Molecular Evidence for Origin, Diversification and Ancient

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OPEN Molecular evidence for origin, diversifcation and ancient gene duplication of plant subtilases Received: 22 January 2019 Accepted: 9 August 2019 (SBTs) Published: xx xx xxxx Yan Xu1,2,3, Sibo Wang2,4,5, Linzhou Li2,6, Sunil Kumar Sahu 2,4, Morten Petersen5, Xin Liu 2,4, Michael Melkonian7, Gengyun Zhang2,4 & Huan Liu 2,4,5

Plant subtilases (SBTs) are a widely distributed family of serine proteases which participates in plant developmental processes and immune responses. Although SBTs are divided into seven subgroups in plants, their origin and evolution, particularly in green algae remain elusive. Here, we present a comprehensive large-scale evolutionary analysis of all subtilases. The plant subtilases SBT1-5 were found to be monophyletic, nested within a larger radiation of bacteria suggesting that they originated from bacteria by a single horizontal gene transfer (HGT) event. A group of bacterial subtilases comprising representatives from four phyla was identifed as a sister group to SBT1-5. The phylogenetic analyses, based on evaluation of novel streptophyte algal genomes, suggested that the recipient of the HGT of bacterial subtilases was the common ancestor of Coleochaetophyceae, Zygnematophyceae and embryophytes. Following the HGT, the subtilase gene duplicated in the common ancestor and the two genes diversifed into SBT2 and SBT1, 3–5 respectively. Comparative structural analysis of homology-modeled SBT2 proteins also showed their conservation from bacteria to embryophytes. Our study provides the frst molecular evidence about the evolution of plant subtilases via HGT followed by a frst gene duplication in the common ancestor of Coleochaetophyceae, Zygnematophyceae, and embryophytes, and subsequent expansion in embryophytes.

Serine proteases are a highly abundant and functionally diverse class of proteins which occupy a notable place in plants1,2. According to the MEROPS peptidase database, the clan SB is one of 13 clans of serine proteases that are widely distributed in Archaea, Bacteria, and eukaryotes2–4. Clan SB contains two families, family S8 (ofen called the subtilase family) and family S53 (the sedolisin family)4. Te catalytic mechanisms of the two families of clan SB are diferent. In family S8 the active site residues form a catalytic triad in the order Asp, His, Ser, whereas family S53 contains a catalytic tetrad in the order Glu, Asp, Asp, Ser. Family S8 is the second largest family of serine proteases and is divided into two subfamilies, S8A (type example subtilisin) and S8B (type example kexin)5. Plant subtilisin-like proteases (also known as plant subtilases, SBTs) belong to subfamiliy S8A. Te frst subtilase cloned from plants was cucumisin from melon fruit6. In the past 20 years, several SBT gene families have been revealed throughout the plant kingdom, in Vitis vinifera7,8, Arabidopsis thaliana9, Oryza sativa10, Solanum lycopersicum11,12, Solanum tuberosum13, and others. Te Arabidopsis proteome alone comprises 56 subtilases. Based on these fndings SBTs have been divided into six subgroups9, and recently one of the subgroups (SBT6.1) was described as a seventh subgroup (SBT7)14. Schaller et al. have summarized most of the functions of plant subtilases, such as in embryogenesis, seed development and germination, cuticle formation and epidermal patterning, vascular development, programmed cell

1BGI Education Center, University of Chinese Academy of Sciences, Shenzhen, 518083, China. 2BGI-Shenzhen, Beishan Industrial Zone, Yantian District, Shenzhen, 518083, China. 3China National GeneBank, Institute of New Agricultural Resources, BGI-Shenzhen, Jinsha Road, Shenzhen, 518120, China. 4State Key Laboratory of Agricultural Genomics, BGI-Shenzhen, Shenzhen, 518083, China. 5Department of Biology, University of Copenhagen, Copenhagen, Denmark. 6School of Biology and Biological Engineering, South China University of Technology, Guangzhou, 510006, China. 7Botanical Institute, Cologne Biocenter, University of Cologne, Cologne, D-50674, Germany. Yan Xu, Sibo Wang and Linzhou Li contributed equally. Correspondence and requests for materials should be addressed to H.L. (email: [email protected])

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a. b.

Embryophyta Alveolata Streptophyte algae Euglenozoa Chlorophyta Stramenopiles Glaucophyta Amoebozoa Rhodophyta Fungi Bacteria Archaea

Chloroflexi Chloroflexi Firmicutes 100 Bacterial S8A genes 100 Caldiserica (S8 cluster 1) SBT1-5 97 Caldiserica 98 Coprothermobacterota 84 Actinobacteria Chloroflexi Chloroflexi 98 Gamma-proteobacteria 83 75 S8 cluster 1 86 100 Delta-proteobacteria Delta-proteobacteria Bacterial Subtilases 79 100 Actinobacteria 69 100 Chloroflexi Actinobacteria

Actinobacteria 40 83 100 Bacterial Subtilases S8 cluster 2 100 100 Gamma-proteobacteria 98 59 Proteinase K 97 Chloroflexi 99 Beta-proteobacteria/Gamma-proteobacteria

Beta-proteobacteria/Gamma-proteobacteria KP43 proteinase 93 100 Gamma-proteobacteria 70 Beta-proteobacteria 88 100 Firmicutes 83 Actinobacteria

