Horizontal gene transfer is more frequent with PNAS PLUS increased heterotrophy and contributes to parasite adaptation

Zhenzhen Yanga,b,c,1, Yeting Zhangb,c,d,1,2, Eric K. Wafulab,c, Loren A. Honaasa,b,c,3, Paula E. Ralphb, Sam Jonesa,b, Christopher R. Clarkee, Siming Liuf, Chun Sug, Huiting Zhanga,b, Naomi S. Altmanh,i, Stephan C. Schusteri,j, Michael P. Timkog, John I. Yoderf, James H. Westwoode, and Claude W. dePamphilisa,b,c,d,i,4 aIntercollege Graduate Program in Biology, Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA 16802; bDepartment of Biology, The Pennsylvania State University, University Park, PA 16802; cInstitute of Molecular Evolutionary Genetics, Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA 16802; dIntercollege Graduate Program in Genetics, Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA 16802; eDepartment of Plant Pathology, Physiology and Weed Science, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061; fDepartment of Plant Sciences, University of California, Davis, CA 95616; gDepartment of Biology, University of Virginia, Charlottesville, VA 22904; hDepartment of Statistics, The Pennsylvania State University, University Park, PA 16802; iHuck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA 16802; and jDepartment of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802

Edited by David M. Hillis, The University of Texas at Austin, Austin, TX, and approved September 20, 2016 (received for review June 7, 2016) Horizontal gene transfer (HGT) is the transfer of genetic material diverse angiosperm lineages (13, 14) and widespread incorpo- across species boundaries and has been a driving force in prokaryotic ration of fragments or entire mitochondrial genomes from algae evolution. HGT involving eukaryotes appears to be much less frequent, or moss sources by the giant Amborella mitochondrial genome and the functional implications of HGT in eukaryotes are poorly (15, 16). Active recombination processes and an absence of ge- understood. We test the hypothesis that parasitic , because of nomic downsizing pressures to remove excess sequences are their intimate feeding contacts with host plant tissues, are especially probably important factors in mitochondrial HGT in plants (11, prone to horizontal gene acquisition. We sought evidence of HGTs in EVOLUTION transcriptomes of three parasitic members of , a plant 16, 17), but additional steps are required to increase the likeli- family containing species spanning the full spectrum of parasitic hood of integrated sequences being propagated through sexual capabilities, plus the free-living Lindenbergia. Following initial phy- reproduction (16). logenetic detection and an extensive validation procedure, 52 high- confidence horizontal transfer events were detected, often from Significance lineages of known host plants and with an increasing number of HGT events in species with the greatest parasitic dependence. Anal- Horizontal gene transfer (HGT) is the nonsexual transfer and yses of intron sequences in putative donor and recipient lineages genomic integration of genetic materials between organisms. In provide evidence for integration of genomic fragments far more eukaryotes, HGT appears rare, but parasitic plants may be ex- often than retro-processed RNA sequences. Purifying selection pre- ceptions, as haustorial feeding connections between parasites dominates in functionally transferred sequences, with a small fraction and their hosts provide intimate cellular contacts that could fa- of adaptively evolving sites. HGT-acquired genes are preferentially cilitate DNA transfer between unrelated species. Through anal- — — expressed in the haustorium the organ of parasitic plants and are ysis of genome-scale data, we identified >50 expressed and strongly biased in predicted gene functions, suggesting that expres- likely functional HGT events in one family of parasitic plants. sion products of horizontally acquired genes are contributing to the HGT reflected parasite preferences for different host plants and unique adaptive feeding structure of parasitic plants. was much more frequent in plants with increasing parasitic de- pendency. HGT was strongly biased toward expression and HGT | phylogenomics | validation pipeline | genomic transfer | parasitism protein types likely to contribute to haustorial function, sug- gesting that functional HGT of host genes may play an impor- orizontal gene transfer (HGT) is the movement and geno- tant role in adaptive evolution of parasites. Hmic integration of genetic material across strong species boundaries. HGT involving prokaryotes (1) has been repeatedly Author contributions: Z.Y., Y.Z., and C.W.d. designed research; Z.Y., Y.Z., and E.K.W. per- associated with adaptive evolution (2), such as the acquisition of formed research; L.A.H., P.E.R., S.J., C.S., H.Z., and S.C.S. contributed new reagents/analytic tools; P.E.R., S.J., S.L., and H.Z. harvested tissues and RNAs and prepared the libraries; S.J. and antibiotic resistance (3), resistance to heavy metal (4), and pes- H.Z. performed validation experiments; S.C.S. provided genomic sequences; C.W.d. super- ticide degradation (5). Although there was a massive endosym- vised the whole process of data analysis; Z.Y., Y.Z., and E.K.W. analyzed data; N.S.A. helped biotic transfer of genes into the nuclear genome from eubacterial design the statistical analyses; and Z.Y., L.A.H., and C.W.d wrote the paper with contributions ancestors of plastids (6) and mitochondria (7), relatively few cases from Y.Z., E.K.W., P.E.R., S.J., C.R.C., S.L., C.S., H.Z., N.S.A., S.C.S., M.P.T., J.I.Y., and J.H.W. of functional eukaryote-to-eukaryote HGTs have been detected or The authors declare no conflict of interest. studied in detail (8). This article is a PNAS Direct Submission. As HGT is detected in eukaryotic genomes with greater frequency, Data deposition: Sequence data are archived at National Center for Biotechnology In- it is becoming increasingly possible to address questions of the formation BioProject (ID codes SRP001053 and SRP083761) and at ppgp.huck.psu.edu. Scripts and alignments were submitted to github user “dePamphilis” under mechanism, function, and potentially adaptive significance of HGTs. directory “HGT_PNAS_2016.” In plants, the horizontal acquisition of genes from microbial sources 1Z.Y. and Y.Z. share senior authorship. has been hypothesized to play an important role in early land plant 2Present address: Department of Genetics and the Human Genetics Institute of New evolution (9). HGT among plant lineages may also occur; a notable Jersey, Rutgers, the State University of New Jersey, Piscataway, NJ 08854. example involves the adaptive transfer of a photoreceptor gene from 3Present address: Tree Fruit Research Laboratory, US Department of Agriculture–Agricultural bryophytes that enabled ferns to adapt to low-light conditions (10). Research Service, Wenatchee, WA 98801. Reported HGT events in plants most commonly involve 4To whom correspondence should be addressed. Email: [email protected]. — mitochondrial sequences (11, 12) for instance, the repeated This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. invasion of mitochondrial coxI by a group I homing intron in 1073/pnas.1608765113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1608765113 PNAS Early Edition | 1of10 A second apparent concentration of horizontally acquired three models, we identified “ancestral” nodes, defined here as the sequences in plant genomes is associated with parasitic plants, node containing exclusively parasite genes and genes in the donor where both mitochondrial (14, 17–23) and nuclear horizontal clade. To allow for incomplete sampling of all donor lineages and transfers (24–29) have been identified. Repeated horizontal ac- complexities of gene family evolution, we used a loose initial quisitions of mitochondrial atpI by parasitic flowering plants bootstrap support (BS) cutoff of 50 in the initial phylogenomic occurred in four extreme parasite lineages where the parasite screening. The first model describes a scenario where genes from lives inside the host for much of its lifespan (14). The intimate the or nonparasitic relative are supported (BS ≥ 50) contact between parasitic plants and their host plants, facilitated as nested within a donor clade (Fig. 1A). The second model (Fig. by the haustorium, a conspicuous adaptation of parasitic plants 1B) describes a case where the parasite’s genes are placed outside (30), may increase the likelihood of genetic exchange, especially of the donor clade. In both cases, two nodes supporting (BS ≥ 50) in parasites with direct phloem–hloem connections (14, 18). The the grouping of parasitic genes with donor clades were required. observation of massive mRNA movement between parasitic The third model (Fig. 1C) requires only one node supporting the Cuscuta and its host (31) suggests a likely route of HGT via placement of the parasite’s gene(s) as sisters of donor clades. mRNA intermediates. We thus hypothesize that parasitic plants act as nexus points for HGT and that HGT will be more frequent High-Confidence HGT Events. Custom scripts searching for topolo- in more nutritionally dependent parasite species. gies consistent with these three models resulted in the identifica- Among all of the parasitic lineages (at least 11 independent ori- tion of a set of 192 gene trees with preliminary evidence for HGT gins) of flowering plants (14), Orobanchaceae is the only family that (143 orthogroups with potential HGTs from rosids and 49 with has a complete spectrum of parasitic capabilities (30) and thus is potential HGTs from monocots) in the three focal species. Only ideal for testing these predictions. Four cases of nuclear gene HGT one orthogroup (3861) was identified as including a potential have been identified in this family to date (26–29),butthisislikely HGT in the nonparasitic “control” species Lindenbergia; all others to be a small fraction of the number of events. This is due to the involved the parasitic species only. We then applied a scoring challenges in HGT discovery and to the lack of a well-established approach for use with complex plant genomes. The previous three cases were discovered by using a BLAST-based detection approach (26–28), which impeded discovery on a large scale because BLAST- AB based predictions are subject to high false positive errors from in- complete sampling in the database as well as extensive follow-up work to identify erroneous results. Inspired by Xi et al. (25), who used a phylogenomic approach for HGT inferences in parasitic Rafflesia, we developed a robust phylogenomic pipeline for HGT detection followed by a comprehensive validation procedure to re- veal the extent of HGT in three parasitic Orobanchaceae with dif- ferent degrees of parasitic dependence. However, HGT inferences C with gene trees can also suffer from high error rates due to factors including misidentification of contaminant sequences, insufficient taxon sampling, complex gene birth and death processes, gene tree errors, inappropriate rooting, and frame-shift errors. We thus took these factors into account in our pipeline and carefully evaluated D E each potential HGT candidate with additional data to identify and reduce potential sources of error. We focus on potentially functional genes acquired by parasitic plants by concentrating our search on extensive transcriptome evidence for members of Orobanchaceae grown on host plants with known genome sequences (30, 32, 33); in addition to greatly improving the accuracy of HGT identification, it offers the opportunity to identify the expression patterns associated with genes derived from HGT events (28). Results and Discussion Analytical Schema for Detection of HGT. An initial phylogenomic Fig. 1. Three models for phylogenomic identification of HGTs and further screen was used to identify putative HGTs from transcriptome as- examination of the preliminary-screened HGT candidates. (Scheme 1) Para- semblies of parasitic plants. This approach used an automated sitic genes (P) are nested inside donor clades (D). (Scheme 2) Parasitic gene pipeline to build phylogenetic trees for each approximate gene group outside of the donor clade. (Scheme 3) Only one node of donor se- family (orthogroup), containing genes from 22 representative se- quence is sister to parasite genes. In this study, donor refers to distantly related monocot and rosid sequences. Ancestral node is defined to be quenced plant genomes and six transcriptomes (32). Four species of composed of exclusively parasitic and donor sequences. In A1, at least two Orobanchaceae were tested, including the nonparasitic Lindenbergia, nodes within the ancestral node (including the ancestral) are required to and three parasitic plants with increasing degrees of parasitic have BS ≥ 50; in A2, both the ancestral node and node 1 are required to have dependence— versicolor (facultative hemiparasite— BS ≥ 50. In scheme 2 (B), the ancestral node and at least one node within the partly heterotrophic when attached to host), hermonthica ancestral node are required to have BS ≥ 50. In scheme 3 (C), only the node (obligate hemiparasite—photosynthetic but most carbon derived that supports the grouping of the parasitic gene and donor sequence is from host), and Phelipanche aegyptiaca (Syn. Orobanche aegyptiaca) required to have BS ≥ 50. “Non-DPs” refers to nonparasitic, nondonor se- (obligate holoparasite—nonphotosynthetic and fully heterotrophic) quences. (D) A total of 192 HGT orthogroup trees from the initial screening (30). Three models illustrate topologies indicative of HGTs we were classified into low-, medium-, and high-confidence categories based on a scoring scheme (SI Appendix, Table S1). Gray colors represent the HGT sought to detect (Fig. 1). With a goal of identifying unambiguous orthogroups identified in the monocots; darker gray colors represent the HGTs, we focused our search on rosid and monocot donors be- rosids. (E) The number of HGT candidate orthogroups manually curated as cause of the relatively large number of finished genomes in these true HGTs (light gray); artifacts resulting from insufficient taxon sampling, groups and the relatively large genetic distance from Orobancha- frame shift errors, or tree inaccuracies (white); or fungal or host contami- ceae (as all the parasites are ) (SI Appendix,Fig.S1). In all nation (dark gray). The 42 “true” HGT orthogroups all fit scheme 1 of A.

