Microbial-type terpene synthase genes occur widely innonseed land plants, but not in seed plantsQidong Jiaa, Guanglin Lib,c, Tobias G. Köllnerd, Jianyu Fub,e, Xinlu Chenb, Wangdan Xiongb, Barbara J. Crandall-Stotlerf,John L. Bowmang, David J. Westonh, Yong Zhangi, Li Cheni, Yinlong Xiei, Fay-Wei Lij, Carl J. Rothfelsj, Anders Larssonk,Sean W. Grahaml, Dennis W. Stevensonm, Gane Ka-Shu Wongi,n,o, Jonathan Gershenzond, and Feng Chena,b,1

aGraduate School of Genome Science and Technology, University of Tennessee, Knoxville, TN 37996; bDepartment of Plant Sciences, University of Tennessee,Knoxville, TN 37996; cCollege of Life Sciences, Shaanxi Normal University, Xian 710062, China; dDepartment of Biochemistry, Max Planck Institute forChemical Ecology, D-07745 Jena, Germany; eTea Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou 310008, China; fDepartment ofPlant Biology, Southern Illinois University, Carbondale IL 62901; gSchool of Biological Sciences, Monash University, Melbourne, VIC 3800, Australia;hBiosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831; iBeijing Genomics Institute-Shenzhen, Beishan Industrial Zone, Yantian District,Shenzhen 518083, China; jDepartment of Integrative Biology, University of California, Berkeley, CA 94720; kSystematic Biology, Uppsala University, 752 36Uppsala, Sweden; lDepartment of Botany, University of British Columbia, Vancouver, BC V6T 1Z4, Canada; mPlant Genomics Program, New York BotanicalGarden, Bronx, NY 10458; nDepartment of Biological Sciences, University of Alberta, Edmonton, AB T6G 2E9, Canada; and oDepartment of Medicine,University of Alberta, Edmonton AB T6G 2E1, Canada

Edited by Jerrold Meinwald, Cornell University, Ithaca, NY, and approved August 29, 2016 (received for review May 26, 2016)

The vast abundance of terpene natural products in nature is due toenzymes known as terpene synthases (TPSs) that convert acyclicprenyl diphosphate precursors into a multitude of cyclic and acycliccarbon skeletons. Yet the evolution of TPSs is not well understoodat higher levels of classification. Microbial TPSs from bacteria andfungi are only distantly related to typical plant TPSs, whereasgenes similar to microbial TPS genes have been recently identifiedin the lycophyte Selaginella moellendorffii. The goal of this studywas to investigate the distribution, evolution, and biochemicalfunctions of microbial terpene synthase-like (MTPSL) genes inother plants. By analyzing the transcriptomes of 1,103 plant spe-cies ranging from green algae to flowering plants, putative MTPSLgenes were identified predominantly from nonseed plants, includ-ing liverworts, mosses, hornworts, lycophytes, and monilophytes.Directed searching for MTPSL genes in the sequenced genomes of awide range of seed plants confirmed their general absence in thisgroup. Among themselves, MTPSL proteins from nonseed plantsform four major groups, with two of these more closely related tobacterial TPSs and the other two to fungal TPSs. Two of the fourgroups contain a canonical aspartate-rich “DDxxD”motif. The thirdgroup has a “DDxxxD” motif, and the fourth group has only thefirst two “DD” conserved in this motif. Upon heterologous expres-sion, representative members from each of the four groups dis-played diverse catalytic functions as monoterpene and sesquiterpenesynthases, suggesting these are important for terpene formation innonseed plants.

terpene synthase | specialized metabolism | nonseed plant | gene evolution

Terpenoids are the largest class of land plant secondary me-tabolites, but they are not uniformly distributed in the plant

kingdom (1). Many seed plants (angiosperms and gymnosperms)produce terpenoids of diverse types in large quantities. However,among nonseed plants, only liverworts are known as copiousproducers of terpenoids (2). Terpenoids have diverse biologicaland ecological functions with many serving as chemical defensesagainst herbivores and pathogens (3, 4). Some have lineage-specific functions, such as the volatile terpenoids in flowers thatare involved in attracting pollinators (5). Characterizing the bio-synthesis of terpenoids in all plant lineages is therefore an importantavenue to understanding their roles in the adaptation of variouslineages of terrestrial plants.Terpene synthases (TPSs) are pivotal enzymes for terpenoid

biosynthesis, forming a distinctive superfamily based on both se-quence identity and structure classification. However, within thisgroup, typical plant and microbial (bacterial and fungal) TPSs sharevery low sequence similarity and are therefore only distantly related(6). The typical plant TPSs form subfamilies with individual

subfamilies generally associated with specific biochemical func-tions, such as monoterpene, sesquiterpene, or diterpene biosyn-thesis (7, 8). Monoterpene synthases and sesquiterpene synthaseshave been proposed to have evolved independently in gymnospermsand angiosperms from diterpene synthase ancestors (7, 9). In-terestingly, the typical plant TPSs in the moss Physcomitrella patens(10) and the lycophyte Selaginella moellendorffii (11–13), twononseed plants, were found to be of the diterpene synthase type.Therefore, the molecular basis underlying the biosynthesis ofmonoterpenes and sesquiterpenes identified in nonseed plantshas long been unclear.Recently, microbial terpene synthase-like (MTPSL) genes were

identified in S. moellendorffii that encode monoterpene andsesquiterpene synthases (13). Unlike typical plant TPSs, whichare composed of either three domains (αβγ) or two domains(αβ) (14, 15), MTPSLs contain only an α-domain. Phylogeneticanalysis indicated that MTPSLs from S. moellendorffii are moreclosely related to microbial TPSs, in particular fungal TPSs,than to typical plant TPSs (13). So far, MTPSLs have only beenidentified in S. moellendorffii (13), raising intriguing questions

Significance

Terpenoids are ubiquitous products made by land plants withdiverse biological functions. Their formation in seed plants iscatalyzed by typical plant terpene synthases (TPSs), a well-characterized group of enzymes. In contrast, our knowledge ofterpenoid biosynthesis in nonseed plants is very limited. Bysystematically analyzing the transcriptomes and/or genomes ofmore than 1000 plant species, we report that microbial terpenesynthase-like genes, which are only distantly related to typicalplant TPS genes, are widely distributed in nonseed plants, butvirtually absent in seed plants. The study provides insights intothe evolution of TPS genes in early land plants and opens thedoor to investigating the diversity and functions of terpenoidsin nonseed plants.

Author contributions: Q.J., G.L., T.G.K., J.G., and F.C. designed research; Q.J., G.L., T.G.K.,J.F., X.C., W.X., and F.C. performed research; B.J.C.-S., J.L.B., D.J.W., Y.Z., L.C., Y.X., F.-W.L.,C.J.R., A.L., S.W.G., D.W.S., and G.K.-S.W. contributed new reagents/analytic tools; Q.J.,G.L., T.G.K., J.G., and F.C. analyzed data; and Q.J., T.G.K., J.G., and F.C. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The sequences for the biochemically characterized MTPSLs reported inthis paper have been deposited in the GenBank database (accession nos. KX230835–KX230843).1To whom correspondence should be addressed. Email: fengc@utk.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1607973113/-/DCSupplemental.

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about the origin, evolution, and function of this type of plantterpene synthase genes. The goal of this study was to investigatethe distribution of MTPSL genes in the green plants, infer theirevolution, and determine their biochemical functions.

Results and DiscussionTerpene Synthase Genes of the Microbial Type Are Widespread in theTranscriptomes of Nonseed Land Plants, but Not in Green Algae andSeed Plants. To determine the distribution ofMTPSL genes in thegreen plants, the transcriptomes of 1,103 species (SI Appendix,Table S1) of green plants (779 species of seed plants, 166 speciesof nonseed land plants, 47 species of charophytes, and 111 spe-cies of chlorophytes) generated from the 1,000 Plant (OneKP)initiative (https://sites.google.com/a/ualberta.ca/onekp/) weresearched for microbial type terpene synthase genes using aHMMER method as previously described (13). A total of 712MTPSL genes were identified from the transcriptomes of 146species. Strikingly, the vast majority of MTPSL genes (706 of the712MTPSL genes or 99.2%) were found in the transcriptomes ofnonseed land plants (Fig. 1).Bryophytes consist of three lineages: hornworts, mosses, and

liverworts, which have 7, 41, and 26 species in the OneKPdataset, respectively. The number of hornwort, moss, and liver-wort species whose transcriptomes contain MTPSL genes was 3,30, and 24, respectively. Among the 22 lycophyte species, 21possessed MTPSL genes in their transcriptomes. For mon-ilophytes, 65 of the 70 species were found to contain MTPSLgenes in their transcriptomes. The median number of MTPSLgenes from the transcriptome of each species for hornworts,mosses, liverworts, lycophytes, and monilophytes was 0, 1, 8, 3,and 4.5, respectively (Fig. 1 and SI Appendix, Table S2). Amongall species, the monilophyte Cystopteris utahensis (a tetraploid)was found to contain the most MTPSL genes with 20 members(SI Appendix, Table S1).On the other hand, extremely low occurrences of MTPSL

genes were found in the transcriptomes of seed plants and

charophytes. Among the 779 species of seed plants, only twospecies, Phytolacca bogotensis and Opuntia sp., both members ofthe Caryophyllales, were found to contain MTPSL genes in theirtranscriptomes with one and four members, respectively. Amongthe 47 species of charophytes, only one species, Micrasteriasfimbriata, contained a MTPSL gene (one member) in its tran-scriptome. NoMTPSL genes were found in the transcriptomes of111 species of chlorophytes.

The Majority of MTPSL Genes Identified in Plant TranscriptomesBelong to Four Groups Clustered with Either Fungal or BacterialTerpene Synthases. Phylogenetic analysis was performed for the712 MTPSL genes identified from plant transcriptomes togetherwith the 48 known MTPSL genes from S. moellendorffii (13) andselected terpene synthase genes from bacteria and fungi. Theresulting phylogenetic tree indicates that the distribution ofMTPSL genes in nonseed plants exhibits lineage-specific charac-teristics and that the majority of them (690 of 712) were clusteredinto four major groups with either similarity to bacterial TPS(groups I and II) or fungal TPS (groups III and IV) (Fig. 2).Group I, the second largest MTPSL gene group, contains

about 86% of MTPSL genes (152 of 177) from 23 species ofliverworts, 34% of MTPSL genes (27 of 79) from 10 speciesof mosses, and 28% ofMTPSL genes (23 of 83) from 9 species oflycophytes. Group II was composed of MTPSL genes primarilyfrom mosses (about 66% of MTPSL genes from 24 species) andhornworts (50% of MTPSL genes from all three species in whichMTPSL genes have been found). There was also one group IIMTPSL gene found in a liverwort species, Scapania nemorea.Members of MTPSL genes in this species were also present ingroups I and III. Group III, the smallest group, contains 4MTPSL genes from 3 species of hornworts and 14 MTPSL genesfrom 7 species of liverworts. These 18 genes were clustered withthe fungal trichodiene synthase (Tri5) genes. Group IV containsalmost all MTPSL genes found from 65 species of monilophytes(352 of 353) and about 70% ofMTPSL genes in lycophytes (58 of83). The knownMTPSL genes from S. moellendorffii were closelyrelated toMTPSL genes from the transcriptomes of other lycophytespecies.Twenty-two MTPSL genes that lie outside of the four major

groups were designated as “unclassified” (SI Appendix, TableS3). For example, the MTPSL genes found in two seed plantspecies, Opuntia sp. (four members) and P. bogotensis (onemember), and one green alga, M. fimbriata (one member), wereincluded in this list (Fig. 2 and SI Appendix, Table S3).