56 57 SBT7 97 Chloroflexi 95 68 Actinobacteria

Mesotaenium endlicherianum S8 cluster 4 99 Streptophyte algae Coleochaete scutata HGT 100 92 Streptophyte algae

100 “Spirotaenia sp.” Plant Subtilases 100 Streptophyte algae (SBT2) 73 Tree scale: 1 Embryophyta SBT6 100

S8 cluster 3

Figure 1. Phylogenetic relationship of the S8A protease gene subfamily. (a) Maximum likelihood unrooted phylogenetic tree of the S8A subfamily from representative Archaea, Bacteria and eukaryote species was constructed with IQ-TREE (model: LG + R10, predicted by Modelfnder) using an ultrafast bootstrap approximation (100,000 bootstrap replicates). Colored domains display eight diferent clusters in the S8A subfamily. Te domains with a continuous line indicate resolved clusters, while domains with dotted lines represent undefned clusters. Te colored circles at the top lef represent the species composition of individual clusters. Te plant subtilases (SBT1-5) apparently originated from bacterial subtilases (red branches in in paraphyletic divergences) through a single HGT. (b) Phylogenetic analysis of plant subtilases using an extended taxon sampling of bacterial subtilases to search for a bacterial sister group to the plant subtilases was constructed by Maximum Likelihood using 500 bootstrap replicates (model: WAG + F + R7, predicted by Modelfnder). Plant subtilases are monophyletic with a clade of bacterial sequences derived from four phyla (Proteobacteria (only Gammaproteobacteria and Betaproteobacteria), Chlorofexi, Actinobacteria and Firmicutes). Te streptophyte algal sequences from Mesotaenium endlicherianum, Coleochaete scutata and “Spirotaenia sp.” diverge paraphyletically from the common ancestor of the plant subtilases with “Spirotaenia sp.” in sister position to embryophytes (the detailed tree with all taxon and species names is shown as Supplementary Fig. S1). Some bacterial S8 genes from the phylogenetic tree of the S8 cluster 1 (a) were selected as an outgroup.

death, organ abscission, and plant responses to their biotic and abiotic environments15–17. However, many specifc functions and physiological substrates of SBTs still need to be explored. Previous studies have found that land plant subtilases were derived from a single HGT (horizontal gene transfer) event18. Horizontal gene transfer is proposed relative to vertical gene transfer, which breaks down the boundaries of kinship and complicates the pos- sibility of gene fow. Many genes found in algae doesn’t have any homologs in higher plants were suggesting their possible bacterial origin. For instance, two alginate-specifc enzymes, MC5E and GDP-mannose dehydrogenase show high similarity with the bacterial genes, indicating that these genes might have undergone a non-canonical evolutionary history. Taylor and Qiu (2017) investigated the evolutionary history of plant subtilases through a phylogenetic analysis using 2,460 subtilase amino acid sequences of 341 species, and identifed 11 new gene lin- eages14. Meanwhile, the presence of plant subtilases in streptophyte algae, the grade of green algae most closely related to land plants (embryophytes), was also frst reported in their study, based on analysis of transcriptomes14. Plant subtilases seem to have experienced several gene duplications, which were accompanied by functional diversifcation7. Due to their multiple duplications and complex evolutionary history, it is important to explore the origin and diversifcation of SBTs among Viridiplantae including the streptophyte algae. We have recently sequenced the genomes of several streptophyte algal species (unpublished data) enabling us to explore the evolution of subtilases including all major lineages of streptophyte algae. We reconstructed the phylogeny of all S8A subtilases existing in archaea, bacteria, and eukaryotes. Ten we focused on the origin and evolution of plant subtilases (SBTs). Here we show that plant subtilases originated from bacteria by HGT into streptophyte algae followed by a gene duplication event in the common ancestor of Coleochaetophyceae, Zygnematophyceae and embryophytes. Our study provides new information about the origin and early diversifcation of plant subtilases and thus contributes to a better understanding of the phylogeny of the S8A protease family. Results Classifcation of the S8A gene family and putative origins of plant-type subtilases. To explore the phylogenetic relationship between plant subtilases and other members of the S8A family, genes related to plant subtilases were selected based on genome functional annotation. We performed a phylogenetic analysis incorpo- rating genes from Archaea, Bacteria, Fungi, Amoebozoa, Stramenopiles, Euglenozoa and Archaeplastida (Fig. 1a;