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1608765113 Yang et al. scheme to assign these 192 candidate HGT trees to low-, medium-, the placement of parasitic genes within the donor clade, indicating PNAS PLUS and high-confidence groups based on tree characteristics in- a clear HGT origin (Figs. 2A and 3A and SI Appendix,Fig.S2). cluding the BS for key nodes surrounding the inferred HGT event, For instance, in Fig. 2A, StHeBC3_10075.1 from S. hermonthica is sampling of the donor clades grouped with the HGT genes, and placed within a grass clade with the proximal node supported with branch length heterogeneity (Fig. 1D;seeSI Appendix,TableS1 BS 100, and two additional deeper nodes with BS 100, supporting for detailed criteria). The authenticity of the 158 HGT trees in the a strong case of HGT. Eleven out of 42 trees suggested a poly- medium- and high-confidence groups was then validated with phyletic origin of HGT genes (multiple distinct transfers within a follow-up analyses, including manual examination of branch gene family). Thus, we used the Shimodaira–Hasegawa (SH) test lengths and sequence alignments, correcting any translation or (37) to evaluate whether more than one transfer was most likely alignment errors, and examining the phylogenetic stability with based on the available data. A single transfer could not be rejected increased taxon sampling. We examined low-coverage tran- for orthogroup 3861 (SI Appendix,Fig.S2.11). Interestingly, the scriptome sequences from eight more parasitic Orobanchaceae other 10 trees each supported more than one transfer (SI Ap- species and also from 10 related nonparasitic taxa from pendix,TableS2), suggesting a propensity for certain gene families the 1kp transcriptome project (34), four sequenced asterid ge- to include successful horizontal transfers. A minimum of 52 hor- nomes (Phytozome) (35), and the Striga asiatica genome (under izontal transfer events were thus inferred from these 42 gene github “dePamphilis/HGT_PNAS_2016”) (see Methods, HGT families. That a majority, fully 78%, of the candidates (67% of Validation by Increased Taxon Sampling). This resulted in a final initial medium and high confidence) were (i) excluded due to low set of 42 HGT orthogroups (SI Appendix, Fig. S2), with the support, (ii) identified as artifacts (via increased taxon sampling), remaining 150 putative HGT orthogroups determined to be arti- or (iii) identified as contamination from host or other organisms factual or merely low confidence (Fig. 1E and SI Appendix,Fig. illustrates the challenge of accurate HGT discovery. S4). The primary source of artifacts (106 out of 150) was insuffi- Our analyses with explicit phylogenetic schema and stringent cient taxon sampling (SI Appendix,Fig.S4A and B), especially of evaluation by the use of increased taxon sampling represent a the order (Lamiales) that contains Orobanchaceae (36). Other robust approach for HGT identification. In addition, it includes artifacts came from frame-shift errors (SI Appendix,Fig.S4D and two of the published high-confidence HGT cases (26, 28). Two E) and contamination, either from the experimental host (nine other published HGTs in Orobanchaceae (27, 29) were detected orthogroups) (SI Appendix,Fig.S4C) or from fungal contamina- in the stage 4-specific assembly (28) but not in the (generally tion (one orthogroup). The topology and BS values for the 42 more complete) combined assembly we examined in this study. EVOLUTION HGT orthogroup trees strongly support (gene trees all fit model 1) In addition, these 42 orthogroups include six orthogroups

A Phelipanche_aegyptiaca_24772.1 B rosid 100 Striga_asiatica_105G00190 asterid Striga_asiatica_147G00190 basal eudicot 100 Striga_asiatica_35G00460 monocot Striga_asiatica_81G00250 Striga_asiatica_79G00260 basal angiosperm Striga_asiatica_71G00860 Striga_asiatica_80G00330 C 100 20 Striga_asiatica_430G00030 25 HGT recipient Lactuca_sativa_28322 2 Lactuca_sativa_45833 Phelipanche (Phe) 94 Lactuca_sativa_1379 3 97 Striga (Str) Lactuca_sativa_6044 20 Lactuca_sativa_9873 Triphysaria (Tri) 69 Lactuca_sativa_20530 82 Lactuca_sativa_46312 Lindenbergia (Lin) 49 Helianthus_annuus_60209 15 100 76 Helianthus_annuus_28195 Lactuca_sativa_42718 20 Lactuca_sativa_19173 8 95 10 Lactuca_sativa_23853 # of orthogroups 79 Lactuca_sativa_51707 7 Medicago_truncatula_7g061520.1 Medicago_truncatula_AC235757_20.1 5 48 Medicago_truncatula_4g116690.1 7 32 Medicago_truncatula_4g116510.1 3 1 1 Medicago_truncatula_2g061140.1 0 2 98 Medicago_truncatula_6g055350.1 Medicago_truncatula_6g079030.1 Poaceae 96 Glycine_max_16289845 RosaceaeFabaceae MalvaceaeSalicaceae 97 Brassicaceae Glycine_max_16290054 Euphorbiaceae Medicago_truncatula_8g054330.1 Striga_hermonthica_10075.1 Inferred HGT Donor 100 Orysa_sativa_01g60580.1 Orysa_sativa_10g41350.1 D Orysa_sativa_10g31130.1 15 15 68 76 Brachypodium_distachyon_3g24430.1 Brachypodium_distachyon_1g64655.1 92 Sorghum_bicolor_1970319 8 Sorghum_bicolor_1983153 100 4 3 Sorghum_bicolor_1969324 2 Brachypodium_distachyon_1g25296.1 1 1 04812 100 Sorghum_bicolor_1958959 # of orthogroups Brachypodium_distachyon_5g16030.1 Phe Str 74 Brachypodium_distachyon_2g07670.1 Brachypodium_distachyon_1g54870.1 Phe, Oro Phe, StrPhe, Tri 99 Brachypodium_distachyon_3g03300.1 Phe, Oro, AleLin, Phe, Oro 89 Brachypodium_distachyon_2g57400.1 Phe, Str, Tri, Ale Brachypodium_distachyon_1g38060.1 0.1

Fig. 2. RAxML-based maximum likelihood (ML) trees supporting HGT, donor families, and recipient taxa inferred from the 42 HGT set. Orthogroup tree (12835) supports a grass-derived Pong-like TE in S. hermonthica. HGT sequence is labeled with “H” and vertically inherited sequences with “V.” The species abbreviations are shown in SI Appendix, Fig. S1.(B) A hypothetical tree illustrates the color-coding system for each angiosperm lineage represented in A and Fig. 3. (C) Mapping of parasitic recipient taxa onto inferred donor family (x axis). Each in HGT recipient is followed with a three-letter code used in D. Total number of HGT orthogroups inferred from each donor family is placed on top of each bar. Numbers within each bar represent number of orthogroups; the number of singletons is not shown due to space limitations (SI Appendix, Table S9). (D) Number of HGT orthogroups supports transfers from shared and unique parasitic genera. Ale, Alectra; Lin, Lindenbergia; Oro, Orobanche; Phe, Phelipanche;Str,Striga; Tri, Triphysaria.

Yang et al. PNAS Early Edition | 3of10 AB

C

D

Fig. 3. Genomic horizontal transfer of a tRNAHis guanylyltransferase from Frave (Fragaria) (or its ancestor) to Phelipanche.(A) A coding-sequence (CDS) tree by RAxML from represented species across angiosperm lineages. D, inferred donor (in Fragaria); H, the parasitic HGT gene; V, vertical parasitic gene; VR, related sequence of the vertical parasitic gene (in Mimulus). Seventy-four percent represents the CDS similarity between the HGT gene and its inferred donor. (B)Gene structure with four selected introns for the four sequences (H, D, V, and VR). Yellow and green bars represent coding sequence; the vertical dashed lines represent the intron positions; the boxes represent introns. At least four conserved intron positions were shown on the gene structure; the third intron was lostintheHGT gene, and the fourth intron on the graph (which is the seventh intron of the Mimulus gene) showed strong sequence similarity between the HGT gene and its donor (marked by red intron boxes with length within). (C) The phylogeny of the seventh intron (marked red in B) from genes on the CDS tree: the HGT gene groups with its donor supported by 98% BS, whereas the vertically inherited gene groups with a close relative (Mimulus sequence). The intron sequence similarity between the HGT (HGT) gene and its donor (D) is 51%, and the intron sequence similarity between the vertical gene (V) and its vertical relative (VR) is 21%. (D) The number of HGT orthogroups that support 25 genomic transfers in 24 HGT orthogroups containing introns in the CDS region (dark blue) and 2 HGT orthogroups containing introns in the UTR region (pink). The remaining orthogroups contain 15 HGT orthogroups (24 transfers) with insufficient genomic data to infer presence of introns (white) and one orthogroup containing HGT gene without introns (light blue). encoding transposable elements (TEs), which is consistent with their All these HGT genes were verified with at least one additional line invasive nature. Four TE-related sequences encode ORFs with of evidence, providing additional validation for HGT not due to abundant transcripts in haustorial tissues (orthogroup 1021, 5002, assembly errors. In addition, the genomic contigs of HGT sequences 14230, and 15149), suggesting a potentially active role in the para- had read depths equivalent to those of genomic contigs of the ver- sites, a scenario similar to the recently identified Brassicaceae- tically transmitted sequences (SI Appendix,Fig.S3), rejecting the derived hobo-Ac-Tam3 transposon (hAT) in P. aegyptiaca (29). possibility that they may represent sample contamination. Finally, We also provided multiple lines of evidence for cross-validation of the use of increased taxon sampling sometimes helped to identify a HGT sequences. This includes sequences that were confirmed more likely donor lineage, as seen in orthogroup 17, where an initial by RT-PCR (22 events) or PCR amplification with genomic DNA donor lineage (Populus, a rosid) gave way to a more likely donor (three events), genomic sequence data (27 events), or present in lineage (Beta, a caryophyllid) after additional taxon sampling (SI more than one parasitic species (11 events) (SI Appendix,TableS3). Appendix,Fig.S2.1).