The Majority of MTPSL Genes Identified from Plant TranscriptomesAre Genuine Plant Genes. The putative MTPSL genes identified inthe plant transcriptomes could have one of two possible origins:from plants or from plant-associated microbes. Three lines ofevidence were used to judge that the vast majority, if not all, ofthese genes in the four major MTPSL clades are plant genes.The first line of evidence comes from the analysis of putative

MTPSL genes from axenic culture. The liverwort S. nemorea wasselected for this purpose. A total of eight putative MTPSL geneswere identified in the transcriptome of S. nemorea (SI Appendix,Table S4), belonging to group I (five genes), group II (one gene),group III (one gene), and unclassified (one gene). An axenicculture of S. nemorea was initiated by germinating isolatedspores, using sterile culture methods. This culture was thereforefree of contamination of endophytic microbes. We extractedgenomic DNA from axenically cultured S. nemorea and usedPCR to amplify DNA fragments for each of the eight putativeMTPSL genes, and the results were compared with thoseobtained from the transcriptome analysis. Six of the eight MTPSLgenes (five from group I and one from group III) were amplifiedand confirmed by sequencing. However, the amplification of themembers from group II and the unclassified group failed. The

Fig. 1. Distribution ofMTPSL genes identified from the transcriptomes of 1,103plant species. The numbers in parentheses represent the number of tran-scriptomes containing putative MTPSLs (in red) and total transcriptomes ana-lyzed in each lineage (in black). The phylogeny of green plants presented wasmodified from refs. 25 and 26. Each boxplot represents the number of MTPSLsfound for individual species in each plant lineage. The solid black lines denotethe median number of MTPSLs from each species. Whiskers represent 1.5 timesthe quantile of the data. Points outside of the range of the whiskers are outliers.

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member from the unclassified group was suggested to be a con-taminant from endophytic fungi (SI Appendix, Fig. S1). Analysis ofthe OneKP transcriptomes has suggested that S. nemorea wascontaminated with an unknown source of plant material (https://pods.iplantcollaborative.org/wiki/display/iptol/Sample+source+and+purity). The putative MTPSL from S. nemorea in group II is theonly MTPSL from liverworts to be assigned to this group, sug-gesting that it was also derived from contamination by other plantmaterial. Overall, this experimental study confirms that the groupI and group III MTPSLs from the S. nemorea transcriptome areendogenous S. nemorea genes.The second line of evidence for the plant origin of the majority

ofMTPSL genes reported is the identification of their immediategenomic neighbors as bona fide plant genes. We obtained suchevidence for group II and III genes of the hornwort Anthocerospunctatus, whose genome has been recently sequenced (13).Assembling the raw genome sequence and then searching forMTPSL genes using the HMMER search, seven putative full-length MTPSL genes were identified; two of the seven genes

belong to group II and the remaining five to group III (SI Ap-pendix, Table S5). ApMTPSL1 from group II and ApMTPSL2from group III were then selected as representatives for thefollowing study. In the assembled genome of A. punctatus,ApMTPSL1 is the immediate neighbor to a cytochrome P450gene, for which the most similar gene is from the plant Oryzabrachyantha (Fig. 3A). Similarly, ApMTPSL2 is the immediateneighbor to a leucine-rich receptor-like kinase gene, for whichthe most similar gene is from the plant Theobroma cacao (Fig.3B). We extracted genomic DNA from A. punctatus grown inaxenic culture and performed PCR to amplify the coding se-quence of ApMTPSL1 and -2 and their respective neighbors. Theamplified DNA fragment was cloned and fully sequenced con-firming that both ApMTPSL1 (Fig. 3A) and ApMTPSL2 genes(Fig. 3B) reside in the A. punctatus genome and are neighbors toa plant gene. Similar evidence for a group I gene was obtainedfrom the moss Sphagnum fallax. The sequenced S. fallax genomecontains 21 MTPSL genes, all of which belong to group I (SIAppendix, Table S6). A representativeMTPSL gene (SfMTPSL1)

Hornworts

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Fig. 2. Phylogeny of MTPSLs identified from OneKP with known MTPSLs from S. moellendorffii, bacterial TPSs, and fungal TPSs. Genes are color-coded basedon their source. The majority of the newly identified MTPSLs are clustered into four major groups (I–IV).

12330 | www.pnas.org/cgi/doi/10.1073/pnas.1607973113 Jia et al.

from S. fallax was confirmed to reside in its genome and neighborwith a Copia-like retrotransposable element, to which the mostsimilar sequence is from the plant Arabidopsis thaliana (Fig. 3C).The genes in group IV form a clade with SmMTPSL genes fromS. moellendorffii, which have already been determined to be plantgenes (13). This evidence that representative MTPSL genes ofgroups I–IV originated from plant genomes supports that theirapparent orthologs/homologs in each group are also of plant origin.The third line of evidence for plant origin is that the overall

evolutionary relationships of MTPSLs are largely congruent withthe relationships of the land plant species from which theMTPSL genes are identified. The MTPSL genes in each of thefour groups are inferred to share a common evolutionary origin(Fig. 2). Within each group, the MTPSLs from the same plantlineage showed higher sequence similarity than with MTPSLsfrom a different plant lineage. For instance, in group I theMTPSLs from three plant groups, liverworts, mosses, and lyco-phytes, form three distinct subclades (Fig. 2). The MTPSLs fromclosely related species are often also most closely related. Forinstance, the analyzed mosses included three species from thesame genus Sphagnum: Sphagnum lescurii, Sphagnum palustre,and Sphagnum recurvatum. In group I, the MTPSLs from thesethree species reside in a clade (Fig. 2). Such fine-scale correla-tions of MTPSL sequence similarity and plant phylogeny alsosupport a plant origin for most of these genes.On the other hand, the 22 unclassified MTPSL genes (SI

Appendix, Table S3) have a high probability of being derivedfrom plant-associated microbes because their similarities withmicrobial TPS genes are extremely high (SI Appendix, Table S3),as in the example of the S. nemorea gene mentioned above, andso were not considered further in this study. Nevertheless, someof these genes may have been obtained from microbes very re-cently through horizontal gene transfer (HGT), which will be asubject of future investigation.

MTPSL Genes Are Patchily Distributed in Green Plants: EvolutionaryImplications. The confirmation that the vast majority of MTPSLgenes identified from the OneKP transcriptomes are plant genesindicates that MTPSL genes occur widely in nonseed plants.Group I contains MTPSL genes from liverworts, mosses, and

lycophytes (Fig. 2), which implies the presence of MTPSL genesin the common ancestor of land plants. However, our surveyfound that MTPSL genes are generally absent from the tran-scriptomes of green algae (Fig. 1). To provide further evidenceon the presence/absence of MTPSL genes in green algae, weconducted a focused search on sequenced genomes for six spe-cies of chlorophytes and one species of charophyte (SI Appendix,Table S7). No MTPSL genes were detected in these sequencedgreen algae. The absence of MTPSL genes in the transcriptomesof a wide range of chlorophytes and the genome of the char-ophyte, Klebsormidium flaccidum (16) suggests that MTPSLgenes may have their origin in an ancestral land plant rather thanan algal ancestor. Broader genome sampling from green algae,especially charophytes, is needed to test this hypothesis. None-theless, the evolution of MTPSL genes may have been associatedwith the transition of plants from aquatic to terrestrial habitats.The pioneer land plants faced a harsh environment replete withmany new biotic and abiotic stresses. Many products of TPSs arevolatile hydrocarbons that may be more useful in a terrestrialenvironment than in an aquatic one.MTPSLs from nonseed land plants exhibited different degrees

of relatedness to bacterial TPSs and fungal TPSs. Group I ismost closely related to bacterial TPSs, whereas groups III and IVare most closely related to fungal TPSs. Group II is most closelyrelated to a number of bacterial TPSs, which, however, residewithin a fungal clade (Fig. 2). These patterns suggest a complexevolutionary history of microbial type TPSs. Whereas it is pos-sible that microbial-type TPS genes are ancestral in all kingdomsof life and took different evolutionary trajectories, their con-finement to bacteria, fungi, and plants implies HGT. Assumingthat bacterial and fungal TPS genes are ancestral toMTPSLs, thedistribution pattern of MTPSLs can be explained by multipleHGT events from bacteria and fungi. However, it is premature tomake strong claims about the donors and recipients of suchtransfer events because our understanding of phylogenetic rela-tionships of TPSs in bacteria and fungi is still very limited. Abetter understanding of relationships among TPS genes in bac-teria and fungi will allow testing of this hypothesis.The absence of MTPSL genes in seed plants is also notable

(Fig. 1). To gain additional evidence about the presence/absenceof MTPSL genes in seed plants, we analyzed the genomic se-quences of 48 species of seed plants (SI Appendix, Table S7): noMTPSL genes were identified in them. As mentioned previously,land plants contain typical plant TPSs, which catalyze similarbiochemical reactions for the production of terpenoids as doMTPSLs, but typical plant TPSs are only distantly related toMTPSLs (13). However, in the nonseed plants that have beenstudied, the typical plant TPSs function as diterpene synthases(7) rather than the full range of monoterpene synthases, ses-quiterpene synthases, and diterpene synthases found in seedplants. Several MTPSL genes from S. moellendorffii have beendemonstrated to encode monoterpene synthases and sesquiter-pene synthases (13), and we hypothesized that most MTPSL genesin nonseed plants function in this way.

Representative MTPSL Genes Encode Active TPSs with DiverseCatalytic Activities. The presence of MTPSL genes only in non-seed land plants poses an intriguing question about their func-tions. In seed plants, TPSs are responsible for the production ofa diversity of terpenoids important for ecological interactions,especially as defenses against herbivores and pathogens. Somenonseed plants, such as liverworts (17) and mosses (18), alsoproduce a vast diversity of terpenoids. However, little is knownabout how such terpenoids are synthesized or about their bio-logical functions.In general, TPSs contain two highly conserved motifs: the

DDxxD and NSD/DTE motifs, which are both involved in sub-strate binding (14, 15, 19). Whereas the NSD/DTE motif is

Fig. 3. Validation that representative MTPSL genes are of plant origin.Schematic genomic organization of representative MTPSL genes with theirneighboring genes is shown. The genomic region spanning each MTPSLgene, its neighboring gene, and the intergenic region were amplified usingPCR from genomic DNA and confirmed by sequencing. (A) ApMTPSL1 withits neighboring gene annotated as a cytochrome P450 71A1-like from thehornwort A. punctatus. (B) ApMTPSL2 and its neighboring gene annotatedas a leucine-rich receptor-like kinase family protein from the hornwortA. punctatus. (C) SfMTPSL1 with its neighboring gene annotated as a Copia-like retrotransposon element from the moss S. fallax. In each schematic figure,underneath the neighboring gene, the ID in parentheses represents the ac-cession number of the best hit of this gene by searching the nonredundantdatabase of the National Center for Biotechnology Information. The plantspecies indicates from which species the best hit was identified.