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Supplementary Tables 1–3 with all genes harboring the conserved S8 domain. Finally, eight (nine: SBT7 and S8 cluster 4 could not be reliably separated in the phylogenetic analysis, Fig. 1a) defned clusters were obtained by combining domains and phylogenetic information (Fig. 1a; for conserved protein domain designations see Supplementary Table S3): Proteinase K, KP43 proteinase, S8 clusters 1, 2, 3, and 4, SBT 6, SBT 7, and cluster SBT 1–5. Among the S8A subfamily genes, the Proteinase K-related cluster fwas well supported. Proteinase K is a known endopeptidase that has previously been found only in fungi and bacteria5 but our analysis revealed that Proteinase K homologs also occur in Archaeplastida [from Rhodophyta, Glaucophyta, to green algae and bryophytes (Marchantia polymoprpha and Physcomitrella patens)] but not in vascular plants (Fig. 1; Supplementary Table S3). Another well-supported cluster is the KP43 protease with the Peptidases_S8_Kp43_protease domain. Te architecture of the Kp43 protease was reported to be similar to kexin and furin, both of which belong to the S8B subfamily19. In addition to Proteinase K and KP43 protease, other S8 family genes were also clustered in diferent groups (Supplementary Table S3). However, it was difcult to defne them precisely because of their non-systematic domain distribution. Plant subtilases possess one of three types of domains: Peptidases_S8_Tripeptidyl_Aminopeptidase_II (SBT6), Peptidases_S8_SKI-1_like (SBT7), and Peptidases_S8_3 (SBT1-5) (Supplementary Table S3). In Fig. 1a, SBT-1-5 occurs only in embryophytes, two clades of streptophyte algae (Coleochaetophyceae and Zygnematophyceae) and a set of diverse bacteria, which suggests horizontal gene transfer between these unrelated organisms. We termed these bacterial genes “bacterial subtilases” or “bacterial SBT”. In order to further explore the phylogenetic relationship between bacterial subtilases and plant subtilases, a detailed tree was reconstructed by using the genes selected from a wider range of species (Fig. 1b; Supplementary Fig. S1). In our preliminary analysis we performed the Blastp against the nr database using the cutof e value 1e-10. We found that only bacteria and streptophyte algae possess plant-like SBTs, suggesting the complete absence of SBTs among Archaea, Fungi and other eukaryote taxa. Te further detailed phylogenetic analyses of the plant algae-type subtilases suggested a single HGT event from a bacterial donor because the streptophyte sequences had a single origin, nested within a larger radiation of bacterial subtilases (Fig. 1a,b; Supplementary Fig. S1). Te search for the bacterial donor of the HGT is compounded by the fact that the SBTs apparently underwent multiple HGTs among bacteria (Supplementary Fig. S1), i.e. the bacterial SBT phylogeny does not refect the species phylogeny. Te bacterial sister group to the plant subtilases (bootstrap support 95%; Fig. 1b; Supplementary Fig. S1) comprised species from four phyla (Proteobacteria (only Gammaproteobacteria and Betaproteobacteria), Chlorofexi, Actinobacteria and Firmicutes). All bacterial SBT sequences in this clade (except Halioglobus sp.20, which is a marine bacterium) correspond to soil bacteria, e.g. Glycomyces xiaoerkulensis21, Longilinea arvoryzae22 and Streptosporangium roseum23. HGT among soil bacteria is rampant involving IncP- and IncPromA-type broad host range plasmids24. Te frst diverging lineage in the bacterial sister clade consists of fve sequences from Gammaproteobacteria and Betaproteobacteria indicating that perhaps one of these two classes of bacteria had provided the donor SBT gene for the plant subtilases. Although a representative set of fve cyanobacterial genomes was included in the analysis, none encoded plant-like SBTs (Supplementary Fig. S1). An HGT of the SBT gene in the opposite direction i.e. from a plant donor to bacteria cannot be ruled out, however, this is unlikely, because the plant subtilases were nested within a larger bacterial radiation (and not the opposite), and no trace of plant subtilases were found in the earlier diverging streptophyte algae (for a recent review on HGT from bacteria to eukaryotes25). Te phylogenetic tree (Fig. 1b; Supplementary Fig. S1) indicated that the recipient of the bacterial subtilase was a streptophyte alga, most likely the common ancestor of Coleochaetophyceae, Zygnematophyceae and embryophytes (Fig. 1b).

Phylogenetic analysis of plant subtilases SBT1 to SBT5. To further explore the phylogenetic relationship among SBT1-5 plant subtilases, an extensive search was conducted by taking 54 Arabidopsis thaliana subtilases as the standard reference9. A total of 314 genes from 3 species of streptophyte algae (Coleochaete scutata, “Spirotaenia sp.” and Mesotaenium endlicherianum) and 7 species of embryophytes (Marchantia polymorpha, Physcomitrella patens, Selaginella moellendorfi, Salvinia cucullata, Oryza sativa, Zea mays and Arabidopsis thaliana) were selected. Tese identifed plant subtilase genes were also classifed into fve subgroups (SBT1-5), similar to the classifcation based on subtilases from Arabidopsis thaliana (Fig. 2). Each gene’s intron number and their average length was calculated as well (Fig. 2; Supplementary Table S5). It allowed us to gain new insights into the evolution of subtilases in Viridiplantae. First, the most interesting observation was the existence of plant-like SBT genes in streptophyte algae. Te possible presence of plant subtilases in algae had been reported before for three species of Zygnematophyceae (Spirogyra sp., Cylindrocystis sp. and Roya obtusa; Taylor & Qiu, 2017; their Fig. 114) based on the evaluation of transcriptomes established by the 1,000 plants transcriptome initiative (1KP project)26, but their distribution within streptophyte algae was not studied. Because of the recent availability of high-quality genome data of several streptophyte algal clades, we were able to provide the frst evidence for the origin of SBT1 to SBT5 in the common ancestor of Coleochaetophyceae, Zygnematophyceae and embryophytes. According to the phylogenetic tree, following their origin by HGT, the plant subtilases likely underwent one gene duplication in the ancestor of Coleochaetophyceae, Zygnematophyceae and embryophytes, one copy evolved into SBT2 and the other was ancestral to SBT1, SBT3, SBT4, and SBT5 (Fig. 2). Other gene or genome duplications possibly occurred in the common ancestor of embryophytes, however, because of the low confdence levels with less than 50% bootstrap support, relationships among SBT3, SBT4, and SBT5 could not be resolved. Te SBT1 subclass is the largest of the subtilase subfamilies according to their large gene copy numbers, and this subclass has undergone multiple gene duplications starting in the monocot plants. Interestingly, we found that the intron numbers vary considerably between diferent groups of plant subtilases, especially in SBT1, almost all the genes had no intron. Tis phenomenon was also reported in grape subtilases, which is inferred that, in order to increase the ftness of an organism, the intragenic recombination is also increased which is further related to the evolutionary rate of genes7,27,28.

