Widespread impact of horizontal gene transfer on plant colonization of land

Abstract

In complex multicellular eukaryotes such as animals and plants, horizontal gene transfer is commonly considered rare with very limited evolutionary significance. Here we show that horizontal gene transfer is a dynamic process occurring frequently in the early evolution of land plants. Our genome analyses of the moss Physcomitrella patens identified 57 families of nuclear genes that were acquired from prokaryotes, fungi or viruses. Many of these gene families were transferred to the ancestors of green or land plants. Available experimental evidence shows that these anciently acquired genes are involved in some essential or plant-specific activities such as xylem formation, plant defence, nitrogen recycling as well as the biosynthesis of starch, polyamines, hormones and glutathione. These findings suggest that horizontal gene transfer had a critical role in the transition of plants from aquatic to terrestrial environments. On the basis of these findings, we propose a model of horizontal gene transfer mechanism in nonvascular and seedless vascular plants.

ARTICLE

Received 10 Jun 2012 | Accepted 20 Sep 2012 | Published 23 Oct 2012 DOI: 10.1038/ncomms2148

Widespread impact of horizontal gene transfer on plant colonization of land

Jipei Yue1,2, Xiangyang Hu1,3, Hang Sun1, Yongping Yang1,3 & Jinling Huang2

In complex multicellular eukaryotes such as animals and plants, horizontal gene transfer is commonly considered rare with very limited evolutionary signicance. Here we show that horizontal gene transfer is a dynamic process occurring frequently in the early evolution of land plants. Our genome analyses of the moss Physcomitrella patens identied 57 families of nuclear genes that were acquired from prokaryotes, fungi or viruses. Many of these gene families were transferred to the ancestors of green or land plants. Available experimental evidence shows that these anciently acquired genes are involved in some essential or plant-specic activities such as xylem formation, plant defence, nitrogen recycling as well as the biosynthesis of starch, polyamines, hormones and glutathione. These ndings suggest that horizontal gene transfer had a critical role in the transition of plants from aquatic to terrestrial environments. On the basis of these ndings, we propose a model of horizontal gene transfer mechanism in nonvascular and seedless vascular plants.

1 Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China. 2 Department of Biology, East Carolina University, Greenville, North Carolina 27858, USA. 3 Institute of Tibet Plateau Research, Chinese Academy of Sciences, Kunming 650201, China. Correspondence and requests for materials should be addressed to J.H. (email: [email protected]).

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2148

Horizontal gene transfer (HGT) is the process of genetic movement between species. Traditionally considered to be predominant in prokaryotes1, HGT now appears to be

widespread in microbial eukaryotes2. As an efficient mechanism to spread evolutionary success, HGT may introduce genetic novelties to recipient organisms, thus facilitating phenotypic variation and adaptation to shiing environments or allowing access to new resources. The novelties introduced by HGT range from virulence factors in pathogens3,4, food digestive enzymes in nematodes and rumen ciliates5,6, to anaerobic metabolism in intracellular parasites6,7.

Although HGT in prokaryotes and unicellular eukaryotes has been under some extensive studies and well documented810, how HGT has contributed to the evolution of complex multicellular eukaryotes, such as animals and plants, remains elusive. Presumably because of the barrier of germline in animals and apical meristem in plants9,11, HGT is generally believed to be rare and insignicant in complex multicellular eukaryotes, except for organisms in a symbiotic relationship12,13 and for plant mitochondrial genes14,15. This belief, however, has been cast in doubt by reports of acquired genes in invertebrates and plants from free-living organisms5,1618. Importantly, because all multicellular eukaryotes are derived from unicellular ancestors, this belief largely discounts the dynamic nature of HGT and the contribution of ancient HGT to the evolution of multicellular lineages19. Therefore, to better understand the role of HGT in eukaryotic evolution, it is critical to reassess the occurrence and biological functions of horizontally acquired genes in multicellular eukaryotes.

Land plants emerged from charophycean green algae about 480490 million years ago20. During their colonization of land, plants gradually evolved complex regulatory systems, body plan and phenotypic novelties that facilitated their adaptation to and radiation in terrestrial environments21. Because of the importance of HGT in the adaptation of organisms to new niches, we decided to investigate whether such habitat and developmental transition was aided by acquisition of novel genes, especially those during early evolution of land plants. Thus far, although the role of HGT in the evolution of land plants, especially owering plants, has long been speculated22, there are very few reported cases of HGT in land plants that are related to nuclear genes2325. We here present evidence for the widespread and signicant impact of HGT of nuclear genes on plant colonization of land based on analyses of the moss Physcomitrella patens, an extant representative of early land plants. We further propose a model for gene acquisition in nonvascular and seedless vascular plants and discuss the cumulative impact of HGT on multicellular eukaryotes.

ResultsHGT-derived genes in land plants. Eukaryotic genomes contain many genes of prokaryotic origin, most of which are derived from mitochondria and plastids26. Gene transfer from these organelles to the nucleus, oen called endosymbiotic gene transfer (EGT), has been studied in many eukaryotic groups2729 and will not be included here. In this study, we identied genes in land plants that were acquired independently from other sources, primarily based on phylogenomic analyses of the moss P. patens. Whenever possible, independent evidence such as restricted taxonomic distribution and uniquely shared genomic characters (for example, indels or domain structures) were also considered. To reduce the complication arising from dierential gene losses, we focused on identifying genes acquired from prokaryotes and viruses. Genes acquired from fungi were also identied because of the role of mycorrhizae in land plant evolution30 and available evidence for HGT between mycorrhizal partners25. Furthermore, because genes acquired by the common ancestor of Plantae (green plants, red algae and glaucophytes) have been under some detailed analyses3133, this study only identied

genes in P. patens that were acquired aer the separation of green plants from red algae and glaucophytes.