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highly conserved in the MTPSLs, the aspartate-rich DDxxDmotif exhibits variations (SI Appendix, Fig. S2). Group I and IVproteins contain the canonical DDxxD motif, but group II pro-teins displayed a conserved DDxxxD motif. In the group IIIproteins, only the first two aspartates (DD) are conserved.To gain an initial assessment of the biochemical functions

of MTPSLs, a total of nine genes representing the four MTPSLgroups (SI Appendix, Table S8) were selected for experimentalwork. Recombinant MTPSLs produced from Escherichia coli weretested for TPS activity with geranyl diphosphate [(E)-GPP], far-nesyl diphosphate [(E,E)-FPP], and geranylgeranyl diphosphate[(E,E,E)-GGPP], the substrates for monoterpenes, sesquiterpenes,and diterpenes, respectively. With the exception of Mos-GOWD-MTPSL2, all tested enzymes were able to convert (E,E)-FPP intoindividual sesquiterpenes or complex sesquiterpene mixtures (Fig.4). Whereas most of the MTPSL sesquiterpene products are alsoknown as products from typical plant TPSs, Mon-UJTT-MTPSL4and Mon-YJJY-MTPSL1, which are closely related to fungal TPS,produced protoillud-6-ene, a sesquiterpene that has only beenreported from a fungus so far (20). Mos-GOWD-MTPSL2 showedexclusively monoterpene synthase activity and converted (E)-GPPinto (Z)-β-ocimene and some minor monoterpene products (SIAppendix, Fig. S3). In addition to sesquiterpene synthase activity,Liv-IRBN-MTPSL2, Mon-UJTT-MTPSL4, and Hon-Ap-MTPSL7were able to produce monoterpenes from (E)-GPP and Liv-IRBN-

MTPSL4 and Mos-VBMM-MTPSL3 were able to convert (E,E,E)-GGPP into diterpenes (SI Appendix, Fig. S4). Recently, it wasshown that certain typical plant TPSs naturally use cis-prenyl di-phosphates as substrates for terpene production (21). Because mostof the representative MTPSLs tested showed sesquiterpene syn-thase activity using (E,E)-FPP (Fig. 4), we performed additionalassays to determine whether (Z,E)-FPP and (Z,Z)-FPP, the cis-isomers of FPP, could serve as substrates for these enzymes. Mos-GOWD-MTPSL2 was inactive with either substrate, whereas all ofthe other eight characterized MTPSLs showed activity with either(Z,E)-FPP or (Z,Z)-FPP or both (SI Appendix, Fig. S5). It is in-teresting to observe that some enzymes, such as Mos-QKQO-MTPSL3, produced the same products using the cis-FPP isomers aswith (E,E)-FPP, whereas others such as Mon-UJTT-MTPSL4 pro-duced different products (Fig. 4 and SI Appendix, Fig. S5). Never-theless, the fact that the Myriopteris eatonii UJTT-MTPSL4 forms asingle distinctive product from (E,E)-FPP and broad mixtures of over10 products from each of the cis-FPP isomers suggests that (E,E)-FPP is the natural substrate. However, more information about theactual occurrence of these assay products in M. eatonii is neededbefore the natural substrates of these enzymes can be determinedwith certainty.The substrates actually used by seed plant TPSs depend on

their subcellular locations because the various prenyl diphosphatesubstrates are restricted to different subcellular compartments

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Group II (DDxxxD) Group III (DDxxx)

22

Fig. 4. Representative MTPSL genes from four groups (I–IV) encode active TPSs. The aspartate-rich motif associated with each group is indicated. MTPSLgenes were heterologously expressed in E. coli and crude protein extracts were incubated with the potential substrate farnesyl diphosphate [(E,E)-FPP].Enzyme products were collected using solid-phase microextraction and analyzed with gas chromatography/mass spectrometry. 1, bicycloelemene; 2, bicy-clogermacrene; 3, α-isocomene; 4, β-elemene*; 5, (E)-β-caryophyllene*; 6, (E)-β-farnesene*; 7, nerolidol*; 8, dactylol; 9, γ-curcumene; 10, α-zingiberene; 11,β-bisabolene*; 12, β-curcumene; 13, sesquiphellandrene*; 14, (E)-α-bisabolene; 15, (Z,E)-α-farnesene; 16, (E,E)-α-farnesene; 17, protoillud-6-ene*; 18, (Z)-γ-bisabolene; 19, (E)-γ-bisabolene; 20, β-bisabolol; 21, α-bisabolol; and 22, β-acoradiene. Compounds marked with an asterisk (*) were identified using authenticstandards. The origin of each MTPSL gene is listed in SI Appendix, Table S8. Ap, A. punctatus; GOWD, S. lescurii; GSXD, M. eatonii; Hon, hornwort; IRBN,S. nemorea; Liv, liverwort; Mon, monilophyte; Mos, moss; QKQO, Pseudotaxiphyllum elegans; UJTT, Pityrogramma trifoliate; VBMM, Anomodon rostratus;and YJJY, Woodsia scopulina.

12332 | www.pnas.org/cgi/doi/10.1073/pnas.1607973113 Jia et al.

(GPP and GGPP to plastids and FPP to the cytosol) (22). Thus,in seed plants, mono- and diterpene synthases are present inplastids and sesquiterpene synthases in the cytosol. However,information on TPS enzyme and substrate localization is not yetavailable for nonseed plants. To learn more, we used an in silicoprotein-targeting program (Target P, www.cbs.dtu.dk/services/TargetP) for Mon-UJTT-MTPSL4, an enzyme that produced asingle sesquiterpene from (E,E)-FPP but also formed monoter-penes from (E)-GPP. Because the program suggests a cytosolic,nonplastid location for Mon-UJTT-MTPSL4, and FPP is knownto be cytosolic in seed plants, this enzyme is likely to act as asesquiterpene synthase in planta. Under steady-state conditions,the apparent KM and kcat values of Mon-UJTT-MTPSL4 using(E,E)-FPP as substrate were determined to be 2.13 ± 0.23 μMand 0.15·s−1, respectively. Such kinetic parameters are very com-parable to those of typical plant TPSs (23), suggesting that MTPSLenzymes function in almost the same way as typical plant TPSs.Based on the in vitro biochemical activities of representative

MTPSLs, one could speculate that the MTPSLs have been theprimary enzymes to make mono- and sesquiterpenes in earlyland plants and that the evolution of monoterpene and sesqui-terpene synthases among the typical plant TPS family allowedthe eventual loss of MTPSL genes in seed plants.

ConclusionsIn this study, microbial-type terpene synthase genes, once thoughtto be confined to bacteria and fungi, were systematically minedfrom large-scale plant transcriptomes. Of 779 seed plant species,only 5MTPSL genes were found in 2 species, whereas 706MTPSLgenes were found in 143 nonseed land plant species. So, in addi-tion to the previous report on S. moellendorffii (13),MTPSL genesare widely distributed in nonseed land plants, but generally absentfrom seed plants and green algae. Although these genes are alsofound in fungi and bacteria, their occurrence in plants is in mostcases not due to microbial contamination of the plant samplesused for sequencing, based on experiments with axenic cultures,

phylogenetic analyses, and their embedment in plant genomeswith bona fide plant genes as neighbors. MTPSL genes form fourlineage-specific groups that exhibit diverse structural features, whichimplies multiple evolutionary origins. Biochemical studies of se-lected MTPSL genes showed that they encode sesquiterpene andmonoterpene synthases. However, much more remains to be doneto investigate the biological functions of their products and howthey have influenced the evolution of the MTPSL gene family innonseed land plants.

Materials and MethodsMTPSL genes were searched against the assembled transcriptomes for1,103 nonmodel plant species derived from the OneKP (sites.google.com/a/ualberta.ca/onekp/) (24). Fresh materials of three axenically cultured plants,A. punctatus, S. nemorea, and S. fallax, were used for the extraction of ge-nomic DNA that served as template for PCR analysis. For TPS activity assays,crude proteins extracted from E. coli expressing individual representativeMTPSL genes were assayed with individual prenyl diphosphates. The kineticproperties of Mon-UJTT-MTPSL4 were measured with its purified recombi-nant enzyme following a radiochemical protocol as previously described (23).Details on transcriptome and genome assembly, sequence searches, phylo-genetic reconstruction, plant cultures, and biochemical analysis of MTPSLsare provided in SI Appendix.

ACKNOWLEDGMENTS. We acknowledge the contributions of Dr. JamesLeebens-Mack’s laboratory at the University of Georgia in obtaining mate-rial for RNA sequencing and developing the infrastructure for distributionof the OneKP data. We thank Natascha Rauch (Max Planck Institute forChemical Ecology) for technical assistance. This project was supported by aUniversity of Tennessee (UT) AgResearch Innovation Grant (to F.C.), the MaxPlanck Society (to T.G.K. and J.G.), and the Program for New Century Excel-lent Talents in University (NCET-12-0896 to G.L.). The OneKP initiative led byG.K.-S.W. was funded by the Alberta Ministry of Advanced Education,Alberta Innovates Technology Futures, Innovates Centres of Research Excel-lence, Musea Ventures, and the China National Genebank. The BGI-Shenzhengroup led by Y.Z. was funded by the Shenzhen Supporting Projects Programunder Grant CXZZ20140421112021913. S. fallax plant and genomic resourceswere supported by the Laboratory-Directed Research and Development Pro-gram of Oak Ridge National Laboratory, managed by UT–Battelle MemorialInstitute, for the US Department of Energy under contract DE-AC05-000R22725.

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Jia et al. PNAS | October 25, 2016 | vol. 113 | no. 43 | 12333

PLANTBIOLO

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Supplementary Information

Microbial-type terpene synthase genes occur widely in nonseed land plants, but not

in seed plants

Qidong Jia, Guanglin Li, Tobias G. Köllner, Jianyu Fu, Xinlu Chen, Wangdan Xiong, Barbara Crandall-Stotler, John L. Bowman, David J. Weston, Yong Zhang, Li Chen, Yinlong Xie, Fay-Wei Li, Carl J Rothfels, Anders Larsson, Sean W. Graham, Dennis W. Stevenson, Gane Ka-Shu Wong, Jonathan Gershenzon, and Feng Chen

Materials and Methods

Identification of terpene synthases of the microbial type from transcriptomes and

sequenced genomes

The One Thousand Plants Project (OneKP; https://sites.google.com/a/ualberta.ca/onekp/)

has sequenced transcriptomes for over 1000 non-model plant species spanning almost all

major plant clades (from green algae to flowering plants) (1). All transcriptomes were

pre-assembled with the SOAPdenovo-Trans (2) assembler. Transcriptomes of 1103

representative species (Table S1) were analyzed in this study. For all the assembled

contigs, the longest regions without stop codons were annotated and translated using the

getorf program from the EMBOSS package (3) with a minimum length of 150 amino

acids. The resulting peptides were searched against the Pfam-A database locally using

HMMER 3.0 hmmsearch (4) with an E-value of 1e-5. Only sequences with best hits from

the following four HMM profiles were considered as putative terpene synthases:

Terpene_synth_C (PF03936) and Terpene synthase N-terminal domain (PF01397), TRI5

(PF06330) and SmMTPSLs (a profile created by using 48 microbial type TPSs identified

from S. moellendorffii). For sequences from the same species that had 100% identity,

only the longest one was retained, to reduce redundancy. All the putative TPS sequences

were subjected to a BLASTP search against the NCBI's non-redundant database using

default parameters. A TPS was annotated as “Microbial TPS-like protein” (MTPSL) if all

the top ten best hits were from bacteria and/or fungi or identical/highly similar to

SmMTPSLs.

