein AT4G34050.1 Lignin biosynthesis Peroxidase superfamily protein AT5G05340.1 Lignin biosynthesis Cotton Golgi-related 2 (pectin methyltransferase) AT3G49720.1 Cell wall modification Vascular related NAC-domain protein 1 AT2G18060.1 Xylem secondary cell wall formation Ion flux and sequestering ATP synthase epsilon chain, mitochondrial AT1G51650.1 Proton-transporting ATPase activity Vacuolar proton ATPase A1 AT2G28520.1 Proton-transporting ATPase activity Calmodulin 4 AT1G66410.1 Calcium ion binding Lipid catabolism Trigalactosyldiacylglycerol 5 AT1G27695.1 Lipid transport GDSL-like lipase/acylhydrolase superfamily protein AT1G29670.1 AT5G45670.1 Lipid catabolic process Peroxin 6 AT1G03000.1 Peroxisomal matrix protein import Alkaline phytoceramidase AT4G22330.1 Ceramide synthase involved in sphingolipid metabolism Transcription-related RNA polymerase subunit beta ATCG00190.1 Constituent of RNA polymerase B Pre-mRNA-splicing factor SPF27 homologue AT3G18165.1 mRNA splicing of resistance genes Ribosome/ translation-related Ribosomal protein L16 ATCG00790.1 Structural constituent of ribosome Ribosomal protein S26e family protein AT2G40510.1 Structural constituent of ribosome Ribosomal protein S8e family protein AT5G59240.1 Structural constituent of ribosome Ribosomal protein S2 ATCG00160.1 Structural constituent of ribosome Eukaryotic translation initiation factor 3A AT4G11420.1 Constituent of eukaryotic initiation factor 3 Protein ubiquitination F-box protein PP2-A13 AT3G61060.1 Protein ubiquitination BTB/POZ domain-containing protein AT1G63850.1 Protein ubiquitination Ubiquitin-conjugating enzyme 28 AT1G64230.1 Protein ubiquitination Ubiquitin-like protein 5 AT5G42300.1 Ubiquitin-like modification Histone Histone H2A.2 AT3G20670.1 Histones/DNA binding/nucleosome assembly Histone H3.3 AT4G40030.2 Histones/DNA binding/nucleosome assembly Others Photosystem II light harvesting complex gene 2.1 AT2G05100.1 Constituent of light harvesting complex II Alternative oxidase 1A AT3G22370.1 Alternative oxidase activity Tubulin folding cofactor D AT3G60740.1 Microtubule stability Asparagine synthetase 2 AT5G65010.2 Asparagine biosynthesis Glutamate-1-semialdehyde 2,1-aminomutase 2 AT3G48730.1 Porphyrin-containing compound metabolism Membrane-associated progesterone binding protein 3 AT3G48890.1 Porphyrin binding Thioredoxin superfamily protein AT3G62950.1 Electron carrier activity DNA polymerase epsilon catalytic subunit AT1G08260.1 DNA replication proofreading NAC domain containing protein 32 AT1G77450.1 Transcription factor DNA-binding protein phosphatase 1 AT2G25620.1 Protein phosphatase activity Protein kinase 1B AT2G28930.1 Serine/threonine kinase activity UDP-glycosyltransferase superfamily protein AT5G04480.1 — Adenine nucleotide alpha hydrolases-like superfamily protein AT1G11360.4 — Protein of unknown function (DUF300) AT1G11200.1 — Protein of unknown function (DUF803) AT1G34470.1 — Gene functions were predicted with corresponding Arabidopsis gene of highest sequence similarity. View Large Fig. 1. View largeDownload slide Significantly enriched cellular component GO terms in seagrass-specific genes. Terms in green are subcomponents or organelles of the intracellular transport pathways. Fig. 1. View largeDownload slide Significantly enriched cellular component GO terms in seagrass-specific genes. Terms in green are subcomponents or organelles of the intracellular transport pathways. Molecular comparison of seagrass ribosomal proteins Ribosomal 50S L16 orthologues from the two Zostera species, H. ovalis, and 12 species in the Alismatales order were aligned, together with predicted proteins from five model land plants (Table S1). We identified nine amino acid positions that appear to be specific to the seagrasses H. ovalis, Z. muelleri, and Z. marina (white arrows in Fig. 2) and conserved among the other 17 angiosperms (12 belong to the Alismatales order, eight are freshwater plants), one charophyte and one chlorophyte. Fig. 2. View largeDownload slide Ribosomal protein L16 multiple sequence alignments between 19 species (AMA, Alocasia macrorhizzos; ATH, Arabidopsis; DSE, Dieffenbachia seguine; EAU, Epiprenum aureum; ECA, Elodia canadensis; LMI, Lemna minor; MAC, Musa acuminata; NFL, Najas flexilis; OSA, Oryza sativa; PDA, Phoenix dactylifera; PPE, Potamogeton perfoliatus; SLI, Sagittaria lichuanensis; SLY, Solanum lycopersicum; SPO, Spirodela polyrhiza; SPU, Spirodela pundata; TTH, Tofieldia thibetica; WAU, Wolfia australiana) together with three seagrasses (HAL, H. ovalis; ZMA, Z. marina; ZMU, Z. muelleri). Species and corresponding IDs are listed in Table S1. Amino acids that were conserved within the non-seagrass group or among seagrasses are coloured according to physicochemical properties based on ‘Zappo’ colour scheme. White arrows indicated seagrass-specific mutations. Fig. 2. View largeDownload slide Ribosomal protein L16 multiple sequence alignments between 19 species (AMA, Alocasia macrorhizzos; ATH, Arabidopsis; DSE, Dieffenbachia seguine; EAU, Epiprenum aureum; ECA, Elodia canadensis; LMI, Lemna minor; MAC, Musa acuminata; NFL, Najas flexilis; OSA, Oryza sativa; PDA, Phoenix dactylifera; PPE, Potamogeton perfoliatus; SLI, Sagittaria lichuanensis; SLY, Solanum lycopersicum; SPO, Spirodela polyrhiza; SPU, Spirodela pundata; TTH, Tofieldia thibetica; WAU, Wolfia australiana) together with three seagrasses (HAL, H. ovalis; ZMA, Z. marina; ZMU, Z. muelleri). Species and corresponding IDs are listed in Table S1. Amino acids that were conserved within the non-seagrass group or among seagrasses are coloured according to physicochemical properties based on ‘Zappo’ colour scheme. White arrows indicated seagrass-specific mutations. A phylogenetic tree for these 22 species based on this protein sequence, which describes the relationships between orthologues of these 22 species, separates the seagrass species (H. ovalis, Z. muelleri, and Z. marina) from the other species (Fig. 3). The separation of the two Zostera orthologues from H. ovalis is also well-supported. Sister genera of both Halophila and Zostera grouped together as members of core alismatids (red in Fig. 3) (Alismatidae sensu; Les and Tippery, 2013). Fig. 3. View largeDownload slide Phylogenetic tree showing distance between ribosome protein L16 sequences of 17 species (AMA, Alocasia macrorhizzos; ATH, Arabidopsis; DSE, Dieffenbachia seguine; EAU, Epiprenum aureum; ECA, Elodia canadensis; LMI, Lemna minor; MAC, Musa acuminata; NFL, Najas flexilis; OSA, Oryza sativa; PDA, Phoenix dactylifera; PPE, Potamogeton perfoliatus; SLI, Sagittaria lichuanensis; SLY, Solanum lycopersicum; SPO, Spirodela polyrhiza; SPU, Spirodela pundata; TTH, Tofieldia thibetica; WAU, Wolfia australiana) together with three seagrasses (HAL, H. ovalis; ZMA, Z. marina; ZMU, Z. muelleri). The order and habitat of species were indicated in the second part of each ID: DL, dicot, land; ML, monocot, land; MLA, monocot, land, Alismatales; MMA, monocot, marine, Alismatales; MWA, monocot, freshwater, Alismatales. Complete details are listed in Table S1. IDs coloured in red are members of core Alismatids, blue are members of Araceae, and black are others. Branches are labelled with bootstrap values (%). Fig. 3. View largeDownload slide Phylogenetic tree showing distance between ribosome protein L16 sequences of 17 species (AMA, Alocasia macrorhizzos; ATH, Arabidopsis; DSE, Dieffenbachia seguine; EAU, Epiprenum aureum; ECA, Elodia canadensis; LMI, Lemna minor; MAC, Musa acuminata; NFL, Najas flexilis; OSA, Oryza sativa; PDA, Phoenix dactylifera; PPE, Potamogeton perfoliatus; SLI, Sagittaria lichuanensis; SLY, Solanum lycopersicum; SPO, Spirodela polyrhiza; SPU, Spirodela pundata; TTH, Tofieldia thibetica; WAU, Wolfia australiana) together with three seagrasses (HAL, H. ovalis; ZMA, Z. marina; ZMU, Z. muelleri). The order and habitat of species were indicated in the second part of each ID: DL, dicot, land; ML, monocot, land; MLA, monocot, land, Alismatales; MMA, monocot, marine, Alismatales; MWA, monocot, freshwater, Alismatales. Complete details are listed in Table S1. IDs coloured in red are members of core Alismatids, blue are members of Araceae, and black are others. Branches are labelled with bootstrap values (%). Discussion The concurrent absence of multiple genes in H. ovalis, Z. muelleri, and Z. marina suggests independently evolved convergent adaptation of seagrasses to the marine environment. Seagrass leaves lack stomata and the flowers have simplified structures when compared with terrestrial angiosperms (Kuo and Hartog, 2006). The loss of genes in stomata patterning and differentiation, and in sepal and petal development was previously described in Zostera, together with the loss of gaseous hormones and metabolites, such as ethylene, methyl jasmonate, and secondary volatile terpenes (Golicz et al., 2015; Lee et al., 2016; Olsen et al., 2016). Consistent with the low diffusion rate of gases underwater, the absence of ethylene production in seagrasses avoids accumulation in the tissues. In flood-adapted land plants, the ethylene signal is used to sense submergence and induces a response to flooding (Voesenek et al., 2015). Ethylene biosynthesis and signalling also play an important role in plant response to salinity (Zhang et al., 2016). There is conflicting evidence of ethylene as a positive or negative regulator during salinity stress in different species at different developmental stages (Tao et al., 2015), suggesting that some species adjust their sensitivity to environmental factors through regulation of the ethylene signalling pathway. As the loss of ethylene genes is observed in both H. ovalis and Zostera, it is likely that ethylene is selected against during seagrass adaptation to a submerged marine lifestyle. The sharing of OGCZ genes in H. ovalis to form a seagrass-specific gene set identifies orthologous relationships that appear to be unique to seagrass adaptation, despite their evolutionary distance and multiple origins. It is important to note that these 57 OGCZ genes are not novel genes but genes that have diverged sufficiently to cluster separately from other plants in orthologue analysis. The functions of these genes were not annotated but inferred by homology using annotated Arabidopsis genes. The majority of these genes are predicted to be involved in intracellular transport and in cell wall organization and modification. In plant cells, secreted proteins are processed through the Golgi apparatus as cargo molecules and sorted by receptors in the trans-Golgi network to different destinations (Brandizzi and Barlowe, 2013). Non-cellulosic cell wall matrix polysaccharides are among the wide range of vesicles synthesized and transported by the Golgi apparatus (Driouich et al., 1993; Lerouxel et al., 2006; Driouich et al., 2012). Besides catalytic mechanisms of glycosyltransferases and nucleotide-sugar conversions for polysaccharide assembly, the Golgi is also responsible for methylation of the cell wall polysaccharides. There are significant differences between cell walls of seagrasses and land plants. Seagrass cell walls contain sulfated polysaccharides (Aquino et al., 2005) and seagrass pectin contains a rare class of apiose-substituted homogalacturonan (Ovodov et al., 1971) with low levels of methyl esterification (Khotimchenko et al., 2012). These two modifications are thought to provide salt tolerance by increasing the polyanionic potential of cell walls (Aquino et al., 2005, 2011; Olsen et al., 2016). An expansion of pectin catabolic and methylesterase genes was observed in the genomes of Z. muelleri (Lee et al., 2016) and Z. marina (Olsen et al., 2016), suggesting complex pectin modification in seagrasses. Interestingly, within the list of seagrass-specific genes conserved in H. ovalis, CGR2 (cotton Golgi-related 2), a methyltransferase, was shown to be involved in pectin methylesterification in Arabidopsis (Weraduwage et al., 2016). Tubulin cofactor, which is responsible for the stability of microtubules (Zhu et al., 2015), is also found to be conserved among seagrasses. A total of five genes that encode RAB GTPases, the key regulators of vesicle trafficking (Miserey-Lenkei et al., 2010; Valente et al., 2010), were also conserved across both seagrass lineages. In Arabidopsis, knockouts of some members of the RAB GTPases have demonstrated roles in salinity stress tolerance (Asaoka et al., 2013). It is likely that this conservation of cell wall-related genes, as well as proteins involved in intracellular transport, in both families of seagrasses is linked to modification of cell wall composition as one of the adaptations to osmotic stress. Multiple salt-tolerance mechanisms have been hypothesized in seagrasses (reviewed in Touchette, 2007), including cell wall rigidity, selective ion flux and vacuolar ion sequestering, and the synthesis of compatible solutes and amino acids (Ye and Zhao, 2003; Carpaneto et al., 2004; Touchette et al., 2014; Cambridge et al., 2017). To avoid salt damage, plant cells adjust osmotic balance through influx and efflux of ions through the transmembrane transport proteins, assisted by H+ pumps (Hasegawa, 2013). Three genes, namely a component of a vacuolar proton pump, ATP synthase and calmodulin, were identified as conserved across the two seagrass lineages. Moreover, vacuolar proton ATPase A1 has been shown to be responsive to salt stress in sugar beet (Kirsch et al., 1996). This collection of genes may have a role in osmotic homeostasis of cells in the marine environment. Lipid transport and catabolism is another important role of the intracellular transport system. The endoplasmic reticulum synthesizes and exports phospholipids, sterols, and storage lipids for various purposes, including formation of membrane structures (van Meer et al., 2008). A total of four genes involved in lipid transport and catabolism were conserved in all three seagrass species, including ceramidase, which is responsible for sphingolipid metabolism. Sphingolipids provide membrane structure and are involved in cellular signal transduction (Hannun and Obeid, 2008). The difference between lipids of seagrasses and land plants is not well understood, but expansion in genes related to sphingolipid metabolism was observed in Z. marina when compared with duckweed (Olsen et al., 2016). Another alkaline ceramidase had been shown to regulate cell turgor pressure in Arabidopsis (Chen et al., 2015), but more evidence is needed to determine whether seagrass-specific lipid metabolism plays a role in marine adaptation. Two members of the core histone family are conserved in seagrasses. The domains in histone families, particularly H2A and H3, demonstrate expansion in numbers and variety, but with strong conservation of each variant across species (Kawashima et al., 2015). Ribosomal constituents were previously identified as modified in Z. muelleri when compared with land plants (Lee