With the annotated protein sequences of P. patens as input, 910 genes were identied using AlienG34 as potentially of prokaryotic, fungal or viral origin. Among these 910 genes, 394 were removed from further analyses because of their locations on short scaolds or their high percent-identities with cyanobacterial sequences, which is oen suggestive of plastid origin. Of the remaining 516 genes, 32 genes of four families had identiable homologues only in prokaryotes or fungi; 96 genes of 53 families showed a monophyletic relationship between sequences of green plants and those from prokaryotes, fungi or viruses in phylogenetic analyses, with bootstrap support of 80% or higher from either maximum likelihood or distance analyses or both. In total, 128 genes of 57 families were identied as derived from prokaryotes, fungi or viruses (Table 1; Figs 1 and 2; Supplementary Information). Twenty-four of these gene families in green plants also share unique indels and amino acid residues with their putative donors. The online Supplementary Data show the taxonomic distributions, multiple sequence alignments, molecular phylogenies and other relevant information for the 57 gene families we have identied in this study.

Of the 57 gene families, 18 are present in both green algae and land plants, suggesting that they were likely acquired before the origin of land plants. The remaining 39 gene families are not found in green algae and might have been acquired during or aer the origin of land plants. Notably, 19 of the identied gene families are only found in P. patens and their putative donors (prokaryotes, viruses or fungi) (Table 1; Supplementary Table S1). All of these 19 families are located on large genomic scaolds, indicating that they are unlikely to be bacterial contaminants. As P. patens is the only moss whose complete genome sequence is available, it is unclear whether these families also exist in other mosses or nonvascular land plants. However, the lack of homologues for these gene families in vascular plants suggests that they were likely transferred more recently to either P. patens or its close relatives.

The vast majority of acquired gene families identied in our analyses are derived from miscellaneous bacterial lineages. Ten families are derived from fungi, and only one family is from archaea and viruses, respectively. As expected for land plants, which have oen undergone frequent duplication events, 25 of identied gene families contain multiple copies in P. patens. In some cases, both acquired genes and endogenous homologues co-exist in P. patens. For instance, the gene family encoding FAD-linked oxidase comprises three identiable copies in P. patens, two of which are closely related to CFB bacterial homologues and one may have been vertically inherited in eukaryotes (Supplementary Fig. S1). A similar evolutionary scenario is also observed for the gene encoding phosphoenolpyruvate carboxylase (PEPCase). In this case, two PEP-Case gene copies exist in P. patens, one of which is clearly related to proteobacterial sequences, whereas the other to those from photo-synthetic eukaryotes, the chytrid fungus Spizellomyces, and other bacteria (Supplementary Fig. S2).

As HGT identication can be prone to errors owing to poor data quality and methodological limitations19, we have taken very cautious measures to alleviate these issues. These measures include construction of a comprehensive database, broad and balanced taxonomic sampling, careful inspection of alignments, determination of optimal protein substitution matrix for each data set and detection of other molecular characters consistent with the identied relationships. Such measures may have reduced most of the artifacts commonly encountered in HGT detection. It is critical to note that, although dierential gene loss, sometimes associated with hidden paralogy, can always be invoked as an alternative explanation, HGT is the most parsimonious interpretation for the genes identied in Table 1. This interpretation is consistent with independent evidence such as shared indels and amino-acid residues for many identied

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Table 1 | Horizontally acquired genes identied in Physcomitrella patens.

Putative gene product Putative donor Functional category Figure Homologous locus in Arabidopsis

Subtilase family (10) Bacteria Proteolysis Figure 1A AT4G30020 Arginase Bacteria Polyamine biosynthesis Figure S3 AT4G08900 Acyl-activating enzyme 18 (AAE18) (3) Bacteria Auxin biosynthesis Figure 2B AT1G55320 YUCCA family monooxygenase (YUC3) (5) Bacteria Auxin biosynthesis Figure S4 AT1G04610 Glutamate-cysteine ligase (GCL) (3) Proteobacteria Glutathione synthesis Figure S5 AT4G23100 Wound-responsive family protein (6) Bacteria Defense response Figure S39 AT1G19660 HAD superfamily, subfamily IIIB acidphosphatase (4)

Alpha-proteobacteria Cadmium stress response Figure S43 AT2G19940

HAD-superfamily hydrolase Bacteria Cold stress response Figure S18 AT5G48960 Killer toxin Protein (KP4) (2) Ascomycetes Pathogen resistance Figure S49 No Flotillin-like protein Ascomycetes Endocytosis Figure S45 AT5G25260 Allantoate amidohydrolase (AAH) (2) Bacteria Purine degradation Figure S7 AT4G20070 Ureidoglycolate amidohydrolase (UAH) Bacteria Purine degradation Figure S7 AT5G43600 Guanine deaminase (GDA) Alpha-proteobacteria Purine degradation Figure S6 NoPfkB family kinase (3) Delta-proteobacteria Vitamin B6 salvaging Figure S36 AT5G58730 Methionine gamma-lyase (MGL) (2) CFB bacteria L-methionine degradation Figure S20 AT1G64660 Glutamine synthetase (GS) CFB bacteria Glutamine biosynthesis Figure S8 No 3,4-Dihydroxy-2-butanone 4-phosphatesynthase (ribB)

Euryarchaeotes Riboavin biosynthesis Figure S13 No

Hemerythrin HHE domain protein Ascomycetes Iron homeostasis Figure S46 No Hydroxypyruvate reductase 2 (HPR2) (2) Bacteria Photorespiration Figure S26 AT1G79870 Inositol 2-dehydrogenase like protein Alpha-proteobacteria Pollen germination and tube growth Figure S27 AT4G17370 Peptidoglycan-binding domain containingprotein