Assembly of hornwort Anthoceros punctatus genome and identification of MTPSL

genes

For Anthoceros punctatus, the Illumina paired-end whole genome sequencing data

(access number: SRR1278954) (5) were retrieved from NCBI's Sequence Read Archive

(SRA) database. The reads were assembled using SPAdes-3.1.1 (6) and the resulting

contigs and singletons were further assembled by CAP3 (7). The final CAP3 assembly

contains 34448 sequences (16272 contigs and 18176 singletons) a total length of 97Mb,

of which 15596 sequences have a minimum length of 500 bp. The N50 contig length

based on these 15596 sequences is 12,462 bp. The assemblies were searched for

occurrences of terpene synthases using homology-based methods and ab initio predictors.

A TBLASTN search was performed with an E-value cutoff of 1e-30 using the 716 MTPSL

genes identified from OneKP transcriptomes. We also ran SNAP (8) trained for

Arabidopsis thaliana on the assembly. The resulting protein sequences of predicted genes

were subsequently subjected to a HMMER search against four HMM profiles (PF03936,

PF01397, PF06330 and SmMTPSLs generated by using 48 microbial type TPSs from S.

moellendorffii).

Phylogenetic analyses of terpene synthases.

Bacterial and fungal terpene synthases were obtained from Pfam (version 27.0).

Considering that certain MTPSLs from plants may contain a transit peptide that is absent

in bacterial and fungal TPSs and certain fungal TPSs contain an extended N-terminal

domain, to reduce ambiguities in sequence alignment, only the terpene synthase C

terminal domains were included. Sequences were aligned using MAFFT (linsi) (9) with

1000 iterations of improvement. ProtTest (10) was used to select the most appropriate

protein evolution model for the protein alignment under the Akaike Information Criterion.

For the maximum likelihood analyses, we used RAxML (11) with 1000 bootstrap

replicates under the best substitution model (LG+G+F) selected by ProtTest.

Plant material, genomic DNA isolation and PCR

Scapania nemorea, Anthoceros punctatus and Sphagnum fallax were cultured axenically

in Hatcher’s medium (12), Knop medium (13) and BCD medium (14), respectively.

Genomic DNA from each species was isolated using the VIOGENE plant genomic DNA

isolation kit (Viogene BioTek Corp., Taiwan) and used for PCR with primers listed in

Table S9. PCR products were cloned into the pGEM®-T Easy Vector (Promega, USA)

and fully sequenced.

Reagents

(E)-GPP, (E,E)-FPP, (Z,Z)-FPP and (E,E,E)-GGPP were purchased from Echelon

Biosciences (Salt Lake City). (Z,E)-FPP was kindly provided by Nathalie Gatto and

Wilhelm Boland from Max Planck Institute for Chemical Ecology, Jena, Germany. [1-3H](E,E)-FPP was a product of American Radiolabeled Chemicals (St. Louis).

Terpene synthase enzyme assays and kinetic measurements

Representative members from each of the four groups of MTPSL genes were selected for

gene synthesis. The synthesized cDNAs were cloned into a protein expression vector

pEXP5/CT-TOPO (Thermo Fisher Scientific, USA). Protein expression in E. coli and

terpene synthase enzyme assays were performed as previously described (15). To

determine the kinetic properties of Mon-UJTT-MTPSL4, its cDNA was first amplified

via PCR using a pair of primers listed in Table S9. The PCR product was cloned into

pET32a, in which the Mon-UJTT-MTPSL4 coding sequence was fused to the his-tag

coding sequence at its N-terminal. E. coli-expressed recombinant Mon-UJTT-MTPSL4

was purified through the his-tag using the HisTrap HP column (GE Lifesciences, USA).

The purified Mon-UJTT-MTPSL4 was used for kinetic measurements using [1-3H](E,E)-

FPP as substrate via a radiochemical method as previously described (16).

References

1. Matasci N, et al. (2014) Data access for the 1,000 Plants (1KP) project. Gigascience 3:17.

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6. Bankevich A, et al. (2012) SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19(5):455-477.

7. Huang X, Madan A (1999) CAP3: A DNA sequence assembly program. Genome Res 9(9):868-877.

8. Korf I (2004) Gene finding in novel genomes. BMC Bioinformatics 5:59. 9. Katoh K, Misawa K, Kuma K, Miyata T (2002) MAFFT: a novel method for

rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res 30(14):3059-3066.

10. Darriba D, Taboada GL, Doallo R, Posada D (2011) ProtTest 3: fast selection of best-fit models of protein evolution. Bioinformatics 27(8):1164-1165.

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12. Hatcher RE (1965) Towards the establishment of a pure culture collection of hepaticae. The Bryologist 68(2):227-231.

13. Reski R, Abel WO (1985) Induction of budding on chloronemata and caulonemata of the moss, Physcomitrella patens, using isopentenyladenine. Planta 165(3):354-358.

14. Ashton NW, Cove DJ (1977) The isolation and preliminary characterisation of auxotrophic and analogue resistant mutants of the moss, Physcomitrella patens. Mol Gen Genet 154(1):87-95.

15. Li G, et al. (2012) Nonseed plant Selaginella moellendorfii has both seed plant and microbial types of terpene synthases. Proc Natl Acad Sci USA 109(36):14711-14715.

16. Tholl D, Chen F, Petri J, Gershenzon J, Pichersky E (2005) Two sesquiterpene synthases are responsible for the complex mixture of sesquiterpenes emitted from Arabidopsis flowers. Plant J 42(5):757-771.

17. Hanssen HP, Sprecher E, Abraham WR (1986) 6-Protoilludene, the major molatile metabolite from ceratocystis piceae liquid cultures. Phytochemistry 25(8):1979-1980.

TableS1.ListofscreenedplantspeciesandthenumberofMTPSLs ineachspecies.Allthese1103transcriptomeswerefrom1KP(https://sites.google.com/a/ualberta.ca/onekp/).MTPSLsarefoundfrom146species.

353 77 1Adiantumaleuticum 5 Dendrolycopodiumobscurum 2 Bambusinaborreri 0 Alliumcommutatum 0 Daphnegeraldii 0Adiantumtenerum 8 Diphasiastrumdigitatum 0 Chaetosphaeridiumglobosum 0 Alliumsativum 0 Daphniphyllummacropodum 0Anemiatomentosa 12 Huperzialucidula 1 Charavulgaris 0 Alnusserrulata 0 Daturametel 0Angiopterisevecta 5 Huperziamyrisinites 2 Chlorokybusatmophyticus 0 Aloevera 0 Delospermaechinatum 0Argyrochosmanivea 10 Huperziaselago 7 Closteriumlunula 0 Alternantherabrasileana 0 Dendropemoncaribaeus 0Aspleniumnidus 6 Huperziasquarrosa 2 Coleochaeteirregularis 0 Alternantheracaracasana 0 Deschampsiacespitosa 0Aspleniumplatyneuron 7 Isoetessp. 6 Coleochaetescutata 0 Alternantherasessilis 0 Desmanthusillinoensis 0Athyriumfilixfemina 2 Isoetessp. 7 Cosmariumbroomei 0 Alternantheratenella 0 Deutziascabra 0Azollacf.caroliniana 5 Isoetestegetiformans 5 Cosmariumgranatum 0 Amaranthuscruentus 0 Dianthussp. 0Blechnumspicant 15 Lycopodiellaapressa 1 Cosmariumochthodes 0 Amaranthuspalmeri 0 Dichroafebrifuga 0Botrypusvirginianus 6 Lycopodiumannotinum 1 Cosmariumsubtumidum 0 Amaranthusretroflexus 0 Digitalispurpurea 0Cheilanthesarizonica 7 Lycopodiumdeuterodensum 4 Cosmariumtinctum 0 Amaryllisbelladonna 0 Dilleniaindica 0Cibotiumglaucum 5 Phylloglossumdrummondii 4 Cosmocladiumcf.constrictum 0 Amborellatrichopoda 0 Dioscoreavillosa 0Crepidomanesvenosum 0 Pseudolycopodiellacaroliniana 3 Cylindrocystisbrebissonii 0 Amelanchiercanadensis 0 Diospyrosmalabarica 0Cryptogrammaacrostichoides 3 Selaginellaacanthonota 3 Cylindrocystiscushleckae 0 Ancistrocladustectorius 0 Dipsacussativum 0Culcitamacrocarpa 2 Selaginellaapoda 2 Cylindrocystissp. 0 Anemonehupenhensis 0 Disporopsispernyi 0Cyatheaspinulosa 1 Selaginellakraussiana 8 Desmidiumaptogonum 0 Anemonepulsatilla 0 Dombeyaburgessiae 0Cystopterisfragilis 8 Selaginellalepidophylla 10 Entransiafimbriat 0 Angelicaarchangelica 0 Drabaaizoides 0Cystopterisprotrusa 5 Selaginellaselaginoides 2 Euastrumaffine 0 Anisacanthusquadrifidas 0 Drabahispida 0Cystopterisreevesiana 1 Selaginellastauntoniana 2 Gonatozygonkinahanii 0 Annonamuricata 0 Drabamagellanica 0Cystopterisutahensis 20 Selaginellawallacei 6 Interfilumparadoxum 0 Anthemistinctoria 0 Drabaoligosperma 0Danaeasp. 7 Selaginellawilldenowii 5 Klebsormidiumsubtile 0 Anthirrhinummajus 0 Drabaossetica 0Davalliafejeensis 8 Mesostigmaviride 0 Anticharisglandulosa 0 Drabasachalinensis 0Dennstaedtia 3 0 Mesotaeniumbraunii 0 Antirrhinumbraun 0 Drakeaelastica 0Deparialobato 8 Acrosiphoniasp. 0 Mesotaeniumcaldariorum 0 Aphanopetalumresinosum 0 Drimiaaltissima 0Didymochlaenatruncatula 2 Ankistrodesmussp. 0 Mesotaeniumendlicherianum 0 Apiosamericana 0 Drimyswinteri 0Diplaziumwichurae 6 Aphanochaeterepens 0 Mesotaeniumkramstei 0 Apocynumandrosaemifolium 0 Dryasoctopetala 0Dipterisconjugata 5 Asteromonasgracilis 0 Micrasteriasfimbriata 1 Arabisalpina 0 Drypetesdeplanchei 0Equisetumdiffusum 2 Bathycoccusprasinos 0 Mougeotiasp. 0 Ardisiahumilis 0 Edgeworthiapapyrifera 0Equisetumhymale 0 Blastophysacf.rhizopus 0 Netriumdigitus 0 Ardisiarevoluta 0 Ehretiaacuminata 0Gymnocarpiumdryopteris 6 Bolbocoleonpiliferum 0 Nucleotaeniumeifelense 0 Argemonemexicana 0 Elaeagnuspungens 0Hemionitisarifolia 3 Botryococcusbraunii 0 Onychonemalaeve 0 Aristidastricta 0 Elaeocarpussylvestris 0Homalosoruspycnocarpos 6 Botryococcussudeticus 0 Peniumexiguum 0 Aristolochiaelegans 0 Eleusinecoracana 0Hymenophyllumbivalve 4 Botryococcusterribilis 0 Peniummargaritaceum 0 Aruncusdioicus 0 Epifagusvirginiana 0Hymenophyllumcupressiforme 1 Brachiomonassubmarina 0 Phymatodocisnordstedtiana 0 Ascarinarubricaulis 0 Epilobiumsp. 0Leucostegiaimmersa 8 Bryopsisplumosa 0 Planotaeniumohtanii 0 Asclepiacurassavica 0 Erigeronspeciosus 0Lindsaealinearis 7 Carteriacrucifera 0 Pleurotaeniumtrabecul 0 Asclepiassyriaca 0 Eriospermumlancifolia 0Lindsaeamicrophylla 2 Carteriaobtusa 0 Royaobtusa 0 Asparagusdensiflorus 0 Erythroxylumcoca 0Lygodiumjaponicum 5 Cephaleurosvirescens 0 Spirogyrasp. 0 Astertataricus 0 Escalloniarubra 0Marattiasp. 3 Chaetopeltisorbicularis 0 Spirotaeniaminuta 0 Astilbechinensis 0 Escalloniasp.cv.Newport 0Myriopteriseatonii 11 Chlamydomonasbilatus 0 Spirotaeniasp. 0 Astragalusmembranaceus 0 Eschscholziacalifornica 0Nephrolepisexaltata 13 Chlamydomonascribrum 0 Staurastrumsebaldi 0 Astragaluspropinquus 0 Eucalyptusleucoxylon 0Notholaenamontieliae 4 Chlamydomonasmoewusii 0 Staurodesmusconvergens 0 Atriplexhortensis 0 Eucommiaulmoides 0Onocleasensibilis 1 Chlamydomonasnoctigama 0 Staurodesmusomearii 0 Atriplexprostrata 0 Euphorbiapekinensis 0Ophioglossumvulgatum 7 Chlamydomonassp. 0 Xanthidiumantilopaeum 0 Atriplexrosea 0 Eupomatiabennettii 0Osmundajavanica 2 Chlorellaminutissima 0 Zygnemasp. 0 Atropabelladonna 0 Eupteleapleiosperma 0Osmundaregalis 0 Chloromonasoogama 0 Zygnemopsissp. 0 Aucubajaponica 0 Exacumaffine 0Osmundasp. 4 Chloromonasperforata 0 Austrobaileyascandens 0 Exocarposcupressiformis 0Osmundastrumcinnamomeum 0 Chloromonasreticulata 0 0 Avenafatua 0 Fagussylvatica 0Pilulariaglobulifera 1 Chloromonasrosa 0 Abieslasiocarpa 0 Azadirachtaindica 0 Ficusreligiosa 0Pityrogrammatrifoliata 7 Chloromonassubdivisa 0 Acmopylepancheri 0 Bacopacaroliniana 0 Flaveriaangustifolia 0Plagiogyriajaponica 2 Chloromonastughillensi 0 Agathisrobusta 0 Balanophorafungosa 0 Flaveriabidentis 0Pleopeltispolypodioides 9 Chlorosarcinopsishalophila 0 Amentotaxusargotaenia 0 Basellaalba 0 Flaveriabrownii 0Polypodiumamorphum 9 Cladophoraglomerata 0 Araucariarulei 0 Batismaritima 0 Flaveriacronquestii 0Polypodiumglycyrrhiza 7 coccoidprasinophyte 0 Arucariasp. 0 Bauhiniatomentosa 0 Flaveriakochiana 0Polypodiumhesperium 4 Coccomyxapringsheimii 0 Athrotaxiscupressoides 0 Begoniasp. 0 Flaveriapalmeri 0Polypodiumplectolens 3 Codiumfragile 0 Austrocedruschilensis 0 Berberidopsisbeckleri 0 Flaveriapringlei 0Polystichumacrostichoides 5 Cylindrocapsageminella 0 Austrotaxusspicata 0 Bergeniasp. 0 Flaveriapubescens 0Psilotumnudum 2 Cymbomonassp. 0 Callitrisgracilis 0 Betamaritima 0 Flaveriasonorensis 0Pterisensigormis 2 Dolichomastixtenuilepi 0 Callitrismacleayana 0 Betulapendula 0 Flaveriatrinervia 0Pterisvittata 10 Dunaliellaprimolecta 0 Calocedrusdecurrens 0 Bischofiajavanica 0 Flaveriavaginata 0Sceptridiumdissectum 4 Dunaliellasalina 0 Cathayaagryrophylla 0 Bituminariabituminosa 0 Forestierasegregata 0Sticheruslobatus 0 Dunaliellatertiolecta 0 Cedruslibani 0 Bixaorellana 0 Fouqueriamacdougalli 0Thelypterisacuminata 3 Entocladiaendozoica 0 Cephalotaxusharringtonia 0 Blutoparonvermiculare 0 Francoaappendiculata 0Thyrsopteriselegans 3 Eremosphaeraviridi 0 Chamaecyparislawsoniana 0 Boehmerianivea 0 Frankenialaevis 0Tmesipterisparva 4 Eudorinaelegans 0 Cryptomeriajaponica 0 Boerhaviacf.spiderwort 0 Freycinetiamultiflora 0Vittariaappalachiana 1 Fritschiellatuberosa 0 Cunninghamialanceolata 0 Boerhaviadominnii 0 Galaxurceolata 0Vittarialineata 3 Geminellasp. 0 Cupressusdupreziana 0 Boryasphaerocephala 0 Galiumboreale 0Woodsiailvensis 3 Golenkinialongispicula 0 Cycasmicholitzii 0 Boswelliasacra 0 Galphimiagracilis 0Woodsiascopulina 10 Goniumpectorale 0 Dacrycarpuscompactus 0 Bougainvilleaspectabilis 0 Garcinialivingstonei 0