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1608765113 Yang et al. PNAS PLUS Table 1. Information of the 42 HGT orthogroups including the HGT recipient, donor, expression, Dn/Ds, functional category, and homology-based annotation

Ortho group Recipient Donor Intron Expression Dn/Ds Functional category Annotation based on homology

226 P Poptr Y/Y 1, 4.2 P Defense Cytochrome P450 1685 P Gyma + Medtr Y/Y >2 P Defense Cysteine-rich receptor-like kinase 2376 P Poptr + Theca Y/Y >2 RP Defense Proteasome subunit alpha type 14624 S Sorbi + Zea N/N-5′UI NA P Defense (disease resistance) BTB/POZ 23343 P Theca — 5.2 P Defense (disease resistance) Disease resistance protein 11841 P Frave Y/Y 5.1 SP Defense Hyoscyamine 6-dioxygenase-like 1886 P Frave — 6.2 RP Defense (immunity) Ankyrin repeat family protein 11437 P Frave — >2 RP Defense and nodule Kelch modif related to development galactose oxidase 8888 P Frave — 2, 4.1 RP Transcription Poly(A) polymerase 18709 P Arath Y/Y Int POS Transcription Nucleolin 2-like 806 P Theca Y/Y Int RP Translation Valyl-tRNA synthetase 2270 P, S Theca Y/Y >2 RP Translation Methionyl-tRNA synthetase 4067 P Frave Y/Y 41 RP Translation tRNAHis guanylyltransferase 10050 P Frave — 42 RP Translation Histidine-tRNA ligase 13892 P Medtr Y/Y Int P Translation Ribosomal protein S13 17 P Betvu Y/Y 4.2, 5.1, 6.2 P Nutrient transport ABC transporter C family member 3 9613 P Glyma Y/Y 0 RP Nodule development Cytosolic purine 5-nucleotidase and cytokinin biosynthesis 15246 P Medtr — 6.2 P Defense-related Albumin I (28) (insect toxin) 1226 P Poptr Y/Y 3, int RP Diverse Alpha/beta-Hydrolases 10143 P Frave — 4.2 RP Diverse Tubulin-specific chaperone D EVOLUTION 3861 P + L Glyma Y/Y >2 POS Diverse Poly(ADP ribose) glycohydrolase 4598 P Medtr — Int RP Diverse Nuclear pore complex protein 19696 P Poptr Y/Y 4.1, 6.2 SP Diverse Ubiquitin-like–specific protease 1 16703 S Orysa Y/Y 52 P Diverse Zinc finger, GRF-type 4572 P Frave — 3, 4.1 P Diverse FBD-associated F-box protein 5896 S Glyma Y/Y 5.2, 6.1 P Plastid-to-nucleus signaling Uroporphyrinogen-III synthase 218 P, S, T Prunus Y/Y (2) >2 P TE hAT transposon 1021 P, T Frave + Malus Y/Y >2 P TE hAT transposon 5002 P Prunus Y/Y Int P TE hAT transposon 12835 S Sorbi N/N 0 P TE Putative harbinger Transposase-derived nuclease 14230 P Prunus Y/Y 42 P TE MULE transposase 15149 P Frave Y/Y Int P TE hAT transposon 13512 P Frave — 51 POS Unknown Unknown 14233 S Sorbi + Orysa Y/Y 6.1 SP Unknown Unknown 14675 P Frave — 6.2 P Unknown Unknown 18354 P Frave — Int P Unknown Unknown 20190 P Frave — int, 2 P Unknown Unknown 23480 P Frave — Int P Unknown Unknown 18774 S Orysa Y/Y 0 P Unknown Unknown 13656 S Sorbi + Orysa — Int SP Unknown Hypothetical protein 19297 S Bradi N/N-3′UI NA P Unknown Unknown (26) 20188 P Frave Y/Y Int P Unknown Unknown

Recipient column: L, Lindenbergia;P,Phelipanche;S,Striga;T,Triphysaria. Intron column: “—”, not determined; 3′UI and 5′UI mean 3′/5′-UTR introns; N/N, absence of introns in both donor and recipient; Y/Y, presence of intron in both donor and recipient gene (orthogroup 218 has two genomic transfers with introns). Expression: >2, means highly expressed in more than two stages; int, interface. Dn/Ds: P, purifying selection; POS, positive selection; RP, relaxed purifying selection; SP, stronger purifying selection. Functional category: TE, transposable element. Donor is represented by the five-letter code of species abbreviations in SI Appendix, Fig. S1. Note that the donor is the indicated taxon or an ancestor. Expression column represents the stages with primary expression for the HGT genes. Haustorial stages are 3, 4 (4.1 and 4.2), and int. For detailed information about these stages, please refer to ref. 32.

Mechanism of HGT transfer was inferred to be unique to one genus (15 are unique in Transfers from Ancestral Host Lineages. A majority of these HGTs Phelipanche, eight are unique in Striga) or in two closely related could be assigned to ancestral donors from known host lineages. genera (15 occurred both in Phelipanche and Orobanche) (Fig. All of the HGTs from grass donors (Poaceae) were discovered in 2D and SI Appendix, Table S9). Striga (Table 1; SI Appendix, Table S9, and Fig. 2C), which (ex- Any HGT events coincident with the origin of parasitism cept for Striga gesnerioides) are specialized parasites of Poaceae would have occurred in a common ancestor of the parasites. (38). In Phelipanche, inferred donors reflected a wide range of Previously reported cases of HGT to microbial parasites or dicot families with the majority from Rosaceae and Fabaceae, pathogens of plants often encode cell wall-degrading enzymes (39) consistent with the host range of this parasite and its congeners and thus are implicated in host invasion. Surprisingly, although (30) (Fig. 2C and SI Appendix, Table S9). In 38 orthogroups, the cell wall-modifying enzymes are well-represented in haustorial

Yang et al. PNAS Early Edition | 5of10 tissues (24), no such proteins were identified in our HGT search. Although these genes lacked CDS introns, two of these had introns in Instead, numerous proteins involved in cell wall modification theirUTRs(Fig.3D). A gene in Orthogroup 14624 (BTB/POZ do- processes in the haustorium were attributed to gene duplications main containing protein) was transferred from an ancestor of that occurred in an ancestor of all parasitic lineages of Orobanchaceae into S. hermonthica,andthe5′-UTR intron (32). Our HGT phylogenies, however, supported predominantly shows 87% sequence identity between the donor and recipient gene recent occurrences that were restricted to individual genera. In only (CDS, 91%; 5′-UTR, 87%; 3′-UTR, 68%) (SI Appendix,Fig.S5). In one case (i.e., orthogroup 218, SI Appendix,Fig.S2.2), the transfer the other case, a gene in orthogroup 19297, a conserved 3′-UTR was detected in almost all of the parasitic taxa (Phelipanche, Striga, intron (3′-UTR intron, 78%; CDS, 85%; 5′-UTR, 54%; 3′-UTR, Triphysaria,andAlectra)(Fig.2D and SI Appendix,TableS9), but 82%), is present in both the donor and recipient (SI Appendix,Fig. the SH test indicated that this likely involved at least two (more S6). It is noteworthy that this HGT event was previously identified recent) transfers instead of a single ancestral HGT event (SI Ap- by Yoshida et al. (26), who speculated, based in part on the pres- pendix,TableS2). Therefore, although gene duplications often ence of a remnant poly-A tail in the cDNA, that this HGT event preceded and underpin the origin of parasitism in Orobanchaceae may have been mediated by integration of a mature mRNA rather (32), HGT events are more recent and are likely to have been fa- than a genomic fragment. Our analyses identified the presence of a cilitated by parasite connections. 284-bp high-identity intron in the 3′-UTR, suggesting that this event (like the majority of cases reported here) was mediated by a ge- Increased Numbers of HGT with Increased Heterotrophic Dependence. nomic fragment rather than an mRNA. Only one orthogroup The absence of HGT events involving the nonparasitic common (12835—a Pong-like TE) lacked introns in both the donor and ancestor of the Orobanchaceae supports the hypothesis that para- recipient gene, and the nonconserved flanking region failed to in- sitic (heterotrophic) interactions lead to more HGT events than form whether genomic or mRNA-mediated transfer was supported occurs between free-living organisms. The number of HGT events (Table 1). The remaining 24 HGT orthogroups contained 25 genes also appears to increase in parasites with greater host dependence. whose donor and recipient contained CDS introns. We further re- We detected only one likely HGT in Lindenbergia, the free-living duced the list to 13 orthogroups with full-length gene assemblies, sister lineage to all parasitic Orobanchaceae. In T. versicolor,the allowing us to examine similarities and differences in intron po- facultative hemiparasite, two HGT events were found (Table 1 and sitions and sequences between donor and recipient. SI Appendix,Fig.S2.2andS2.5). In Striga, the obligate hemiparasite, All 13 orthogroups showed congruence of CDS structure be- 10 orthogroup trees support HGTs and seven were from grasses tween donor and recipient, suggesting a transfer of a genomic (Poaceae). A majority (34 orthogroups) of the HGTs were detected fragment containing the gene, rather than an mRNA intermediate. in Phelipanche, the obligate holoparasite with the strongest host Intron positions are highly conserved (SI Appendix,TablesS4and dependence (Figs. 2D and 3A and SI Appendix,Fig.S2). S5 and Fig. S7) (although with occasional intron loss; Fig. 3B), Several factors could account for the increasing number of suggesting maintenance of intron structure for functional tran- HGTs in parasites with increased host dependence. First, the scription. These sequences provide support for genomic fragments lifestyles of facultative and obligate parasites are quite different as HGT intermediates but do not help to diagnose the source of the with respect to the timing of host invasion and parasite gamete horizontally acquired sequence. We constructed phylogenies using formation. The seedlings of the obligate parasites, which require the intron sequences only and compared them to phylogenies host plant-induced germination stimulation, are in contact with constructed with exons only, finding that three of the orthogroup host plants from a very young developmental stage, thus increasing phylogenies were well-resolved (orthogroup 4067, 806, and 2270) the chances that cells that experience HGT events will develop (Table 1). The intron phylogenies were congruent with the CDS into germ-line tissues (40). In contrast, facultative parasites like phylogenies, indicating the same donor lineage as inferred from Triphysaria develop roots and aboveground parts before parasitism exon sequence and providing strong support of a genomic fragment- occurs. In these plants, host-derived gene fragments that cross the mediated HGT (Fig. 3 A–C and SI Appendix,Fig.S7). The strongest haustorium need to be subsequently transported into developing example, orthogroup 4067 [tRNAHis guanylyltransferase—required flowers to be captured in germ-line cells. There is also clear evi- for translation (45)], not only exhibits strong CDS similarity with its dence for phloem connections between host and P. aegyptiaca inferred Fragaria donor (∼74%) (Fig. 3A), but the intron sequences (41), allowing for more HGTs along with the genetic exchange of maintain ∼51% similarity (Fig. 3 B and C), even higher than that nucleic acids via phloem (31). Similar phloem connections have between the vertically inherited parasite gene and its close relative not been observed in Triphysaria (42). in Mimulus (∼21%) (Fig. 3 B and C). These results show that all of the resolvable HGT events were likely mediated by ge- Integration of Genomic Fragments. Signatures of the donor molecule nomic fragments containing the donor genes rather than by should persist in the genome, giving clues to the mechanism of RT-mediated transfer. transfer. For instance, a nuclear HGT reported in Striga supports a possible mRNA-mediated transfer, as the HGT lacked introns and Functional HGT seemed to contain a remnant poly-A tail, whereas the donor Sor- Tissue-Specific HGT Expression. A total of 37/49 HGT genes show ghum gene lacked a poly-A tail (26). Documented translocation of expression with maximum Fragments Per Kilobase of Exon Per host RNA into Triphysaria (43) and Phelipanche (44) as well as the Million Fragments Mapped (FPKM) greater than 5 in at least one massivemovementofhostRNAintoCuscuta would support an developmental stage, indicating that most are actively transcribed. RNA-based mechanism for HGT in parasitic plants (31). In con- In addition, 36/42 HGT orthogroups contain HGT genes from trast, a horizontally acquired albumin 1 gene in Phelipanche and more than one parasitic taxon (SI Appendix,Fig.S2), suggesting related taxa (28) and horizontally acquired Brassicaceae-specific evolutionary conservation in the parasites. The species with the strictosidine synthase-like (SSL) genes contained introns in ge- most HGTs is P. aegyptiaca, and a majority of the candidate genes nomic sequences of both donor and the parasites (Phelipanche and show tissue-specific expression (Fig. 4; for Striga and Triphysaria, Cuscuta), all consistent with direct genomic transfers without an see SI Appendix,Fig.S9). The expression profiles of P. aegyptiaca RNA intermediate (27). To test the hypothesis of mRNA-mediated HGT genes (Table 1) revealed a distinctive cluster of interface- transfer, we examined coding sequence structure (exon–intron specific expression (Fig. 4) and an equal number with abundant boundaries) in the 42 HGT orthogroups. We had sufficient genomic expression in haustorial tissues. A subset of these genes encodes data to examine 28 genes from 52 horizontal transfer events (27 functions related to transcription and protein synthesis (Table 1), orthogroups) (Table 1), although three HGT genes lacked coding and in each of these gene trees, HGT has added an extra gene along sequence (CDS) introns in both donor and recipient (Fig. 3D). with the vertically inherited gene family member. This additional