et al., 2016) and positively selected in Z. marina and P. oceanica (Wissler et al., 2011), and our results demonstrate that these genes are also conserved in H. ovalis. The basis for the observed differences in ribosomal gene sequences is not known, but it is postulated to be related to salt tolerance. Translation, and consequently protein synthesis are known to be salt-sensitive in yeast and plants (Rausell et al., 2003). For example, the expression of genes encoding the translation apparatus was lower when the transcriptome of Arabidopsis was compared with the halophyte salt cress (Taji et al., 2004). If seagrass ribosomes are adapted to relatively high salinity, this may have an application for improvement of salt tolerance in crop species. Sequence variations were identified in chloroplast-encoded 50S ribosomal protein L16. Nine amino acid mutations were shared by the three seagrass species despite belonging to two separate clades (Les et al., 1997; Li and Zhou, 2009; Les and Tippery, 2013; Petersen et al., 2016; Ross et al., 2016). The possible convergence is highlighted by the absence of these mutations in representatives of sister genera for both clades. Potamogeton perfoliatus belongs to the tepaloid clade together with Zosteraceae, whereas Najas flexilis, Elodea canadensis and Sagittaria lichuanensis belong to the petaloid clade together with H. ovalis (Les et al., 1997; Li and Zhou, 2009; Les and Tippery, 2013; Petersen et al., 2016; Ross et al., 2016). Protein sequences of L16 in these non-marine species have greater similarity with other monocots and dicots than with the seagrasses (Figs 2, 3) suggesting selection and convergent evolution to the marine habitat in seagrasses. Since N. flexilis and P. perfoliatus shared submergence characteristics with seagrasses, the mutations may be linked to salinity tolerance, rather than an ability to survive underwater. These results complement the seagrass clustering of OGCZ through OrthoMCL analysis and provided further molecular evidence of convergent evolution of seagrasses. Differences between H. ovalis and the two Zosteraceae species were identified in genes encoding NDH, a major protein complex residing in the thylakoid membrane of chloroplasts that participates in cyclic electron flow pathways as an oxidoreductase (reviewed in Peltier et al., 2016). As the NDH complex is only present in the Streptophyta lineage, which includes charophyte algae and land plants, acquisition of novel NDH genes likely occurred during terrestrial transition, and NDH is hypothesized to be one of the innovations enabling land plant evolution (Martín et al., 2009; Ruhlman et al., 2015). The absence of genes encoding NDH subunits and proteins required for complex formation in H. ovalis points to a total loss of the NDH complex in the H. ovalis thylakoid. Rare evidence of loss or pseudogenization of plastid NDH genes has been reported in independent lineages (Wolfe et al., 1992; Haberhausen and Zetsche, 1994; Funk et al., 2007; Braukmann et al., 2009; Logacheva et al., 2011), including several genera in the Hydrocharitaceae family (Iles et al., 2013; Peredo et al., 2013; Wilkin and Mayo, 2013; Ross et al., 2016). The observed loss of NDH genes in H. ovalis is the first report of their dispensability among Alismatales. Several reasons for NDH dispensability have been suggested (Stefanović and Olmstead, 2005; Iles et al., 2013; Peredo et al., 2013; Xu et al., 2013). Ross et al. (2016) suggest that NDH loss enabled low N investment as an adaptation to nutrient deficiency in the submerged environment. This is plausible, as H. ovalis is adapted to grow in low nutrient sediments (Carruthers et al., 2007), and examples of nitrogen sources affecting NDH expression have been reported in green algae (Peltier and Schmidt, 1991). Interestingly, two proteins related to nitrate uptake, nitrogen reductase 1 (NR1) and nitrate transporter (NRT3.1) are also lost in H. ovalis (Table S4). One limitation of seagrass nitrogen uptake studies is that the potential contribution of microbial communities is not accounted for, and may be compensating for the loss of NDH in H. ovalis, as demonstrated in myco-heterotrophic liverworts (Wickett et al., 2008a,b). Cyanobacteria on leaves have been shown to contribute to nitrogen uptake in Posidonia (Jeremy Bougoure, personal communication). However, further targeted studies are required to determine whether the loss of the NDH complex in H. ovalis is related to nitrogen uptake. Conclusion Together, the conservation of gene loss and the sharing of seagrass-specific orthologues in these two independent lineages, despite the phylogenetic distance, has shed light on the genetics of marine adaptation in angiosperms of land plant ancestry. These results also present another example of habitat-driven parallel evolution in the plant kingdom. Supplementary data Supplementary data are available at JXB online. Fig. S1. Venn diagram showing the number of shared orthologous clusters among six species (Arabidopsis, M. acuminata, O. sativa, S. polyrhiza, and two Zosteraceae species). Table S1. Species selected for multiple sequence alignment of orthologous proteins. Table S2. Number of H. ovalis reads sequenced and remaining after filtering process. Table S3. List of TAIR genes that were conserved in OGCsM and at least one species among H. ovalis, Z. muelleri, and Z. marina. Table S4. List of TAIR genes that were conserved in OGCsM but absent in at least one species among H. ovalis, Z. muelleri, and Z. marina. Table S5. Significantly enriched biological process GO terms in the genes that were lost in H. ovalis, but present in Z. muelleri, Z. marina, and five other plant species. Table S6. List of orthologous groups that are conserved in Z. muelleri and Z. marina (OGCZ). Table S7. List of OGCZ orthologous groups that are conserved in H. ovalis and the best corresponding TAIR ID hit; each protein ID is followed by InterProScan IDs of domains found or no domain found (NA). Abbreviations: Abbreviations: Mya million years ago NDH NADH dehydrogenase-like OGC orthologous gene cluster OGCsM orthologous gene cluster with at least one gene originating from a monocot species OGCZ orthologous gene cluster unique to Zosteraceae TAIR The Arabidopsis Information Resource. Acknowledgements The authors would like to acknowledge funding support from the Australian Research Council (Projects LP160100030, LP140100537, LP130100925, LP110100200, and LP0989200). Support is also acknowledged from the Queensland Cyber Infrastructure Foundation (QCIF), the Pawsey Supercomputing Centre with funding from the Australian Government and the Government of Western Australia and resources from the National Computational Infrastructure (NCI), which is supported by the Australian Government. References Alexa A , Rahnenfuhrer J . 2010 . topGO: Enrichment analysis for Gene Ontology . R package version 2.26.0. http://bioconductor.uib.no/2.7/bioc/html/topGO.html. 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Genomic comparison of two independent seagrass lineages reveals habitat-driven convergent evolution

Journal of Experimental Botany , Volume Advance Article (15) – Apr 18, 2018

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Experimental Biology.
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0022-0957
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1460-2431
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10.1093/jxb/ery147
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Abstract

Abstract Seagrasses are marine angiosperms that live fully submerged in the sea. They evolved from land plant ancestors, with multiple species representing at least three independent return-to-the-sea events. This raises the question of whether these marine angiosperms followed the same adaptation pathway to allow them to live and reproduce under the hostile marine conditions. To compare the basis of marine adaptation between seagrass lineages, we generated genomic data for Halophila ovalis and compared this with recently published genomes for two members of Zosteraceae, as well as genomes of five non-marine plant species (Arabidopsis, Oryza sativa, Phoenix dactylifera, Musa acuminata, and Spirodela polyrhiza). Halophila and Zosteraceae represent two independent seagrass lineages separated by around 30 million years. Genes that were lost or conserved in both lineages were identified. All three species lost genes associated with ethylene and terpenoid biosynthesis, and retained genes related to salinity adaptation, such as those for osmoregulation. In contrast, the loss of the NADH dehydrogenase-like complex is unique to H. ovalis. Through comparison of two independent return-to-the-sea events, this study further describes marine adaptation characteristics common to seagrass families, identifies species-specific gene loss, and provides molecular evidence for convergent evolution in seagrass lineages. Gene loss, Halophila ovalis, marine adaptation, NDH complex, osmoregulation, seagrass, Zostera muelleri Introduction Seagrasses are a polyphyletic group of flowering plants that live fully submerged in the marine environment and form monospecific meadows resembling terrestrial grasses. The morphology of seagrasses varies among species, though common features include long, strap-shaped leaves and simple flowers. Seagrasses belong to a basal lineage that diverged around 140 million years ago (Mya), before the divergence of the Poaceae within the monocotyledon clade. Although similar in form, seagrass species represent at least three independent return-to-the-sea events (Les et al., 1997). The convergent evolution of seagrasses is characterized by common physiological and morphological features that possibly represent a collection of marine adaptation traits. For example, seagrass leaves lack stomata, and gas exchange occurs through permeable cuticles, while seagrass roots and rhizomes have aerenchyma to enhance gas transport. Seagrasses have also adapted to variable quality and low levels of light, which attenuates quickly in seawater (Larkum et al., 2006; Strydom et al., 2017) and have effective osmoregulation to survive in the saline aqueous environment (Koch et al., 2007; Touchette, 2007). Seagrasses are adapted to aquatic reproduction, where the transport and capture of pollen grains is carried out on or below the water surface. Current seagrass taxonomy contains around 72 species forming three families, Zosteraceae, Hydrocharitaceae and Cymodoceaceae complex (Les et al., 1997; Short et al., 2011; Nguyen et al., 2015). Recent genome-wide comparative studies of two species in the Zosteraceae provided the first insight into genomic adaptation to the marine environment (Golicz et al., 2015; Lee et al., 2016; Olsen et al., 2016). Genes associated with the synthesis and signalling of volatile substances, including ethylene, methyljasmonate, and terpenoids, were lost in both Zostera muelleri and Z. marina. Genes associated with morphological adaptation, including those for stomatal cell differentiation, flower development and pollen formation, were also absent or greatly reduced in number. An increase in gene families associated with low light harvesting and cell wall modification was observed and postulated to contribute to survival in the light-attenuated and high salinity environment. These gene losses, gene modifications, and gene family expansions in the two Zostera species may not reflect the independent adaptation of other seagrass lineages to the marine environment, and analysis of a second lineage is required to answer the question whether they share a common adaptation pathway to the ocean. Halophila ovalis is a seagrass species in the family Hydrocharitaceae, and is an ideal model for comparison with the Zostera species. The seagrass subclade in Hydrocharitaceae is embedded within branches of largely diverse aquatic angiosperms, including freshwater species (Larkum et al., 2006), indicating the independent rise of marine adaption phenotypes. As the likelihood of convergent evolution is predicted to decrease with phylogenetic distance (Ord and Summers, 2015), the divergence time difference between the seagrass subclade in Hydrocharitaceae (55 Mya; Chen et al., 2012) and Zosteraceae (25 Mya; Coyer et al., 2013) highlights the importance of this study. Moreover, since examples of parallel evolution, where similar phenotypes are generated from a similar genetic process of independent convergent evolution (Ord and Summers, 2015), are not abundant in plants (examples include carnivorous species (Fukushima et al., 2017), recurrence of C4 photosynthesis (reviewed in Washburn et al., 2016) and convergent mutations in loci during domestication (Paterson et al., 1995)), and that habitat is the most common factor associated with reported examples of repeated evolution (Ord and Summers, 2015), independent seagrass lineages are excellent subjects for study. In this work, a genomic comparison between seagrasses of Hydrocharitaceae and Zosteraceae was explored to determine whether the gene loss previously identified in Z. muelleri and Z. marina is also observed in H. ovalis. We also attempt to identify any seagrass-specific genes that are present in one or both lineages. Halophila ovalis genome sequencing data were compared with the annotated genomes of Z. marina and Z. muelleri, together with representative land plants. Our study demonstrates that lost genes associated with the synthesis and signalling of volatile substances, as well as stomatal development, are shared by both seagrass lineages. Genes that are uniquely conserved across the two lineages are enriched in pathways related to cell osmoregulation, and provide molecular evidence for independent marine colonization. Results also revealed the loss of the NADH dehydrogenase-like (NDH) protein complex in H. ovalis, a characteristic that is not shared by the other two seagrass species. This study provides a more complete description of marine adaptation, and suggest a parallel convergent evolution of two independent return-to-the-sea events in seagrasses separated by 30 million years. Materials and methods Genome sequencing of H. ovalis One H. ovalis plant sample was collected at Swan River, Claremont, Perth, Western Australia (coordinates: 32° 0′ 3.98″ S, 115° 45′ 18.31″ E). The growth tips of the seagrass thalli were carefully removed, rinsed in sterile water, and inspected for visible external contamination. Seven hundred milligrams of tissue was placed in 5 ml tubes, flash frozen in liquid nitrogen, and bead-pulverized using a 2010 Geno/Grinder (SPEX SamplePrep, USA). The Qiagen DNeasy Plant Mini Kit was used for the extraction of the DNA. The frozen powdered plant material was suspended in 3 ml of Buffer AP1 and 28 µl of RNAse A was added. After incubating at 65 °C, 910 µl of Buffer AP2 was added. The tubes were incubated on ice for 5 min and centrifuged