Haematococcuspluviali 0 Dacrydiumbalansae 0 Boykiniajamesii 0 Garciniaoblongiflolia 0183 Haematococcuspluvialis 0 Dioonedule 0 Brassicanigra 0 Gelsemiumsempervirens 0

Barbilophoziabarbata 11 Hafniomonasreticulata 0 Diselmaarcheri 0 Brocchiniareducta 0 Gentianaacaulis 0Bazzaniatrilobata 0 Halochlorococcummarinum 0 Encephalartosbarteri 0 Brodiaeasierrae 0 Geraniumcarolinianum 0Blasiasp. 1 Helicodictyonplanctonicum 0 Ephedrasinica 0 Brugmansiasanguinea 0 Geraniummaculatum 0Calypogeiafissa 4 Heterochlamydomonasinaequalis 0 Falcatifoliumtaxoides 0 Buddlejalindleyana 0 Geumquellyon 0Conocephalumconicum 9 Hormidiellasp. 0 Fokieniahodginsii 0 Buddlejasp. 0 Gleditsiasinensis 0Frullania 8 Ignatiustetrasporus 0 Ginkgobiloba 0 Burserasimaruba 0 Gleditsiatriacanthos 0Lejeuneaceaesp. 14 Leptosiraobovata 0 Glyptostrobuspensilis 0 Buxussempervirens 0 Gloriosasuperba 0Lunulariacruciata 9 Lobochlamyssegnis 0 Gnetummontanum 0 Byblisgigantea 0 Glycinesoja 0Marchantiaemarginata 4 Lobomonasrostrata 0 Halocarpusbidwillii 0 Caiophorachuquitensis 0 Glycyrrhizaglabra 0Marchantiapaleacea 8 Mantoniellasquamata 0 Juniperusscopulorum 0 Calceolariapinifolia 0 Glycyrrhizalepidota 0Marchantiapolymorpha 7 Microsporacf.tumidula 0 Keteleeriaevelyniana 0 Calycanthusfloridus 0 Gomortegakeule 0Metzgeriacrassipilis 1 Microthamnionkuetzigianum 0 Lagarostrobosfranklinii 0 Camptothecaacuminata 0 Gompholobiumpolymorphum 0Monocleagottschei 0 Monomastixopisthostigma 0 Larixspeciosa 0 Canellawinterana 0 Goodyerapubescens 0Odontoschismaprostratum 10 Nannochlorisatomus 0 Manoaocolensoi 0 Cannasp. 0 Grevillearobusta 0Pallavicinialyellii 4 Neochlorisoleoabundans 0 Metasequoiaglyptostroboides 0 Cannabissativa 0 Greyiasutherlandii 0Pelliacf.Epiphylla 9 Neochlorissp. 0 Microbiotadecussata 0 Capnoidessempervirens 0 Griselinialittoralis 0Pellianeesiana 16 Neochlorosarcinasp. 0 Microcachrystetragona 0 Carthamuslanatus 0 Griseliniaracemosa 0Plagiochilaasplenioides 9 Nephroselmisolivace 0 Microstrobosfitzgeraldii 0 Caryaglabra 0 Gunneramanicata 0Porellanavicularis 8 Nephroselmispyriformis 0 Nageianagi 0 Cassythafiliformis 0 Gylcinesoja 0Porellapinnata 8 Ochlochaetesp. 0 Neocallitropsispancheri 0 Castaneacrenata 0 Gymnocladusdioicus 0Ptilidiumpulcherrimum 11 Oedogoniumcardiacu 0 Nothotsugalongibracteata 0 Castaneapumila 0 Gyrocarpusamericanus 0Radulalindenbergia 8 Oedogoniumfoveolatum 0 Papuacedruspapuana 0 Casuarinaglauca 0 Gyrostemonramulosus 0Ricciaberychiana 4 Oltmannsiellopsisviridis 0 Parasitaxususta 0 Catharanthusroseus 0 Haemariadiscolor 0Scapanianemorea 8 Oogamochlamysgigantea 0 Phyllocladushypohyllus 0 Cavendishiacuatrecasasii 0 Hakeadrupaceae 0Schistochilasp. 3 Pandorinamorum 0 Piceaengelmanii 0 Celsiaarcturus 0 Hakeaprostrata 0Sphaerocarpostexanus 3 Parachlorellakessleri 0 Pilgerodendronuviferum 0 Celtisoccidentalis 0 Hamamelisvirginiana 0

Pediastrumduplex 0 Pinusjeffreyi 0 Centellaasiatica 0 Hederahelix 079 Pedinomonasminor 0 Pinusparviflora 0 Cephalotusfollicularis 0 Heleniumautumnale 0