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1608765113 Yang et al. PNAS PLUS 123 Value −1 Color Key

after HIF (2) −3 beforegerminated HIF (1) seedling undergroundundergroundabovegroundaboveground imbibedgerminated seed (0) seedlinghaustoriahaustoria (3) haustoria (4.1) interface (4.2) (int) shoots (5.1) roots (5.2) shoots (6.1) floral buds (6.2) PhAeBC5_31016.1_hAT transposon PhAeBC5_14888.1_cytochrome P450 PhAeBC5_22555.2_Poly (ADP-ribose) glycohydrolase PhAeBC5_15086.1_polyA polymerase PhAeBC5_34854.1_MULE transposase PhAeBC5_4284.2_histidyl-tRNA synthetase PhAeBC5_17034.1_peptidase PhAeBC5_16890.17_hydroxysteroid dehydrogenase PhAeBC5_15353.2_cysteine-rich receptor-like kinase PhAeBC5_11914.4_alpha/beta-Hydrolase PhAeBC5_9762.1_tRNAHis guanylyltransferase PhAeBC5_8623.1_LRR-containing protein PhAeBC5_15496.1_Ankyrin repeat family protein PhAeBC5_2525.1_albumin I PhAeBC5_8480.1_ABC transporter PhAeBC5_12620.5_Kelch motif PhAeBC5_3356.1_methionyl-tRNA synthetase PhAeBC5_270.3_proteasome subunit A PhAeBC5_6791.1_cysteine-rich receptor-like kinase PhAeBC5_4800.1_NB-ARC disease resistance protein PhAeBC5_3740.1_Galactose oxidase PhAeBC5_9914.1_Poly (ADP-ribose) glycohydrolase PhAeBC5_14056.1_Tubulin-specific chaperone D PhAeBC5_3756.1_2OG-Fe(II) oxygenase PhAeBC5_1584.1_Galactose oxidase PhAeBC5_2529.1_DNA polymerase phi PhAeBC5_9781.2_hAT transposon PhAeBC5_5786.1_C2H2 zinc finger PhAeBC5_1574.1_unknown PhAeBC5_654.1_RNA recognition motif PhAeBC5_8832.4_hAT transposon PhAeBC5_13756.1_nuclear pore complex protein PhAeBC5_4266.1_ribosomal protein S13 EVOLUTION PhAeBC5_5395.1_GAG-polyprotein PhAeBC5_4239.2_valyl-tRNA synthetase PhAeBC5_20413.1_hAT transposon

0G 1G 2G 3G 4.1G 4.2G 5.1G 5.2G 6.1G 6.2G

IntArathG

Fig. 4. Heat map showing the expression of HGT transgenes in P. aegyptiaca. Expression is shown with FPKM-transformed z scores to ensure even signal intensity across stages. Rows represent HGT genes, with their identified domain shown on the right; columns represent stages (below) or tissues (above). Haustorial and interface tissues are colored in green. Genes were clustered on the left to show similarity. gene may increase the rate of transcription and protein synthesis In summary, three primary lines of evidence support a functional specifically at the haustorium interface, where such processes go on role for these horizontally acquired sequences in parasitic at elevated levels (46, 47). Orobanchaceae: (i)HGTsequencesaredetectedandcom- monly conserved across species boundaries; (ii) the sequences HGTs Are Evolving Under Constraint and Are Likely Functional. For are actively and differentially transcribed, frequently with a bias each of the HGT orthogroup phylogenies, we estimated Ds (the toward haustorial expression; and (iii) all of the high-confidence frequency of synonymous substitutions per synonymous site) HGT genes are evolving under purifying selection, consistent with and Dn (the frequency of nonsynonymous substitutions per the conservation of functional protein structures. Notably, a group nonsynonymous site) and ω (the ratio Dn/Ds; values less than 1 of HGTs related to transcription and translation are highly indicate purifying selection) for each of the HGT protein-coding expressed in haustoria of P. aegyptiaca (Table 1), the species with sequences and related genes. A branch test, implemented in the greatest host dependence. As haustoria have a high metabolic PAML (Phylogenetic Analysis by Maxiumum Likelihood) (48), rate associated with loading host nutrients (46, 47), it is possible compares the Dn/Ds estimate for the foreground (HGT genes) that horizontal transfers of such gene functions help Phelipanche to the background (non-HGT orthogroup members). The same efficiently mobilize host resources transported through haustorial or even stronger levels of purifying selection in parasitic HGT tissues. Noteworthy, although not serving as direct evidence, no genes were observed in 27 orthogroups (Table 1 and SI Appen- parasite-to-host transfers were identified using the same ap- dix, Table S6). An additional test—RELAX (49)—was imple- proach. If the intimate contact between the parasite and the host— mented to identify whether HGT sequences experienced stronger the haustorium—provides a mechanism for the exchange of levels of selection (purifying or adaptive) or relaxed selective genetic elements facilitating HGT, we expect that horizontal constraint (compared with the background). The same levels of transfersalsooccurredfromparasite to host. A lack of nuclear selection were observed between HGT sequences and the back- transfers from parasite to host suggests that such transfers are ground genes in 23 orthogroups, whereas stronger levels of selec- generally not functional. tion were observed for HGT sequences of 12 orthogroups (SI Appendix,TableS8). These results show that HGT-encoded pro- Evidence of Adaptive Evolution of HGTs. Our observation of haus- teins are largely evolving under strong constraint, indicating a torial expression in a majority of the HGT genes suggests a likely likely functional role in parasitic plants. Additional evidence comes contribution of HGT to parasitic adaptation. To corroborate this from conservation of a predicted 3D structure for HGT proteins idea, we examined the possibility of adaptive signatures on in comparison with their nonparasitic orthologs in Arabidopsis protein sequences of these HGTs (see Methods, Selective Con- thaliana (SI Appendix,Fig.S8). straint Analyses). Out of 15 HGT orthogroups (SI Appendix,

Yang et al. PNAS Early Edition | 7of10 Table S6), 13 contain potentially adaptive sites present in HGT These hypotheses could be tested experimentally by comparing genes of parasites. Interestingly, 9 out of 13 orthogroups were the capacity of Orobanchaceae parasites to recognize and tran- identified by RELAX (49) that show a different level of selective scribe sequences with foreign promoters (from other constraint in HGT sequences compared with the background. and from monocots) versus the likelihood of substantial tran- Five orthogroups show stronger levels of selection, and four scription of a randomly inserted cDNA. show relaxed constraint; both patterns could be associated with Functional roles conferred by these HGT genes have identi- the presence of adaptive sites (SI Appendix, Tables S6 and S8). fied HGT as a mechanism contributing to the adaptive evolution These sites are unchanged in nonparasitic species, including of parasitic plants. Our methods likely have underestimated the Mimulus, the close nonparasitic relative of Orobanchaceae number of horizontally transferred genes because (i) the phylo- (SI Appendix,TableS7). Of these, six orthogroups have genes genomic approach in this study relies on an fairly complete and encoding functions related to transcription and translation accurate construction of gene family phylogenies, (ii) large and (orthogroup 8888, 18709, 806, 4067, 10050, and 13512), and four complex gene families do not always produce well-resolved trees, orthogroups contain genes with abundant haustorial expression and (iii) we restricted our search of possible donor lineages to (orthogroup 1226, 8888, 18709, and 806) (Table 1). The signa- distantly related monocot and rosid groups for enhanced signal- tures of adaptive sites and their retention as haustorial genes in to-noise ratio. With the increasing availability of genome se- the genome suggest that these changes in HGT proteins are quences and other genomic-scale data, along with increasingly under positive selection and may have provided novel functions rigorous standards for discovery and evaluation, many more ex- contributing to increased parasite fitness. amples of functional HGT are likely to be revealed. Similar to the “you are what you eat” model in explaining the HGT of Defense-Related Genes. Our list of HGT events contains a eubacterial origin of nuclear genes of phagotrophic protists (64), group of genes in orthogroups related to defense responses— the massive HGT we identified in parasitic plants from their hosts orthogroup 226 (50), 1685 (51), 2376 (52), 14624 (53), 23343 again reflect the feeding habit of parasitic organisms. The hy- (54), 11841 (55), 1886 (56), and 11437 (57) (Table 1). For in- pothesis of increased HGT frequency in endoparasites compared stance, orthogroup 23343 contains an ortholog of an Arabidopsis with exoparasites was proposed in studies of HGT in the parasitic gene encoding an NB-ARC domain-containing disease resistance plants Rafflesia (20, 25) and Cynomorium (22). Our study revealed protein (SI Appendix,TableS10) (58, 59). Orthogroup 1685 encodes increased numbers of HGT among related species with increased a cysteine-rich receptor-like protein kinase, and the ortholog in heterotrophic dependence, a pattern that could be corroborated Arabidopsis (AT1G70520) is up-regulated during pathogen infection with rigorous HGT identification in a much larger sampling of and rapid cell death (51). Orthogroups 2376 and 14624 contain parasitic taxa from Orobanchaceae and other parasitic lineages genes in Arabidopsis and that are up-regulated in response to (65) of varying ages and degrees of nutritional dependence. pathogens and parasite attack, respectively [based on analyses with PLEXdb (60)]. Methods Genes involved in defense that have been obtained by hori- Transcriptome sequencing, de novo assembly (including read cleaning and adapter zontal transfer could have been co-opted by the parasites for de- filtering), postprocessing, expression quantification (using CLC workbench), and fense against pathogens that attack it as well as its host. However, annotation [predicted protein sequences were used to search against Swissprot, six of the HGT genes in defense-related orthogroups show ele- TAIR10 (The Arabidopsis Information Resource 10), tremble, and Pfam domain vated expression in haustoria, suggesting that these genes may play databases] followed Yang et al. (32). a role in the parasite–host interactions. HGTs related to defense responses may provide a mechanism to attenuate the attack of the Removal of Contamination. Sequences were cleaned by removing nonplant host plant immune system against the parasite during haustorial transcripts and transcripts of the host plants used for growing the parasites formation. It is also possible that parasite invasion sites are more [Medicago or Zea for Triphysaria, Sorghum for Striga,andArabidopsis for susceptible to microbial pathogens, in which case enhanced defense Phelipanche (30)] with BLASTN (nucleotide BLAST) (E-value of 1e-10). responses may reduce the risk of infection. This model is also a Phylogenomic Construction of Parasite Gene Trees. ORFs and protein se- potential explanation for the haustorial up-regulation of the putative quences encoded by assembled transcripts were predicted with ESTScan defense-related HGTs. The specific role of defense-related genes in version 2.0 (66). A total of 586,228 protein coding genes of 22 representatives this potentially multitrophic interaction remains to be discovered. of sequenced land plant genomes were classified into 53,136 orthogroups using OrthoMCL (67). The selected taxa include nine rosids (A. thaliana, Conclusion Thellungiella parvula, Carica papaya, Theobroma cacao, Populus trichocarpa, In this study, we developed a phylogenomic pipeline that parses Fragaria vesca, Glycine max, Medicago truncatula,andVitis vinifera), three large-scale phylogenetic trees for preliminary HGT identifica- asterids (Solanum lycopersicum, Solanum tuberosum,andMimulus guttatus), tion, followed by careful validation with further analyses and two basal eudicots (Nelumbo nucifera and Aquilegia coerulea), five monocots increased taxon sampling. Our final 42 HGT trees (52 high- (Oryza sativa, Brachypodium distachyon, S. bicolor, Musa acuminate,and Phoenix dactylifera), one basal angiosperm (Amborella trichopoda) (68), one confidence HGT events in three parasites of Orobanchaceae) lycophyte (Selaginella moellendorffii), and one moss (Physcomitrella patens). support the placement of focal HGT gene(s) being nested within Unigenes from Lindenbergia, Triphysaria, Striga, Phelipanche, and two Aster- donor clades with at least two strong nodes, instead of just aceae species, Lactuca sativa and Helianthus annuus, were assigned into the appearing as a sister to a putative donor lineage. This approach 22-genome orthogroup classifications by BLASTP (69) with e-value ≤ 1e-5 and proves to be stringent but also robust to the challenges of Hidden Markov Models (HMMs) (70). This resulted in 13,125 orthogroup phy- genome-scale HGT discovery. Our analyses of intron sequences logenetic trees containing at least one parasitic species in the phylogeny. and structure support genomic fragment integration of HGTs Orthogroup phylogenies were generated with an automated approach (ref. 32 rather than RNA-mediated retroprocessing events. Although and https://github.com/dePamphilis/PlantTribes) where codon alignments were unexpected, considering the well-documented mRNA transfer used to estimate a maximum likelihood tree using RAxML version 7.2.7 with the that occurs between parasitic plants and their hosts (31), we GTRGAMMA model (71). hypothesize that compared with mRNA, transfers of genomic HGT Screening on Phylogenetic Trees. Customized Python scripts were de- fragments will more often result in functional transfers because veloped to screen incongruent phylogenies. The python script used the tree- genomic regions can contain intact promoters that may be rec- parsing functions available in the ete2 libraries (72) to traverse one node at a ognized by the recipient plant species. Cross-species promoter time and extract members above each node. To decrease the false positive recognition is common in experimental transformation studies rate for HGT discovery, the script searched for donors in distantly related (61, 62), even among very distantly related plant species (63). rosid and monocot groups rather than more closely related asterid lineages,