Ascomycetes Peptidoglycan binding Figure S50 No

Sugar isomerase (SIS) family (2) Alpha-proteobacteria Sugar binding Figure S24 AT5G52190 Limit dextrinase (LDA) Bacteria Starch biosynthesis Figure S14 AT5G04360 Beta-glucosidase (2) Bacteria Cellulose degradation Figure S15 AT5G04885 Gycosyl hydrolase family (2) Ascomycetes Carbohydrate metabolism Figure S47 AT3G26140 Glycoside hydrolase Delta-proteobacteria Carbohydrate metabolism Figure S23 No Glycoside hydrolase family 2 Gamma-proteobacteria Carbohydrate metabolism Figure S25 AT3G54440 Alpha-L-rhamnosidase CFB bacteria Carbohydrate metabolism Figure S33 No FAD-linked oxidase (2) CFB bacteria Oxygen-dependent oxidoreductases Figure S1 No Short-chain dehydrogenase/reductase SDR Proteobacteria Oxidation-reduction Figure S28 NoFatty acyl-ACP thioesterases B (FATB)(5) Bacteria Fatty acid biosynthesis Figure S41 AT1G08510 1,4-dihydroxy-2-naphthoateoctaprenyltransferase

High GC gram + Glycolysis Figure S16 AT5G63620

Pyruvate kinase (2) Bacteria Glycolysis Figure S21 AT3G49160 Phosphoglycerate kinase (PGK) (2) Delta-proteobacteria Glycolysis Figure S17 No ATP-binding cassette I1 (ABCI1) transporter Bacteria Molecular transport Figure S29 AT1G63270 Uracil permease (2) Bacteria Nucleobase transport Figure S30 AT5G03555

L-fucose permease* Ascomycetes Sugar transport Figure S51 No Beta-1,4-mannosyl-glycoprotein (2) Basidiomycetes Glycosyl transferring Figure S48 AT5G14480 DNA repair family protein Ascomycetes DNA replication Figure S12 NoToprim domain-containing protein Bacteria DNA replication Figure S9 AT1G30680 DNA topoisomerase I Proteobacteria DNA replication Figure S10 AT4G31210 Phage/plasmid primase, P4 family (5) Viruses DNA replication Figure S11 No Ribosomal protein S6 Beta-proteobacteria RNA binding Figure S40 NoM6 family peptidase (3) Bacteria Peptidase activity Figure S35 No Amidohydrolase family Bacteria Hydrolase activity Figure S31 No Amidase family protein (2) Bacteria Acrylonitrile metabolism Figure S32 AT5G07360

D-alanine-D-alanine ligase family Chlamydiae/CFB bacteria

Peptidoglycan biosynthesis Figure S34 AT3G08840

Dienelactone hydrolase family Bacteria Hydrolase activity Figure S38 NoVein Patterning 1 (VEP1) Bacteria Vascular development Figure 1B AT4G24220 Heterokaryon incompatibility (HET)superfamily (20)

Fungi Heterokaryon formation No gure No

ybiU protein High GC gram + Unknown Figure S22 No Acyl-CoA N-acyltransferase Alpha-proteobacteria Unknown Figure S19 At2G23390 Hypothetical protein* Ascomycetes Unknown Figure S52 No

Note: numbers in the brackets indicate the numbers of genes within each family. The heterokaryon incompatibility (HET) superfamily was identied based on its restricted taxonomic distribution. *Genes that were also reported by earlier studies.

Bacteria Herbivorous insect resistance Figure S42 AT4G29260

NRPS-like enzyme Fungi Oxidative stress resistance Figure S44 AT4G18540 N-acetyl-gamma-glutamyl-phosphatereductase (argC) (2)

Delta-proteobacteria Menaquinone biosynthesis Figure S37 No

Phosphoenolpyruvate carboxylase (PEPCase) Gamma-proteobacteria Carbon xation Figure S2 No GroES-like zinc-binding alcoholdehydrogenase family

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a b

Selaginella

Arabidopsis Physcomitrella

69/55 Selaginella Physcomitrella

70/71

100/92 Oryza Oryza

100/100 Arabidopsis Arabidopsis Physcomitrella Selaginella Selaginella

75/80

Land plant

61/61 Arabidopsis Selaginella Physcomitrella

Physcomitrella Physcomitrella

Land plants

52/69

87/94

100/100 Arabidopsis Arabidopsis

100/100 Physcomitrella Physcomitrella

100/100 Physcomitrella Physcomitrella

79/72 Selaginella Physcomitrella

97/99 Colwellia Glaciecola

68/70

87/68

97/93

*/58

87/64

94/94 Halomonas Xanthomonas

Granulicella Acidobacteria Chondromyces Delta-proteobacteria

100/99 Arthrobacter Clavibacter

Yersinia Gamma-proteobacteria Burkholderia Beta-proteobacteria Methylobacterium Alpha-proteobacteria

Singulisphaera Planctomycetes

Gamma-proteobacteria

High GC Gram+

100/100

99/97

Gamma-proteobacteria

98/98 Reinekea Gamma-proteobacteria Haliangium Delta-proteobacteria

71/63

53/50 Pseudomonas Gamma-proteobacteria Opitutaceae Verrucomicrobia Brevundimonas Alpha-proteobacteria