Andreaearupestris 0 Pedinomonastuberculata 0 Pinusponderosa 0 Ceratocapnosvesicaria 0 Heliconiasp. 0Anomodonattenuatus 1 Persursariapercursa 0 Pinusradiata 0 Ceratophyllumdemersum 0 Heliotropiumcalcicola 0Anomodonrostratus 4 Phacotuslenticularis 0 Platycladusorientalis 0 Cercidiphyllumjaponicum 0 Heliotropiumconvolvulaceum 0Atrichumangustatum 1 Picocystissalinarum 0 Podocarpuscoriaceus 0 Cerciscanadensis 0 Heliotropiumfiliforme 0Aulacomniumheterostichum 1 Pirulasalina 0 Podocarpusrubens 0 Cercocarpusledifolius 0 Heliotropiumgreggii 0Bryumargenteum 0 Planophilalaetevirens 0 Prumnopitysandina 0 Chamaseycemesebyranthemum 0 Heliotropiumkarwinsky 0Buxbaumiaaphylla 1 Planophilaterrestris 0 Pseudolarixamabilis 0 Chelidoniummajus 0 Heliotropiummendocinum 0Calliergoncordifolium 0 Pleurastruminsigne 0 Pseudotaxuschienii 0 Chenopodiumquinoa 0 Heliotropiumracemosum 0Ceratodonpurpureus 0 Prasinococcuscapsulatus 0 Pseudotsugamenziesii 0 Chionanthusretusus 0 Heliotropiumsp. 0cf.Physcomicromitriumsp. 0 Prasinodermacoloniale 0 Retrophyllumminus 0 Chlorogalumpomeridianum 0 Heliotropiumtenellum 0Climaciumdendroides 1 Prasiolacrispa 0 Saxegothaeaconspicua 0 Chondropetalumtectorum 0 Heliotropiumtexanum 0Dicranumscoparium 3 Protothecawickerhamii 0 Sciadopitysverticillata 0 Chrysobalanusicaco 0 Heloniasbullata 0Diphysciumfoliosum 3 Pseudoscourfieldiamarina 0 Sequoiasempervirens 0 Cicerbitaplumieri 0 Helwingiajaponica 0Encalyptastreptocarpa 0 Pteromonasangulosa 0 Sequoiadendrongiganteum 0 Cimicifugaracemosa 0 Hemerocallissp. 0Fontinalisantipyretica 0 Pteromonassp. 0 Stangeriaeriopus 0 Cinnamomumcamphora 0 Hemerocallisspp. 0Funaria 1 Pycnococcusprovasolii 0 Sundacarpusamarus 0 Cissusquadrandularis 0 Heracleumlanatum 0Hedwigiaciliata 0 Pyramimonasparkeae 0 Taiwaniacryptomerioides 0 Cistusinflatus 0 Hesperaloeparviflora 0Hypnumsubimponens 5 Scenedesmusdimorphus 0 Taxodiumdistichum 0 Citrusxparadisi 0 Heteropyxisnatalensis 0Leucobryumalbidum 2 Scherffeliadubia 0 Taxusbaccata 0 Cladrastislutea 0 Heucherasanguinea 0Leucobryumglaucum 3 Scourfieldiasp. 0 Taxuscuspidata 0 Cleomegynandra 0 Hibbertiagrossulariifolia 0Leucodonsciuroides 2 Spermatozopsisexsultans 0 Tetraclinissp. 0 Cleomeviolaceae 0 Hibiscuscannabinus 0Neckeradouglasii 2 Spermatozopsissimilis 0 Thujaplicata 0 Cleomeviscosa 0 Hillerialatifolia 0Niphotrichumelongatum 1 Stephanosphaerapluvialis 0 Thujopsisdolabrata 0 Cocculuslaurifolius 0 Hoheriaangustifolia 0Orthotrichumlyellii 0 Stichococcusbacillaris 0 Torreyanucifera 0 Cochleareaofficinalis 0 Holarrhenapubescens 0Philonotisfontana 5 Stigeocloniumhelveticum 0 Torreyataxifolia 0 Cocosnucifera 0 Houttuyniacordata 0Plagiomniuminsigne 2 Tetraselmischui 0 Tsugaheterophylla 0 Codariocalyxmotorius 0 Humuluslupulus 0Polytrichumcommune 1 Tetraselmiscordiformis 0 Welwitschiamirabilis 0 Colchicumautumnale 0 Hydrangeaquercifolia 0Pseudotaxiphyllumelegans 3 Tetraselmisstriata 0 Widdringtoniacedarbergensis 0 Conopholisamericana 0 Hydrastiscanadensis 0Racomitriumvarium 4 Trebouxiaarboricola 0 Wollemianobilis 0 Convolvulusarvensis 0 Hydrocotyleumbellata 0Rhynchostegiumserrulatum 4 Trentepohliaannulata 0 Conzyacanadensis 0 Hypecoumprocumbens 0Rhytidiadelphusloreus 1 UnidentifiedspeciesCCMP1205 0 5 Copaiferaofficianalis 0 Hypericumperforatum 0Schwetschkeopsisfabronia 3 Uronemabelka 0 Acaciaargyrophylla 0 Coriarianepalensis 0 Idiospermumaustraliense 0Scouleriaaquatica 0 Vitreochlamyssp. 0 Acaciapycnantha 0 Cornusfloridana 0 Ilexparaguariensis 0Sphagnumlescurii 5 Volvoxaureus 0 Acernegundo 0 Corokiacotoneaster 0 Ilexsp. 0Sphagnumpalustre 7 Volvoxglobator 0 Acorusamericanus 0 Corydalislinstowiana 0 Ilexvomitoria 0Sphagnumrecurvatum 3 Actinidiachinensis 0 Cotoneastertranscaucasicus 0 Illiciumfloridanum 0Syntrichiaprinceps 1 Aervalanata 0 Crassulaperforata 0 Illiciumparviflorum 0Takakialepidozioides 2 Aervapersica 0 Crossopetalumrhacoma 0 Impatiensbalsamifera 0Tetraphispellucida 0 Aesculuspavia 0 Crotontiglium 0 Inulahelenium 0Thuidiumdelicatulum 5 Aextoxiconpunctatum 0 Cunoniacapensis 0 Ipomoeacoccinea 0Timmiaaustriaca 2 Agapanthusafricanus 0 Curculigosp. 0 Ipomoeahederacea 0

Agastacherugosa 0 Curcumaolena 0 Ipomoeaindica 014 Agavetequilana 0 Curtisiadentata 0 Ipomoealindheimeri 0

Anthocerosformosae 0 Ailanthusaltissima 0 Cuscutapentagonia 0 Ipomoealobata 0Leiosporocerosdussii 0 Ajugareptans 0 Cyanastrumcordifolium 0 Ipomoeanil 0Megacerostosanus 0 Akanialucens 0 Cyanellaorchidofromis 0 Ipomoeapubescens 0Megacerosvincentianus 2 Akebiatrifoliata 0 Cymbopogonnardus 0 Ipomoeapurpurea 0Nothocerosaenigmaticus 0 Alangiumchinense 0 Cyperuspapyrus 0 Ipomoeaquamoclit 0Paraphymatoceroshallii 3 Allamandacathartica 0 Cypseleahumifusum 0 Iteavirginica 0Phaeoceroscarolinianus 9 Allioniaincarnata 0 Cyrillaracemiflora 0 Jacquiniasp. 0

Allioniaspp. 0 Daenikerasp. 0 Johnsoniapubescens 0

TableS1.ListofscreenedtranscriptomesandthenumberofMTPSineachsample.Allthese1103transcriptomeswerefrom1KP(www.onekp.com).MTPSLsarefoundfrom146species.

Monilophytes Lycophytes Charophytes Angiosperms(Continued) Angiosperms(Continued)