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1608765113 Yang et al. which would be more prone to false-positive HGT. An ancestral node was intron phylogenies, introns were concatenated to increase the number of in- PNAS PLUS determined when traversing to a node whose left and right branches were formative sites for tree reconstruction with the same approach as building the exclusively composed of parasite and donor sequences. The script then ex- tree of CDS. amined all of the inner nodes within the ancestral node for BS values that support the grouping of parasite and donor sequences. Three models of Selective Constraint Analyses. To identify signatures of adaptive or purifying topology (Fig. 1) represent HGTs with decreasing degrees of confidence. The selection, we conducted two likelihood ratio tests using the branch model and script reported orthogroups that match any of them. After the automated branch-site model in PAML (48). In each test, we identified the HGT genes on a screening, the HGT candidate orthogroups were further classified into three phylogenetic tree as the foreground branches, which were compared with the categories: low-confidence, medium-confidence, and high-confidence trees. remaining background sequences on the tree. The branch model tested if the The classification criteria were based on a scoring scheme that considered foreground HGT branches had the same level of protein sequence constraint as whether the donor clade contained at least two donor sequences, bootstrap the background nonparasitic sequences. Levels of sequence constraint were values supporting the grouping of the parasite gene and donor sequences, measured by estimating a D /D ratio (omega, ω) for both foreground and and the presence of long-branch clades. Each of these three factors was n s background. The null model (a one-ratio model) estimated a single ω for all assigned a score, and the summed score was used to assign trees to each of sequences. A likelihood ratio test was performed to infer if the branch model fit the confidence levels (SI Appendix, Table S1). The medium- and high-confi- the data significantly better than the one-ratio model. A nonsignificant result dence orthogroup trees were then examined carefully for possible sources of from the test indicated the same level of protein constraint between the HGT errors, including contamination, potential for long-branch artifacts, and genes and nonparasitic background; a significant result can reflect either (i)a insufficient taxon sampling. Frame-shift errors were fixed by manually in- ω troducing 1–2 bp to achieve translations that were much more conserved in higher in the HGT branch than the background, indicating either relaxed ω < ω > ω comparison with other species. constraint (if 1) or adaptive evolution (if 1) or (ii)alower in the HGT branch than the background, indicating stronger levels of purifying selection. HGT Validation by Increased Taxon Sampling. For HGT validation, we added HGT sequences that had relaxed constraint were further tested with a branch- more taxa from related species, including five sequenced asterid ge- site model to identify the presence of adaptive sites for the indicated foreground “ = = nomes and 10 transcriptomes from 1kp in the Lamiales order (34). The lineages. To perform the branch-site test, parameters model 2, NSsites 2, = ” genomes include the following: Beta vulgaris (beet), Actinidia chinensis fix_omega 0 were indicated in the codeml files for both the branch-site model (kiwifruit), Utricularia gibba, Sesamum indicum,andS. asiatica (parasite and null model in PAML. The former model used an additional parameter in Orobanchaceae). The transcriptomes include the following: Strobilanthes “fix_omega=1,” whereas the latter model used “fix_omega=0.” Sites with dyeriana (Acanthaceae), (), Sinningia tuberosa probability greater than 0.95 from a Bayes Empirical Bayes analysis represented (Gesneriaceae), Salvia spp. (Lamiaceae), Olea europaea (Oleaceae), Epifagus likely adaptive sites. RELAX analyses (49) were also performed to evaluate virginiana (Orobanchaceae), Paulownia fargesii (Paulowniaceae), Antirrhi- whether selective constraints are stronger in foreground (HGT) branches com- EVOLUTION num majus (Plantaginaceae), Rehmannia glutinosa (Rehmanniaceae), and pared with background (non-HGT) branches. The analyses was performed using Verbena hastata (Verbenaceae). Also, we added genes from transcriptomes of the RELAX tool on datamonkey server (test.datamonkey.org/relax/). An input file above-ground tissues derived from eight additional parasitic Orobanchaceae: that contains the codon sequence alignment and the RAxML tree was provided, Alectra vogelii, Myzorrhiza californica, Orobanche minor, Phelipanche mutelii, and the foreground braches (test branches) and background branches (reference Phelipanche ramosa, Striga gesneroides, Triphysaria eriantha,andTriphysaria branches) were then indicated before running. A likelihood ratio test was also pusilla. To make sure that all of the HGTs were captured from these added taxa, performed to evaluate if selection varied between test and reference branches. we used HMMs (70) (hmmsearch with 1e-5). For lineage-specific HGT orthogroups, A significant result (P value < 0.05) indicates a stronger level of selection if K is a superorthogroup tree (68) was constructed to ensure the inclusion of all greater than 1 or a weaker selection or relaxed constraint if K is less than 1. homologous sequences. Genome Assembly of Parasite Species. Illumina data for S. hermonthica and Validation of HGT Sequences. RT-PCR was used to verify transcriptome assembly P. aegyptiaca were de novo assembled using CLC Assembly Cell v 4.1 (https:// for HGT sequences. HGT sequences (from combined builds) were aligned with www.qiagenbioinformatics.com/products/clc-assembly-cell/): BLASTN against haustoria stage-specific assemblies to identify corresponding transcripts; these were then used as templates to design primers for PCR ampli- “novo_assemble -o contigs.fasta -p fb ss 180 250 -q -i reads1.fq reads2.fq.” fication with haustorial cDNA. Genome PCR used HGT transcriptome sequences as templates to design primers for subsequent PCR with genomic DNA. Verification Estimation of Number Of Transfers. We used a SH test (37) to estimate the was confirmed when the sequenced genomic PCR product matched the assembled number of transfer events from 42 HGT orthogroup trees. Trees in which transcript, and occasionally introns were revealed (SI Appendix,TableS3). HGT genes did not form a monophyletic clade were constrained to represent one event, and a RAxML tree with constrained HGT clade was produced. An Intron Analyses. Intron positions were extracted to examine if they were con- SH test was performed in RAxML version 7.2.7 (71) to test if the likelihood of served in multiple sequence alignments (MSAs). For each orthogroup, the peptide the constrained tree was significantly worse than that of the original tree. sequences were aligned using MAFFT version 7 (73), which were then forced onto coding sequences (CDS) to generate the CDS alignment. A customized Perl script ACKNOWLEDGMENTS. We thank Dr. Craig Praul and the Huck Genomics Core was used to extract the intron positions in each coding sequence, and the cor- Facility for transcriptome sequencing and the gift of Striga genome sequences responding positions were mapped onto the CDS alignment. To identify intron generated on a trial run of the Illumina HiSeq2500 sequencer that was pur- positions in Orobanchaceae genes, we generated and assembled shallow ge- chased as NSF Equipment Grant MRI-1229046 (to C.W.d.); Tony Omeis for grow- nomic sequences from S. hermonthica and P. aegyptiaca. Coding sequence was ing M. californica (with Grindelia host) in the Biology Greenhouse at Penn State predicted for each transcript using ESTScan(66)andwasthenalignedtoge- University from material originally provided by Alison Colwell; K. Shirasu and nomic sequences using BLASTN with an e-value cutoff of 1e-05. Manual curation S. Yoshida for access to the S. asiatica genome sequence and annotation; was then performed for each CDS-genomic DNA alignment to make sure introns D. E. Soltis, M. K. Deyholos, M. W. Chase, and C. Wang for collecting nine of the 10 1KP samples used for HGT validation in this study; Ning Jiang (Michigan State start with GT, and end with AG. To extract intron sequences for each gene from University) for discussion of Pong-like TEs; and J. Naumann, J. Der, J. Palmer, fully sequenced plant genomes, we used the gff file for the intron regions of M. Axtell, D. Cosgrove, S. Maximova, and M. Guiltinan for helpful suggestions. each gene. Intron sequences of genes in sequenced genomes were extracted This research was supported by NSF Plant Genome Research Program Awards from genomic sequences in Phytozome 10 (35) using samtools (74) and betools DBI-0701748 and IOS-1238057 (to J.H.W., C.W.d., M.P.T., and J.I.Y.), with addi- “ ”“ ” [index command samtools faidx, fastaFromBed in BEDTools (75) was used tional support from the Plant Biology graduate program (Z.Y., L.A.H., S.J., and by indicating the genome reference using “-fi” and the gff file using “-bed,” H.Z.) and from the Genetics graduate program as well as the Biology Depart- which generated an output file using “-fo”]. Intron sequences for parasite genes ment (Y.Z.) at Penn State University; National Institute of Food and Agriculture were obtained by blasting the coding sequence onto genomic sequences. For Project 131997 (to J.H.W.); and NSF Grant IOS-1213059 (to M.P.T.).

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10 of 10 | www.pnas.org/cgi/doi/10.1073/pnas.1608765113 Yang et al. Supplementary information Phelipanche_aegyptiaca_PhAe Striga_hermonthica_StHe Triphysaria_versicolor_TrVe Lindenberiga_philippensis_LiPh Mimulus_guttatus_gnl_Mimgu Solanum_tuberosum_gnl_Soltu Solanum_lycopersicum_gnl_Solly Helianthus_annuus_HeAn Lactuca_sativa_LaSa Arabidopsis_thaliana_gnl_Arath Thellungiella_parvula_gnl_Thepa Carica_papaya_gnl_Carpa Theobroma_cacao_gnl_Theca Populus_trichocarpa_gnl_Poptr Fragaria_vesca_gnl_Frave Medicago_truncatula_gnl_Medtr Glycine_max_gnl_Glyma Vitis_vinifera_gnl_Vitvi Nelumbo_nucifera_gnl_Nelnu Aquilegia_coerulea_gnl_Aquco Sorghum_bicolor_gnl_Sorbi Brachypodium_distachyon_gnl_Bradi Oryza_sativa_gnl_Orysa Musa_acuminata_gnl_Musac Phoenix_dactylifera_gnl_Phoda Amborella_trichopoda_gnl_Ambtr Selaginella_moellendorffii_gnl_Selmo Physcomitrella_patens_gnl_Phypa Figure S1. The species tree for HGT discovery. This tree includes 22 sequenced plant genomes containing “gnl_” followed by the first three letters of the genus name, and first two letters of the species name. It also includes four transcriptomes generated in this study – one nonparasitic outgroup Lindenbergia, and three parasitic plants Triphysaria, Striga, and Phelipanche, as well as two additional asterids with extensive EST sequence datasets – Helianthus and Lactuca. Color codes: asterid (green); rosid (red); basal eudicot (purple); monocot (gold); basal angiosperm (blue); outgroup (black).