thereafter to collect plant debris. Lysate (450 µl) was transferred to each of five to six QIAshredder tubes. The remainder of the DNA extraction steps were followed according to the kit protocol. The extracted DNA of each repetition was pooled after elution. DNA concentration was quantified using a Qubit 3.0 Fluorometer (Thermo Fisher Scientific) and visualized using a Labchip GX Touch 24 (PerkinElmer). The extracted DNA was submitted to the Australian Genome Research Facility (AGRF) for library preparation and whole genome sequencing. The libraries for genome sequencing were prepared using the Illumina TruSeq Nano DNA HT Library Preparation Kit, according to the manufacturer’s instructions. Genomic DNA was sequenced using an Illumina HiSeqX sequencer with 150 bp paired-end technology at the Garvan Institute of Medical Research. A total of 510485779 paired-end reads were sequenced. Based on previous flow cytometry analysis of two other Hydrocharitaceae members, Najas minor (2C=7.28) and Eldodea Canadensis (2C=7.54) (Hidalgo et al., 2015), as well as genome size prediction (3628962593 bp, k=45) using the software Kmergenie (Chikhi and Medvedev, 2014) the sequencing coverage was estimated as ~40×. The sequences were deposited in a public repository (NCBI BioProject Accession PRJNA396090). Clones and low quality reads were removed using Sickle (Joshi and Fass, 2011). Pipeline to identify lost and conserved genes The identification of lost and conserved genes was achieved using the mapping of whole genome shotgun sequencing reads against reference genomes based on a previous approach (Golicz et al., 2015). The reads were mapped to coding sequences (CDS) of reference species using dc-megaBLAST (Camacho et al., 2009) with e-value 1e−5. A custom python script, calculate_blast_coverage.py (downloadable at https://github.com/AppliedBioinformatics/H_ovalis_supplementary.git), was used to calculate the horizontal coverage of each CDS. The average coverage of each CDS across multiple reference species was calculated. If the average coverage was <2%, which means that mapped reads covered less than 2% of the length of a CDS, the orthologue was considered lost. If the average coverage was >50%, the orthologue was conserved. Orthologous gene cluster construction A set of 16007 orthologous gene clusters (OGCs) conserved between seven model species with at least one gene originating from a monocot species, termed OGCsM (as defined in Table S1 in Golicz et al., 2015), was used to represent orthologues highly conserved in plants. Gene clusters unique to Zosteraceae were identified using all-against-all comparison with BLASTP (Camacho et al., 2009) using the following parameters: ‘blastp -evalue 1e-5’, followed by OrthoMCL (Li et al., 2003) between Z. muelleri, Z. marina, one dicot (Arabidopsis), and three other monocots (Oryza sativa, Musa acuminata, and Spirodela polyrhiza) (species selection based on Lee et al., 2016). This group of orthologous genes was termed OGCZ. Lost and conserved H. ovalis, Z. muelleri and Z. marina genes in OGCsM Primary transcript CDSs of five species (four land plants: Arabidopsis, Oryza sativa, Musa acuminata, and Phoenix dactylifera; one floating freshwater plant: Spirodela polyrhiza; versions as listed in Golicz et al., 2015) were used as references for mapping of reads from H. ovalis. Presence and absence results from a previous publication were used for Z. muelleri and Z. marina (Golicz et al., 2015). For each orthologue in OGCsM, lost or conserved status was assigned in each species. Lost and conserved H. ovalis genes in OGCZ Primary transcript CDSs of Z. muelleri (http://www.appliedbioinformatics.com.au/index.php/Seagrass_Zmu_Genome; Lee et al., 2016) and Z. marina (Phytozome 10; Olsen et al., 2016) were used as references for H. ovalis read mapping. For each orthologue in OGCZ, lost or conserved status was assigned in H. ovalis. Gene ontology enrichment and word cloud plotting GO annotation and enrichment were performed using the topGO package (Alexa and Rahnenfuhrer, 2010) based on a previous approach (Golicz et al., 2015). OGCsM was used as background, except for the GO enrichment of OGCZ genes where Arabidopsis whole proteome (TAIR10) was used. A word cloud was generated and coloured to represent the enriched significance of GO terms using the wordcloud package (Fellows, 2014). Inferring gene function through the level of protein domain conservation OGCZ proteins of Z. muelleri, Z. marina and Arabidopsis were compared with TIGRFAM, ProDom, Panther, PfamA and PrositePatterns using InterProScan (version 5.14, Jones et al., 2014) for motif and domain annotation. Domains of each protein were assigned with InterProScan IDs. The InterProScan IDs were compared between Arabidopsis and Zosteraceae genes for each OGCZ cluster. Assembly of H. ovalis protein and multiple sequence alignments with orthologues of other species Halophila ovalis reads aligned to CDS of 50S ribosomal protein L16 were extracted and assembled using Spades v3.10.1 (Bankevich et al., 2012) with the following commands: spades.py, only-assembler, 1 reads_1.fasta, 2 reads_2.fasta. Corresponding protein was aligned to the assembled contigs using Exonerate (Slater and Birney, 2005) with the following parameters: exonerate, model protein2genome, E 1, bestn 1, score 100, softmaskquery no, softmasktarget yes, minintron 20, maxintron 20000, ryo “>HAL_%qi_%qd\n%tas”. The aligned target regions were translated to protein sequences using the translate tool in ExPASy (Gasteiger et al., 2003). Each H. ovalis protein sequence obtained was aligned with orthologues of selected species (Table S1 at JXB online) using MAFFT (Katoh et al., 2002). A phylogenetic tree was plotted with PhyML (Guindon et al., 2009) assuming the JTT model for amino acid substitution and gamma parameter for invariable sites (based on Huang et al., 2016) using the alignments excluding the outgroup (charophyte and chlorophyte). The multiple-sequence alignments were visualized and coloured using Jalview (Waterhouse et al., 2009). Results Read alignment of H. ovalis to reference species CDS A total of 112202319 H. ovalis reads (10.9%) were discarded in the process of clonal removal and quality-based filtering (Table S2). Out of the remaining 908769239 H. ovalis reads, 2.7% (24495631) aligned to Arabidopsis CDS, 5.6% (50565060) aligned to Oryza sativa CDS, 1.3% (11617255) aligned to Musa acuminata CDS, 0.8% (7367361) aligned to Phoenix dactylifera CDS and 1.8% (16600802) aligned to Spirodela polyrhiza CDS. For the seagrass reference species, 1.8% (16727940) and 0.5% (5005993) of H. ovalis reads aligned to Z. muelleri and Z. marina CDSs, respectively. Conservation of core biological processes A total of 4367 OGCsM genes, out of 16007, were conserved in H. ovalis. When compared with conserved genes previously described in Z. muelleri and Z. marina (Golicz et al., 2015; Lee et al., 2016; Olsen et al., 2016), 3335 (76.4%) genes were conserved in all three seagrass species, 377 genes were shared with either Z. muelleri or Z. marina, and 655 genes were only conserved in H. ovalis. A total of 508 genes were only conserved in the Zosteraceae species. A full list of genes conserved in H. ovalis and their presence in other seagrass species is presented in Table S3. The GO terms enriched in these 4367 OGCsM genes conserved in H. ovalis involved core biological pathways such as photosynthesis, chlorophyll biosynthesis, and glycolytic processes, as well as response to stresses such as cadmium (Table 1). Table 1. Significantly enriched biological process GO terms in the genes conserved in H. ovalis compared with five other plant species (Arabidopsis, Oryza sativa, Musa acuminata, Phoenix dactylifera, and Spirodela polyrhiza) GO ID Term P value GO:0046686 Response to cadmium ion 3.0 × 10−30 GO:0006412 Translation 4.1 × 10−28 GO:0046496 Nicotinamide nucleotide metabolic process 3.9 × 10−16 GO:0006099 Tricarboxylic acid cycle 1.7 × 10−13 GO:0015991 ATP hydrolysis-coupled proton transport 1.0 × 10−12 GO:1901566 Organonitrogen compound biosynthetic process 7.9 × 10−11 GO:0043039 tRNA aminoacylation 1.2 × 10−10 GO:0006090 Pyruvate metabolic process 1.9 × 10−10 GO:1901293 Nucleoside phosphate biosynthetic process 2.3 × 10−10 GO:0009156 Ribonucleoside monophosphate biosynthetic process 2.7 × 10−10 GO:0009225 Nucleotide-sugar metabolic process 5.4 × 10−10 GO:0007264 Small GTPase-mediated signal transduction 8.2 × 10−9 GO:0046034 ATP metabolic process 8.4 × 10−9 GO:0006108 Malate metabolic process 1.4 × 10−8 GO:0006006 Glucose metabolic process 1.8 × 10−8 GO:0034622 Cellular macromolecular complex assembly 2.3 × 10−8 GO:0071702 Organic substance transport 2.4 × 10−8 GO:0018105 Peptidyl-serine phosphorylation 3.2 × 10−8 GO:0009250 Glucan biosynthetic process 2.0 × 10−7 GO:0016192 Vesicle-mediated transport 2.0 × 10−7 GO:0010499 Proteasomal ubiquitin-independent protein catabolic process 3.4 × 10−7 GO:0043094 Cellular metabolic compound salvage 6.7 × 10−7 GO:0015994 Chlorophyll metabolic process 1.2 × 10−6 GO:0034613 Cellular protein localization 1.4 × 10−6 GO:0006536 Glutamate metabolic process 1.9 × 10−6 GO:0005985 Sucrose metabolic process 5.3 × 10−6 GO:0098656 Anion transmembrane transport 5.7 × 10−6 GO:0015672 Monovalent inorganic cation transport 7.5 × 10−6 GO:0009932 Cell tip growth 9.9 × 10−6 GO:0006081 Cellular aldehyde metabolic process 1.0 × 10−5 GO:0018298 Protein–chromophore linkage 1.0 × 10−5 GO:0030163 Protein catabolic process 1.3 × 10−5 GO:0048588 Developmental cell growth 1.3 × 10−5 GO:0006102 Isocitrate metabolic process 2.4 × 10−5 GO:0006607 NLS-bearing protein import into nucleus 2.5 × 10−5 GO:0015977 Carbon fixation 5.1 × 10−5 GO:0015979 Photosynthesis 5.4 × 10−5 GO:0006563 L-Serine metabolic process 6.0 × 10−5 GO:0006268 DNA unwinding involved in DNA replication 6.9 × 10−5 GO:0007035 Vacuolar acidification 6.9 × 10−5 GO:0009768 Photosynthesis, light harvesting in photosystem I 7.2 × 10−5 GO:0016197 Endosomal transport 8.1 × 10−5 GO:0030048 Actin filament-based movement 0.00012 GO:0009651 Response to salt stress 0.00012 GO:0006206 Pyrimidine nucleobase metabolic process 0.00013 GO:0030243 Cellulose metabolic process 0.00014 GO:0097164 Ammonium ion metabolic process 0.00015 GO:0010315 Auxin efflux 0.00015 GO:0006551 Leucine metabolic process 0.00017 GO:0006085 Acetyl-CoA biosynthetic process 0.00018 GO:0045899 Positive regulation of RNA polymerase II transcriptional preinitiation complex assembly 0.00020 GO:0032012 Regulation of ARF protein signal transduction 0.00020 GO:0009735 Response to cytokinin 0.00027 GO:0006782 Protoporphyrinogen IX biosynthetic process 0.00030 GO:0009846 Pollen germination 0.00032 GO:1901679 Nucleotide transmembrane transport 0.00041 GO:0030042 Actin filament depolymerization 0.00048 GO:0006558 L-Phenylalanine metabolic process 0.00050 GO:0006544 Glycine metabolic process 0.00057 GO:0035999 Tetrahydrofolate interconversion 0.00096 GO:0009066 Aspartate family amino acid metabolic process 0.00103 GO:0006222 UMP biosynthetic process 0.00113 GO:0046500 S-Adenosylmethionine metabolic process 0.00125 GO:0015804 Neutral amino acid transport 0.00125 GO:0006097 Glyoxylate cycle 0.00125 GO:0030433 Endoplasmic reticulum-associated ubiquitin- dependent protein degradation 0.00132 GO:0019627 Urea metabolic process 0.00169 GO:0015800 Acidic amino acid transport 0.00169 GO:0000398 mRNA splicing, via spliceosome 0.00170 GO:0010540 Basipetal auxin transport 0.00235 GO:0035435 Phosphate ion transmembrane transport 0.00258 GO:0006457 Protein folding 0.00262 GO:0051259 Protein oligomerization 0.00265 GO:0006525 Arginine metabolic process 0.00265 GO:0016482 Cytoplasmic transport 0.00284 GO:0016036 Cellular response to phosphate starvation 0.00288 GO:0043604 Amide biosynthetic process 0.00295 GO:0019395 Fatty acid oxidation 0.00317 GO:0006570 Tyrosine metabolic process 0.00326 GO:0052646 Alditol phosphate metabolic process 0.00326 GO:0010043 Response to zinc ion 0.00346 GO:0055085 Transmembrane transport 0.00377 GO:0000338 Protein deneddylation 0.00436 GO:0006002 Fructose 6-phosphate metabolic process 0.00436 GO:0043650 Dicarboxylic acid biosynthetic process 0.00439 GO:0010501 RNA secondary structure unwinding 0.00439 GO:0009629 Response to gravity 0.00447 GO:0006103 2-Oxoglutarate metabolic process 0.00490 GO:0009833 Plant-type primary cell wall biogenesis 0.00490 GO:0006610 Ribosomal protein import into nucleus 0.00490 GO:0010541 Acropetal auxin transport 0.00540 GO:0006012 Galactose metabolic process 0.00540 GO:0006511 Ubiquitin-dependent protein catabolic process 0.00630 GO:0051275 β-Glucan catabolic process 0.00735 GO:0009141 Nucleoside triphosphate metabolic process 0.00770 GO:0018208 Peptidyl-proline modification 0.00803 GO:0006486 Protein glycosylation 0.00804 GO:0009624 Response to nematode 0.00847 GO:1904659 Glucose transmembrane transport 0.00872 GO:0046323 Glucose import 0.00872 GO:0006631 Fatty acid metabolic process 0.00878 GO:0044282 Small molecule catabolic process 0.00886 GO ID Term P value GO:0046686 Response to cadmium ion 3.0 × 10−30 GO:0006412 Translation 4.1 × 10−28 GO:0046496 Nicotinamide nucleotide metabolic process 3.9 × 10−16 GO:0006099 Tricarboxylic acid cycle 1.7 × 10−13 GO:0015991 ATP hydrolysis-coupled proton transport 1.0 × 10−12 GO:1901566 Organonitrogen compound biosynthetic process 7.9 × 10−11 GO:0043039 tRNA aminoacylation 1.2 × 10−10 GO:0006090 Pyruvate metabolic process 1.9 × 10−10 GO:1901293 Nucleoside phosphate biosynthetic process 2.3 × 10−10 GO:0009156 Ribonucleoside monophosphate biosynthetic process 2.7 × 10−10 GO:0009225 Nucleotide-sugar metabolic process 5.4 × 10−10 GO:0007264 Small GTPase-mediated signal transduction 8.2 × 10−9 GO:0046034 ATP metabolic process 8.4 × 10−9 GO:0006108 Malate metabolic process 1.4 × 10−8 GO:0006006 Glucose metabolic process 1.8 × 10−8 GO:0034622 Cellular macromolecular complex assembly 2.3 × 10−8 GO:0071702 Organic substance transport 2.4 × 10−8 GO:0018105 Peptidyl-serine phosphorylation 3.2 × 10−8 GO:0009250 Glucan biosynthetic process 2.0 × 10−7 GO:0016192 Vesicle-mediated transport 2.0 × 10−7 GO:0010499 Proteasomal ubiquitin-independent protein catabolic process 3.4 × 10−7 GO:0043094 Cellular metabolic compound salvage 6.7 × 10−7 GO:0015994 Chlorophyll metabolic process 1.2 × 10−6 GO:0034613 Cellular protein localization 1.4 × 10−6 GO:0006536 Glutamate metabolic process 1.9 × 10−6 GO:0005985 Sucrose metabolic process 5.3 × 10−6 GO:0098656 Anion transmembrane transport 5.7 × 10−6 GO:0015672 Monovalent inorganic cation transport 7.5 × 10−6 GO:0009932 Cell tip growth 9.9 × 10−6 GO:0006081 Cellular aldehyde metabolic process 1.0 × 10−5 GO:0018298 Protein–chromophore linkage 1.0 × 10−5 GO:0030163 Protein catabolic process 1.3 × 10−5 GO:0048588 Developmental cell growth 1.3 × 10−5 GO:0006102 Isocitrate metabolic process 2.4 × 10−5 GO:0006607 NLS-bearing protein import into nucleus 2.5 × 10−5 GO:0015977 Carbon