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SBT3 AT1G6621 112 63 89 100 Zm00001d 0 123.43 020627 T5G11940.1 25 average length of intron 9 A Os10t0394 70 100 200-01 460 90.86 AT4G21326 1 98 53 Zm00001d0330 100 39 0 7 AT4G21323.1 95 100 Streptophyte algae 105.75 AT5G5175 21283 100 0.1 1 Zm00001d0 17 Os04t057330 98 7 100 861 7 02089 100 0-00 Zm00001d Zm00001d 0 0 5 90.88 026068 01d00620 100 Bacteria 22 100 AT5G67360 Zm000 0 intron number 0 8 40 1 .1 97 50 1 100 Os03t06 1 Os09t0530800-0 100 59 74 05300-0 0 83 Zm00001d 1 6 0 PACid 154160 276 50 033194 0 99 22 Os03t 0 PACid 15415 100 10 12 0761500-0 107.56 50 37 78 Zm0 2 8 PACid 15415415 32 0001d034 0 0 horizontal gene transfer and ancient gene duplication 82 P 1 5 ACid 1 12 15.7 Cid 15404306 1 PA 54083 0 9 402669 PACid 1 37 0 59.88 PACid 15 907 100 99 5418128 0 PACid 0 2 154191 PACid 15406 85 100 58.75 Mapoly0 4 0 2 6 0 Zm00001d02773 100 PA 013s008 Tree scale: 1 Cid 15 51.14 4.1. Os03t0158700-01 9 PA 41 p 0 0 9 Cid 1387 AT4G26330.1 43 28 100 15 54.63 9V6.2 Phypa2 411963 4 100 0 0 Sacu v1.1 18s0 Phypa70 24 10 115V6.1 55.88 p 26 .2. 85 PACid 15418 79. Phypa70 249V6.1 52 100 068.g0164 23 s0026 .p 100 92 46 0 8 026.1 34 97 AT 57.5 34 5G4564 22 22 Mapoly0098 98 24 100 1 603.44 100 AT5G45650. oly0098s0 8 41 99 0.1 0 Map 8 284.86 ACid 154077 100 46 Os01t07 P 100 1 407. 98 82 91 15 65 Zm00 27800-0 55.1 5 PACid 1541889 0 7 100 001d 3 Cid 15404373 100 PACid 154127 1 PA 5418903 20 0435 54.8 122.14 20 33 58 57 397 3 41 950.13 PA 81 9 ACid 1 100 Cid 0 P 100 38 83 8 34 PACid 15420 11 243.86 59 100 PACid 15404 20 291.62 80 15405672589 97 43 PACid 196 ACid 1541890654227 44 85 100 P 34 Mapoly0040s0059.1. 1542 8 98 100 5404398 0 PACid 1 92 100 Mapoly004 0093 524.63 99 114.3 234.7 Cid 1 90 100 2 PA 94 Mapoly 8 6 9 79 0s0057.1. ACid 1540438 9 Mapo 13 234.7 P 99 0004s01 232.88 100 100 p 100 9 58.13 ACid 15422486 84 Mapol ly00 P 49 100 7 1 100 p 0 Sacu 40s0 74.1.p ACid 1540180 6230 71 y0059s0 361. P 9

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Figure 2. Phylogenetic relationship of diferent classes of plant subtilases. Te tree was constructed with IQ- TREE by employing the Maximum Likelihood method (model: WAG + R8, predicted by Modelfnder). Te circles around the phylogenetic tree (from center to periphery) represent SBT1-5, and the respective species distribution, indicated by diferent colors. Some bacterial subtilase genes from the phylogenetic tree of the S8A subfamily (Supplementary Fig. S1) were selected as outgroup. Histograms of two outer rings are numbers (red histogram) and average length of introns (green histogram) respectively. For each clade, numbers above branches indicate bootstrap values based on 200 replications.

SBT6 and SBT7 are highly conserved among various lineages. SBT7 and SBT6 are reported as homologs of human protein convertases and are characterized by a stronger similarity to the mammalian kexins and pyrolysins than to plant subtilases9. According to the phylogenetic tree in Fig. 1, SBT7 and SBT6 are indeed very distinct from SBT1-5 with diferent S8 domains (Supplementary Table S3). We conducted a broad search among animals (Homo sapiens, Drosophila melanogaster, Mus musculus, and Bactrocera dorsalis) and found that both SBT6 and SBT7 have a broad distribution with one or two gene copies. Te phylogeny of SBT7 and SBT6 among eukaryotic species is shown in Fig. 3. It seems that these genes are ubiquitously present among all species we selected. Interestingly, the presence of SBT6 has been reported in only two bacterial species (Blastopirellula marina, Rubinisphaera brasiliensis)29. Both species are related members of Planctomycetes occurring in saline/ marine environments. Based on these observations, and our phylogenetic tree, we hypothesize that SBT7 and SBT6 had likely a eukaryotic origin and SBT6 might have been transferred to the two species of Planctomycetes via HGT, although the donor remains unknown as the support values in this region of the SBT6 tree are extremely low. In spite of their broad distribution, the motifs of both SBT7 and SBT6 genes displayed a consistently high similarity among eukaryotes, showing their conservation among various lineages.