Spirosoma CFB group bacteria Zymomonas Alpha-proteobacteria

50/*

99/100

94/95 Nakamurella High GC Gram+Roseiflexus

Herpetosiphon Colwellia Gamma-proteobacteria Intrasporangium High GC Gram+

Ralstonia Beta-proteobacteriaChitinophaga CFB group bacteria

55/74 Kytococcus Arthrobacter Clavibacter

59/60 Gloeobacter Cyanobacteria

100/100 Thermococcus Pyrococcus

Solibacter

100/100 Thermobaculum Intrasporangium High GC Gram+

100/100

GNS bacteria

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Euryarchaeota

Bacteria

Paenibacillus

Paenibacillus

Rhodococcus High GC Gram+ Ktedonobacter GNS bacteria

Saccharomonospora High GC Gram+

Enterobacter Gamma-proteobacteria Streptomyces

Gordonia

alpha proteobacterium BAL199 alpha proteobacterium BAL199 Azotobacter Gamma-proteobacteriaChlorella Green algae

100/100 Aspergillus

Aspergillus

100/100

91/98

Firmicutes

71/51

High GC Gram+

Spizellomyces Fungi

*/55 Allomyces Rhizopus

Chaetomium Meiothermus Deinococci Bacillus

Streptococcus

Fungi

84/84

52/*

53/*

81/73

Firmicutes

Ascomycetes

0.2

0.2

Figure 1 | Molecular phylogenies of subtilases (a) and vein patterning 1 (VEP1) (b). Numbers above branches show bootstrap values from maximum likelihood and distance analyses, respectively. Asterisks indicate values < 50%.

gene families (Fig. 2; Supplementary Information). On the other hand, the number of acquired genes in P. patens may have been underestimated in this study for several reasons. First, our study is primarily based on phylogenetic analysis, which, despite being considered the most reliable approach for HGT detection35, tends to have more false negatives owing to the lack of sufficient phylo-genetic signal in many data sets. Second, only genes transferred from prokaryotes, viruses and fungi to plants were included in our results, those from other eukaryotes were not detected. Third, our results only include genes derived from a single HGT event (that is, genes transferred directly from their ultimate donors to mosses or to recent common ancestors of green plants). This might overlook genes involved in secondary or recurrent transfer events, which oen lead to complex and patchy distributions36,37. Finally, our results are based solely on the analyses of the P. patens genome. Acquired genes in other land plants or secondarily lost in P. patens are not included. Therefore, our current results may only be viewed as a glimpse of acquired genes in land plants.

HGT in plant development and adaptation. Many of the genes identied in our analyses are related to essential or plant-specic metabolic and developmental processes (Table 1). Multiple gene families related to carbohydrate metabolism were acquired from bacteria, and they are involved in starch biosynthesis, cellulose degradation, pollen and seed germination as well as other activities in

Arabidopsis. Another notable example is the large and versatile subtilase gene family. With subtilases of P. patens as queries, we were able to identify homologues only in bacteria and other land plants. Such sequence similarity is consistent with earlier reports that plant subtilases dier signicantly from those of fungi and animals38. Further phylogenetic analyses indicate that land plant subtilases are derived from a single HGT event from bacteria, followed by rapid gene duplication (Fig. 1a).

Our analyses also identied genes related to biosynthesis of plant polyamines and hormones. The gene encoding arginase is responsible for degrading arginine into ornithine, a major precursor for the biosynthesis of polyamines. Sequences of land plant arginase share 3248% identities with those of bacterial agmatinase, but only 2528% identities with arginase of other organisms. Consistent with the results of sequence comparisons, phylogenetic analyses indicate that land plant arginase evolved from bacterial agmatinase (Supplementary Fig. S3). At least two acquired gene families, including those encoding acyl-activating enzyme 18 (AAE18) and YUCCA avin monooxygenase (YUC3), are involved in the biosynthesis of auxin39,40, a hormone that regulates abscission suppression, apical dominance, cell elongation and xylem dierentiation. Both AAE18 and YUC3 families were likely acquired from bacteria (Fig. 2; Supplementary Fig. S4). In particular, plant AAE18 sequences share multiple conserved amino-acid residues and indels with homo-logues from planctomycetes, verrucomicrobia and CFB bacteria

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ARTICLE

a

PIRCTCDTWAHLDIQPQDIFCWPTNLGWVMG-PILLYSCFLSGATLALYHGSPLGRGFCKF---VQDAGVTILGSVP 318Selaginella IL

Oryza IL

PIRSACDGWAHLDVQVGHTYCWPTNLGWVMG-PTLMFSCFLTGATLALYSGSPLGRGFGKF---VQDAGVTVLGTVP 451Picea IM

SIRSGAESWAHLDVKAGDIFCWPTNFGWIMG-SVLVYSCFLSGATAALYHGSPLDRGFGKF---VQDAGVNVLGTVP 459Physcomitrella_1 IL

PLRCAADAWAHLDAREGDIICWPTNLGWVVG-HLVLYAAFLNGATLALFNGSPLDQEFGKF---VQDANISILGTVP 453Physcomitrella_2 IL

PLRCAADSWAHLDSRQGDVLCWPTNLGWMVG-PMIVYSAFVNGATLALYNGSPLDRGFGKF---VQDAKVTMLGTVP 453Physcomitrella_3 IL

PIKAAADAWAHHDIRHRDVVAWPTNLGWMMG-PWLIYAALLNRASIALYNGAPLGYGFAKF---VQDAKVTMLGLVP 459Micromonas VL

PLHGVSDGRLHMDIKHGDVVSWPTNLGWMMG-SWLIY-QLANGACLGVYEGAPTTRGFCDF---VSDANVTHLGLVP 266Planctomyces IL

PIKSAGDGYLHHDIHAGDIVCWPTNLGWMMG-PWLVYAALINDATIALSDAVPTSRRFCEF---VQNAGVTMLGLVP 415Pedosphaera IL

PIKCAADAHFHQDIHPGDVLVWPTNLGWMMG-PWLVFASLLNRATMGLYYGAPTGAEFGRF---VQQAKATMLGVVP 355Chthoniobacter IL

PIKCAVDAHFHVNVQPADVVVWPTNLGWMMG-PWLIFGALMNRAAIGLYCGAPTGKGFGQF---VEASGATVLGLVP 356Cytophaga_1 IL

PIKCAVDGKLLQDIHAGDVVTWTSGMGWMMA-PWLIFAALLNKASIAVYGGAYSKKEFLDF---TVQTHVTVLGTIP 386Cytophaga_2 ILYTSGSTGKPKGVVHTIGGYMVYTAFSFANVFQYNEGDVYFCTADIGWITGHSYLVYGPLLQGATQVMFEGIPTYPDAGRFWSIIDKYAVTHFYTAP 342