fixation 5.1 × 10−5 GO:0015979 Photosynthesis 5.4 × 10−5 GO:0006563 L-Serine metabolic process 6.0 × 10−5 GO:0006268 DNA unwinding involved in DNA replication 6.9 × 10−5 GO:0007035 Vacuolar acidification 6.9 × 10−5 GO:0009768 Photosynthesis, light harvesting in photosystem I 7.2 × 10−5 GO:0016197 Endosomal transport 8.1 × 10−5 GO:0030048 Actin filament-based movement 0.00012 GO:0009651 Response to salt stress 0.00012 GO:0006206 Pyrimidine nucleobase metabolic process 0.00013 GO:0030243 Cellulose metabolic process 0.00014 GO:0097164 Ammonium ion metabolic process 0.00015 GO:0010315 Auxin efflux 0.00015 GO:0006551 Leucine metabolic process 0.00017 GO:0006085 Acetyl-CoA biosynthetic process 0.00018 GO:0045899 Positive regulation of RNA polymerase II transcriptional preinitiation complex assembly 0.00020 GO:0032012 Regulation of ARF protein signal transduction 0.00020 GO:0009735 Response to cytokinin 0.00027 GO:0006782 Protoporphyrinogen IX biosynthetic process 0.00030 GO:0009846 Pollen germination 0.00032 GO:1901679 Nucleotide transmembrane transport 0.00041 GO:0030042 Actin filament depolymerization 0.00048 GO:0006558 L-Phenylalanine metabolic process 0.00050 GO:0006544 Glycine metabolic process 0.00057 GO:0035999 Tetrahydrofolate interconversion 0.00096 GO:0009066 Aspartate family amino acid metabolic process 0.00103 GO:0006222 UMP biosynthetic process 0.00113 GO:0046500 S-Adenosylmethionine metabolic process 0.00125 GO:0015804 Neutral amino acid transport 0.00125 GO:0006097 Glyoxylate cycle 0.00125 GO:0030433 Endoplasmic reticulum-associated ubiquitin- dependent protein degradation 0.00132 GO:0019627 Urea metabolic process 0.00169 GO:0015800 Acidic amino acid transport 0.00169 GO:0000398 mRNA splicing, via spliceosome 0.00170 GO:0010540 Basipetal auxin transport 0.00235 GO:0035435 Phosphate ion transmembrane transport 0.00258 GO:0006457 Protein folding 0.00262 GO:0051259 Protein oligomerization 0.00265 GO:0006525 Arginine metabolic process 0.00265 GO:0016482 Cytoplasmic transport 0.00284 GO:0016036 Cellular response to phosphate starvation 0.00288 GO:0043604 Amide biosynthetic process 0.00295 GO:0019395 Fatty acid oxidation 0.00317 GO:0006570 Tyrosine metabolic process 0.00326 GO:0052646 Alditol phosphate metabolic process 0.00326 GO:0010043 Response to zinc ion 0.00346 GO:0055085 Transmembrane transport 0.00377 GO:0000338 Protein deneddylation 0.00436 GO:0006002 Fructose 6-phosphate metabolic process 0.00436 GO:0043650 Dicarboxylic acid biosynthetic process 0.00439 GO:0010501 RNA secondary structure unwinding 0.00439 GO:0009629 Response to gravity 0.00447 GO:0006103 2-Oxoglutarate metabolic process 0.00490 GO:0009833 Plant-type primary cell wall biogenesis 0.00490 GO:0006610 Ribosomal protein import into nucleus 0.00490 GO:0010541 Acropetal auxin transport 0.00540 GO:0006012 Galactose metabolic process 0.00540 GO:0006511 Ubiquitin-dependent protein catabolic process 0.00630 GO:0051275 β-Glucan catabolic process 0.00735 GO:0009141 Nucleoside triphosphate metabolic process 0.00770 GO:0018208 Peptidyl-proline modification 0.00803 GO:0006486 Protein glycosylation 0.00804 GO:0009624 Response to nematode 0.00847 GO:1904659 Glucose transmembrane transport 0.00872 GO:0046323 Glucose import 0.00872 GO:0006631 Fatty acid metabolic process 0.00878 GO:0044282 Small molecule catabolic process 0.00886 View Large Table 1. Significantly enriched biological process GO terms in the genes conserved in H. ovalis compared with five other plant species (Arabidopsis, Oryza sativa, Musa acuminata, Phoenix dactylifera, and Spirodela polyrhiza) GO ID Term P value GO:0046686 Response to cadmium ion 3.0 × 10−30 GO:0006412 Translation 4.1 × 10−28 GO:0046496 Nicotinamide nucleotide metabolic process 3.9 × 10−16 GO:0006099 Tricarboxylic acid cycle 1.7 × 10−13 GO:0015991 ATP hydrolysis-coupled proton transport 1.0 × 10−12 GO:1901566 Organonitrogen compound biosynthetic process 7.9 × 10−11 GO:0043039 tRNA aminoacylation 1.2 × 10−10 GO:0006090 Pyruvate metabolic process 1.9 × 10−10 GO:1901293 Nucleoside phosphate biosynthetic process 2.3 × 10−10 GO:0009156 Ribonucleoside monophosphate biosynthetic process 2.7 × 10−10 GO:0009225 Nucleotide-sugar metabolic process 5.4 × 10−10 GO:0007264 Small GTPase-mediated signal transduction 8.2 × 10−9 GO:0046034 ATP metabolic process 8.4 × 10−9 GO:0006108 Malate metabolic process 1.4 × 10−8 GO:0006006 Glucose metabolic process 1.8 × 10−8 GO:0034622 Cellular macromolecular complex assembly 2.3 × 10−8 GO:0071702 Organic substance transport 2.4 × 10−8 GO:0018105 Peptidyl-serine phosphorylation 3.2 × 10−8 GO:0009250 Glucan biosynthetic process 2.0 × 10−7 GO:0016192 Vesicle-mediated transport 2.0 × 10−7 GO:0010499 Proteasomal ubiquitin-independent protein catabolic process 3.4 × 10−7 GO:0043094 Cellular metabolic compound salvage 6.7 × 10−7 GO:0015994 Chlorophyll metabolic process 1.2 × 10−6 GO:0034613 Cellular protein localization 1.4 × 10−6 GO:0006536 Glutamate metabolic process 1.9 × 10−6 GO:0005985 Sucrose metabolic process 5.3 × 10−6 GO:0098656 Anion transmembrane transport 5.7 × 10−6 GO:0015672 Monovalent inorganic cation transport 7.5 × 10−6 GO:0009932 Cell tip growth 9.9 × 10−6 GO:0006081 Cellular aldehyde metabolic process 1.0 × 10−5 GO:0018298 Protein–chromophore linkage 1.0 × 10−5 GO:0030163 Protein catabolic process 1.3 × 10−5 GO:0048588 Developmental cell growth 1.3 × 10−5 GO:0006102 Isocitrate metabolic process 2.4 × 10−5 GO:0006607 NLS-bearing protein import into nucleus 2.5 × 10−5 GO:0015977 Carbon fixation 5.1 × 10−5 GO:0015979 Photosynthesis 5.4 × 10−5 GO:0006563 L-Serine metabolic process 6.0 × 10−5 GO:0006268 DNA unwinding involved in DNA replication 6.9 × 10−5 GO:0007035 Vacuolar acidification 6.9 × 10−5 GO:0009768 Photosynthesis, light harvesting in photosystem I 7.2 × 10−5 GO:0016197 Endosomal transport 8.1 × 10−5 GO:0030048 Actin filament-based movement 0.00012 GO:0009651 Response to salt stress 0.00012 GO:0006206 Pyrimidine nucleobase metabolic process 0.00013 GO:0030243 Cellulose metabolic process 0.00014 GO:0097164 Ammonium ion metabolic process 0.00015 GO:0010315 Auxin efflux 0.00015 GO:0006551 Leucine metabolic process 0.00017 GO:0006085 Acetyl-CoA biosynthetic process 0.00018 GO:0045899 Positive regulation of RNA polymerase II transcriptional preinitiation complex assembly 0.00020 GO:0032012 Regulation of ARF protein signal transduction 0.00020 GO:0009735 Response to cytokinin 0.00027 GO:0006782 Protoporphyrinogen IX biosynthetic process 0.00030 GO:0009846 Pollen germination 0.00032 GO:1901679 Nucleotide transmembrane transport 0.00041 GO:0030042 Actin filament depolymerization 0.00048 GO:0006558 L-Phenylalanine metabolic process 0.00050 GO:0006544 Glycine metabolic process 0.00057 GO:0035999 Tetrahydrofolate interconversion 0.00096 GO:0009066 Aspartate family amino acid metabolic process 0.00103 GO:0006222 UMP biosynthetic process 0.00113 GO:0046500 S-Adenosylmethionine metabolic process 0.00125 GO:0015804 Neutral amino acid transport 0.00125 GO:0006097 Glyoxylate cycle 0.00125 GO:0030433 Endoplasmic reticulum-associated ubiquitin- dependent protein degradation 0.00132 GO:0019627 Urea metabolic process 0.00169 GO:0015800 Acidic amino acid transport 0.00169 GO:0000398 mRNA splicing, via spliceosome 0.00170 GO:0010540 Basipetal auxin transport 0.00235 GO:0035435 Phosphate ion transmembrane transport 0.00258 GO:0006457 Protein folding 0.00262 GO:0051259 Protein oligomerization 0.00265 GO:0006525 Arginine metabolic process 0.00265 GO:0016482 Cytoplasmic transport 0.00284 GO:0016036 Cellular response to phosphate starvation 0.00288 GO:0043604 Amide biosynthetic process 0.00295 GO:0019395 Fatty acid oxidation 0.00317 GO:0006570 Tyrosine metabolic process 0.00326 GO:0052646 Alditol phosphate metabolic process 0.00326 GO:0010043 Response to zinc ion 0.00346 GO:0055085 Transmembrane transport 0.00377 GO:0000338 Protein deneddylation 0.00436 GO:0006002 Fructose 6-phosphate metabolic process 0.00436 GO:0043650 Dicarboxylic acid biosynthetic process 0.00439 GO:0010501 RNA secondary structure unwinding 0.00439 GO:0009629 Response to gravity 0.00447 GO:0006103 2-Oxoglutarate metabolic process 0.00490 GO:0009833 Plant-type primary cell wall biogenesis 0.00490 GO:0006610 Ribosomal protein import into nucleus 0.00490 GO:0010541 Acropetal auxin transport 0.00540 GO:0006012 Galactose metabolic process 0.00540 GO:0006511 Ubiquitin-dependent protein catabolic process 0.00630 GO:0051275 β-Glucan catabolic process 0.00735 GO:0009141 Nucleoside triphosphate metabolic process 0.00770 GO:0018208 Peptidyl-proline modification 0.00803 GO:0006486 Protein glycosylation 0.00804 GO:0009624 Response to nematode 0.00847 GO:1904659 Glucose transmembrane transport 0.00872 GO:0046323 Glucose import 0.00872 GO:0006631 Fatty acid metabolic process 0.00878 GO:0044282 Small molecule catabolic process 0.00886 GO ID Term P value GO:0046686 Response to cadmium ion 3.0 × 10−30 GO:0006412 Translation 4.1 × 10−28 GO:0046496 Nicotinamide nucleotide metabolic process 3.9 × 10−16 GO:0006099 Tricarboxylic acid cycle 1.7 × 10−13 GO:0015991 ATP hydrolysis-coupled proton transport 1.0 × 10−12 GO:1901566 Organonitrogen compound biosynthetic process 7.9 × 10−11 GO:0043039 tRNA aminoacylation 1.2 × 10−10 GO:0006090 Pyruvate metabolic process 1.9 × 10−10 GO:1901293 Nucleoside phosphate biosynthetic process 2.3 × 10−10 GO:0009156 Ribonucleoside monophosphate biosynthetic process 2.7 × 10−10 GO:0009225 Nucleotide-sugar metabolic process 5.4 × 10−10 GO:0007264 Small GTPase-mediated signal transduction 8.2 × 10−9 GO:0046034 ATP metabolic process 8.4 × 10−9 GO:0006108 Malate metabolic process 1.4 × 10−8 GO:0006006 Glucose metabolic process 1.8 × 10−8 GO:0034622 Cellular macromolecular complex assembly 2.3 × 10−8 GO:0071702 Organic substance transport 2.4 × 10−8 GO:0018105 Peptidyl-serine phosphorylation 3.2 × 10−8 GO:0009250 Glucan biosynthetic process 2.0 × 10−7 GO:0016192 Vesicle-mediated transport 2.0 × 10−7 GO:0010499 Proteasomal ubiquitin-independent protein catabolic process 3.4 × 10−7 GO:0043094 Cellular metabolic compound salvage 6.7 × 10−7 GO:0015994 Chlorophyll metabolic process 1.2 × 10−6 GO:0034613 Cellular protein localization 1.4 × 10−6 GO:0006536 Glutamate metabolic process 1.9 × 10−6 GO:0005985 Sucrose metabolic process 5.3 × 10−6 GO:0098656 Anion transmembrane transport 5.7 × 10−6 GO:0015672 Monovalent inorganic cation transport 7.5 × 10−6 GO:0009932 Cell tip growth 9.9 × 10−6 GO:0006081 Cellular aldehyde metabolic process 1.0 × 10−5 GO:0018298 Protein–chromophore linkage 1.0 × 10−5 GO:0030163 Protein catabolic process 1.3 × 10−5 GO:0048588 Developmental cell growth 1.3 × 10−5 GO:0006102 Isocitrate metabolic process 2.4 × 10−5 GO:0006607 NLS-bearing protein import into nucleus 2.5 × 10−5 GO:0015977 Carbon fixation 5.1 × 10−5 GO:0015979 Photosynthesis 5.4 × 10−5 GO:0006563 L-Serine metabolic process 6.0 × 10−5 GO:0006268 DNA unwinding involved in DNA replication 6.9 × 10−5 GO:0007035 Vacuolar acidification 6.9 × 10−5 GO:0009768 Photosynthesis, light harvesting in photosystem I 7.2 × 10−5 GO:0016197 Endosomal transport 8.1 × 10−5 GO:0030048 Actin filament-based movement 0.00012 GO:0009651 Response to salt stress 0.00012 GO:0006206 Pyrimidine nucleobase metabolic process 0.00013 GO:0030243 Cellulose metabolic process 0.00014 GO:0097164 Ammonium ion metabolic process 0.00015 GO:0010315 Auxin efflux 0.00015 GO:0006551 Leucine metabolic process 0.00017 GO:0006085 Acetyl-CoA biosynthetic process 0.00018 GO:0045899 Positive regulation of RNA polymerase II transcriptional preinitiation complex assembly 0.00020 GO:0032012 Regulation of ARF protein signal transduction 0.00020 GO:0009735 Response to cytokinin 0.00027 GO:0006782 Protoporphyrinogen IX biosynthetic process 0.00030 GO:0009846 Pollen germination 0.00032 GO:1901679 Nucleotide transmembrane transport 0.00041 GO:0030042 Actin filament depolymerization 0.00048 GO:0006558 L-Phenylalanine metabolic process 0.00050 GO:0006544 Glycine metabolic process 0.00057 GO:0035999 Tetrahydrofolate interconversion 0.00096 GO:0009066 Aspartate family amino acid metabolic process 0.00103 GO:0006222 UMP biosynthetic process 0.00113 GO:0046500 S-Adenosylmethionine metabolic process 0.00125 GO:0015804 Neutral amino acid transport 0.00125 GO:0006097 Glyoxylate cycle 0.00125 GO:0030433 Endoplasmic reticulum-associated ubiquitin- dependent protein degradation 0.00132 GO:0019627 Urea metabolic process 0.00169 GO:0015800 Acidic amino acid transport 0.00169 GO:0000398 mRNA splicing, via spliceosome 0.00170 GO:0010540 Basipetal auxin transport 0.00235 GO:0035435 Phosphate ion transmembrane transport 0.00258 GO:0006457 Protein folding 0.00262 GO:0051259 Protein oligomerization 0.00265 GO:0006525 Arginine metabolic process 0.00265 GO:0016482 Cytoplasmic transport 0.00284 GO:0016036 Cellular response to phosphate starvation 0.00288 GO:0043604 Amide biosynthetic process 0.00295 GO:0019395 Fatty acid oxidation 0.00317 GO:0006570 Tyrosine metabolic process 0.00326 GO:0052646 Alditol phosphate metabolic process 0.00326 GO:0010043 Response to zinc ion 0.00346 GO:0055085 Transmembrane transport 0.00377 GO:0000338 Protein deneddylation 0.00436 GO:0006002 Fructose 6-phosphate metabolic process 0.00436 GO:0043650 Dicarboxylic acid biosynthetic process 0.00439 GO:0010501 RNA secondary structure unwinding 0.00439 GO:0009629 Response to gravity 0.00447 GO:0006103 2-Oxoglutarate metabolic process 0.00490 GO:0009833 Plant-type primary cell wall biogenesis 0.00490 GO:0006610 Ribosomal protein import into nucleus 0.00490 GO:0010541 Acropetal auxin transport 0.00540 GO:0006012 Galactose metabolic process 0.00540 GO:0006511 Ubiquitin-dependent protein catabolic process 0.00630 GO:0051275 β-Glucan catabolic process 0.00735 GO:0009141 Nucleoside triphosphate metabolic process 0.00770 GO:0018208 Peptidyl-proline modification 0.00803 GO:0006486 Protein glycosylation 0.00804 GO:0009624 Response to nematode 0.00847 GO:1904659 Glucose transmembrane transport 0.00872 GO:0046323 Glucose import 0.00872 GO:0006631 Fatty acid metabolic process 0.00878 GO:0044282 Small molecule catabolic process 0.00886 View Large Gene loss in H. ovalis and comparison of lost genes between the three seagrass species A total of 1822 OGCsM genes were lost in H. ovalis, and these were compared with those previously reported as lost in both Z. muelleri and Z. marina (Golicz et al., 2015; Lee et al., 2016; Olsen et al., 2016) (Table S4). A total of 1197 (65.6%) lost genes were shared between all three seagrass species, 187 were shared with either Z. muelleri or Z. marina, and 412 were only lost in H. ovalis. In comparison, 743 genes were only lost in the Zosteraceae lineage. Enriched GO terms for the 1822 OGCsM genes highlighted the loss of genes associated with ethylene synthesis and perception, and stomatal development (Table 2). The presence or absence of genes involved in stomatal development, ethylene synthesis and signalling, and terpenoid biosynthesis in