Taxonomic distribution of S8A genes and plant subtilases expansion in Embryophyta. To intu- itively show the overall distribution of S8A genes, we performed a quantitative statistic combing all explored

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Schizosaccharomyces pombe TPP2 Motif 10 96 Malassezia pachydermatis TPP2 Fungi Motif 17 100 Moesziomyces antarcticus TPP2 Motif 19 Stramenopiles-Fragilariopsis-OEU13959.1 Stramenopiles Motif 20 SBT6 32 Porum69557.1 Red algae Drosophila melanogaster TPP2 Motif 3 Caenorhabditis elegans TPP2 80 Animal Motif 11 40 Homo sapiens TPP2 Motif 1 19 100 Mus musculus TPP2 32 bac-Blastopirellula marina DSM 3645 TPP2 Motif 12 93 bac-Rubinisphaera brasiliensis TPP2 Bacteria Motif 2 Amoebozoa-Planoprotostelium-PRP81954.1 24 35 Amoeba Motif 13 Stramenopiles-Ectocarpus-ES0004G00070 Motif 16 25 Stramenopiles-Phytophthora-XP 002906072.1 Stramenopiles 20 100 Stramenopiles-Phytophthora-XP 008899999.1 Motif 5 Chlre5586 Motif 8 100 Volca7180 Motif 6 100 Chlva7429 100 Cocsu1782 Motif 9 99 Mesovi-comp25362 c0 seq1 m.23508 Motif 4 100 Mesovi-comp7206 c0 seq1 m.2217 Motif 7 KleflGAQ87799.1 84 PACid 15412976 Green lineage Motif 15 96 Mapoly0069s0029.1.p Motif 18 69 100 Phypa2 123V6.1 Motif 14 100 Phypa304 1V6.1 36 Sacu v1.1 s0149.g023286 Os02t0664300-01 96 100 Zm00001d017522 100 AT4G20850.1 Euglenozoa-Trypanosoma cruzi XP 806372.1 Euglenozoa SBT7 Stramenopiles-Ectocarpus siliculosus ES0273G00010 100 Stramenopiles-Phytophthora parasitica XP 008898115.1 Stramenopiles 100 Stramenopiles-Phytophthora infestans XP 002904862.1 81 Porum68732.1 Red algae Bactrocera dorsalis MBTPS1 24 100 Homo sapiens MBTPS1 Animal 52 100 Mus musculus MBTPS1 39 Amoebozoa-Planoprotostelium fungivorum PRP88795.1 48 Amoebozoa-Acytostelium subglobosum XP 012754371.1 Amoeba 100 Amoebozoa-Acytostelium subglobosum XP 012758124.1 24 Cyanophora paradoxa ConsensusfromContig10185-SBT6.1Glaucophyte algae MicRCC9363 MBTPS1 77 Batpr7359 MBTPS1 71 Ostta7021 MBTPS1 46 55 54 Chlva756 MBTPS1 Cocsu148 MBTPS1 78 Chlre8114 MBTPS1 100 38 Volca24 53 Chlat66S07212 Mesvi29S04991 KleflGAQ86362.1 36 Spisp1455S07574 Green lineage 88 100 Spisp369S13987 80 Mesen371S07005 76 Colsc4866S0T10164 82 Mapoly0155s0018.1.p 57 Phypa55 133V6.1 92 PACid 15422900 Sacu v1.1 s0003.g001892 75 Os06t0163500-01 92 100 Zm00001d045217 Tree scale: 1 100 AT5G19660.1 5' 3' 0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 1550 1600 1650 1700

Figure 3. Phylogenetic relationship of SBT7 and SBT6 and their motif composition. Te phylogenetic tree was constructed by IQ-TREE based on the Maximum Likelihood method (model: LG + F + R5, predicted by Modelfnder). For each clade, bootstrap values are labeled on each branch based on 200 replications. 20 conserved motifs were identifed through MEME analysis.

genes with their phylogenetic relationship above. Statistical analyses of the copy numbers showed the distribution of S8A genes among Archaeplastida (Fig. 4; Supplementary Table S4). Te S8 gene clusters revealed a discontinuous distribution among the lineages, and we could only detect their presence up to early-diverging embryophytes (Bryophyta). For example, we found that genes of the S8 cluster 3 exist in species of Chlorophyta, Sreptophyta, in bryophytes (Physcomitrella patens) and in the red alga Porphyra umbilicales but not in vascular plants. In proteinease K, both Porphyra umbilicalis (Rhodophyta) and Cyanophora paradoxa (Glaucophyta) have a larger copy number than the Viridiplantae combined. Plant subtilases (SBT2 and the ancestor of SBT1, SBT3, SBT4, and SBT5) frst appeared in derived streptophyte algae (Coleochaetophyceae and Zygnematophyceae) with the exception of SBT6 and SBT7 that occur throughout Archaeplastida albeit with a discontinuous distribution in SBT 6 (Fig. 4). Signifcant expansions of copy numbers were observed in SBT1 among monocot species and in SBT 4 and SBT 5 in Selaginella moellendorfi although their signifcance remains unknown (Figs 2,4). Unfortunately, the function of these genes remains uncertain without experimental validation, although we inferred that the expansions may be connected with it and the species’ living environment (habitat).