Sorangium ILYTSGSTGKPKGVLHTTAGYLVGAHVTTKYVFDLRDDDVYWCTADVGWVTGHSYIVYGPLSNGATCLMYEGAPNFPDWGRFWRLIEKHGVTILYTAP 367Glaciecola ILYTSGSTGQPKGVVHSSGGYALYTAMTFKYGFDYREDDIYWCTADVGWITGHSYMTYGPLINGATQVFFEGVPTYPDVRRIAQVVEKYKVNSLYTAP 357Citrobacter ILYTSGSTGKPKGVLHTTGGYLVYAATTFKYVFDYHPGDIYWCTADVGWVTGHSYLLYGPLACGATTLMFEGVPNWPTPARMAQVVDKHQVNILYTAP 358Synechococcus VLYTSGSTGKPKGVVHTTAGYNLWAHLTFQWIFDIRDNDVYWCTADVGWITGHSYIVYGPLSNGATTVMYEGAPRPSKPGAFWELIQKHGITIFYTAP 400

Allomyces MLYTSGSTGKPKGLMHTTAGYLLGAALSTKYVFDVHEGDKFACVADVGWITGHSYIVYGPLALGTTTLVFEGVPTWPEPDMYWRLIQKHKLTQFYTSP 375Polysphondylium ILYTSGSTGKPKGLVHTQAGYLLYTTMTHRYVFDIQENDIYACVADVGWITGHSYIVYGPLSNGTTTFIFEGTPLHPTPSRYWEMVERHKITQFYTAP 408Cyanidioschyzon LLYTSGSTGTPKGVLHTTGGYMVNAALTFKYSFNYQPGDVYFCTADCGWITGHSYVVYGPMLNAATQVLFEGVPTWPDPGRLWAIVDKYQVTHLYTAP 468

Guillardia LLYTSGSTGKPKGVLHTTAGYMVWSATTFKYVFDYRPGDVYWCTADCGWITGHSYITYGPMINGATQVLFEGVPTHPTPARCWEIIDKYKVNLFYTAP 366Bigelowiella MLYTSGSTGRPKGILHTTGGYMVWSSLTHKWVFDYQEGDIYACVADIGWITGHSYIVYGPLCNGATSFMFESTPLYPDAGRYWDMVQRHKISSFYTAP 336

Candida LLYTSGSTGTPKGVVHTTAGYLLGAALTTKYIFDIHQQDVLFTAGDVGWITGHTYALYGPLLLGVTSVVFEGTPAYPDFGRLFKIIDDHKVTHFYIAP 371Sphaeroforma LLYTSGSTGTPKGVVHTTGGYMVYAYTTFKYVFDYQENDVFWCTADCGWITGHSYITYGPLFAGSTSIVFEGIPTYPDVGRFWEVCEKYKVTQFYTAP 368Thecamonas VLYTSGSTGRPKGVLHTSAGYLVWAMMTHKYTFDLRDDDVFACMADIGWITGHTYNCYGPLANGATSMLFESTPLYPDAGRYWDVVERHGVTQLYTAP 367

Hydra ILYTSGSTGKPKGVQHSTGGYLLWAKLTMDWTFDLKPEDVFWCTADIGWITGHTYVAYGPLAAGATQIIFEGVPTFPNAGRFWQMIERHKCTIFYTAP 336Antonospora CLYTSGSTGRPKGIIHTTAGYLLYVSMTLKTCFDFQEDDVMGCTADLGWITGHSYSMYAPLLLNGTTIIFGGSPLFPSEFRLFEMIDKHRVTHLYTAP 355

Bacillus LLYTSGTTGKPKGAVHTHSGFPIKAAFDAGIGMDVKREDVLFWYTDMGWMMG-PFLVYGGLVNGATILLYEGTPDFPNPDRIWELVAKHNVSHLGISP 361Thermus LIYTSGTTGRPKGTVHYHAGFPLKAALDLALLFDLREGDRLFWFTDLGWMMG-PWAILGGLILGATVFLYDGAPDYPGPERLWRMVEAHRLTHLGLSP 346Desmospora IIYTSGTTGRPKGTVHVHAGFPVKSGFDAGYSMDVKAGDILFWMTDMGWMMG-PWMVFGTLMNGATMLLFEGTPDYPEPDRLWKLVDAHGVTHLGVSP 362Burkholderia LMYTSGTTGKPKGTVHSHCGLITKLALDMGLCADMRAGDRLMWLSDMGWLVG-PMLIYGTTLLGGTIVMAEGAHDFPDSGRFWRLMEQHRVSVLGIAP 372Sphaerobacter IIYTSGTTGRPKGAVHTHAGFPIKAAHDLAFCFDLQPDDTLFWITDLGWMMG-PWAIEGTLMLGATLLLYEGTPDYPEPDRLWQVVERHGATVLGVSP 373Haloterrigena LLYSSGTTGKPKGIVHTHAGVQVQCAKEVYFGMDLKPSDRFFWVSDIGWMMG-PWTLIGTHTFGGTVFMYEGAPDHPEPDRFWEMIDRHELTQFGISP 407Pyrobaculum IIYTSGTTGKPKGTVHTHDGFPVKAAADVYFHFDVSEGETLSWVTDMGWMMG-PWMVFAAYLLRGSMAFFEGAPDYPK-DRLWRFVERFKVNALGLAA 343