H. ovalis, Z. marina, and Z. muelleri are listed in Table 3. Table 2. Significantly enriched biological process GO terms in the genes conserved in five other plant species (Arabidopsis, Oryza sativa, Musa acuminata, Phoenix dactylifera and Spirodela polyrhiza) but absent in H. ovalis Function GO ID Term P value Ethylene synthesis and signalling GO:0009835 Fruit ripening 4.1 × 10−10 GO:0042218 1-Aminocyclopropane-1-carboxylate biosynthetic process 6.3 × 10−10 GO:0009693 Ethylene biosynthetic process 1.9 × 10−8 GO:0010105 Negative regulation of ethylene-activated signalling pathway 1.2 × 10−6 Stomata development GO:0010375 Stomatal complex patterning 0.00012 GO:2000038 Regulation of stomatal complex development 0.00608 Others GO:0045168 Cell–cell signalling involved in cell fate commitment 3.1 × 10−6 GO:0006952 Defence response 9.4 × 10−6 GO:0009626 Plant-type hypersensitive response 1.0 × 10−5 GO:0031640 Killing of cells of other organism 8.4 × 10−5 GO:0010039 Response to iron ion 0.00011 GO:0034644 Cellular response to UV 0.00014 GO:0071484 Cellular response to light intensity 0.00018 GO:0009773 Photosynthetic electron transport in photosystem I 0.00023 GO:0080027 Response to herbivore 0.00034 GO:0033473 Indoleacetic acid conjugate metabolic process 0.00034 GO:0009696 Salicylic acid metabolic process 0.00059 GO:0033609 Oxalate metabolic process 0.00093 GO:0050832 Defence response to fungus 0.00113 GO:0071423 Malate transmembrane transport 0.00209 GO:0042542 Response to hydrogen peroxide 0.00234 GO:1900426 Positive regulation of defence response to bacterium 0.00239 GO:0010876 Lipid localization 0.00313 GO:0018106 Peptidyl-histidine phosphorylation 0.00404 GO:0046688 Response to copper ion 0.00558 GO:0010257 NADH dehydrogenase complex assembly 0.00608 GO:0009838 Abscission 0.00704 GO:0071732 Cellular response to nitric oxide 0.00791 Function GO ID Term P value Ethylene synthesis and signalling GO:0009835 Fruit ripening 4.1 × 10−10 GO:0042218 1-Aminocyclopropane-1-carboxylate biosynthetic process 6.3 × 10−10 GO:0009693 Ethylene biosynthetic process 1.9 × 10−8 GO:0010105 Negative regulation of ethylene-activated signalling pathway 1.2 × 10−6 Stomata development GO:0010375 Stomatal complex patterning 0.00012 GO:2000038 Regulation of stomatal complex development 0.00608 Others GO:0045168 Cell–cell signalling involved in cell fate commitment 3.1 × 10−6 GO:0006952 Defence response 9.4 × 10−6 GO:0009626 Plant-type hypersensitive response 1.0 × 10−5 GO:0031640 Killing of cells of other organism 8.4 × 10−5 GO:0010039 Response to iron ion 0.00011 GO:0034644 Cellular response to UV 0.00014 GO:0071484 Cellular response to light intensity 0.00018 GO:0009773 Photosynthetic electron transport in photosystem I 0.00023 GO:0080027 Response to herbivore 0.00034 GO:0033473 Indoleacetic acid conjugate metabolic process 0.00034 GO:0009696 Salicylic acid metabolic process 0.00059 GO:0033609 Oxalate metabolic process 0.00093 GO:0050832 Defence response to fungus 0.00113 GO:0071423 Malate transmembrane transport 0.00209 GO:0042542 Response to hydrogen peroxide 0.00234 GO:1900426 Positive regulation of defence response to bacterium 0.00239 GO:0010876 Lipid localization 0.00313 GO:0018106 Peptidyl-histidine phosphorylation 0.00404 GO:0046688 Response to copper ion 0.00558 GO:0010257 NADH dehydrogenase complex assembly 0.00608 GO:0009838 Abscission 0.00704 GO:0071732 Cellular response to nitric oxide 0.00791 View Large Table 2. Significantly enriched biological process GO terms in the genes conserved in five other plant species (Arabidopsis, Oryza sativa, Musa acuminata, Phoenix dactylifera and Spirodela polyrhiza) but absent in H. ovalis Function GO ID Term P value Ethylene synthesis and signalling GO:0009835 Fruit ripening 4.1 × 10−10 GO:0042218 1-Aminocyclopropane-1-carboxylate biosynthetic process 6.3 × 10−10 GO:0009693 Ethylene biosynthetic process 1.9 × 10−8 GO:0010105 Negative regulation of ethylene-activated signalling pathway 1.2 × 10−6 Stomata development GO:0010375 Stomatal complex patterning 0.00012 GO:2000038 Regulation of stomatal complex development 0.00608 Others GO:0045168 Cell–cell signalling involved in cell fate commitment 3.1 × 10−6 GO:0006952 Defence response 9.4 × 10−6 GO:0009626 Plant-type hypersensitive response 1.0 × 10−5 GO:0031640 Killing of cells of other organism 8.4 × 10−5 GO:0010039 Response to iron ion 0.00011 GO:0034644 Cellular response to UV 0.00014 GO:0071484 Cellular response to light intensity 0.00018 GO:0009773 Photosynthetic electron transport in photosystem I 0.00023 GO:0080027 Response to herbivore 0.00034 GO:0033473 Indoleacetic acid conjugate metabolic process 0.00034 GO:0009696 Salicylic acid metabolic process 0.00059 GO:0033609 Oxalate metabolic process 0.00093 GO:0050832 Defence response to fungus 0.00113 GO:0071423 Malate transmembrane transport 0.00209 GO:0042542 Response to hydrogen peroxide 0.00234 GO:1900426 Positive regulation of defence response to bacterium 0.00239 GO:0010876 Lipid localization 0.00313 GO:0018106 Peptidyl-histidine phosphorylation 0.00404 GO:0046688 Response to copper ion 0.00558 GO:0010257 NADH dehydrogenase complex assembly 0.00608 GO:0009838 Abscission 0.00704 GO:0071732 Cellular response to nitric oxide 0.00791 Function GO ID Term P value Ethylene synthesis and signalling GO:0009835 Fruit ripening 4.1 × 10−10 GO:0042218 1-Aminocyclopropane-1-carboxylate biosynthetic process 6.3 × 10−10 GO:0009693 Ethylene biosynthetic process 1.9 × 10−8 GO:0010105 Negative regulation of ethylene-activated signalling pathway 1.2 × 10−6 Stomata development GO:0010375 Stomatal complex patterning 0.00012 GO:2000038 Regulation of stomatal complex development 0.00608 Others GO:0045168 Cell–cell signalling involved in cell fate commitment 3.1 × 10−6 GO:0006952 Defence response 9.4 × 10−6 GO:0009626 Plant-type hypersensitive response 1.0 × 10−5 GO:0031640 Killing of cells of other organism 8.4 × 10−5 GO:0010039 Response to iron ion 0.00011 GO:0034644 Cellular response to UV 0.00014 GO:0071484 Cellular response to light intensity 0.00018 GO:0009773 Photosynthetic electron transport in photosystem I 0.00023 GO:0080027 Response to herbivore 0.00034 GO:0033473 Indoleacetic acid conjugate metabolic process 0.00034 GO:0009696 Salicylic acid metabolic process 0.00059 GO:0033609 Oxalate metabolic process 0.00093 GO:0050832 Defence response to fungus 0.00113 GO:0071423 Malate transmembrane transport 0.00209 GO:0042542 Response to hydrogen peroxide 0.00234 GO:1900426 Positive regulation of defence response to bacterium 0.00239 GO:0010876 Lipid localization 0.00313 GO:0018106 Peptidyl-histidine phosphorylation 0.00404 GO:0046688 Response to copper ion 0.00558 GO:0010257 NADH dehydrogenase complex assembly 0.00608 GO:0009838 Abscission 0.00704 GO:0071732 Cellular response to nitric oxide 0.00791 View Large Table 3. Presence and absence of genes involved in stomatal development, ethylene synthesis and signalling, and terpenoid biosynthesis in OGCsM, H. ovalis, Z. marina, and Z. muelleri Gene ID Protein name Function Conserved in OGCsM Presence in H. ovalis Presence in Z. muelleri Presence in Z. marina Stomata development AT1G04110 SBT1.2 Spacing and patterning + NA − − AT4G12970 EPFL9 Spacing and patterning + − − − AT2G20875 EPF1 Spacing and patterning + − − − AT1G80080 TMM Spacing and patterning + − − − AT1G34245 EPF2 Spacing and patterning + − − − AT2G02820 MYB88 Differentiation − NA NA − AT3G06120 MUTE Differentiation + − − − AT5G53210 SPCH Differentiation + − − − AT3G24140 FAMA Differentiation + NA NA − AT1G12860 SCRM2 Differentiation − NA NA − AT1G14350 FLP Differentiation + − − − Ethylene synthesis and signalling AT2G19590 ACO1 ACC oxidase + − − − AT1G62380 ACO2 ACC oxidase + − − − AT1G05010 ACO4 ACC oxidase + − − − AT1G77330 ACO5 ACC oxidase + − − − AT3G61510 ACS1 ACC synthase + − − − AT1G01480 ACS2 ACC synthase + − − − AT2G22810 ACS4 ACC synthase + − − − AT5G65800 ACS5 ACC synthase + − − − AT4G11280 ACS6 ACC synthase + − − − AT4G26200 ACS7 ACC synthase + − − − AT4G37770 ACS8 ACC synthase + − − − AT3G49700 ACS9 ACC synthase + − − − AT4G08040 ACS11 ACC synthase + − − − AT2G40940 ERS1 Ethylene receptor + − − − AT1G66340 ETR1 Ethylene receptor + − − − AT3G23150 ETR2 Ethylene receptor + − − − AT3G04580 EIN4 Ethylene receptor + − − − AT5G03730 CTR1 Raf-like kinase + NA NA − AT5G03280 EIN2 Signal transducer + NA − − AT2G25490 EBF1 EIN2 degradation + − − − AT5G25350 EBF2 EIN2 degradation + − − − Terpenoid biosynthesis AT3G25820 TPS-CIN Terpene synthase + − − − AT3G25830 TPS23 Terpene synthase + − − − AT4G16740 TPS03 Terpene synthase − − − − AT2G24210 TPS10 Terpene synthase + − − − AT3G25810 TPS24 Terpene synthase − − − − Gene ID Protein name Function Conserved in OGCsM Presence in H. ovalis Presence in Z. muelleri Presence in Z. marina Stomata development AT1G04110 SBT1.2 Spacing and patterning + NA − − AT4G12970 EPFL9 Spacing and patterning + − − − AT2G20875 EPF1 Spacing and patterning + − − − AT1G80080 TMM Spacing and patterning + − − − AT1G34245 EPF2 Spacing and patterning + − − − AT2G02820 MYB88 Differentiation − NA NA − AT3G06120 MUTE Differentiation + − − − AT5G53210 SPCH Differentiation + − − − AT3G24140 FAMA Differentiation + NA NA − AT1G12860 SCRM2 Differentiation − NA NA − AT1G14350 FLP Differentiation + − − − Ethylene synthesis and signalling AT2G19590 ACO1 ACC oxidase + − − − AT1G62380 ACO2 ACC oxidase + − − − AT1G05010 ACO4 ACC oxidase + − − − AT1G77330 ACO5 ACC oxidase + − − − AT3G61510 ACS1 ACC synthase + − − − AT1G01480 ACS2 ACC synthase + − − − AT2G22810 ACS4 ACC synthase + − − − AT5G65800 ACS5 ACC synthase + − − − AT4G11280 ACS6 ACC synthase + − − − AT4G26200 ACS7 ACC synthase + − − − AT4G37770 ACS8 ACC synthase + − − − AT3G49700 ACS9 ACC synthase + − − − AT4G08040 ACS11 ACC synthase + − − − AT2G40940 ERS1 Ethylene receptor + − − − AT1G66340 ETR1 Ethylene receptor + − − − AT3G23150 ETR2 Ethylene receptor + − − − AT3G04580 EIN4 Ethylene receptor + − − − AT5G03730 CTR1 Raf-like kinase + NA NA − AT5G03280 EIN2 Signal transducer + NA − − AT2G25490 EBF1 EIN2 degradation + − − − AT5G25350 EBF2 EIN2 degradation + − − − Terpenoid biosynthesis AT3G25820 TPS-CIN Terpene synthase + − − − AT3G25830 TPS23 Terpene synthase + − − − AT4G16740 TPS03 Terpene synthase − − − − AT2G24210 TPS10 Terpene synthase + − − − AT3G25810 TPS24 Terpene synthase − − − − Categories are: gene present (+), gene absent (−), and information not available (NA). View Large Table 3. Presence and absence of genes involved in stomatal development, ethylene synthesis and signalling, and terpenoid biosynthesis in OGCsM, H. ovalis, Z. marina, and Z. muelleri Gene ID Protein name Function Conserved in OGCsM Presence in H. ovalis Presence in Z. muelleri Presence in Z. marina Stomata development AT1G04110 SBT1.2 Spacing and patterning + NA − − AT4G12970 EPFL9 Spacing and patterning + − − − AT2G20875 EPF1 Spacing and patterning + − − − AT1G80080 TMM Spacing and patterning + − − − AT1G34245 EPF2 Spacing and patterning + − − − AT2G02820 MYB88 Differentiation − NA NA − AT3G06120 MUTE Differentiation + − − − AT5G53210 SPCH Differentiation + − − − AT3G24140 FAMA Differentiation + NA NA − AT1G12860 SCRM2 Differentiation − NA NA − AT1G14350 FLP Differentiation + − − − Ethylene synthesis and signalling AT2G19590 ACO1 ACC oxidase + − − − AT1G62380 ACO2 ACC oxidase + − − − AT1G05010 ACO4 ACC oxidase + − − − AT1G77330 ACO5 ACC oxidase + − − − AT3G61510 ACS1 ACC synthase + − − − AT1G01480 ACS2 ACC synthase + − − − AT2G22810 ACS4 ACC synthase + − − − AT5G65800 ACS5 ACC synthase + − − − AT4G11280 ACS6 ACC synthase + − − − AT4G26200 ACS7 ACC synthase + − − − AT4G37770 ACS8 ACC synthase + − − − AT3G49700 ACS9 ACC synthase + − − − AT4G08040 ACS11 ACC synthase + − − − AT2G40940 ERS1 Ethylene receptor + − − − AT1G66340 ETR1 Ethylene receptor + − − − AT3G23150 ETR2 Ethylene receptor + − − − AT3G04580 EIN4 Ethylene receptor + − − − AT5G03730 CTR1 Raf-like kinase + NA NA − AT5G03280 EIN2 Signal transducer + NA − − AT2G25490 EBF1 EIN2 degradation + − − − AT5G25350 EBF2 EIN2 degradation + − − − Terpenoid biosynthesis AT3G25820 TPS-CIN Terpene synthase + − − − AT3G25830 TPS23 Terpene synthase + − − − AT4G16740 TPS03 Terpene synthase − − − − AT2G24210 TPS10 Terpene synthase + − − − AT3G25810 TPS24 Terpene synthase − − − − Gene ID Protein name Function Conserved in OGCsM Presence in H. ovalis Presence in Z. muelleri Presence in Z. marina Stomata development AT1G04110 SBT1.2 Spacing and patterning + NA − − AT4G12970 EPFL9 Spacing and patterning + − − − AT2G20875 EPF1 Spacing and patterning + − − − AT1G80080 TMM Spacing and patterning + − − − AT1G34245 EPF2 Spacing and patterning + − − − AT2G02820 MYB88 Differentiation − NA NA − AT3G06120 MUTE Differentiation + − − − AT5G53210 SPCH Differentiation + − − − AT3G24140 FAMA Differentiation + NA NA − AT1G12860 SCRM2 Differentiation − NA NA − AT1G14350 FLP Differentiation + − − − Ethylene synthesis and signalling AT2G19590 ACO1 ACC oxidase + − − − AT1G62380 ACO2 ACC oxidase + − − − AT1G05010 ACO4 ACC oxidase + − − − AT1G77330 ACO5 ACC oxidase + − − − AT3G61510 ACS1 ACC synthase + − − − AT1G01480 ACS2 ACC synthase + − − − AT2G22810 ACS4 ACC synthase + − − − AT5G65800 ACS5 ACC synthase + − − − AT4G11280 ACS6 ACC synthase + − − − AT4G26200 ACS7 ACC synthase + − − − AT4G37770 ACS8 ACC synthase + − − − AT3G49700 ACS9 ACC synthase + − − − AT4G08040 ACS11 ACC synthase + − − − AT2G40940 ERS1 Ethylene receptor + − − − AT1G66340 ETR1 Ethylene receptor + − − − AT3G23150 ETR2 Ethylene receptor + − − − AT3G04580 EIN4 Ethylene receptor + − − − AT5G03730 CTR1 Raf-like kinase + NA NA − AT5G03280 EIN2 Signal transducer + NA − − AT2G25490 EBF1 EIN2 degradation + − − − AT5G25350 EBF2 EIN2 degradation + − − − Terpenoid biosynthesis AT3G25820 TPS-CIN Terpene synthase + − − − AT3G25830 TPS23 Terpene synthase + − − − AT4G16740 TPS03 Terpene synthase − − − − AT2G24210 TPS10 Terpene synthase + − − − AT3G25810 TPS24 Terpene synthase − − − − Categories are: gene present (+), gene absent (−), and information not available (NA). View Large Halophila ovalis lost genes encoding NADH dehydrogenase-like complex assembly The five most significantly enriched GO terms in the 412 genes that were only lost in H. ovalis were cellular response to light intensity (GO:0071484), cellular response to UV (GO:0034644), photosynthetic electron transport in photosystem I (GO:0009773), NADH dehydrogenase complex assembly (GO:0010257), and cellular response to salt stress (GO:0071472). A complete list of all significantly enriched terms is given in Table S5. Closer examination revealed the loss of 23 (15 nuclear and 8 chloroplast) genes that encode the five subcomplexes in the NDH complex (Table 4). In addition, 17 genes required for the supercomplex formation, including tethering of NDH to photosystem I, assembly of subunits, accessory proteins, and transcription factors, were absent in H. ovalis. Two proteins required for nitrate uptake and assimilation, nitrogen