Fig. S2.1 - orthogroup 17 gnl_Vitvi12X_PACid_17825572 gnl_Vitvi12X_PACid_17825575 gnl_Vitvi12X_PACid_17825577 100 gnl_Vitvi12X_PACid_17825573 gnl_Vitvi12X_PACid_17825576 gnl_Vitvi12X_PACid_17842305 gnl_Theca1.0_Tc05_g023350 gnl_Theca1.0_Tc07_g001750 gnl_Theca1.0_Tc07_g001760 gnl_Theca1.0_Tc07_g001770 53 gnl_Carpa1.181_PACid_16429035 100 gnl_Carpa1.181_PACid_16406731 99 gnl_Thepa2.0_Tp3g11250 gnl_Arath10_AT3G13080.1 100 gnl_Thepa2.0_Tp3g11260 100 gnl_Arath10_AT3G13090.1 gnl_Arath10_AT3G13100.1 92 gnl_Thepa2.0_Tp3g11270 Populus_euphratica_gi_743932868_ref_XP_011010733.1 gnl_Poptr2.2_PACid_18239169 100 100Populus_trichocarpa_gi_224061172_ref_XP_002300362.1 Populus_euphratica_gi_743932874_ref_XP_011010735.1 gnl_Poptr2.2_PACid_18235364 Populus_trichocarpa_gi_566153266_ref_XP_006369995.1 Spinacia_oleracea_gi_902205074_gb_KNA15081.1 Beta_vulgaris_subsp._vulgaris_gi_731335801_ref_XP_010678911.1 100 67 Beta_vulgaris_subsp._vulgaris_gi_731335315_ref_XP_010678665.1 Caryophyllales - 46 Beta_vulgaris_subsp._vulgaris_gi_731335309_ref_XP_010678661.1_ABC_transporterC_family_member_3-like Amaranthaceae 100 Spinacia_oleracea_gi_902205073_gb_KNA15080.1 Spinacia_oleracea_gi_902205072_gb_KNA15079.1 Phelipanche_ramosa_PhRa5_2879 H 41 66 Phelipanche_aegyptiaca_PhAeBC5_8480.1 gnl_Poptr2.2_PACid_18217322 gnl_Poptr2.2_PACid_18216599 gnl_Poptr2.2_PACid_18216258 gnl_Poptr2.2_PACid_18218339 gnl_Poptr2.2_PACid_18216037 Citrus_sinensis_gi_568841974_ref_XP_006474929.1 Prunus_mume_gi_645279093_ref_XP_008244542.1 100 gnl_Frave2.0_gene21745 gnl_Frave2.0_gene21684 gnl_Medtr3.5_AC151738_58.1 100 gnl_Glyma1.01_PACid_16310972 95 94 gnl_Glyma1.01_PACid_16308351 100 gnl_Glyma1.01_PACid_16273742 gnl_Glyma1.01_PACid_16251948 gnl_Glyma1.01_PACid_16308337 95 gnl_Medtr3.5_AC151738_59.1 100 gnl_Glyma1.01_PACid_16273746 gnl_Glyma1.01_PACid_16273745 Cicer_arietinum_gi_502099483_ref_XP_004491490.1 100gnl_Medtr3.5_Medtr5g033030.1 100 gnl_Medtr3.5_Medtr5g033320.1 gnl_Medtr3.5_Medtr5g094830.1 gnl_Medtr3.5_Medtr5g094810.1 100 gnl_Glyma1.01_PACid_16294023 Glycine_max_gi_356553519_ref_XP_003545103.1 gnl_Glyma1.01_PACid_16250440 gnl_Glyma1.01_PACid_16250439 gnl_Glyma1.01_PACid_16250438 Glycine_soja_gi_734376178_gb_KHN21276.1 Glycine_max_gi_571546314_ref_XP_006602474.1 gnl_Glyma1.01_PACid_16309499 gnl_Glyma1.01_PACid_16273999 gnl_Betvu_v1.1_Bv9_226360_ioks.t1 gnl_Solly2.3_Solyc07g065320.2.1 89 gnl_Soltu3.4_PGSC0003DMP400038391 Gesneriaceae-DTNC-0020920-Sinningia_tuberosa Striga_gesneroides_StGe3_3378 100 100 Alectra_vogelii_AlVo2_9241 85 Striga_gesneroides_StGe3_3377 StHeBC3_7719.1 92 Triphysaria_eriantha_TrEr3_8586 V 100 TrVeBC3_7110.1 63 Triphysaria_pusilla_TrPu1_14058 100 Orobanche_californica_OrCa3_17005 96 PhAeBC5_4110.1 Phelipanche_ramosa_PhRa5_16589 90 Phelipanche_mutelii_PhMu2_4110 100 95 LiPhGnB2_4546.1 gnl_Mimgu1.0_PACid_17671584 StHeBC3_2218.1 LaSa_22056 100 LaSa_50482 gnl_Aquco1.0_PACid_18140201 gnl_Aquco1.0_PACid_18151015 100 gnl_Aquco1.0_PACid_18149012 98 100 gnl_Aquco1.0_PACid_18149029 100 gnl_Aquco1.0_PACid_18142242 gnl_Aquco1.0_PACid_18153760 gnl_Aquco1.0_PACid_18147866 100 gnl_Aquco1.0_PACid_18149399 gnl_Aquco1.0_PACid_18147904 gnl_Nelnu1.0_NNU_005197-RA gnl_Nelnu1.0_NNU_003673-RA 100 gnl_Bradi1.2_Bradi4g39174.1 100 gnl_Bradi1.2_Bradi2g00417.1 100 gnl_Orysa6.0_PACid_16839560 100 gnl_Orysa6.0_PACid_16839558 gnl_Orysa6.0_PACid_16830163 100gnl_Bradi1.2_Bradi2g04577.2 gnl_Bradi1.2_Bradi2g54830.1 55 gnl_Sorbi1.4_PACid_1960518 100 79 gnl_Sorbi1.4_PACid_1954651 gnl_Sorbi1.4_PACid_1953811 gnl_Musac1.0_GSMUA_Achr5T19290_001 gnl_Musac1.0_GSMUA_Achr5T19280_001 95 gnl_Musac1.0_GSMUA_AchrUn_randomT24700_001 88 gnl_Phoda3.0_PDK_30s663101g004 gnl_Musac1.0_GSMUA_Achr10T28510_001 gnl_Ambtr1.0.27_AmTr_v1.0_scaffold00090.107 gnl_Ambtr1.0.27_AmTr_v1.0_scaffold00090.108 0.1