KAAT

FS

FS

FS

FS

FS

FS

FS

FS

FS

FS

FS

FS

SGTTGEPKAI

PW

PW

PW

PW

PW

PW

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PW

PW

PW

PW

PW

SQLS

NQTT

-

-

-

-

-

-

-

-

-

-

-

-

PMRCAADSWAHFDLQAGDIYCWPTNLGWMVG-PYIISACLLSGATMALYNGSPLGRSFGRF---VQDARVTILGTVP 459Arabidopsis IL

SGTTAEPKAI

SGTTGEPKAI

TQLS

SGTSGEPKAI

THVA

SGTTGEPKAI

TQHA

SGTTGEPKAI

TQHT

SGTTGDPKAI

THAT

SGTTGAPKAI

DHSA

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DQTT

SGTTGEPKAM

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b

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90/78 Selaginella Physcomitrella

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57/* Selaginella 100/70 Physcomitrella

Physcomitrella 100/100 Arabidopsis

Arabidopsis

Micromonas Planctomyces Planctomycetes 98/93 Chthoniobacter

Pedosphaera

Cytophaga CFB group bacteria Burkholderia Beta-proteobacteria 59/* Pyrobaculum Crenarchaeotes

Rhodothermus CFB group bacteria 61/* Bacillus

Desmospora Sphaerobacter GNS bacteria

Thermus Deinococci */64 Rhodococcus High GC Gram+

Haloterrigena Euryarchaeotes

Nitrosoarchaeum Archaea

52/74

50/*

Green plants

Verrucomicrobia

57/*

100/100

56/81

51/*

73/96

57/*

92/87

Figure 2 | Multiple sequence alignment (a) and molecular phylogeny (b) of acyl-activating enzymes 18 (AAE18). Boxed columns indicate the amino-acid residues and indels shared by bacterial and green plant AAE18 sequences. Numbers above branches show bootstrap values from maximum likelihood and distance analyses, respectively. Asterisks indicate values < 50%.

97/100

Firmicutes

98/98 Phytophthora OomycetesEctocarpus Brown algae Bigelowiella Rhizaria

Polysphondylium Mycetozoa

Candida Ascomycetes Capitella Metazoa

Acetobacter Alpha-proteobacteria

98/100 Edwardsiella Citrobacter

Pseudomonas

Glaciecola 79/90 Parvibaculum

56/* Labrenzia Rhodothermus Cytophaga

Cyanidioschyzon Red algae Euglena EuglenozoaHydra Hydrozoans Sulfurihydrogenibium Aquificales

Gamma-proteobacteria

Gamma-proteobacteria

Alpha-proteobacteria

CFB group bacteria

60/58 Lyngbya Nostoc

Cyanothece Synechococcus

Sorangium Delta-proteobacteria Thermus Deinococci

85/94

100/100

Cyanobacteria

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0.2

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(Fig. 2a). Intriguingly, both the production and inhibition of auxin may be aected by the expression of acquired genes. In Arabidopsis, the bacteria-derived arginase (see above) may negatively regulate the production of auxin by reducing the level of nitric oxide, which in turn mediates the induction of auxin in roots41.

Several other acquired gene families identied in our analyses are related to plant defence and stress tolerance (Table 1). Notably, glutathione is essential for plant disease resistance, photo-oxidative stress defence and heavy metal detoxication42. Glutamatecysteine ligase (GCL) is the rst of the two enzymes catalysing the formation of glutathione. Identiable homologues of P. patens GCL are only present in green plants and bacteria. Our phylogenetic analyses also show that the GCL gene was acquired from bacteria (Supplementary Fig. S5), which is consistent with an earlier report23. In addition, at least three gene families acquired from bacteria, including guanine deaminase, allantoate amidohydrolase and ureidoglycolate amidohydrolase43, are involved in purine degradation and nitrogen recycling (Table 1; Supplementary Figs S6 and S7). Furthermore, another acquired gene, glutamine synthetase, is directly responsible for assimilating ammonia into amino acids in plants (Supplementary Fig. S8).

Discussion

Conventional belief is that HGT is frequent in unicellular eukaryotes but rare in multicellular eukaryotes because of the barriers of germline and apical meristem. Although evidence of HGT in multicellular eukaryotes is still limited, there have been numerous reports of acquired genes (including those of viral and bacterial origins) in mitochondrial genomes of seed plants44,45. These viral and bacterial genes were integrated into mitochondria and passed onto descendants ultimately through the apical meristem. Such observations, combined with other relatively recent HGT events reported in plants13,17 and animals16,18,46, suggest that neither germline nor apical meristem constitutes an insurmountable barrier to HGT.

The nding of 18 recently acquired gene families in mosses also raises questions why more foreign genes exist in this lineage and whether recent HGT of nuclear genes also occurs in other land plants. We reason that the acquisition of genes by mosses might largely be attributed to the unique evolutionary position and biological features of this lineage. As mosses were among the rst dwellers on land, they might have encountered hostile environments with intense ultraviolet radiation47, which could break large DNA molecules into small fragments and release them into the environment. It is also known that mosses are eective in DNA transformation48. This ability to uptake foreign DNA, including benecial genes from co-inhabitants, likely facilitated the establishment of these early land plants in a hostile and shiing environment. In addition, these early land plants formed mycorrhizal association with diverse fungal species30,49, and this symbiotic relationship provided further opportunities for gene transfer between fungi and early land plants25.