Liverworts

Mosses

Hornworts

Chlorophytes

Angiosperms

Gymnosperms

TableS1(Continued)Joinvilleaascendens 0 Phaceliacampanularia 0 Tiarellapolyphylla 0Juglansnigra 0 Phellinelucida 0 Tragopogoncastellanus 0Juncusinflexus 0 Phellodendronamurense 0 Tragopogondubius 0Kadsuraheteroclite 0 Philadelphusinodorus 0 Tragopogonporrifolius 0Kalanchoecrenatodiagremontiana 0 Phloxdrummondii 0 Tragopogonpratensis 0Kaliphoramadagascariensis 0 Phloxsp. 0 Traubiamodesta 0Kerriajaponica 0 Pholismaarenarium 0 Trianthemumportulacastrum 0Kigeliaafricana 0 Phoradendronserotinum 0 Triglochinmaritimum 0Kirkiawilmsii 0 Phormiumtenax 0 Triodiaaff.bynoei 0Kochiascoparia 0 Phycellaaff.cyrtanthoides 0 Trochodendronaraliodes 0Koeberlinaspinosa 0 Phyladulcis 0 Tropaeolumperegrinum 0Kramerialanceolata 0 Phyllanthussp. 0 Trubuluseichlerianus 0Lactucagraminifolia 0 Physenamadagascariensis 0 Typhaangustifolia 0Lagerstroemiaindica 0 Physocarpusopulifolius 0 Typhalatifolia 0Lantanacamara 0 Phytolaccaamericana 0 Typhoniumblumei 0Larreatridentata 0 Phytolaccabogotensis 1 Ulmusalata 0Lathyrussativus 0 Pilostylesthunbergii 0 Uncarinagrandidieri 0Laureliasempervirens 0 Pinguiculaagnata 0 Uniolapaniculata 0Lavandulaangustifolia 0 Pinguiculacaudata 0 Urgineamaritima 0Ledumpalustre 0 Piperauritum 0 Urticadioica 0Lennoamadreporoides 0 Pistiastratioides 0 Utriculariasp. 0Leontopodiumalpinum 0 Pittosporumresiniferum 0 Uvariamicrocarpa 0Leonurusjaponicus 0 Pittosporumsahnianum 0 Valerianaofficianalis 0Lepidospermagibsonii 0 Plantagocoronopis 0 Vanillaplanifolia 0Licaniamichauxii 0 Plantagomaritima 0 Verbascumsp. 0Ligustrumsinense 0 Plantagovirginica 0 Verbenahastata 0Liliumsargentiae 0 Platantheraclavellata 0 Viburnumodoratissimum 0Limnanthesdouglassii 0 Platanusoccidentalis 0 Violacanadensis 0Limoniumspectabile 0 Platycodongrandiflorus 0 Violatricolor 0Lindenbergiaphilippensis 0 Platyspermationcrassifolium 0 Vitexagnuscastus 0Linderabenzoin 0 Plumbagoauriculata 0 Wikstroemiaindica 0Linumbienne 0 Podophyllumpeltatum 0 Wisteriafloribunda 0Linumflavum 0 Pogostemonsp. 0 Wrightianatalensis 0Linumgrandiflorum 0 Polansiatrachysperma 0 Xanthicerciszambesiaca 0Linumhirsutum 0 Poliominthabustamanta 0 Xanthuiumstrumarium 0Linumleonii 0 Polycarpaearepens 0 Xeronemacallistemon 0Linumlewisii 0 Polygalalutea 0 Xerophyllumasphodeloides 0Linummacraei 0 Polygonumconvolvulus 0 Xerophytavillosa 0Linumperenne 0 Polypremumprocumbens 0 Ximeniaamericana 0Linumstrictum 0 Polysciasfruticosa 0 Yuccabrevifolia 0Linumtenuifolium 0 Portulacaamilis 0 Yuccafilamentosa 0Linumusitatissimum 0 Portulacacryptopetala 0 Zaleyapentandra 0Liquidambarstyraciflua 0 Portulacagrandiflora 0 Zephyranthestreatiae 0Litchichinensis 0 Portulacamauii 0 Zingiberofficinale 0Lobeliasiphilitica 0 Portulacamolokaiensis 0 Ziziphusjujuba 0Lomandralongifolia 0 Portulacaoleracea 0Lonicerajaponica 0 Portulacapilosa 0Lophophorawilliamsii 0 Portulacasuffruticosa 0Loropetalumchinense 0 Portulacaumbraticola 0Ludoviasp. 0 Posidoniaaustralis 0Lupinusangustifolius 0 Prunellavulgaris 0Lupinuspolyphyllus 0 Prunusprostrata 0Lyciumbarbarum 0 Psychotriadouarrei 0Lyciumsp. 0 Psychotriaipecacuanha 0Lycopersiconcheesmanii 0 Psychotriamarginata 0Maesalanceolata 0 Punicagranatum 0Magnoliagrandiflora 0 Pycnanthemumtenuifolium 0Maianthemumcanadense 0 Pyrenacanthamalvifolia 0Maianthemumsp. 0 Quassiaamara 0Malesherbiafasiculata 0 Quercusshumardii 0Malusbaccata 0 Quillajasaponaria 0Manihotgrahamii 0 Rauvolfiatetraphyla 0Manilkarazapota 0 Rehmanniaglutinosa 0Mansoaalliacea 0 Resedaodorata 0Mapaniapalustris 0 Rhamnuscaroliniana 0Marantaleuconeura 0 Rhamnusjaponica 0Marrubiumvulgare 0 Rhizophoramangle 0Masdevalliayuangensis 0 Rhodiolarosea 0Matricariamatricariodes 0 Rhododendronscopulorum 0Medinillamagnifica 0 Rhodophialapratensis 0Melaleucaquinquenervia 0 Rhusradicans 0Meliaazedarach 0 Ribesaff.giraldii 0Meliosmacuneifolia 0 Ricinuscommunis 0Melissaofficinalis 0 Roridulagorgonias 0Menyanthestrifoliata 0 Rosapalustris 0Mertensiapaniculata 0 Rosmarinusofficinalis 0Micheliamaudiae 0 Ruelliabrittoniana 0Micromeriafruticosa 0 Ruscussp. 0Microstegiumvimineum 0 Sabalbermudana 0Microteadebilis 0 Sagittarialatifolia 0Mirabilisjalapa 0 Saintpauliaionantha 0Mitellapentandra 0 Salixacutifolia 0Mollugocerviana 0 Salixdasyclados 0Mollugonudicaulis 0 Salixeriocephala 0Mollugopentaphylla 0 Salixfargesii 0Mollugoverticillata 0 Salixpurpurea 0Monotropauniflora 0 Salixsachalinensis 0Morindacitrifolia 0 Salixviminalis 0Moringaoleifera 0 Salvadorasp. 0Morusnigra 0 Salviaspp. 0Mumeaamericana 0 Sambucuscanadensis 0Muntingiacalabura 0 Sancheziasp. 0Mydocarpussp. 0 Sanguinariacanadensis 0Myricacerifera 0 Sanguisorbaminor 0Myriophyllumaquaticum 0 Sansevieriatrifasciata 0Myristicafragrans 0 Santalumacuminatum 0Nandinadomestica 0 Saponariaofficianalis 0Narcissusviridiflorus 0 Sarcandraglabra 0Nelumbonucifera 0 Sarcobatusvermiculatus 0Nelumbosp. 0 Sarcodessanguinea 0Nepenthesalata 0 Sarumahenryi 0Nepetacataria 0 Sassafrasalbidum 0Neurachnealopecuroidea 0 Saururuscernuus 0Neurachneannularis 0 Saxifragastolonifera 0Neurachnelanigera 0 Scaevolasp. 0Neurachneminor 0 Schiedeamembranacea 0Neurachnemunroi 0 Schizolaenasp. 0Neurachnetenuifolia 0 Schlegeliaparasitica 0Nicotianasylvestris 0 SchlegeliaparasiticaB 0Nolinaatopocarpa 0 Schlegeliaviolacea 0Nolinabigelorii 0 Scutellariamontana 0Nothofagusobliqua 0 Seneciorowleyanus 0Nupharadvena 0 Sennahebecarpa 0Nypafruticans 0 Serenoarepens 0Nyssaogeche 0 Sessuviumportulacastrum 0Ochnamossambicensis 0 Sessuviumventricosum 0Ochnaserrulata 0 Sideroxylonreclinatum 0Oenotheraaffinis 0 Silenelatifolia 0Oenotheraberlandieri 0 Silybummarianum 0Oenotherabiennis 0 Simmondsiachinensis 0Oenotheraclelandii 0 Sinapisalba 0Oenotheraelata 0 Sinningiatuberosa 0Oenotheraelatahookeri 0 Sinojackiaxylocarpa 0Oenotherafiliformis 0 Sisyrinchiumangustifolium 0Oenotheragaura 0 Smilaxbonanox 0Oenotheragrandiflora 0 Solanumdulcamara 0Oenotheragrandis 0 Solanumlasiophyllum 0Oenotheralaciniata 0 Solanumptychanthum 0Oenotheralongituba 0 Solanumsisymbriifolium 0Oenotheranana 0 Solanumxanthocarpum 0Oenotherapicensis 0 Solenostemonscutellarioides 0Oenotherarhombipetala 0 Solidagocanadensis 0Oenotherarosea 0 Sorbuskoehneana 0Oenotheraserrulata 0 Souroubeaexauriculata 0Oenotheraspeciosa 0 Spergulariamedia 0Oenotherasuffultasuffulta 0 Stachyuruspraecox 0Oenotheravillaricae 0 Stackhousiaspathulata 0Oleaeuropaea 0 Staphyleatrifolia 0Oncidiumsphacelatum 0 Stemonatuberosa 0Oncothecabalansae 0 Strelitziareginae 0Opuntiasp. 4 Strobilanthesdyerianus 0Orchidanthamaxillaroides 0 Strychnosspinosa 0Oresitropherupifraga 0 Stylidiumadnatum 0Orobanchefasciculata 0 Symphoricarpossp. 0Oxalissp. 0 Symplocussp. 0Oxeraneriifolia 0 Synsepalumdulcificum 0Oxerapulchella 0 Syzygiummacranthum 0Paeonialactiflora 0 Syzygiumpaniculatum 0PanicummiliaceumA 0 Tabebuiaumbellate 0Papaverbracteatum 0 Talbotiaelegans 0Papaverrhoeas 0 Talinumsp. 0Papaversetigerum 0 Tamarixchinensis 0Papaversomniferum 0 Tanacetumparthenium 0Paraneurachnemuelleri 0 Tapisciasinensis 0Passifloracaerulea 0 Tellimabreviflora 0Passifloraedulis 0 Terminalianeotaliala 0Paulowniafargesii 0 Ternstroemiagymnanthera 0Peganumharmala 0 Tetrastigmaobtectum 0Peliosantheseminor 0 Tetrastigmavoinierianum 0Peltoboykiniawatanabei 0 Tetrazygiabicolor 0Pennantiacorymbosa 0 Teucriumchamaedrys 0Peperomiafraseri 0 Thalictrumthalictroides 0Pereskiaaculeata 0 Thladianthavillosula 0Perseaborbonia 0 Thymusvulgaris 0Petiveriaalliacea 0 Thyridolepismitchelliana 0Peumusboldus 0 Thyridolepismulticulmis 0

Angiosperms(Continued) Angiosperms(Continued) Angiosperms(Continued)

TableS2.SummarystaEsEcsofMTPSLsin9plantlineages.

Lineage Species Count MTPSL Count Mean Median St. Dev. Min Max Angiosperms 699 5 0.01 0 0.16 0 4 Gymnosperms 80 0 0 0 0 0 0 Monilophytes 70 353 5.04 4.50 3.81 0 20 Lycophytes 22 83 3.77 3 2.65 0 10 Hornworts 7 14 2 0 3.32 0 9 Mosses 41 79 1.93 1 1.82 0 7 Liverworts 26 177 6.81 8 4.15 0 16 Charophytes 47 1 0.02 0 0.15 0 1 Chlorophytes 111 0 0 0 0 0 0

MTPSL_id Lineages NR_top_hit evalue bit_score Scientific_name Kingdom

(RXRQ_WCZB)_MTPSL7 Hornworts gi|751680917|gb|KIM31075.1| 0 1019 Serendipita vermifera MAFF 305830 Fungi

(RXRQ_WCZB)_MTPSL8 Hornworts gi|353240956|emb|CCA72799.1| 0 981 Piriformospora indica DSM 11827 Fungi

(RXRQ_WCZB)_MTPSL9 Hornworts gi|353240956|emb|CCA72799.1| 7E-130 391 Piriformospora indica DSM 11827 Fungi

MCHJ_MTPSL1 Charophytes gi|913451420|ref|WP_050430829.1| 0.001 50.8 Chondromyces crocatus Bacteria

MRKX_MTPSL1 Angiosperms gi|588255974|ref|XP_006957163.1| 0 558 Wallemia mellicola CBS 633.66 Fungi

QAIR_MTPSL1 Angiosperms gi|751178290|gb|KIL64254.1| 5E-177 508 Amanita muscaria Koide BX008 Fungi

QAIR_MTPSL2 Angiosperms gi|751175026|gb|KIL61028.1| 0 620 Amanita muscaria Koide BX008 Fungi

QAIR_MTPSL3 Angiosperms gi|751174784|gb|KIL60790.1| 7E-143 427 Amanita muscaria Koide BX008 Fungi

QAIR_MTPSL4 Angiosperms gi|751181225|gb|KIL67171.1| 0 691 Amanita muscaria Koide BX008 Fungi

JKAA_MTPSL1 Lycophytes gi|927407765|ref|XP_013949969.1| 0 541 Trichoderma virens Gv29-8 Fungi

ZYCD_MTPSL1 Lycophytes gi|238496645|ref|XP_002379558.1| 1E-130 390 Aspergillus flavus NRRL3357 Fungi

AEXY_MTPSL1 Liverworts gi|389636521|ref|XP_003715910.1| 6E-178 513 Magnaporthe oryzae 70-15 Fungi

IRBN_MTPSL6 Liverworts gi|751680917|gb|KIM31075.1| 0 934 Serendipita vermifera MAFF 305830 Fungi

JHFI_MTPSL16 Liverworts gi|751680917|gb|KIM31075.1| 6E-101 313 Serendipita vermifera MAFF 305830 Fungi

LGOW_MTPSL3 Liverworts gi|629725325|ref|XP_007822988.1| 7E-56 196 Metarhizium robertsii Fungi

NWQC_MTPSL8 Liverworts gi|629725325|ref|XP_007822988.1| 6E-36 144 Metarhizium robertsii Fungi

OFTV_MTPSL7 Liverworts gi|751680917|gb|KIM31075.1| 0 931 Serendipita vermifera MAFF 305830 Fungi

RTMU_MTPSL4 Liverworts gi|667838359|ref|XP_007783348.1| 3E-128 386 Coniosporium apollinis CBS 100218 Fungi

WJLO_MTPSL3 Liverworts gi|549052256|emb|CCX30236.1| 4E-68 231 Pyronema omphalodes CBS 100304 Fungi

WJLO_MTPSL4 Liverworts gi|549052256|emb|CCX30236.1| 1E-71 240 Pyronema omphalodes CBS 100304 Fungi

YBQN_MTPSL8 Liverworts gi|648165817|gb|KDR79494.1| 0 596 Galerina marginata CBS 339.88 Fungi

QIAD_MTPSL2 Monilophytes gi|629662947|ref|XP_007805277.1| 2E-77 251 Endocarpon pusillum Z07020 Fungi

TableS3.22MTPSLgenesdesignatedasUnclassifiedandtheirtophitsfromNonredundant(NR)databaseofNCBI

Gene Group Liv-IRBN-MTPSL1 I Liv-IRBN-MTPSL2 I Liv-IRBN-MTPSL3 I Liv-IRBN-MTPSL4 I Liv-IRBN-MTPSL5 I Liv-IRBN-MTPSL6 II Liv-IRBN-MTPSL7 III Liv-IRBN-MTPSL8 Ua

aunclassified

TableS4.EightMTPSLsidenEfiedfromthetranscriptomeoftheliverwortScapanianemorea

TableS5.MTPSLgenesfromthegenomeofthehornwortAnthocerospunctatus

Gene Protein GroupApMTPSL1 408 IIApMTPSL2 430 IIIApMTPSL3 436 IIApMTPSL4 401 IIIApMTPSL5 413 IIIApMTPSL6 421 IIIApMTPSL7 427 III