Fig. S2.2 - orthogroup 218 gnl_kiwi_Achn065441 97 gnl_kiwi_Achn271061 gnl_kiwi_Achn337111 gnl_Stras_v1.02_SGA1.0.scaffold134G00240 98 gnl_Stras_v1.02_SGA1.0.scaffold5G00860 100 Orobanche_minor_OrMi2_13559 Phelipanche_mutelii_PhMu2_41903 gnl_Stras_v1.02_SGA1.0.scaffold176G00080 gnl_Stras_v1.02_SGA1.0.scaffold2490G00010 V 83 Phelipanche_mutelii_PhMu2_26589 51 Triphysaria_pusilla_TrPu1_13120 89 85 gnl_Stras_v1.02_SGA1.0.scaffold107G00180 Orobanche_minor_OrMi2_24497 98 gnl_Stras_v1.02_SGA1.0.scaffold1059G00030 62 98 Striga_gesneroides_StGe3_6709 Orobanche_minor_OrMi2_174 gnl_Stras_v1.02_SGA1.0.scaffold79G00050 91 Alectra_vogelii_AlVo2_15797 Verbenaceae-GCFE-2004467-Verbena_hastata Graimondii_cotton_002G232100.1 Graimondii_cotton_011G212800.1 Graimondii_cotton_005G104500.1 62 gnl_kiwi_Achn321401 gnl_kiwi_Achn298471 100 gnl_kiwi_Achn267851 gnl_kiwi_Achn139471 gnl_kiwi_Achn315811 gnl_kiwi_Achn307181 gnl_kiwi_Achn218791 gnl_kiwi_Achn346511 Mulberry_Morus_natabilis006840 Mulberry_Morus_natabilis006012 75 Cannabis_sativa_PK25767.1 100 85 Cannabis_sativa_PK12026.1 Prunus_persica_ppa016213m Prunus_persica_ppa024297m Prunus_persica_ppa016227m Prunus_persica_ppa018881m Prunus_persica_ppa019206m Prunus_persica_ppa015420m Prunus_persica_ppa018763m 99 Prunus_persica_ppa025795m 95 Prunus_persica_ppa016010m 96 Prunus_persica_ppa024136m 64 Prunus_persica_ppa024909m Prunus_persica_ppa026657m Prunus_persica_ppa022981m Prunus_persica_ppa026648m 48 Prunus_persica_ppa025773m Phelipanche_aegyptiaca_PhAeBC5_31016.1 Phelipanche_mutelii_PhMu2_2986 100 Phelipanche_mutelii_PhMu2_17480 100 gnl_Stras_v1.02_SGA1.0.scaffold72G00100 H 100 68 gnl_Stras_v1.02_SGA1.0.scaffold628G00020 98 Striga_hermonthica_StHeBC3_32425.1 Striga_gesneroides_StGe3_13149 gnl_Stras_v1.02_SGA1.0.scaffold280G00090 88 Phelipanche_aegyptiaca_PhAeBC5_20413.1 Phelipanche_mutelii_PhMu2_19024 Prunus_persica_ppa026428m 98 Prunus_persica_ppa024848m Prunus_persica_ppa016147m Orobanche_californica_OrCa3_28196 Triphysaria_versicolor_TrVeBC3_29569.1 100 Alectra_vogelii_AlVo2_24277 H 100 100 Triphysaria_pusilla_TrPu1_8076 Prunus_persica_ppa023051m 61 Prunus_persica_ppa025415m 49 Prunus_persica_ppa020692m 100 97 Prunus_persica_ppa017579m Prunus_persica_ppa020338m Prunus_persica_ppa003125m Phelipanche_mutelii_PhMu2_4123 H 69 gnl_Frave2.0_gene35080 gnl_Frave2.0_gene25378 Prunus_persica_ppa021175m Prunus_persica_ppa022011m Prunus_persica_ppa021590m Prunus_persica_ppa020871m 100 Spolyrhiza_duckweed_Lemna4G0087300 Spolyrhiza_duckweed_Lemna2G0037800 100 Spolyrhiza_duckweed_Lemna3G0068200 Spolyrhiza_duckweed_Lemna24G0000500 89 1.0 Spolyrhiza_duckweed_Lemna28G0019700 Fig. S2.3- orthogroup 226 Mesculenta_cassava.12G132800.1 Mesculenta_cassava.12G132900.1 gnl_Carpa1.181_PACid_16429432 72 gnl_Theca1.0_Tc01_g008800 98 gnl_Theca1.0_Tc01_g013850 gnl_Vitvi12X_PACid_17832884 100 gnl_Vitvi12X_PACid_17832886 gnl_Mimgu1.0_PACid_17680354 94 Sesamum_indicumSIN_1004308 88 51 Verbenaceae-GCFE-2012538-Verbena_hastata 100 Verbenaceae-GCFE-2012539-Verbena_hastata Striga_hermonthica_StHeBC3_27187.1 V gnl_Stras_v1.02_SGA1.0.scaffold4758G00010 98 LaSa_51117 100 100 LaSa_55227 100 LaSa_17032 100 LaSa_3756379 LaSa_13170 HeAn_34956 68 LaSa_10142 Pvulgaris_common_bean.006G039800.1 100 Pvulgaris_common_bean.010G079100.1 100 Pvulgaris_common_bean.010G011000.1 Pvulgaris_common_bean.010G010900.1 Graimondii_cotton_011G262400.1 100 Graimondii_cotton_011G225200.1 94 99 Graimondii_cotton_011G225300.1 77 61 Graimondii_cotton_011G224900.1 Graimondii_cotton_011G225000.1 96 Graimondii_cotton_011G225500.1 Mesculenta_cassava.S058500.1 Mesculenta_cassava.05G150100.1 100 Mesculenta_cassava.05G139700.1 Mesculenta_cassava.05G151100.1 81 Mesculenta_cassava.05G140000.1 Mesculenta_cassava.05G150700.1 Mesculenta_cassava.05G150400.1 Mesculenta_cassava.05G150900.1 100 Mesculenta_cassava.05G150000.1 66 Mesculenta_cassava.05G139400.1 Mesculenta_cassava.05G139600.1 Csinensis.orange1.1g009872m_PAC_18132875 100 Csinensis.orange1.1g035827m_PAC_18114153 100 Csinensis.orange1.1g010479m_PAC_18116477 Csinensis.orange1.1g038171m_PAC_18116437 Csinensis.orange1.1g010727m_PAC_18093086 gnl_kiwi_Achn230171 gnl_kiwi_Achn028781 97 gnl_kiwi_Achn028771 gnl_kiwi_Achn028761 91 Paulawniaceae-UMUL-2004160-Paulownia_fargesii 83 Paulawniaceae-UMUL-2006956-Paulownia_fargesii 100 Lamiaceae-EQDA-2057599-Salvia_spp. 98 Phelipanche_mutelii_PhMu2_9609 52 97 gnl_Stras_v1.02_SGA1.0.scaffold50G001160 100 90 Alectra_vogelii_AlVo2_2404 99 Triphysaria_pusilla_TrPu1_6841 V 66 gnl_Stras_v1.02_SGA1.0.scaffold9G00500 Gesneriaceae-DTNC-0020412-Sinningia_tuberosa 100 Striga_gesneroides_StGe3_28914 99 Sesamum_indicumSIN_1007705 Sesamum_indicumSIN_1007703 Egrandis.eucalyptus.B03255.1 Egrandis.eucalyptus.B03258.1 98 Egrandis.eucalyptus.B03262.1 53 Egrandis.eucalyptus.B03265.1 54 Egrandis.eucalyptus.K00784.1 Egrandis.eucalyptus.B03261.1 Egrandis.eucalyptus.B03247.1 30 Egrandis.eucalyptus.B03252.1 Mesculenta_cassava.02G135900.1 Mesculenta_cassava.02G135800.1 100 Mesculenta_cassava.02G135700.1 Mesculenta_cassava.11G116900.1 100 75 Mesculenta_cassava.04G045800.1 gnl_Theca1.0_Tc00_g049380 Csativus_cucumber.286740.1_PACid_16975055 Prunus_persica_ppa004343m 58 99 Prunus_persica_ppa004390m Mulberry_Morus_natabilis027659 Csinensis.orange1.1g010614m_PAC_18101431 88 73 gnl_Theca1.0_Tc02_g025270 Graimondii_cotton_008G161500.1 100 Mesculenta_cassava.13G021800.1 Mesculenta_cassava.13G021700.1 60 100 Phelipanche_mutelii_PhMu2_16484 H 32 Phelipanche_aegyptiaca_PhAeBC5_14888.1 100 gnl_Poptr2.2_PACid_18241666 gnl_Poptr2.2_PACid_18248808 gnl_Poptr2.2_PACid_18247775 73 gnl_Poptr2.2_PACid_18248470 SIZE-2008354-Passiflora_caerulea 99 Mulberry_Morus_natabilis027660 100 Mulberry_Morus_natabilis027664 51 Csativus_cucumber.286750.1_PACid_16975056 100 Csativus_cucumber.286700.1_PACid_16975051 Csativus_cucumber.286720.1_PACid_16975053 77 Csativus_cucumber.286680.1_PACid_16975049 78 Csativus_cucumber.286650.1_PACid_16975045 Csativus_cucumber.286710.1_PACid_16975052 gnl_Carpa1.181_PACid_16417696 gnl_Carpa1.181_PACid_16417697 gnl_Thepa2.0_Tp6g18990 gnl_Arath10_AT5G57260.1 gnl_Thepa2.0_Tp2g15460 66 gnl_Thepa2.0_Tp2g15440 100 37 gnl_Thepa2.0_Tp2g15450 47 gnl_Arath10_AT3G26290.1 gnl_Arath10_AT3G26300.1 95 68 gnl_Arath10_AT3G26310.1 gnl_Thepa2.0_Tp2g15470 83 gnl_Arath10_AT3G26320.1 gnl_Arath10_AT3G26330.1 100 gnl_Thepa2.0_Tp2g15480 37 gnl_Arath10_AT2G02580.1 gnl_Carpa1.181_PACid_16417698 gnl_Carpa1.181_PACid_16409039 99 gnl_Carpa1.181_PACid_16409041 gnl_Aquco1.0_PACid_18150014 gnl_Aquco1.0_PACid_18150046 100 gnl_Aquco1.0_PACid_18151700 gnl_Aquco1.0_PACid_18145010 gnl_Aquco1.0_PACid_18152276 gnl_Aquco1.0_PACid_18150156 gnl_Orysa6.0_PACid_16851105 gnl_Bradi1.2_Bradi1g22340.1 100 gnl_Bradi1.2_Bradi1g22370.1 69 gnl_Sorbi1.4_PACid_1949422 gnl_Sorbi1.4_PACid_1970662 gnl_Sorbi1.4_PACid_1949502 0.1 gnl_Sorbi1.4_PACid_1949506 gnl_Sorbi1.4_PACid_1956460 Fig. S2.4 - orthogroup 806 LaSa_53606 LaSa_3856381 LaSa_35681 LaSa_45473 42 LaSa_53826 36 HeAn_2351 98 HeAn_40477 HeAn_342 57 gnl_Solly2.3_Solyc09g007540.2.1 96 gnl_Mimgu1.0_PACid_17678221 gnl_Mimgu1.0_PACid_17684099 99 gnl_Mimgu1.0_PACid_17693905 100Lindenbergia_philippensis_LiPhGnB2_3240.1 60 99 Alectra_vogelii_AlVo2_3101 Striga_hermonthica_StHeBC3_1171.3 V 90 Triphysaria_versicolor_TrVeBC3_3608.1 68 Phelipanche_aegyptiaca_PhAeBC5_4810.1 68 LaSa_11347 50 24 gnl_Betvu_v1.1_Bv_30900_jfnu.t1 gnl_Vitvi12X_PACid_17822742 gnl_Frave2.0_gene13331 100 gnl_Medtr3.5_Medtr1g101620.1 100 gnl_Glyma1.01_PACid_16281404 gnl_Glyma1.01_PACid_16281403 gnl_Poptr2.2_PACid_18244025 100 gnl_Poptr2.2_PACid_18207203 gnl_Thepa2.0_Tp1g12970 99 100 gnl_Arath10_AT1G14610.1 73 gnl_Carpa1.181_PACid_16412864 94 gnl_Theca1.0_Tc09_g018620 100 Orobanche_minor_OrMi2_4015 Phelipanche_aegyptiaca_PhAeBC5_4239.1 H Phelipanche_ramosa_PhRa5_26371 Phelipanche_mutelii_PhMu2_16115 gnl_Nelnu1.0_NNU_017995-RA gnl_Aquco1.0_PACid_18144325 gnl_Aquco1.0_PACid_18157961 gnl_Aquco1.0_PACid_18164810 gnl_Sorbi1.4_PACid_1975126 99 gnl_Orysa6.0_PACid_16846845 100 80 gnl_Bradi1.2_Bradi2g04960.1 64 gnl_Orysa6.0_PACid_16844494 gnl_Orysa6.0_PACid_16844493 100 gnl_Orysa6.0_PACid_16849430 gnl_Orysa6.0_PACid_16849429 gnl_Bradi1.2_Bradi3g30350.1 100 100 gnl_Orysa6.0_PACid_16886435 gnl_Phoda3.0_PDK_30s728481g001 89 gnl_Musac1.0_GSMUA_AchrUn_randomT06440_001 gnl_Ambtr1.0.27_AmTr_v1.0_scaffold00038.43 0.1

Fig. S2.5 - orthogroup 1021 gnl_Thepa2.0_Tp6g14040 gnl_Thepa2.0_Tp5g19130 100 gnl_Thepa2.0_Tp4g01170 gnl_Thepa2.0_Tp6g08560 100 gnl_Arath10_AT1G43260.1 gi_727440336_ref_XM_010503284.1_PREDICTED_Camelina_sativa_uncharacterized_LOC104778868 gi_727543855_ref_XM_010446890.1_PREDICTED_Camelina_sativa_uncharacterized_LOC104727821 55 Phelipanche_aegyptiaca_PhAeBC5_17559.1 H 100 100 gi_658064228_ref_XM_008369839.1_PREDICTED_Malus_x_domestica_uncharacterized_LOC103431666 gi_658047128_ref_XM_008361018.1_PREDICTED_Malus_x_domestica_uncharacterized_LOC103422954 90 gi_470147643_ref_XM_004309350.1_PREDICTED_Fragaria_vesca_subsp_vesca_uncharacterized_LOC101299050 100 100 gi_764640492_ref_XM_004309311.2_PREDICTED_Fragaria_vesca_subsp_vesca_uncharacterized_LOC101303082 100 Triphysaria_versicolor_TrVeBC3_24500.1 H gnl_Medtr3.5_Medtr8g041360.1 100 gnl_Medtr3.5_Medtr6g029340.1 gnl_Poptr2.2_PACid_18212122 90 gnl_Poptr2.2_PACid_18215743 gnl_Poptr2.2_PACid_18230486 gnl_Poptr2.2_PACid_18215448 gnl_Poptr2.2_PACid_18236154 100 gnl_Solly2.3_Solyc12g021300.1.1 gnl_Solly2.3_Solyc11g042510.1.1 gnl_Solly2.3_Solyc00g005150.1.1 gnl_Solly2.3_Solyc11g020250.1.1 gnl_Solly2.3_Solyc12g056870.1.1 88 gnl_Solly2.3_Solyc06g016740.1.1 OrAeBC5_15158.1 V 100 StHeBC3_25113.1 86 LiPhGnB2_13002.22 78 StHeBC3_30495.1 V LiPhGnB2_24076.1 gnl_Sorbi1.4_PACid_1975701 66 gnl_Sorbi1.4_PACid_1968732 gnl_Sorbi1.4_PACid_1960022 0.1