Mosses also have distinct and dominant gametophytes in their lifecycle. As one of the earliest plant groups on land, mosses lack true vascular systems and complex protective structures for gametes and zygotes. We hypothesize that at least two entry points exist for foreign genes to be acquired and integrated into the moss nucleus (Fig. 3). The rst entry point for acquired genes is the stage of spore germination and early gametophyte development. Moss gametophytes are developed from haploid spores through mitosis. These gametophytes are simple, oen relatively undierentiated and prostrate in direct contact with soil surface, thus providing ample opportunities to uptake foreign DNA. In such cases, any genes acquired during spore germination and the early stage of gametophyte development could potentially be propagated into adult gametophytes, which bear either antheridia or archegonia or both. In the latter case, fertilization may also occur on the same gametophyte and lead to the xation of acquired genes into zygotes and sporophytes.

The second likely entry point for acquired genes in mosses is the stage of fertilization and early embryo development. Unlike seed plants where eggs are protected within ovules and fertilization entails a precise mechanism for pollen tube elongation and sperm delivery, mosses conceal eggs in single-layered and hollow archegonia, which are open during fertilization. Any foreign genes transferred from the exterior environment to exposed zygotes and young embryos will likely be xed and passed onto adult sporophytes.

The above model presumes that organisms with unprotected or weakly protected zygotes in their lifecycles are prone to HGT. This model predicts the existence of recently acquired genes in plants with independent, though sometimes reduced, gametophytes such as nonvascular and seedless vascular plants. Given the gradual transition of these early-branching land plants toward seed plants, this model also predicts the existence of anciently acquired genes in gymnosperms and angiosperms, where fertilization and embryogenesis are structurally internalized. It should be noted here that even such structural internalization might not entirely exclude recent HGT in gymnosperms and angiosperms. It is conceivable that pollen grains from distantly related plants may be deposited on the stigma of another plant, allowing foreign pollen DNA the chance to be transformed into the zygote and the young embryo17.

The increasing structural complexity of land plants has been accompanied by diversied metabolic pathways and their chemical output. Like other complex multicellular eukaryotes, plants are able to form distinctive structures and coordinate development throughout their lifecycle. Our data clearly show that HGT contributed greatly to the metabolism, development and regulation of land plants. For example, members of the subtilase family participate in many biological processes, including protein degradation in seeds and fruits, lateral root formation, xylem dierentiation, cuticle and epidermal development and stomata pattern formation5052. Likewise, polyamines are involved in numerous important biological activities in plants such as translation, cell proliferation and signalling, ion channel regulation, and stress response53,54. Furthermore, plant hormones have a vital role in regulating cell dierentiation and structural development.

Land plants are also diverse in morphology, life history and habitat, and they have evolved many adaptive traits essential for their survival and development. Particularly during their transition from aquatic to terrestrial environments, plants evolved features to not only tolerate abiotic stress such as desiccation, uctuating temperature and nutrient limitation, but also defend themselves against herbivory and microbial infection. Many of the acquired genes identied in our analyses are either directly or indirectly related to plant defence and stress tolerance. For instance, polyamines not only regulate calcium homeostasis and stomatal closure, but also are involved in plant tolerance to abiotic stress such as drought, salt and cold53. Given the role of arginase in polyamine biosynthesis, the acquisition of the arginase gene might benet plants greatly as they adapt to water shortages, salinity and uctuating temperatures on land. Similarly, the involvement of subtilases in the development of lateral roots, cuticle and stomatal cells also points to an important role of this gene family in water conduction as well as protection from desiccation and microbial infection in land plants. Additionally, several gene families identied in our analyses are functionally related to DNA replication and repair (Table 1; Supplementary Figs S9, S10, S11 and S12). Given the fact that early land plants faced ubiquitous and intense ultraviolet radiation on earth surface47 (which might cause DNA damage and consequently interrupt the normal cell cycle of plants), the acquisition of these genes may have conferred early land plants additional abilities to x DNA damage and facilitate their survival. Such DNA repair-related genes have also been demonstrated to be of preferential uptake in some bacteria55.

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Sporophyte

Sporophyte cell

HGT

Gametophyte cell

Female gametophyte

Foreign DNA

Zygote

Gametophyte cell

Female gametophyte

Fertilization

Diploid stage

Haploid stage

Meiosis

Spores

Sperm

Antheridium

Mitosis

Young gametophyte

Gametophyte cell

Gametophyte cell

Mature gametophyte

Egg

HGT

Archegonium

Foreign DNA

Figure 3 | A hypothetical scheme of HGT in mosses. Two entry points for foreign genes into the moss genome are proposed. The rst entry point is spore germination and the early stage of gametophyte development. The second entry point is fertilization and the early stage of embryo development. This model is also applicable to other nonvascular plants and seedless vascular plants that have independent gametophytes. DNA acquired from foreign sources through the two entry points is shown in red and blue, respectively. Dash lines show the status of acquired genes in different stages of the lifecycle.

The cumulative impact of acquired genes depends critically on the number of such genes accumulated in a taxon. Genes acquired by any ancestral organism, if benecial, are likely to be retained in descendent lineages. Indeed, 35 gene families identied in P. patens are also present in seed plants. Likewise, a considerable number of genes were transferred independently from bacteria during the early evolution of Plantae31,32. These data indicate that HGT is a dynamic process with foreign genes gradually accumulating over time (Fig. 4). Such gradual accumulation of foreign genes in plants also suggests that anciently acquired genes are more frequent than commonly expected.