TableS6.MTPSLgenesfromthegenomeofthemossSphagnumfallax

Gene Proteinsize GroupSfMTPSL1 472 ISfMTPSL2 341 ISfMTPSL3 328 ISfMTPSL4 482 ISfMTPSL5 489 ISfMTPSL6 477 ISfMTPSL7 481 ISfMTPSL8 484 ISfMTPSL9 377 ISfMTPSL10 481 ISfMTPSL11 440 ISfMTPSL12 487 ISfMTPSL13 455 ISfMTPSL14 456 ISfMTPSL15 472 ISfMTPSL16 472 ISfMTPSL17 341 ISfMTPSL18 457 ISfMTPSL19 453 ISfMTPSL20 455 ISfMTPSL21 455 I

Species DataversionAmaranthushypochondriacus v1.0Amborellatrichopoda v1.0Ananascomosus v3Aquilegiacoerulea v1.1Aquilegiacoerulea v3.1Arabidopsishalleri v1.1Arabidopsislyrata v1.0Arabidopsisthaliana TAIR10Boecherastricta v1.2Brachypodiumdistachyon v3.1Brachypodiumstacei v1.1Brassicarapa FPscv1.3Capsellagrandiflora v1.1Capsellarubella v1.0Caricapapaya ASGPBv0.4ChlamydomonasreinhardPi v5.5CitrusclemenPna v1.0Citrussinensis v1.1CoccomyxasubellipsoideaC-169 v2.0CucumissaPvus v1.0Eucalyptusgrandis v2.0Eutremasalsugineum v1.0Fragariavesca v1.1Glycinemax Wm82.a2.v1Gossypiumraimondii v2.1Kalanchoemarnieriana v1.1Klebsormidiumflaccidum v1.0LinumusitaPssimum v1.0MalusdomesPca v1.0Manihotesculenta v6.1Medicagotruncatula Mt4.0v1MicromonaspusillaCCMP1545 v3.0Micromonassp.RCC299 v3.0MimulusguVatus v2.0Musaacuminata v1OryzasaPva v7_JGIOstreococcuslucimarinus v2.0Panicumhallii v2.0Panicumvirgatum v1.1Phaseolusvulgaris v1.0Physcomitrellapatens v3.3Populustrichocarpa v3.0Prunuspersica v2.1Ricinuscommunis v0.1Salixpurpurea v1.0Selaginellamoellendorffii v1.0Setariaitalica v2.2Setariaviridis v1.1Solanumlycopersicum iTAG2.3Solanumtuberosum v3.4Sorghumbicolor v3.1Sphagnumfallax v0.5Spirodelapolyrhiza v2Theobromacacao v1.1TriPcumaesPvum v2.2ViPsvinifera Genoscope.12XVolvoxcarteri v2.1Zeamays 6aZosteramarina v2.2

TableS7.AlistofsequencedgenomessearchedforMTPSLgenes

SequenceID Lineage Species GroupLiv-IRBN-MTPSL2 Liverworts Scapanianemorea ILiv-IRBN-MTPSL4 Liverworts Scapanianemorea IMos-GOWD-MTPSL2 Mosses Sphagnumlescurii IMos-QKQO-MTPSL3 Mosses Pseudotaxiphyllumelegans IIMos-VBMM-MTPSL3 Mosses Anomodonrostratus IIHon-ApMTPSL7 Hornworts Anthocerospunctatus IIIMon-GSXD-MTPSL3 Monilophytes Myriopteriseatonii IVMon-UJTT-MTPSL4 Monilophytes Pityrogrammatrifoliata IVMon-YJJY-MTPSL1 Monilophytes Woodsiascopulina IV

TableS8.AlistofrepresentaEveMTPSLgenesexperimentallystudied.

TableS9.PrimersusedinthisstudyRegion amplified Primer DNA sequences ApMTPSL1 and its Forward 5'-CACTACTGCGTCGGCTTCATG-3' neighoring gene Reverse 5'-CGCACAGCATTCACAATTTCACTT-3' ApMTPSL2 and its Forward 5'-CAGGTAGGAGCCCGCGATTT-3' neighoring gene Reverse 5'-AGGGAAAAGGAGGGTGGTG-3' SfMTPSL1 and its Forward 5'-CAGAAGCAAAGTATCGGTCTCTTAC -3' neighoring gene Reverse 5'-CACTGTTAGCAGGGTATGGTGAAC-3'

Liv-IRBN-MTPSL1 Forward 5'-TTCTGAGGACGAGCGTATTCTTC-3' Reverse 5'-GCAAAACGTCAACTAAACGAGAAG-3'

Liv-IRBN-MTPSL2 Forward 5'-TCATACTCGCCTCCATATCCTGTG-3' Reverse 5'-GATTTGAAATGTCAGTCATGTGTGC-3'

Liv-IRBN-MTPSL3 Forward 5'-GATGCCAACGCAGCCATACAGAC-3' Reverse 5'-GCCTGATACCCAGTTTCTGACGG-3'

Liv-IRBN-MTPSL4 Forward 5'-CAGTATGTGTGAACTCCTCTTGGGTC-3' Reverse 5'-GCACTCCTTTTCTGTACCGACTGG-3'

Liv-IRBN-MTPSL5 Forward 5'-TCAAAGGCATCACCTGAAGTCTG-3' Reverse 5'-ATATTATCGGTGTTCCAATCCTCC-3'

Liv-IRBN-MTPSL6 Forward 5'-AATGCTTGGTGTGTGTTCGTCTC-3' Reverse 5'-CCTCCATGTGATTTCGCAAAGTAG-3'

Liv-IRBN-MTPSL7 Forward 5'-CTGGCAGATGATTTAGATGAGATAGC-3' Reverse 5'-CAGAAACAACCCGCAAACCATTC-3'

Liv-IRBN-MTPSL8 Forward 5'-TCTCCCTGTTGCCACTGCTTTCC-3' Reverse 5'-GTTGGTCCTGGTACGGCGACTGA-3'

Mon-UJTT-MTPSL4-pET32a Forward 5'-CATGCCATGGCATCCATTATATTAGGAAGCTC-3' Reverse 5'-CCCAAGCTTAGTTAAAGGCCATCATGACAC-3'

Fig. S1. Sequence alignment of IRBN_MTPSL6 identified from the transcriptome of the liverwort Scapania nemorea with its top hit in nr database at NCBI. “gi:751680917” is putative terpene synthase gene identified in the fungus Serendipita vermifera. The two sequences are 91% identical.

IV

III

II

I

Fig. S2. Identification of conserved motifs among the four groups (I, II, III and IV) of microbial type (MTPSL) terpene synthases. The ‘NDxxSxxxD/E’motif is highly conserved among all MTPSLs. The canonical ‘DDxxD’ motif was present in group I and IV proteins, but group II proteins displayed a conserved ‘DDxxxD’ motif, whereas in the group III enzymes only the first two aspartates (‘DD’) are conserved. Sequence motif logos made using weblogo 3.0, showing the conserved motifs found in each group of terpene synthase genes of microbial type.

13 14 15 16 17 18

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Mon-UJTT-MTPSL4 product

Ceratocystis piceae headspace volatiles

protoillud-6-ene

protoillud-6-ene

unidentified sesquiterpene

alcohols

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4

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175.1 119.1

105.1

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41.1 189.1 77.1 147.1 55.1 161.1

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175.1 119.1

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41.1 204.1 77.0 189.1 55.1 147.1 161.1

Retention time (min)

Retention time (min)

mass spectrum of C. piceae protoillud-6-ene

Fig. S3. Product identification of Mon-UJTT-MTPSL4. (A) The gene was heterologously expressed in Escherichia coli and the crude bacterial protein extract was incubated with FPP. The enzyme product was collected using solid phase micro extraction (SPME) and analyzed with gas chromatography/mass spectrometry (GC-MS). (B) Volatiles from the headspace of a liquid culture of Ceratocystis piceae were collected using SPME and analyzed with GC-MS. C. piceae has been reported to produce protoillud-6-ene as main sesquiterpene hydrocarbon (17).

mass spectrum of Mon-UJTT-MTPSL4 product

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24

Mos-GOWD- MTPSL2

Liv-IRBN- MTPSL2

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Mon-UJTT- MTPSL4

1

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A Monoterpene synthase activity

B Diterpene synthase activity

Liv-IRBN-MTPSL4

Mos-VBMM-MTPSL3

20 22 24 26

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20 22 24 26

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300

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95.1

41.1 272.1 77.1 177.1

122.1

229.1

15

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60 119.1

41.1 95.0

69.0 229.2 272.1

136.0

185.1

280

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105.1

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peak B

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m/z Retention time (min)

mass spectra of major diterpene product

Fig. S4. Monoterpene synthase and diterpene synthase activities of MTPSLs. MTPSL genes were heterologously expressed in Eschericha coli and and crude protein extracts were incubated with the potential substrates GPP (A) and GGPP (B), respectively. Monoterpene products were collected using solid-phase micro-extraction and diterpene products were extracted with hexane. Products were analyzed using gas chromatography/mass spectrometry. 1, myrcene*; 2, limonene*; 3, (Z)-β-ocimene; 4, (E)-β-ocimene*; 5, linalool*; 6, allo-ocimene; 6, α-pinene; 7, β-phellandrene. A-F, unidentified diterpenes. Compounds marked with an asterisk (*) were identified using authentic standards.

Retention time (min)

Hon-Ap- MTPSL7

7.0 8.0 9.0 10.0 11.0 12.0 4 8 12 16

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Mon-YJJY-MTPSL1

Mon-UJTT-MTPSL4

Mon-GSXD-MTPSL3

Mos-QKQO-MTPSL3

Mos-GOWD-MTPSL2

Mos-VBMM-MTPSL3

Liv-IRBN-MTPSL4

Liv-IRBN-MTPSL2

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Hon-ApMTPSL7

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no activity

no activity

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(Z,Z)-FPP

13 14 15 16 17 Retention time (min)

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no activity

no activity

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(Z,E)-FPP

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unidentified sesquiterpene hydrocarbons

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unidentified sesquiterpene hydrocarbons

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unidentified sesquiterpene hydrocarbons

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11

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Fig. S5. Sesquiterpene synthase activity of representative MTPSLs with (Z,E)-FPP and (Z,Z)-FPP. MTPSL genes were heterologously expressed in Eschericha coli and crude protein extracts were incubated with the potential substrates (Z,E)-FPP and (Z,Z)-FPP. Enzyme products were collected using solid-phase micro-extraction and analyzed with gas chromatography/mass spectrometry. 1, (E)-β-farnesene; 2, β-acoradiene; 3, β-bisabolene; 4, (Z)-γ-bisabolene; 5, unidentified ST; 6, unidentified ST; 7, (Z)-α-bisabolene; 8, nerolidol; 9, unidentified oxygenated ST; 10, unidentified oxygenated ST; 11, γ-curcumene; 12, zingiberene; 13, unidentified ST; 14, unidentified ST; 15, β-sesquiphellandrene; 16, unidentified ST; 17, epi-bicyclosesquiphellandrene; 18, γ-cadinene; 19, δ-cadinene; 20, α-cadinene; 21, unidentified ST; 22, unidentified ST; 23, (E)-γ-bisabolene; 24, (E,E)-α-farnesene. ST, sesquiterpene.