reductase 1 (NR1) and nitrate transporter (NRT3.1), were also lost in H. ovalis. Table 4. Presence and absence of 40 nuclear and chloroplast-encoded genes involved in formation of the NDH complex Gene ID Protein name Function Presence in OGCsM Presence in H. ovalis Presence in Z. muelleri Presence in Z. marina Nuclear encoded AT1G70760 NDHL Subunit A + NA + + AT4G37925 NDHM Subunit A + − + + AT5G58260 NDHN Subunit A + NA + + AT1G74880 NDHO Subunit A + − + + AT4G23890 NDHS Subunit ED + − NA + AT4G09350 NDHT Subunit ED + − + + AT5G21430 NDHU Subunit ED + − + + AT1G15980 PNSB1 Subunit B + − NA + AT1G64770 PNSB2 Subunit B + − + + AT3G16250 PNSB3 Subunit B + − + + AT1G18730 PNSB4 Subunit B + − + + AT2G39470 PNSL1 Subunit B + − + + AT1G14150 PNSL2 Subunit L + − + + AT3G01440 PNSL3 Subunit L + NA + + AT4G39710 PNSL4 Subunit L + − + + AT5G13120 PNSL5 Subunit L + + + + AT2G47910 CRR6 Complex formation + − + + AT5G39210 CRR7 Complex formation + − + + AT1G45474 Lhca5 Complex formation + − + + AT1G19150 Lhca6 Complex formation + NA + + AT1G26230 CRR27 Complex formation + NA + + AT1G51100 CRR41 Complex formation + NA + + AT2G05620 PGR5 Proton gradient regulation + NA + + AT4G22890 PGRL1A Proton gradient regulation + + + + AT3G46790 CRR2 Unknown + NA + + AT2G01590 CRR3 Unknown + − − + AT5G20935 CRR42 Unknown + − + + AT2G01918 PQL3 Unknown + − − + AT1G55370 NDF5 Unknown + − + + Chloroplast encoded ATCG00890 ATCG01250 NDHB Subunit M + NA + + ATCG01010 NDHF Subunit M + NA + + ATCG00440 NDHC Subunit M + − + + ATCG01050 NDHD Subunit M + − + + ATCG01070 NDHE Subunit M + − + + ATCG01100 NDHA Subunit M + − + + ATCG01080 NDHG Subunit M + NA + + ATCG01110 NDHH Subunit A + + + + ATCG00420 NDHJ Subunit A + − + + ATCG00430 NDHK Subunit A + − + + ATCG01090 NDHI Subunit A + − + + Gene ID Protein name Function Presence in OGCsM Presence in H. ovalis Presence in Z. muelleri Presence in Z. marina Nuclear encoded AT1G70760 NDHL Subunit A + NA + + AT4G37925 NDHM Subunit A + − + + AT5G58260 NDHN Subunit A + NA + + AT1G74880 NDHO Subunit A + − + + AT4G23890 NDHS Subunit ED + − NA + AT4G09350 NDHT Subunit ED + − + + AT5G21430 NDHU Subunit ED + − + + AT1G15980 PNSB1 Subunit B + − NA + AT1G64770 PNSB2 Subunit B + − + + AT3G16250 PNSB3 Subunit B + − + + AT1G18730 PNSB4 Subunit B + − + + AT2G39470 PNSL1 Subunit B + − + + AT1G14150 PNSL2 Subunit L + − + + AT3G01440 PNSL3 Subunit L + NA + + AT4G39710 PNSL4 Subunit L + − + + AT5G13120 PNSL5 Subunit L + + + + AT2G47910 CRR6 Complex formation + − + + AT5G39210 CRR7 Complex formation + − + + AT1G45474 Lhca5 Complex formation + − + + AT1G19150 Lhca6 Complex formation + NA + + AT1G26230 CRR27 Complex formation + NA + + AT1G51100 CRR41 Complex formation + NA + + AT2G05620 PGR5 Proton gradient regulation + NA + + AT4G22890 PGRL1A Proton gradient regulation + + + + AT3G46790 CRR2 Unknown + NA + + AT2G01590 CRR3 Unknown + − − + AT5G20935 CRR42 Unknown + − + + AT2G01918 PQL3 Unknown + − − + AT1G55370 NDF5 Unknown + − + + Chloroplast encoded ATCG00890 ATCG01250 NDHB Subunit M + NA + + ATCG01010 NDHF Subunit M + NA + + ATCG00440 NDHC Subunit M + − + + ATCG01050 NDHD Subunit M + − + + ATCG01070 NDHE Subunit M + − + + ATCG01100 NDHA Subunit M + − + + ATCG01080 NDHG Subunit M + NA + + ATCG01110 NDHH Subunit A + + + + ATCG00420 NDHJ Subunit A + − + + ATCG00430 NDHK Subunit A + − + + ATCG01090 NDHI Subunit A + − + + Categories are: gene present (+), gene absent (−), and information not available (NA). View Large Table 4. Presence and absence of 40 nuclear and chloroplast-encoded genes involved in formation of the NDH complex Gene ID Protein name Function Presence in OGCsM Presence in H. ovalis Presence in Z. muelleri Presence in Z. marina Nuclear encoded AT1G70760 NDHL Subunit A + NA + + AT4G37925 NDHM Subunit A + − + + AT5G58260 NDHN Subunit A + NA + + AT1G74880 NDHO Subunit A + − + + AT4G23890 NDHS Subunit ED + − NA + AT4G09350 NDHT Subunit ED + − + + AT5G21430 NDHU Subunit ED + − + + AT1G15980 PNSB1 Subunit B + − NA + AT1G64770 PNSB2 Subunit B + − + + AT3G16250 PNSB3 Subunit B + − + + AT1G18730 PNSB4 Subunit B + − + + AT2G39470 PNSL1 Subunit B + − + + AT1G14150 PNSL2 Subunit L + − + + AT3G01440 PNSL3 Subunit L + NA + + AT4G39710 PNSL4 Subunit L + − + + AT5G13120 PNSL5 Subunit L + + + + AT2G47910 CRR6 Complex formation + − + + AT5G39210 CRR7 Complex formation + − + + AT1G45474 Lhca5 Complex formation + − + + AT1G19150 Lhca6 Complex formation + NA + + AT1G26230 CRR27 Complex formation + NA + + AT1G51100 CRR41 Complex formation + NA + + AT2G05620 PGR5 Proton gradient regulation + NA + + AT4G22890 PGRL1A Proton gradient regulation + + + + AT3G46790 CRR2 Unknown + NA + + AT2G01590 CRR3 Unknown + − − + AT5G20935 CRR42 Unknown + − + + AT2G01918 PQL3 Unknown + − − + AT1G55370 NDF5 Unknown + − + + Chloroplast encoded ATCG00890 ATCG01250 NDHB Subunit M + NA + + ATCG01010 NDHF Subunit M + NA + + ATCG00440 NDHC Subunit M + − + + ATCG01050 NDHD Subunit M + − + + ATCG01070 NDHE Subunit M + − + + ATCG01100 NDHA Subunit M + − + + ATCG01080 NDHG Subunit M + NA + + ATCG01110 NDHH Subunit A + + + + ATCG00420 NDHJ Subunit A + − + + ATCG00430 NDHK Subunit A + − + + ATCG01090 NDHI Subunit A + − + + Gene ID Protein name Function Presence in OGCsM Presence in H. ovalis Presence in Z. muelleri Presence in Z. marina Nuclear encoded AT1G70760 NDHL Subunit A + NA + + AT4G37925 NDHM Subunit A + − + + AT5G58260 NDHN Subunit A + NA + + AT1G74880 NDHO Subunit A + − + + AT4G23890 NDHS Subunit ED + − NA + AT4G09350 NDHT Subunit ED + − + + AT5G21430 NDHU Subunit ED + − + + AT1G15980 PNSB1 Subunit B + − NA + AT1G64770 PNSB2 Subunit B + − + + AT3G16250 PNSB3 Subunit B + − + + AT1G18730 PNSB4 Subunit B + − + + AT2G39470 PNSL1 Subunit B + − + + AT1G14150 PNSL2 Subunit L + − + + AT3G01440 PNSL3 Subunit L + NA + + AT4G39710 PNSL4 Subunit L + − + + AT5G13120 PNSL5 Subunit L + + + + AT2G47910 CRR6 Complex formation + − + + AT5G39210 CRR7 Complex formation + − + + AT1G45474 Lhca5 Complex formation + − + + AT1G19150 Lhca6 Complex formation + NA + + AT1G26230 CRR27 Complex formation + NA + + AT1G51100 CRR41 Complex formation + NA + + AT2G05620 PGR5 Proton gradient regulation + NA + + AT4G22890 PGRL1A Proton gradient regulation + + + + AT3G46790 CRR2 Unknown + NA + + AT2G01590 CRR3 Unknown + − − + AT5G20935 CRR42 Unknown + − + + AT2G01918 PQL3 Unknown + − − + AT1G55370 NDF5 Unknown + − + + Chloroplast encoded ATCG00890 ATCG01250 NDHB Subunit M + NA + + ATCG01010 NDHF Subunit M + NA + + ATCG00440 NDHC Subunit M + − + + ATCG01050 NDHD Subunit M + − + + ATCG01070 NDHE Subunit M + − + + ATCG01100 NDHA Subunit M + − + + ATCG01080 NDHG Subunit M + NA + + ATCG01110 NDHH Subunit A + + + + ATCG00420 NDHJ Subunit A + − + + ATCG00430 NDHK Subunit A + − + + ATCG01090 NDHI Subunit A + − + + Categories are: gene present (+), gene absent (−), and information not available (NA). View Large Co-evolution of genes for intracellular transport, cell wall, and ion transport-related genes in H. ovalis and Zostera We identified a set of 1748 genes that are unique to Z. muelleri and Z. marina, termed OGCZ (Fig. S1; Table S6), with 57 also found to be conserved in H. ovalis (Table S7). Putative functions of these 57 genes were inferred by matching their protein domains to the corresponding best aligned Arabidopsis gene. Out of 57 OGCZ groups, 45 have identical domains (indicated as InterProScan IDs) to the corresponding Arabidopsis genes. A total of eight of them have fewer or different domains from Arabidopsis, and four of them have no domains assigned (Table S7). The majority of the 57 OGCZ genes conserved in H. ovalis are predicted to be involved in protein secretion and intracellular transport, with significantly enriched terms annotated with cellular component ontology, including organelles of the intracellular transport pathways, namely Golgi apparatus, trans-Golgi network, and endosome, and nearly half of the remaining terms intracellular transport-related (Fig. 1). A total of 13 genes are predicted to function in protein secretion and intracellular transport, mainly as transport proteins or transport regulators. Nine genes are associated with cell wall construction, organization, and modification, while other predicted functions include ion or proton transport, lipid catabolism, transcription and translation-related, protein ubiquitination, and histone assembly (Table 5). Table 5. Fifty-seven orthologous groups of seagrass-specific genes shared in two Zosteraceae species (Z. muelleri and Z. marina) and Halophila categorized by predicted function Category of related function Name of best TAIR10 hit corresponding to Zostera orthologue ID of best TAIR10 hit corresponding to Zostera orthologue Putative gene function Protein secretion and intracellular transport Endoplasmic reticulum retention defective 2B AT3G25040.1 Retention mechanism Endoplasmic reticulum-type calcium-transporting ATPase 3 AT1G10130.1 Calcium and manganese ion transport RAB GTPase homologue A1F AT5G60860.1 GTPase activity RAB GTPase homologue A2B AT1G07410.1 GTPase activity Secretory carrier 3 AT1G61250.1 Integral membrane protein NOD26-like intrinsic protein 1;2 AT4G18910.1 Aquaporin Mitochondrial substrate carrier family protein AT3G53940.1 Substrate transport Mitochondrial import inner membrane translocase subunit Tim17/Tim22/Tim23 family protein AT5G63000.1 Protein transport Transducin/WD40 repeat-like superfamily protein AT3G01340.1 Protein transport Protein of unknown function AT1G09330.1 — Cell wall Expansin A16 AT3G55500.1 Cell wall loosening Expansin A1 AT1G69530.2 Cell wall loosening Galacturonosyltransferase-like 2 AT3G50760.1 Cell wall organization Xyloglucan endotransglucosylase/hydrolase 5 AT5G13870.1 Cell wall organization Glucan synthase-like 8 AT2G36850.1 Callose synthesis S-Adenosyl-L-methionine-dependent methyltransferases superfamily protein AT4G34050.1 Lignin biosynthesis Peroxidase superfamily protein AT5G05340.1 Lignin biosynthesis Cotton Golgi-related 2 (pectin methyltransferase) AT3G49720.1 Cell wall modification Vascular related NAC-domain protein 1 AT2G18060.1 Xylem secondary cell wall formation Ion flux and sequestering ATP synthase epsilon chain, mitochondrial AT1G51650.1 Proton-transporting ATPase activity Vacuolar proton ATPase A1 AT2G28520.1 Proton-transporting ATPase activity Calmodulin 4 AT1G66410.1 Calcium ion binding Lipid catabolism Trigalactosyldiacylglycerol 5 AT1G27695.1 Lipid transport GDSL-like lipase/acylhydrolase superfamily protein AT1G29670.1 AT5G45670.1 Lipid catabolic process Peroxin 6 AT1G03000.1 Peroxisomal matrix protein import Alkaline phytoceramidase AT4G22330.1 Ceramide synthase involved in sphingolipid metabolism Transcription-related RNA polymerase subunit beta ATCG00190.1 Constituent of RNA polymerase B Pre-mRNA-splicing factor SPF27 homologue AT3G18165.1 mRNA splicing of resistance genes Ribosome/ translation-related Ribosomal protein L16 ATCG00790.1 Structural constituent of ribosome Ribosomal protein S26e family protein AT2G40510.1 Structural constituent of ribosome Ribosomal protein S8e family protein AT5G59240.1 Structural constituent of ribosome Ribosomal protein S2 ATCG00160.1 Structural constituent of ribosome Eukaryotic translation initiation factor 3A AT4G11420.1 Constituent of eukaryotic initiation factor 3 Protein ubiquitination F-box protein PP2-A13 AT3G61060.1 Protein ubiquitination BTB/POZ domain-containing protein AT1G63850.1 Protein ubiquitination Ubiquitin-conjugating enzyme 28 AT1G64230.1 Protein ubiquitination Ubiquitin-like protein 5 AT5G42300.1 Ubiquitin-like modification Histone Histone H2A.2 AT3G20670.1 Histones/DNA binding/nucleosome assembly Histone H3.3 AT4G40030.2 Histones/DNA binding/nucleosome assembly Others Photosystem II light harvesting complex gene 2.1 AT2G05100.1 Constituent of light harvesting complex II Alternative oxidase 1A AT3G22370.1 Alternative oxidase activity Tubulin folding cofactor D AT3G60740.1 Microtubule stability Asparagine synthetase 2 AT5G65010.2 Asparagine biosynthesis Glutamate-1-semialdehyde 2,1-aminomutase 2 AT3G48730.1 Porphyrin-containing compound metabolism Membrane-associated progesterone binding protein 3 AT3G48890.1 Porphyrin binding Thioredoxin superfamily protein AT3G62950.1 Electron carrier activity DNA polymerase epsilon catalytic subunit AT1G08260.1 DNA replication proofreading NAC domain containing protein 32 AT1G77450.1 Transcription factor DNA-binding protein phosphatase 1 AT2G25620.1 Protein phosphatase activity Protein kinase 1B AT2G28930.1 Serine/threonine kinase activity UDP-glycosyltransferase superfamily protein AT5G04480.1 — Adenine nucleotide alpha hydrolases-like superfamily protein AT1G11360.4 — Protein of unknown function (DUF300) AT1G11200.1 — Protein of unknown function (DUF803) AT1G34470.1 — Category of related function Name of best TAIR10 hit corresponding to Zostera orthologue ID of best TAIR10 hit corresponding to Zostera orthologue Putative gene function Protein secretion and intracellular transport Endoplasmic reticulum retention defective 2B AT3G25040.1 Retention mechanism Endoplasmic reticulum-type calcium-transporting ATPase 3 AT1G10130.1 Calcium and manganese ion transport RAB GTPase homologue A1F AT5G60860.1 GTPase activity RAB GTPase homologue A2B AT1G07410.1 GTPase activity Secretory carrier 3 AT1G61250.1 Integral membrane protein NOD26-like intrinsic protein 1;2 AT4G18910.1 Aquaporin Mitochondrial substrate carrier family protein AT3G53940.1 Substrate transport Mitochondrial import inner membrane translocase subunit Tim17/Tim22/Tim23 family protein AT5G63000.1 Protein transport Transducin/WD40 repeat-like superfamily protein AT3G01340.1 Protein transport Protein of unknown function AT1G09330.1 — Cell wall Expansin A16 AT3G55500.1 Cell wall loosening Expansin A1 AT1G69530.2 Cell wall loosening Galacturonosyltransferase-like 2 AT3G50760.1 Cell wall organization Xyloglucan endotransglucosylase/hydrolase 5 AT5G13870.1 Cell wall organization Glucan synthase-like 8 AT2G36850.1 Callose synthesis S-Adenosyl-L-methionine-dependent methyltransferases superfamily protein AT4G34050.1 Lignin biosynthesis Peroxidase superfamily protein AT5G05340.1 Lignin biosynthesis Cotton Golgi-related 2 (pectin methyltransferase) AT3G49720.1 Cell wall modification Vascular related NAC-domain protein 1 AT2G18060.1 Xylem secondary cell wall formation Ion flux and sequestering ATP synthase epsilon chain, mitochondrial AT1G51650.1 Proton-transporting ATPase activity Vacuolar proton ATPase A1 AT2G28520.1 Proton-transporting ATPase activity Calmodulin 4 AT1G66410.1 Calcium ion binding Lipid catabolism Trigalactosyldiacylglycerol 5 AT1G27695.1 Lipid transport GDSL-like lipase/acylhydrolase superfamily protein AT1G29670.1 AT5G45670.1 Lipid catabolic process Peroxin 6 AT1G03000.1 Peroxisomal matrix protein import Alkaline phytoceramidase AT4G22330.1 Ceramide synthase involved in sphingolipid metabolism Transcription-related RNA polymerase subunit beta ATCG00190.1 Constituent of RNA polymerase B Pre-mRNA-splicing