Conserved structures of SBT2 during their evolution. With the exception of gene copy numbers and amino acid site mutation, variant gene structure also refects evolutionary diference among the classifcation of diverse species. Te tertiary structures of subtilases have been reported earlier, i.e. the cucumisin from melon fruits (Protein Data Bank (PDB) code 3VTA and 4YN3)30,31, and SlSBT3 from tomato (PDB code 3I6S)32. However, the protein structural information of SBT2 (the earliest-diverging subtilase of embryophytes; Fig. 2) is unavailable. Terefore, we frst homology-modeled the 3D structure of SBT2, and then performed a structural comparison of this modelled protein among prokaryotes and eukaryotes (Fig. 5). Te analyses revealed the signif- icant structural similarity between bacteria vs algae, algae vs bryophytes, and bryophytes vs dicots/monocots. In fact, SBT2 showed a high similarity between bacteria and embryophytes as well. Moreover, when we analyzed the combined SBT2 sequences among all the fve species, they still exhibited highly conserved regions (pink color) (Fig. 5b, lef panel), which was also evident in the MSA (multiple sequence alignment) (Fig. 5b, right panel). Tese observations confrmed the highly conserved nature of SBT2 throughout the evolution, and its likely origin and transfer via HGT from bacteria. Discussion Proteases play key roles in the developmental regulation of plants. While plant genomes encode hundreds of proteases, the largest class of them are represented by serine proteases33. However, despite being the dominant class, the complex evolutionary history and function of serine proteases are not yet fully explored. In this study, we performed a comprehensive phylogenetic analysis of the S8A gene peptidase family using 835 genes from Archaea, Bacteria, Fungi, Amoebozoa, Stramenopiles, Euglenozoa, and Archaeplastida. All the genes were clustered into several groups, including genes that clustered into seven groups of plant subtilases (SBT1 to SBT7). Genes corresponding to plant subtilases were also selected to build a refned phylogenetic tree to show the relationships among them. Previous studies have shown that some plant-like subtilisins in fungi have been acquired from embryophytes34, and another study implicated a single HGT event involved in the origin of plant subti- lases18. However, none of these studies addressed the possible recipient of the HGT among Viridiplantae or the early evolution of plant subtilases. Based on our phylogenetic analyses and the copy number variation among subtilases, we concluded that the evolution of plant subtilases began with a single HGT event followed by a

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Arabidopsis thaliana

Oryza sativa

Zea mays

Salvinia cucullata

Selaginella moellendorffii

Physcomitrella patens

Marchantia polymorpha

Mesotaenium endlicherianum

“Spirotaenia sp.”

Coleochaete scutata

Chara braunii

Klebsormidium nitens

Chlorokybus atmophyticus

Mesostigma viride

Chlorella variabilis

Coccomyxa sorokiniana

Chlamydomonas reinhardtii

Volvox carteri

Ostreococcus tauri Embryophyta 30

Streptophyte algae 25 Micromonas commoda Chlorophyta 15 Bathycoccus prasinos Rhodophyta 5

Cyanophora paradoxa Glaucophyta 1

Porphyra umbilicalis

e K SBT7 SBT6 SBT2 SBT1 SBT5 SBT4 SBT3

S8 cluster 1S8 cluster 2S8 cluster S83 cluster 4 Proteinase KP43 proteinas

Figure 4. Copy number variation of subtilases among Archaeplastida. Te copy numbers were calculated based on the phylogenetic tree (Figs 1a and 2) and functional annotation. Te colors corresponding to respective group of taxa are highlighted at appropriate positions.

frst gene duplication in the common ancestor of Coleochaetophyceae, Zygnematophyceae, and embryophytes. Interestingly, the putative donor taxa of the plant subtilases (SBT1-5) genes belong to four phyla (Proteobacteria [only Gammaproteobacteria and Betaproteobacteria], Chlorofexi, Actinobacteria and Firmicutes) (Fig. 1b; Supplementary Fig. S1) and with one exception (Halioglobus sp.) are all soil bacteria. Tis may suggest that the HGT occurred in a terrestrial environment corroborating the mounting evidence that streptophyte algae throughout their evolution underwent increasing adaptation to subaerial/terrestrial habitats although many extant streptophyte algae thrive in aquatic habitats35 (and unpublished results). Subsequently, one of the algae-type SBTs gradually evolved into the present-day plant SBT2s, another was split into two parts evolving into SBT1 and SBT3, 4 and 5 respectively via an extra gene or genome duplication event that presumably occurred in the ancestor of the embryophytes (Fig. 6). However, our hypothesis difers from that proposed by Taylor and Qiu14. According to their hypothesis, the lineages of SBT 1,3,4, and 5 originated before the divergence of Embryophyta and Zygnematophyceae, and the SBT2 lineage originated early in embryophytes. In this study, we also predicted that subtilases in SBT 3,4, and 5 might have undergone more complicated species-specifc duplications or gene losses, and therefore require more detailed phylogenetic information. As SBT6 and SBT7 could not be clustered into the same group with SBT1 to SBT5, we built another tree to show their distribution among eukaryotes. Both SBT6 and SBT7 were also found to be distinct from each other, which can be also judged by their distinct roles in plants9. For instance, in Arabidopsis, the activation of two membrane-bound transcription factors bZIP28 and bZIP17 depends upon the cleavage by SBT7 during ER (endoplasmic reticulum) stress signaling and salt stress, respectively36,37. In addition to membrane-bound transcription factors, other proteins have also been identifed as substrates of SBT738,39. SBT6 acts in a proteolytic pathway downstream of the proteasome during cadmium stress40. Interestingly, two bacterial species that have likely obtained SBT6 genes from plants (Fig. 3) can both survive in a saline environment41,42, suggesting that SBT6 may also act under salt stress.