Eukaryotic evolution has been signicantly shaped by the origins of mitochondria and plastids, which routed numerous bacterial genes to the nucleus. Although such EGT events are oen considered to be a dominant force in eukaryotic genome evolution, the sources of transferred genes are intrinsically constrained by the gene pool of mitochondria and plastids. With organellar genomes becoming increasingly reduced, the process of EGT will eventually approach to a dead end. The lack of such constraint for HGT, on the other hand, may potentially introduce genes of numerous sources and functions. The acquired genes identied in our analyses and their participation in diverse biological processes of land plants suggest a widespread and profound impact of HGT on the evolution of multicellular eukaryotes.

Methods

Data sources and genome screening. The annotated genome of P. patens was downloaded from the Joint Genome Institute. A customized database was created

to search for P. patens gene homologues. In addition to NCBI non-redundant(nr) protein sequences, this customized database also included other sequenced genomes and expressed sequence tags from diverse eukaryotes (Supplementary Table S2). Assembling of expressed sequence tag sequences was carried out using CAP3, and the resulting consensus sequences were translated using the OrfPredictor web server (http://proteomics.ysu.edu/tools/OrfPredictor.html). Genome screening for candidates of acquired genes was performed using a newly developed soware package AlienG34 with P. patens annotated protein sequences as query. AlienG presumes that sequence similarity is correlated to sequence relatedness. Therefore, if a query sequence is signicantly more similar to homologues from distantly related organisms than to those from close relatives, it will be considered a candidate of acquired genes. Genes that are only detected in the query and potential donor groups (default E-value cuto 1e-6) will also be identied. In this study, the signicantly higher sequence similarity to homologues from a donor group was empirically set to a bit score ratio of over 1.5. All candidate genes identiedby AlienG were subject to further sequence re-sampling and manual phylogenetic analyses to determine their evolutionary origins.

Determining the origin for candidates of acquired genes. For each candidateof acquired genes identied by AlienG, we rst checked the scaold on whichthe gene was located. Because of the potential contamination in the process of genome sequencing, any candidate of acquired genes located on a short scaold was removed from further consideration. Detailed phylogenetic analyses, including sequence re-sampling from our internal customized sequence database, were performed for each of the remaining candidates. Taxonomic distribution of sequence homologues was also investigated. Because of the bacterial nature of mitochondria and plastids, we also investigated if other eukaryotic homologues were mitochondrial or plastid precursors, which oen suggest a bacterial origin (see Supplementary Information). Additionally, each alignment was carefully inspected for rare genomic characters that might indicate a close affinity between the candidate gene and homologues from the putative donor. A candidate gene was determined to be horizontally acquired based on (1) gene tree topology that shows a green plant/donor clade with bootstrap support of over 80% from maximum likelihood

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Eukaryotes

Green plants

Amoebozoa

Fungi

Animals

Euglenozoa

Apicomplexa

Diatoms

Red algae

Green algae

Bacteria Archaea Land plants

Mosses

Gene functions

DNA replication and repair Pathogen resistance

Gene functions

Vascular development Cuticle and epidermal developmentAuxin biosynthesis Lateral root formation Stomatal pattern formation Herbivore resistance Abiotic stress tolerance

Gene functions

Plastid biogenesis and development Alcohol fermentation

Figure 4 | Diagram illustrating the dynamics of HGT in plants. Horizontal lines and arrows show HGT donors and recipients. Information about HGT in the ancestor of red algae and green plants is based on31,32.

or distance analyses or both, (2) taxonomic distribution of homologues only in the putative donor group (bacteria, archaea, viruses or fungi) and (3) unique domain structures, indels or amino-acid residues shared with homologues from the putative donor group.

Phylogenetic analyses. Multiple protein sequence alignments were performed using MUSCLE and clustalX, followed by manual renement. Gaps and ambiguously aligned sites were removed manually (alignments are available from the authors on request). Sequences that caused aberrant alignments and whose real identity could not be conrmed were also removed from alignments. Phylogenetic analyses were performed with a maximum likelihood method using PhyML 3.0 (ref. 56) and a distance method using neighbour of PHYLIPNEW v.3.68 (ref. 57) in EMBOSS package. ModelGenerator58 was used to select the available model of protein substitution and rate heterogeneity that best t each data set. Bootstrap support values were estimated using 100 pseudo-replicates. Maximum likelihood distances for distance analyses were calculated using TREE-PUZZLE v.5.2 (ref. 59) and PUZZLEBOOT v.1.03 (A. Roger and M. Holder, www.tree-puzzle.de). The models used in maximum likelihood and distance analyses are the same in most cases. If the best model selected by ModelGenerator was not implemented in TREE-PUZZLE, the second best model was used. All other parameters in the analyses used default settings.

Functional annotation. Whenever possible, functional annotation of the acquired genes followed the information provided by The Arabidopsis Information Resources (TAIR) (www.arabidopsis.org) and published experimental data. Homologous gene loci in Arabidopsis were also obtained from TAIR.

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Acknowledgements

This work is supported in part by a NSF Assembling the Tree of Life (ATOL) grant (DEB 0830024) and the CAS/SAFEA International Partnership Program for Creative Research Teams.

Author contributions

J.H. conceived and designed the study and wrote the manuscript. J.Y. performed the analyses and wrote the manuscript. X.H., H.S. and Y.Y. contributed to data analyses and manuscript writing. All authors read and approved the nal manuscript.

Additional information

Supplementary Information accompanies this paper at http://www.nature.com/ naturecommunications

Competing nancial interests: The authors declare no competing nancial interests.

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How to cite this article: Yue, J. et al. Widespread impact of horizontal gene transfer on plant colonization of land. Nat. Commun. 3:1152 doi: 10.1038/ncomms2148 (2012).

License: This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/

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