factor SPF27 homologue AT3G18165.1 mRNA splicing of resistance genes Ribosome/ translation-related Ribosomal protein L16 ATCG00790.1 Structural constituent of ribosome Ribosomal protein S26e family protein AT2G40510.1 Structural constituent of ribosome Ribosomal protein S8e family protein AT5G59240.1 Structural constituent of ribosome Ribosomal protein S2 ATCG00160.1 Structural constituent of ribosome Eukaryotic translation initiation factor 3A AT4G11420.1 Constituent of eukaryotic initiation factor 3 Protein ubiquitination F-box protein PP2-A13 AT3G61060.1 Protein ubiquitination BTB/POZ domain-containing protein AT1G63850.1 Protein ubiquitination Ubiquitin-conjugating enzyme 28 AT1G64230.1 Protein ubiquitination Ubiquitin-like protein 5 AT5G42300.1 Ubiquitin-like modification Histone Histone H2A.2 AT3G20670.1 Histones/DNA binding/nucleosome assembly Histone H3.3 AT4G40030.2 Histones/DNA binding/nucleosome assembly Others Photosystem II light harvesting complex gene 2.1 AT2G05100.1 Constituent of light harvesting complex II Alternative oxidase 1A AT3G22370.1 Alternative oxidase activity Tubulin folding cofactor D AT3G60740.1 Microtubule stability Asparagine synthetase 2 AT5G65010.2 Asparagine biosynthesis Glutamate-1-semialdehyde 2,1-aminomutase 2 AT3G48730.1 Porphyrin-containing compound metabolism Membrane-associated progesterone binding protein 3 AT3G48890.1 Porphyrin binding Thioredoxin superfamily protein AT3G62950.1 Electron carrier activity DNA polymerase epsilon catalytic subunit AT1G08260.1 DNA replication proofreading NAC domain containing protein 32 AT1G77450.1 Transcription factor DNA-binding protein phosphatase 1 AT2G25620.1 Protein phosphatase activity Protein kinase 1B AT2G28930.1 Serine/threonine kinase activity UDP-glycosyltransferase superfamily protein AT5G04480.1 — Adenine nucleotide alpha hydrolases-like superfamily protein AT1G11360.4 — Protein of unknown function (DUF300) AT1G11200.1 — Protein of unknown function (DUF803) AT1G34470.1 — Gene functions were predicted with corresponding Arabidopsis gene of highest sequence similarity. View Large Table 5. Fifty-seven orthologous groups of seagrass-specific genes shared in two Zosteraceae species (Z. muelleri and Z. marina) and Halophila categorized by predicted function Category of related function Name of best TAIR10 hit corresponding to Zostera orthologue ID of best TAIR10 hit corresponding to Zostera orthologue Putative gene function Protein secretion and intracellular transport Endoplasmic reticulum retention defective 2B AT3G25040.1 Retention mechanism Endoplasmic reticulum-type calcium-transporting ATPase 3 AT1G10130.1 Calcium and manganese ion transport RAB GTPase homologue A1F AT5G60860.1 GTPase activity RAB GTPase homologue A2B AT1G07410.1 GTPase activity Secretory carrier 3 AT1G61250.1 Integral membrane protein NOD26-like intrinsic protein 1;2 AT4G18910.1 Aquaporin Mitochondrial substrate carrier family protein AT3G53940.1 Substrate transport Mitochondrial import inner membrane translocase subunit Tim17/Tim22/Tim23 family protein AT5G63000.1 Protein transport Transducin/WD40 repeat-like superfamily protein AT3G01340.1 Protein transport Protein of unknown function AT1G09330.1 — Cell wall Expansin A16 AT3G55500.1 Cell wall loosening Expansin A1 AT1G69530.2 Cell wall loosening Galacturonosyltransferase-like 2 AT3G50760.1 Cell wall organization Xyloglucan endotransglucosylase/hydrolase 5 AT5G13870.1 Cell wall organization Glucan synthase-like 8 AT2G36850.1 Callose synthesis S-Adenosyl-L-methionine-dependent methyltransferases superfamily protein AT4G34050.1 Lignin biosynthesis Peroxidase superfamily protein AT5G05340.1 Lignin biosynthesis Cotton Golgi-related 2 (pectin methyltransferase) AT3G49720.1 Cell wall modification Vascular related NAC-domain protein 1 AT2G18060.1 Xylem secondary cell wall formation Ion flux and sequestering ATP synthase epsilon chain, mitochondrial AT1G51650.1 Proton-transporting ATPase activity Vacuolar proton ATPase A1 AT2G28520.1 Proton-transporting ATPase activity Calmodulin 4 AT1G66410.1 Calcium ion binding Lipid catabolism Trigalactosyldiacylglycerol 5 AT1G27695.1 Lipid transport GDSL-like lipase/acylhydrolase superfamily protein AT1G29670.1 AT5G45670.1 Lipid catabolic process Peroxin 6 AT1G03000.1 Peroxisomal matrix protein import Alkaline phytoceramidase AT4G22330.1 Ceramide synthase involved in sphingolipid metabolism Transcription-related RNA polymerase subunit beta ATCG00190.1 Constituent of RNA polymerase B Pre-mRNA-splicing factor SPF27 homologue AT3G18165.1 mRNA splicing of resistance genes Ribosome/ translation-related Ribosomal protein L16 ATCG00790.1 Structural constituent of ribosome Ribosomal protein S26e family protein AT2G40510.1 Structural constituent of ribosome Ribosomal protein S8e family protein AT5G59240.1 Structural constituent of ribosome Ribosomal protein S2 ATCG00160.1 Structural constituent of ribosome Eukaryotic translation initiation factor 3A AT4G11420.1 Constituent of eukaryotic initiation factor 3 Protein ubiquitination F-box protein PP2-A13 AT3G61060.1 Protein ubiquitination BTB/POZ domain-containing protein AT1G63850.1 Protein ubiquitination Ubiquitin-conjugating enzyme 28 AT1G64230.1 Protein ubiquitination Ubiquitin-like protein 5 AT5G42300.1 Ubiquitin-like modification Histone Histone H2A.2 AT3G20670.1 Histones/DNA binding/nucleosome assembly Histone H3.3 AT4G40030.2 Histones/DNA binding/nucleosome assembly Others Photosystem II light harvesting complex gene 2.1 AT2G05100.1 Constituent of light harvesting complex II Alternative oxidase 1A AT3G22370.1 Alternative oxidase activity Tubulin folding cofactor D AT3G60740.1 Microtubule stability Asparagine synthetase 2 AT5G65010.2 Asparagine biosynthesis Glutamate-1-semialdehyde 2,1-aminomutase 2 AT3G48730.1 Porphyrin-containing compound metabolism Membrane-associated progesterone binding protein 3 AT3G48890.1 Porphyrin binding Thioredoxin superfamily protein AT3G62950.1 Electron carrier activity DNA polymerase epsilon catalytic subunit AT1G08260.1 DNA replication proofreading NAC domain containing protein 32 AT1G77450.1 Transcription factor DNA-binding protein phosphatase 1 AT2G25620.1 Protein phosphatase activity Protein kinase 1B AT2G28930.1 Serine/threonine kinase activity UDP-glycosyltransferase superfamily protein AT5G04480.1 — Adenine nucleotide alpha hydrolases-like superfamily protein AT1G11360.4 — Protein of unknown function (DUF300) AT1G11200.1 — Protein of unknown function (DUF803) AT1G34470.1 — Category of related function Name of best TAIR10 hit corresponding to Zostera orthologue ID of best TAIR10 hit corresponding to Zostera orthologue Putative gene function Protein secretion and intracellular transport Endoplasmic reticulum retention defective 2B AT3G25040.1 Retention mechanism Endoplasmic reticulum-type calcium-transporting ATPase 3 AT1G10130.1 Calcium and manganese ion transport RAB GTPase homologue A1F AT5G60860.1 GTPase activity RAB GTPase homologue A2B AT1G07410.1 GTPase activity Secretory carrier 3 AT1G61250.1 Integral membrane protein NOD26-like intrinsic protein 1;2 AT4G18910.1 Aquaporin Mitochondrial substrate carrier family protein AT3G53940.1 Substrate transport Mitochondrial import inner membrane translocase subunit Tim17/Tim22/Tim23 family protein AT5G63000.1 Protein transport Transducin/WD40 repeat-like superfamily protein AT3G01340.1 Protein transport Protein of unknown function AT1G09330.1 — Cell wall Expansin A16 AT3G55500.1 Cell wall loosening Expansin A1 AT1G69530.2 Cell wall loosening Galacturonosyltransferase-like 2 AT3G50760.1 Cell wall organization Xyloglucan endotransglucosylase/hydrolase 5 AT5G13870.1 Cell wall organization Glucan synthase-like 8 AT2G36850.1 Callose synthesis S-Adenosyl-L-methionine-dependent methyltransferases superfamily protein AT4G34050.1 Lignin biosynthesis Peroxidase superfamily protein AT5G05340.1 Lignin biosynthesis Cotton Golgi-related 2 (pectin methyltransferase) AT3G49720.1 Cell wall modification Vascular related NAC-domain protein 1 AT2G18060.1 Xylem secondary cell wall formation Ion flux and sequestering ATP synthase epsilon chain, mitochondrial AT1G51650.1 Proton-transporting ATPase activity Vacuolar proton ATPase A1 AT2G28520.1 Proton-transporting ATPase activity Calmodulin 4 AT1G66410.1 Calcium ion binding Lipid catabolism Trigalactosyldiacylglycerol 5 AT1G27695.1 Lipid transport GDSL-like lipase/acylhydrolase superfamily protein AT1G29670.1 AT5G45670.1 Lipid catabolic process Peroxin 6 AT1G03000.1 Peroxisomal matrix protein import Alkaline phytoceramidase AT4G22330.1 Ceramide synthase involved in sphingolipid metabolism Transcription-related RNA polymerase subunit beta ATCG00190.1 Constituent of RNA polymerase B Pre-mRNA-splicing factor SPF27 homologue AT3G18165.1 mRNA splicing of resistance genes Ribosome/ translation-related Ribosomal protein L16 ATCG00790.1 Structural constituent of ribosome Ribosomal protein S26e family protein AT2G40510.1 Structural constituent of ribosome Ribosomal protein S8e family protein AT5G59240.1 Structural constituent of ribosome Ribosomal protein S2 ATCG00160.1 Structural constituent of ribosome Eukaryotic translation initiation factor 3A AT4G11420.1 Constituent of eukaryotic initiation factor 3 Protein ubiquitination F-box protein PP2-A13 AT3G61060.1 Protein ubiquitination BTB/POZ domain-containing protein AT1G63850.1 Protein ubiquitination Ubiquitin-conjugating enzyme 28 AT1G64230.1 Protein ubiquitination Ubiquitin-like protein 5 AT5G42300.1 Ubiquitin-like modification Histone Histone H2A.2 AT3G20670.1 Histones/DNA binding/nucleosome assembly Histone H3.3 AT4G40030.2 Histones/DNA binding/nucleosome assembly Others Photosystem II light harvesting complex gene 2.1 AT2G05100.1 Constituent of light harvesting complex II Alternative oxidase 1A AT3G22370.1 Alternative oxidase activity Tubulin folding cofactor D AT3G60740.1 Microtubule stability Asparagine synthetase 2 AT5G65010.2 Asparagine biosynthesis Glutamate-1-semialdehyde 2,1-aminomutase 2 AT3G48730.1 Porphyrin-containing compound metabolism Membrane-associated progesterone binding protein 3 AT3G48890.1 Porphyrin binding Thioredoxin superfamily protein AT3G62950.1 Electron carrier activity DNA polymerase epsilon catalytic subunit AT1G08260.1 DNA replication proofreading NAC domain containing protein 32 AT1G77450.1 Transcription factor DNA-binding protein phosphatase 1 AT2G25620.1 Protein phosphatase activity Protein kinase 1B AT2G28930.1 Serine/threonine kinase activity UDP-glycosyltransferase superfamily protein AT5G04480.1 — Adenine nucleotide alpha hydrolases-like superfamily protein AT1G11360.4 — Protein of unknown function (DUF300) AT1G11200.1 — Protein of unknown function (DUF803) AT1G34470.1 — Gene functions were predicted with corresponding Arabidopsis gene of highest sequence similarity. View Large Fig. 1. View largeDownload slide Significantly enriched cellular component GO terms in seagrass-specific genes. Terms in green are subcomponents or organelles of the intracellular transport pathways. Fig. 1. View largeDownload slide Significantly enriched cellular component GO terms in seagrass-specific genes. Terms in green are subcomponents or organelles of the intracellular transport pathways. Molecular comparison of seagrass ribosomal proteins Ribosomal 50S L16 orthologues from the two Zostera species, H. ovalis, and 12 species in the Alismatales order were aligned, together with predicted proteins from five model land plants (Table S1). We identified nine amino acid positions that appear to be specific to the seagrasses H. ovalis, Z. muelleri, and Z. marina (white arrows in Fig. 2) and conserved among the other 17 angiosperms (12 belong to the Alismatales order, eight are freshwater plants), one charophyte and one chlorophyte. Fig. 2. View largeDownload slide Ribosomal protein L16 multiple sequence alignments between 19 species (AMA, Alocasia macrorhizzos; ATH, Arabidopsis; DSE, Dieffenbachia seguine; EAU, Epiprenum aureum; ECA, Elodia canadensis; LMI, Lemna minor; MAC, Musa acuminata; NFL, Najas flexilis; OSA, Oryza sativa; PDA, Phoenix dactylifera; PPE, Potamogeton perfoliatus; SLI, Sagittaria lichuanensis; SLY, Solanum lycopersicum; SPO, Spirodela polyrhiza; SPU, Spirodela pundata; TTH, Tofieldia thibetica; WAU, Wolfia australiana) together with three seagrasses (HAL, H. ovalis; ZMA, Z. marina; ZMU, Z. muelleri). Species and corresponding IDs are listed in Table S1. Amino acids that were conserved within the non-seagrass group or among seagrasses are coloured according to physicochemical properties based on ‘Zappo’ colour scheme. White arrows indicated seagrass-specific mutations. Fig. 2. View largeDownload slide Ribosomal protein L16 multiple sequence alignments between 19 species (AMA, Alocasia macrorhizzos; ATH, Arabidopsis; DSE, Dieffenbachia seguine; EAU, Epiprenum aureum; ECA, Elodia canadensis; LMI, Lemna minor; MAC, Musa acuminata; NFL, Najas flexilis; OSA, Oryza sativa; PDA, Phoenix dactylifera; PPE, Potamogeton perfoliatus; SLI, Sagittaria lichuanensis; SLY, Solanum lycopersicum; SPO, Spirodela polyrhiza; SPU, Spirodela pundata; TTH, Tofieldia thibetica; WAU, Wolfia australiana) together with three seagrasses (HAL, H. ovalis; ZMA, Z. marina; ZMU, Z. muelleri). Species and corresponding IDs are listed in Table S1. Amino acids that were conserved within the non-seagrass group or among seagrasses are coloured according to physicochemical properties based on ‘Zappo’ colour scheme. White arrows indicated seagrass-specific mutations. A phylogenetic tree for these 22 species based on this protein sequence, which describes the relationships between orthologues of these 22 species, separates the seagrass species (H. ovalis, Z. muelleri, and Z. marina) from the other species (Fig. 3). The separation of the two Zostera orthologues from H. ovalis is also well-supported. Sister genera of both Halophila and Zostera grouped together as members of core alismatids (red in Fig. 3) (Alismatidae sensu; Les and Tippery, 2013). Fig. 3. View largeDownload slide Phylogenetic tree showing distance between ribosome protein L16 sequences of 17 species (AMA, Alocasia macrorhizzos; ATH, Arabidopsis; DSE, Dieffenbachia seguine; EAU, Epiprenum aureum; ECA, Elodia canadensis; LMI, Lemna minor; MAC, Musa acuminata; NFL, Najas flexilis; OSA, Oryza sativa; PDA, Phoenix dactylifera; PPE, Potamogeton perfoliatus; SLI, Sagittaria lichuanensis; SLY, Solanum lycopersicum; SPO, Spirodela polyrhiza; SPU, Spirodela pundata; TTH, Tofieldia thibetica; WAU, Wolfia australiana) together with three seagrasses (HAL, H. ovalis; ZMA, Z. marina; ZMU, Z. muelleri). The order and habitat of species were indicated in the second part of each ID: DL, dicot, land; ML, monocot, land; MLA, monocot, land, Alismatales; MMA, monocot, marine, Alismatales; MWA, monocot, freshwater, Alismatales. Complete details are listed in Table S1. IDs coloured in red are members of core Alismatids, blue are members of