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C. psychrerythraea M. endlicherianum P. patens C. psychrerythraea M. endlicherianum P. patens A. thaliana A. thaliana

β2 η3 α3 β3 b 230 240 250 260 270 Colwellia psychrerythraea NK LI GA RY F DASFSSQYEV Q YALG EF DSP RD ADGHG SHT A ST A GG N EG V S Mesotaenium endlicherianum KK VI GA NY F KDGFEYSVGAIAKK. D SP SP LD VDGHG SHT A ST A AG N AG Y P Physcomitrella patens GK VI GA KY F ARGIMAADMF N ETY. DF ASP FD GDGHG THT SSI A AG S SG VP Oryza sativa GK IV GA QH F AKAAIAAGAF N PDV. DF ASP LD GDGHG SHT A AI A AG N NG IP Arabidopsis thaliana GK II GA QH F AEAAKAAGAF N PDI. DF ASP MD GDGHG SHT A AI A AG N NG IP β4 β5 η4 TT 280 290 300 310 320 Colwellia psychrerythraea A M L S G ANA G T VSG MAPRARIA AYK A C W NSDYKNPEGGDEA G ....C F YG D Mesotaenium endlicherianum V GS K ELPA G L ASG MAPRARIA AYK VIW YN...... EA G EGPY G ES S D Physcomitrella patens V V VKG YNY G T ASG IAPRARIA VYK VIY RD...... G .... GF LS D Oryza sativa V R MHG HEF G K ASG MAPRARIA VYK VLY R...... LF G .... GY VS D Arabidopsis thaliana V R MHG YEF G K ASG MAPRARIA VYK ALY R...... LF G .... GF VA D

η11β18 α8 α9

590 600 610 620 630 Colwellia psychrerythraea ..SAPMFGTQ GE T F KY LQ GTSM SS PH IAG MA AL FK ESNSS WS P AQ I KSA M Mesotaenium endlicherianum .. G RQTLNGK P LLSN VLS GTSM A APH LAG LA AL VK QK Y P RWS P F AI KSA I Physcomitrella patens QN G IDVTGFV GE S F A MIS GTSM A TPH IAG VV AL VK QK H P TWS TS AI HSA L Oryza sativa PN G TDEANYA GE G F A MVS GTSM A APH IAG IA AL IK QK N P KWS P S AI KSA L Arabidopsis thaliana AN G TDEANYI GE G F A LIS GTSM A APH IAG IA AL VK QK H P QWS P A AI KSA L

Figure 5. SBT2 is conserved during the course of evolution from bacteria to embryophytes. (a) Comparative structural analysis of the homology-modelled SBT2 protein between Colwellia psychrerythraea, Mesotaenium endlicherianum, Physcomitrella patens, and Arabidopsis thaliana or Oryza sativa by superimposition. Te colors corresponding to individual species are labelled in the fgure. (b) Te compiled analysis of the SBT2 protein among all the above-mentioned species by using the CONSURF tool. Te pink color at the core region indicates the highly conserved nature of SBT2 throughout evolution (lef panel). Te right panel displays the multiple sequence alignment of the representative protein sequence from the fve species along with the information regarding secondary structures revealing conserved amino acid blocks.

In conclusion, large-scale phylogenetic analyses of subtilases among species in Archaea, Bacteria, and eukaryotes were performed to better understand their complex evolutionary history. Phylogenetic trees of subtilase genes showed the diversifcation of the S8A superfamily and the origin of plant subtilases through a single HGT event likely from a soil bacterium to the common ancestor of Coleochaetophyceae, Zygnematophyceae and embryophytes, suggesting that this ancestor may have thrived in a subaerial/terrestrial environment. Methods Data retrieval and fundamental analyses of plant subtilases. Te whole genome sequences and structure annotation of 143 species were downloaded from the NCBI database (https://www.ncbi.nlm.nih.gov/), including 5 species from Archaea, 101 species from Bacteria, and 37 species from eukaryotes (Supplementary Table S1). We included six newly sequenced and representative genomes of streptophyte algae: Mesostigma viride (strain CCAC 1140), Chlorokybus atmophyticus (strain CCAC 0220), Entransia fmbriata (strain UTEX 2353), Coleochaete scutata (strain SAG 110.80), “Spirotaenia sp.” (strain CCAC 0214), and Mesotaenium endlicherianum (strain SAG 12.94). Tese algae were obtained as axenic strains from the Culture Collection of Algae at the University of Cologne (http://www.ccac.uni-koeln.de/), and the DNA as extracted by a modifed CTAB method43. All genomes were processed for functional annotation using the SWISS-PROT database (cutoff: e-value of 1e-5) to selected possible subtilisin-like proteases by using the keywords “Subtilisin-like protease”, “Subtilase”, “Tripeptidyl-peptidase II”, “TPP2”, or “Membrane-bound transcription factor site-1 protease”. In all these species, only 2 species of Archaea, 53 species of Bacteria, 41 species of eukaryotes were found to encode a total of 835 S8A genes (including subtilases). To further explore phylogenetic relationship between bacterial subtilases and plant subtilases, we rebuilt a detailed HGT tree (Fig. 1b; Supplementary Fig. S1). Bacterial subtilases in the detailed HGT tree (Fig. 1b) were selected by comparing every algal subtilases with nr databases using a 1e-10 e-value and 1,000 max target sequences as the cutof followed by a process of removing redundancy. According to NCBI bacterial taxonomy, the species sources of these bacterial subtilases were classifed and for each taxon we only selected some representative species in order to cover all the bacterial taxonomy, fnally 80 genes were used in the analysis. Parts of genes from cluster 1 of the S8A gene subfamily tree were also added to this analysis as the outgroup and

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Coleochaetophyceae Algae-type SBT2

Duplication Zygnematophyceae SBT2

Embryophyta

HGT

ancestors of SBT1, 3, 4 and5 Coleochaetophyceae Bacterial SBT-like

Duplication Zygnematophyceae

SBT1

Embryophyta

SBT3, 4, 5

Embryophyta

Figure 6. An overview about the evolution of plant subtilases. Te recipient of the HGT of bacterial subtilases was the common ancestor of Coleochaetophyceae, Zygnematophyceae and embryophytes. Following the HGT the subtilase gene duplicated in the common ancestor and the two genes diversifed into SBT2 and SBT1,3–5 respectively, and subsequent expansion in embryophytes.

SBT2s were selected as the representative plant subtilases since they are one of the earliest-diverging subtilases of embryophytes. For SBT6 and SBT7, 13 genes from animals (Homo sapiens, Caenorhabditis elegans, Drosophila melanogaster, Mus musculus, and Bactrocera dorsalis), fungi (Schizosaccharomyces pombe, Malassezia pachydermatis, Moesziomyces antarcticus) and bacteria (Blastopirellula marina, Rubinisphaera brasiliensis) were selected from the UniProt database (https://www.uniprot.org/). Tese gene were also included in the phylogenetic analysis of the S8A gene subfamily. Te conserved domains of selected genes were searched using NCBI’s CDD database (conserved domain database, https://www.ncbi.nlm.nih.gov/cdd/, cutof: e-value of 1e-5), the genes which do not contain the domain belonging to the Peptidases S8/S53 superfamily or contain a domain named Peptidases S8 Protein convertases Kexins Furin-like (S8B subfamily) were excluded from our analysis. Finally, 915 genes were selected for the downstream analysis (Supplementary Tables S2, S3). Te intron numbers and the intron’s average length of plant subtilases were also calculated (Supplementary Table S5).

Phylogenetic tree construction. Multiple sequence alignments were performed by MAFFT using high accuracy method (parameters:–maxiterate 1000–localpair). We removed the sites having the gap ratio higher than 50%. Phylogenetic analyses were conducted by using the IQ-TREE sofware44 (model: LG + R10, WAG + F + R7, WAG + R8 and LG + F + R5 for phylogenetic trees from Fig. 1a, Fig. 1b, Fig. 2 and Fig. 3 respectively, all these models were predicted and selected by Modelfnder). Due to the large gene number of sequences in the S8A gene family tree, we used an ultrafast bootstrap approximation (UFBoot, parameter: -bb) to assess branch support45, UFBoot overcomes the computational burden required by the nonparametric bootstrap and is faster than the standard procedure providing relatively unbiased branch support values. Te reliability of diferent trees was assessed using diferent bootstrap replicates and except for these, all parameters were set as default. Unexpectedly long branches in all the trees were removed because they ofen refer to erroneous sequences.

Identification of conserved motifs. The local Multiple Em for Motif Elicitation (MEME, http:// meme-suite.org/) tool was used to identify conserved motifs. All SBT6.1 and SBT6.2 genes were analyzed in our study using the classical model. Te number of motifs MEME should fnd was set to 20.

Homology modeling and comparative structural analyses of plant subtilases. Five homolo- gous proteins were selected from Colwellia psychrerythraea, Mesotaenium endlicherianum, Physcomitrella patens, Oryza sativa, and Arabidopsis thaliana respectively. Te tertiary structure of these fve proteins were modelled by SWISS-MODEL (https://swissmodel.expasy.org/), which is a fully automated structure homology-modelling server. Comparative structural analysis of the homology modelled SBT2 protein was carried out using the CONSURF tool (http://consurf.tau.ac.il/). Finally, the protein superimposition was done by employing Biovia Discovery Studio (2017, R2).

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Data Availability Te sequences of S8A genes which we identifed from the green algae (Mesostigma viride, Chlorokybus atmophyticus, Klebsormidium nitens, Chara braunii, Coleochaete scutata, “Spirotaenia sp”., Mesotaenium endlicherianum) are available in the CNGB Nucleotide Sequence Archive (CNSA: http://db.cngb.org/cnsa; accession number CNP0000252). Te specifc details regarding other genes which were used in this study are available in Supple- mentary Table S3. References 1. Schaller, A. A cut above the rest: the regulatory function of plant proteases. Planta 220, 183–197 (2004). 2. Di Cera, E. Serine proteases. IUBMB life 61, 510–515 (2009). 3. Page, M. J. & Di Cera, E. Serine peptidases: classifcation, structure and function. Cellular and Molecular Life Sciences 65, 1220–1236 (2008). 4. Rawlings, N. D. et al. Te MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database. 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