Araceae, and black are others. Branches are labelled with bootstrap values (%). Fig. 3. View largeDownload slide Phylogenetic tree showing distance between ribosome protein L16 sequences of 17 species (AMA, Alocasia macrorhizzos; ATH, Arabidopsis; DSE, Dieffenbachia seguine; EAU, Epiprenum aureum; ECA, Elodia canadensis; LMI, Lemna minor; MAC, Musa acuminata; NFL, Najas flexilis; OSA, Oryza sativa; PDA, Phoenix dactylifera; PPE, Potamogeton perfoliatus; SLI, Sagittaria lichuanensis; SLY, Solanum lycopersicum; SPO, Spirodela polyrhiza; SPU, Spirodela pundata; TTH, Tofieldia thibetica; WAU, Wolfia australiana) together with three seagrasses (HAL, H. ovalis; ZMA, Z. marina; ZMU, Z. muelleri). The order and habitat of species were indicated in the second part of each ID: DL, dicot, land; ML, monocot, land; MLA, monocot, land, Alismatales; MMA, monocot, marine, Alismatales; MWA, monocot, freshwater, Alismatales. Complete details are listed in Table S1. IDs coloured in red are members of core Alismatids, blue are members of Araceae, and black are others. Branches are labelled with bootstrap values (%). Discussion The concurrent absence of multiple genes in H. ovalis, Z. muelleri, and Z. marina suggests independently evolved convergent adaptation of seagrasses to the marine environment. Seagrass leaves lack stomata and the flowers have simplified structures when compared with terrestrial angiosperms (Kuo and Hartog, 2006). The loss of genes in stomata patterning and differentiation, and in sepal and petal development was previously described in Zostera, together with the loss of gaseous hormones and metabolites, such as ethylene, methyl jasmonate, and secondary volatile terpenes (Golicz et al., 2015; Lee et al., 2016; Olsen et al., 2016). Consistent with the low diffusion rate of gases underwater, the absence of ethylene production in seagrasses avoids accumulation in the tissues. In flood-adapted land plants, the ethylene signal is used to sense submergence and induces a response to flooding (Voesenek et al., 2015). Ethylene biosynthesis and signalling also play an important role in plant response to salinity (Zhang et al., 2016). There is conflicting evidence of ethylene as a positive or negative regulator during salinity stress in different species at different developmental stages (Tao et al., 2015), suggesting that some species adjust their sensitivity to environmental factors through regulation of the ethylene signalling pathway. As the loss of ethylene genes is observed in both H. ovalis and Zostera, it is likely that ethylene is selected against during seagrass adaptation to a submerged marine lifestyle. The sharing of OGCZ genes in H. ovalis to form a seagrass-specific gene set identifies orthologous relationships that appear to be unique to seagrass adaptation, despite their evolutionary distance and multiple origins. It is important to note that these 57 OGCZ genes are not novel genes but genes that have diverged sufficiently to cluster separately from other plants in orthologue analysis. The functions of these genes were not annotated but inferred by homology using annotated Arabidopsis genes. The majority of these genes are predicted to be involved in intracellular transport and in cell wall organization and modification. In plant cells, secreted proteins are processed through the Golgi apparatus as cargo molecules and sorted by receptors in the trans-Golgi network to different destinations (Brandizzi and Barlowe, 2013). Non-cellulosic cell wall matrix polysaccharides are among the wide range of vesicles synthesized and transported by the Golgi apparatus (Driouich et al., 1993; Lerouxel et al., 2006; Driouich et al., 2012). Besides catalytic mechanisms of glycosyltransferases and nucleotide-sugar conversions for polysaccharide assembly, the Golgi is also responsible for methylation of the cell wall polysaccharides. There are significant differences between cell walls of seagrasses and land plants. Seagrass cell walls contain sulfated polysaccharides (Aquino et al., 2005) and seagrass pectin contains a rare class of apiose-substituted homogalacturonan (Ovodov et al., 1971) with low levels of methyl esterification (Khotimchenko et al., 2012). These two modifications are thought to provide salt tolerance by increasing the polyanionic potential of cell walls (Aquino et al., 2005, 2011; Olsen et al., 2016). An expansion of pectin catabolic and methylesterase genes was observed in the genomes of Z. muelleri (Lee et al., 2016) and Z. marina (Olsen et al., 2016), suggesting complex pectin modification in seagrasses. Interestingly, within the list of seagrass-specific genes conserved in H. ovalis, CGR2 (cotton Golgi-related 2), a methyltransferase, was shown to be involved in pectin methylesterification in Arabidopsis (Weraduwage et al., 2016). Tubulin cofactor, which is responsible for the stability of microtubules (Zhu et al., 2015), is also found to be conserved among seagrasses. A total of five genes that encode RAB GTPases, the key regulators of vesicle trafficking (Miserey-Lenkei et al., 2010; Valente et al., 2010), were also conserved across both seagrass lineages. In Arabidopsis, knockouts of some members of the RAB GTPases have demonstrated roles in salinity stress tolerance (Asaoka et al., 2013). It is likely that this conservation of cell wall-related genes, as well as proteins involved in intracellular transport, in both families of seagrasses is linked to modification of cell wall composition as one of the adaptations to osmotic stress. Multiple salt-tolerance mechanisms have been hypothesized in seagrasses (reviewed in Touchette, 2007), including cell wall rigidity, selective ion flux and vacuolar ion sequestering, and the synthesis of compatible solutes and amino acids (Ye and Zhao, 2003; Carpaneto et al., 2004; Touchette et al., 2014; Cambridge et al., 2017). To avoid salt damage, plant cells adjust osmotic balance through influx and efflux of ions through the transmembrane transport proteins, assisted by H+ pumps (Hasegawa, 2013). Three genes, namely a component of a vacuolar proton pump, ATP synthase and calmodulin, were identified as conserved across the two seagrass lineages. Moreover, vacuolar proton ATPase A1 has been shown to be responsive to salt stress in sugar beet (Kirsch et al., 1996). This collection of genes may have a role in osmotic homeostasis of cells in the marine environment. Lipid transport and catabolism is another important role of the intracellular transport system. The endoplasmic reticulum synthesizes and exports phospholipids, sterols, and storage lipids for various purposes, including formation of membrane structures (van Meer et al., 2008). A total of four genes involved in lipid transport and catabolism were conserved in all three seagrass species, including ceramidase, which is responsible for sphingolipid metabolism. Sphingolipids provide membrane structure and are involved in cellular signal transduction (Hannun and Obeid, 2008). The difference between lipids of seagrasses and land plants is not well understood, but expansion in genes related to sphingolipid metabolism was observed in Z. marina when compared with duckweed (Olsen et al., 2016). Another alkaline ceramidase had been shown to regulate cell turgor pressure in Arabidopsis (Chen et al., 2015), but more evidence is needed to determine whether seagrass-specific lipid metabolism plays a role in marine adaptation. Two members of the core histone family are conserved in seagrasses. The domains in histone families, particularly H2A and H3, demonstrate expansion in numbers and variety, but with strong conservation of each variant across species (Kawashima et al., 2015). Ribosomal constituents were previously identified as modified in Z. muelleri when compared with land plants (Lee et al., 2016) and positively selected in Z. marina and P. oceanica (Wissler et al., 2011), and our results demonstrate that these genes are also conserved in H. ovalis. The basis for the observed differences in ribosomal gene sequences is not known, but it is postulated to be related to salt tolerance. Translation, and consequently protein synthesis are known to be salt-sensitive in yeast and plants (Rausell et al., 2003). For example, the expression of genes encoding the translation apparatus was lower when the transcriptome of Arabidopsis was compared with the halophyte salt cress (Taji et al., 2004). If seagrass ribosomes are adapted to relatively high salinity, this may have an application for improvement of salt tolerance in crop species. Sequence variations were identified in chloroplast-encoded 50S ribosomal protein L16. Nine amino acid mutations were shared by the three seagrass species despite belonging to two separate clades (Les et al., 1997; Li and Zhou, 2009; Les and Tippery, 2013; Petersen et al., 2016; Ross et al., 2016). The possible convergence is highlighted by the absence of these mutations in representatives of sister genera for both clades. Potamogeton perfoliatus belongs to the tepaloid clade together with Zosteraceae, whereas Najas flexilis, Elodea canadensis and Sagittaria lichuanensis belong to the petaloid clade together with H. ovalis (Les et al., 1997; Li and Zhou, 2009; Les and Tippery, 2013; Petersen et al., 2016; Ross et al., 2016). Protein sequences of L16 in these non-marine species have greater similarity with other monocots and dicots than with the seagrasses (Figs 2, 3) suggesting selection and convergent evolution to the marine habitat in seagrasses. Since N. flexilis and P. perfoliatus shared submergence characteristics with seagrasses, the mutations may be linked to salinity tolerance, rather than an ability to survive underwater. These results complement the seagrass clustering of OGCZ through OrthoMCL analysis and provided further molecular evidence of convergent evolution of seagrasses. Differences between H. ovalis and the two Zosteraceae species were identified in genes encoding NDH, a major protein complex residing in the thylakoid membrane of chloroplasts that participates in cyclic electron flow pathways as an oxidoreductase (reviewed in Peltier et al., 2016). As the NDH complex is only present in the Streptophyta lineage, which includes charophyte algae and land plants, acquisition of novel NDH genes likely occurred during terrestrial transition, and NDH is hypothesized to be one of the innovations enabling land plant evolution (Martín et al., 2009; Ruhlman et al., 2015). The absence of genes encoding NDH subunits and proteins required for complex formation in H. ovalis points to a total loss of the NDH complex in the H. ovalis thylakoid. Rare evidence of loss or pseudogenization of plastid NDH genes has been reported in independent lineages (Wolfe et al., 1992; Haberhausen and Zetsche, 1994; Funk et al., 2007; Braukmann et al., 2009; Logacheva et al., 2011), including several genera in the Hydrocharitaceae family (Iles et al., 2013; Peredo et al., 2013; Wilkin and Mayo, 2013; Ross et al., 2016). The observed loss of NDH genes in H. ovalis is the first report of their dispensability among Alismatales. Several reasons for NDH dispensability have been suggested (Stefanović and Olmstead, 2005; Iles et al., 2013; Peredo et al., 2013; Xu et al., 2013). Ross et al. (2016) suggest that NDH loss enabled low N investment as an adaptation to nutrient deficiency in the submerged environment. This is plausible, as H. ovalis is adapted to grow in low nutrient sediments (Carruthers et al., 2007), and examples of nitrogen sources affecting NDH expression have been reported in green algae (Peltier and Schmidt, 1991). Interestingly, two proteins related to nitrate uptake, nitrogen reductase 1 (NR1) and nitrate transporter (NRT3.1) are also lost in H. ovalis (Table S4). One limitation of seagrass nitrogen uptake studies is that the potential contribution of microbial communities is not accounted for, and may be compensating for the loss of NDH in H. ovalis, as demonstrated in myco-heterotrophic liverworts (Wickett et al., 2008a,b). Cyanobacteria on leaves have been shown to contribute to nitrogen uptake in Posidonia (Jeremy Bougoure, personal communication). However, further targeted studies are required to determine whether the loss of the NDH complex in H. ovalis is related to nitrogen uptake. Conclusion Together, the conservation of gene loss and the sharing of seagrass-specific orthologues in these two independent lineages, despite the phylogenetic distance, has shed light on the genetics of marine adaptation in angiosperms of land plant ancestry. These results also present another example of habitat-driven parallel evolution in the plant kingdom. Supplementary data Supplementary data are available at JXB online. Fig. S1. Venn diagram showing the number of shared orthologous clusters among six species (Arabidopsis, M. acuminata, O. sativa, S. polyrhiza, and two Zosteraceae species). Table S1. Species selected for multiple sequence alignment of orthologous proteins. Table S2. Number of H. ovalis reads sequenced and remaining after filtering process. Table S3. List of TAIR genes that were conserved in OGCsM and at least one species among H. ovalis, Z. muelleri, and Z. marina. Table S4. List of TAIR genes that were conserved in OGCsM but absent in at least one species among H. ovalis, Z. muelleri, and Z. marina. Table S5. Significantly enriched biological process GO terms in the genes that were lost in H. ovalis, but present in Z. muelleri, Z. marina, and five other plant species. Table S6. List of orthologous groups that are conserved in Z. muelleri and Z. marina (OGCZ). Table S7. List of OGCZ orthologous groups that are conserved in H. ovalis and the best corresponding TAIR ID hit; each protein ID is followed by InterProScan IDs of domains found or no domain found (NA). Abbreviations: Abbreviations: Mya million years ago NDH NADH dehydrogenase-like OGC orthologous gene cluster OGCsM orthologous gene cluster with at least one gene originating from a monocot species OGCZ orthologous gene cluster unique to Zosteraceae TAIR The Arabidopsis Information Resource. Acknowledgements The authors would like to acknowledge funding support from the Australian Research Council (Projects LP160100030, LP140100537, LP130100925, LP110100200, and LP0989200). Support is also acknowledged from the Queensland Cyber Infrastructure Foundation (QCIF), the Pawsey Supercomputing Centre with funding from the Australian Government and the Government of Western Australia and resources from the National Computational Infrastructure (NCI), which is supported by the Australian Government. References Alexa A , Rahnenfuhrer J . 2010 . topGO: Enrichment analysis for Gene Ontology . R package version 2.26.0. http://bioconductor.uib.no/2.7/bioc/html/topGO.html. 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Journal of Experimental BotanyOxford University Press

Published: Apr 18, 2018

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