Fig. S2.6 - orthogroup 1226 gnl_Aquco1.0_PACid_18161157 100 gnl_Aquco1.0_PACid_18161093 gnl_Aquco1.0_PACid_18161289 gnl_Nelnu1.0_NNU_010765-RA gnl_Vitvi12X_PACid_17825988 gnl_Vitvi12X_PACid_17825987 Phelipanche_aegyptiaca_PhAeBC5_11914.4 Phelipanche_ramosa_PhRa5_49596 100 Phelipanche_ramosa_PhRa5_27280 H Phelipanche_mutelii_PhMu2_10869 32 97 Phelipanche_mutelii_PhMu2_12766 93 Ricinus_communis_hit gnl_Poptr2.2_PACid_18215261 gnl_Poptr2.2_PACid_18221811 9 15 Populus_tremula_hit Eucalyptus_grandis_hit gnl_Medtr3.5_Medtr2g039570.1 25 gnl_Medtr3.5_Medtr2g039480.1 89 27 gnl_Medtr3.5_Medtr2g039510.1 100 gnl_Glyma1.01_PACid_16290482 gnl_Glyma1.01_PACid_16304596 64 gnl_Glyma1.01_PACid_16290485 gnl_Glyma1.01_PACid_16290483 22 gnl_Glyma1.01_PACid_16290486 gnl_Glyma1.01_PACid_16290484 gnl_Aquco1.0_PACid_18163570 gnl_Aquco1.0_PACid_18163552 75 gnl_Aquco1.0_PACid_18140211 gnl_Frave2.0_gene12656 46 gnl_Frave2.0_gene35181 17 gnl_Arath10_AT1G29840.1 gnl_Arath10_AT3G47590.1 13 77 5 gnl_Thepa2.0_Tp5g14320 gnl_Frave2.0_gene12652 46 gnl_Carpa1.181_PACid_16430012 Gossypium_raimondii_hit 24 85 64 gnl_Theca1.0_Tc09_g007740 gnl_Theca1.0_Tc09_g007750 100 LaSa_1782 99 HeAn_18181 66 HeAn_37527 86 HeAn_51139 HeAn_33084 HeAn_27676 99 HeAn_43436 LaSa_21324 36 gnl_Solly2.3_Solyc07g039510.2.1 Paulawniaceae-UMUL-2018120-Paulownia_fargesii 97 Lamiaceae-EQDA-2055135-Salvia_spp. Lindenbergia_philippensis_LiPhGnB2_2721.1 57 93 99 Orobanchaceae-URZI-0010918-Epifagus_virginiana Triphysaria_versicolor_TrVeBC3_3710.1 32 Triphysaria_pusilla_TrPu1_1859 Striga_gesneroides_StGe3_11506 V 31 100 36 Orobanche_californica_OrCa3_18018 46 Phelipanche_aegyptiaca_PhAeBC5_9820.1 Phelipanche_mutelii_PhMu2_4393 Lamiaceae-EQDA-2056965-Salvia_spp. Lindenbergia_philippensis_LiPhGnB2_17941.1 40 71 Striga_hermonthica_StHeBC3_10384.1 V gnl_Mimgu1.0_PACid_17694864 gnl_kiwi_Achn156101 gnl_Soltu3.4_PGSC0003DMP400033146 gnl_Phoda3.0_PDK_30s1146301g002 gnl_Sorbi1.4_PACid_1962205 95 gnl_Orysa6.0_PACid_16833322 gnl_Bradi1.2_Bradi2g41780.1 29 97 100 gnl_Bradi1.2_Bradi2g41807.1 51 gnl_Bradi1.2_Bradi1g56955.1 100 gnl_Orysa6.0_PACid_16833320 71 gnl_Orysa6.0_PACid_16833321 56 73 gnl_Sorbi1.4_PACid_1962202 84 gnl_Sorbi1.4_PACid_1962204 gnl_Musac1.0_GSMUA_Achr7T24060_001 gnl_Bradi1.2_Bradi1g71420.1 gnl_Musac1.0_GSMUA_Achr7T25930_001 85 gnl_Phoda3.0_PDK_30s867601g004 gnl_Ambtr1.0.27_AmTr_v1.0_scaffold00059.29 gnl_Ambtr1.0.27_AmTr_v1.0_scaffold00052.63 0.1 Fig. S2.7 - orthogroup 1685 HeAn_33941 HeAn_59663 45 LaSa_50847 53 LaSa_18050 84 HeAn_47622 HeAn_60614 LaSa_2472 LaSa_47990 75 LaSa_26769 LaSa_14312 HeAn_33070 99 100 LaSa_49575 LaSa_46835 97 94 LaSa_11493 LaSa_31036 HeAn_19514 28 LaSa_54481 86 89 LaSa_54461 LaSa_6072 HeAn_57321 69 HeAn_49826 LaSa_55798 HeAn_26844 LaSa_24942 99 89 53 LaSa_1998 LaSa_17404 57 LaSa_10890 LaSa_45152 LaSa_31572 gnl_Solly2.3_Solyc11g011880.1.1 Bignoniaceae-TKEK-0102668-Mansoa_alliacea Paulawniaceae-UMUL-2019118-Paulownia_fargesii gnl_Mimgu1.0_PACid_17686838 48 Lamiaceae-EQDA-2013088-Salvia_spp. 39 82 Phelipanche_aegyptiaca_PhAeBC5_8212.1 97 Phelipanche_mutelii_PhMu2_15981 Phelipanche_ramosa_PhRa5_11694 V 99 33 Phelipanche_mutelii_PhMu2_43769 Sesamum_indicumSIN_1011984 82 16 Acanthaceae-WEAC-0012587-Strobilanthes_dyerianus Lindenbergia_philippensis_LiPhGnB2_5053.1 61 Orobanchaceae-URZI-0023293-Epifagus_virginiana V 91 43 Plantaginaceae-EBOL-0070915-Anthirrhinum_majus-B Verbenaceae-GCFE-2056468-Verbena_hastata 100 Gesneriaceae-DTNC-0067054-Sinningia_tuberosa Gesneriaceae-DTNC-0018277-Sinningia_tuberosa Oleaceae-TORX-0019889-Olea_europaea gnl_kiwi_Achn191641 gnl_Theca1.0_Tc00_g049550 gnl_Carpa1.181_PACid_16413274 95 77 gnl_Theca1.0_Tc01_g013100 gnl_Poptr2.2_PACid_18210298 32 gnl_Poptr2.2_PACid_18209787 gnl_Poptr2.2_PACid_18237968 98 gnl_Frave2.0_gene11688 gnl_Glyma1.01_PACid_16285106 100 gnl_Glyma1.01_PACid_16285111 91 gnl_Medtr3.5_Medtr3g064080.1 gnl_Glyma1.01_PACid_16285113 gnl_Glyma1.01_PACid_16285114 90 gnl_Glyma1.01_PACid_16285144 gnl_Glyma1.01_PACid_16285136 100 31 gnl_Glyma1.01_PACid_16285124 98 38gnl_Glyma1.01_PACid_16307988 gnl_Glyma1.01_PACid_16285115 gnl_Glyma1.01_PACid_16307983 gnl_Glyma1.01_PACid_16285137 60 gnl_Glyma1.01_PACid_16285142 97 gnl_Glyma1.01_PACid_16285164 73 gnl_Glyma1.01_PACid_16285127 34gnl_Glyma1.01_PACid_16285154 gnl_Glyma1.01_PACid_16285155 gnl_Glyma1.01_PACid_16285126 100 84 gnl_Glyma1.01_PACid_16307982 gnl_Glyma1.01_PACid_16307984 gnl_Glyma1.01_PACid_16285165 100 gnl_Glyma1.01_PACid_16285157 gnl_Glyma1.01_PACid_16285158 gnl_Medtr3.5_Medtr3g064090.1 gnl_Medtr3.5_Medtr3g064110.1 Phelipanche_ramosa_PhRa5_19003 Phelipanche_mutelii_PhMu2_13255 Phelipanche_aegyptiaca_PhAeBC5_15353.2 H 100 Phelipanche_ramosa_PhRa5_7678 Phelipanche_mutelii_PhMu2_11925 100 Phelipanche_aegyptiaca_PhAeBC5_9731.1 Phelipanche_mutelii_PhMu2_20785 Phelipanche_ramosa_PhRa5_7970 Phelipanche_aegyptiaca_PhAeBC5_6791.2 gnl_Vitvi12X_PACid_17840416 99 gnl_Vitvi12X_PACid_17840415 gnl_Vitvi12X_PACid_17819441 gnl_Aquco1.0_PACid_18143181 gnl_Aquco1.0_PACid_18163597 gnl_Aquco1.0_PACid_18143776 gnl_Ambtr1.0.27_AmTr_v1.0_scaffold00029.148 0.1 Fig. S2.8 - orthogroup 1886 gnl_Mimgu1.0_PACid_17676918 Lindenbergia_philippensis_LiPhGnB2_1939.4 gnl_Mimgu1.0_PACid_17676671 gnl_Mimgu1.0_PACid_17673310 Orobanche_minor_OrMi2_545 63 Alectra_vogelii_AlVo2_1000 gnl_Stras_v1.02_SGA1.0.scaffold68G00120 57 Striga_hermonthica_StHeBC3_5733.1 Striga_gesneroides_StGe3_8910 40 Phelipanche_ramosa_PhRa5_10825 Phelipanche_mutelii_PhMu2_2489 Phelipanche_mutelii_PhMu2_23970 Phelipanche_mutelii_PhMu2_20426 Phelipanche_aegyptiaca_PhAeBC5_392.1 V Orobanche_californica_OrCa3_4710 Orobanche_californica_OrCa3_4709 Orobanchaceae-URZI-0089123-Epifagus_virginiana Orobanchaceae-URZI-0088146-Epifagus_virginiana Triphysaria_eriantha_TrEr3_9866 Triphysaria_eriantha_TrEr3_19164 Triphysaria_pusilla_TrPu1_24566 100 Triphysaria_eriantha_TrEr3_21764 Triphysaria_eriantha_TrEr3_9865 Triphysaria_versicolor_TrVeBC3_4307.1 Triphysaria_pusilla_TrPu1_7560 Acanthaceae-WEAC-0134774-Strobilanthes_dyerianus HeAn_30129 75 100 HeAn_54132 LaSa_10259 LaSa_53791 gnl_Soltu3.4_PGSC0003DMP400011475 gnl_Soltu3.4_PGSC0003DMP400011476 gnl_Solly2.3_Solyc12g017540.1.1 gnl_Solly2.3_Solyc08g015780.2.1 gnl_Soltu3.4_PGSC0003DMP400024257 34 gnl_Glyma1.01_PACid_16303903 gnl_Glyma1.01_PACid_16269401 79 gnl_Glyma1.01_PACid_16269402 gnl_Frave2.0_gene10164 79 Phelipanche_ramosa_PhRa5_103413 Phelipanche_ramosa_PhRa5_58721 H Phelipanche_aegyptiaca_PhAeBC5_15496.1 96 gnl_Arath10_AT2G44090.1 gnl_Thepa2.0_Tp4g26170 gnl_Thepa2.0_Tp5g03260 94 gnl_Arath10_AT3G59910.1 91 gnl_Carpa1.181_PACid_16428134 71gnl_Theca1.0_Tc00_g035340 68 gnl_Poptr2.2_PACid_18242821 gnl_Poptr2.2_PACid_18210308 gnl_Poptr2.2_PACid_18210307 82 gnl_Vitvi12X_PACid_17842635 gnl_Nelnu1.0_NNU_004481-RA gnl_Nelnu1.0_NNU_020768-RA gnl_Aquco1.0_PACid_18158782 99 gnl_Phoda3.0_PDK_30s721731g001 gnl_Phoda3.0_PDK_30s970671g002 50 gnl_Musac1.0_GSMUA_Achr3T02760_001 gnl_Musac1.0_GSMUA_Achr10T16350_001 gnl_Musac1.0_GSMUA_Achr6T12560_001 100 gnl_Musac1.0_GSMUA_Achr6T18250_001 gnl_Musac1.0_GSMUA_AchrUn_randomT20880_001 gnl_Bradi1.2_Bradi4g20950.1 gnl_Sorbi1.4_PACid_1969730 gnl_Orysa6.0_PACid_16888805 gnl_Ambtr1.0.27_AmTr_v1.0_scaffold00169.18 gnl_Ambtr1.0.27_AmTr_v1.0_scaffold00066.79 1.0 Fig. S2.9 - orthogroup 2270 LaSa_20765 97 HeAn_34763 LaSa_48077 59 LaSa_5371 87 HeAn_54649 46 HeAn_35531 100 HeAn_15017 HeAn_15065 gnl_Solly2.3_Solyc07g008950.2.1 gnl_Soltu3.4_PGSC0003DMP400050083 100 Acanthaceae-WEAC-0133725-Strobilanthes_dyerianus 100 Sesamum_indicumSIN_1001287 100 gnl_Mimgu1.0_PACid_17691323 75 gnl_Mimgu1.0_PACid_17669561 gnl_Mimgu1.0_PACid_17695503 94 Phelipanche_aegyptiaca_PhAeBC5_4515.1 V 100 Alectra_vogelii_AlVo2_8771 Lindenbergia_philippensis_LiPhGnB2_3847.1 97 gnl_Poptr2.2_PACid_18210733 gnl_Poptr2.2_PACid_18239126 gnl_Theca1.0_Tc04_g020790 Phelipanche_ramosa_PhRa5_6157 Phelipanche_mutelii_PhMu2_12619 Phelipanche_mutelii_PhMu2_44156 H 81 100 Phelipanche_mutelii_PhMu2_20937 Phelipanche_mutelii_PhMu2_5516 Phelipanche_aegyptiaca_PhAeBC5_3356.1 Phelipanche_mutelii_PhMu2_14031 Orobanche_minor_OrMi2_4206 98 Striga_gesneroides_StGe3_7730 96 Striga_hermonthica_StHeBC3_2868.1 100 Striga_gesneroides_StGe3_20099 gnl_Arath10_AT4G13780.1 100 73 gnl_Thepa2.0_Tp7g11570 gnl_Carpa1.181_PACid_16427157 gnl_Glyma1.01_PACid_16286058 100 gnl_Glyma1.01_PACid_16290193 100 gnl_Medtr3.5_Medtr4g122580.1 99 82 gnl_Medtr3.5_Medtr4g077810.1 gnl_Medtr3.5_Medtr4g077700.1 92 gnl_Frave2.0_gene06519 gnl_Vitvi12X_PACid_17830170 gnl_Vitvi12X_PACid_17842684 100 gnl_Carpa1.181_PACid_16426409 gnl_Aquco1.0_PACid_18150130 49 gnl_Nelnu1.0_NNU_026444-RA gnl_Nelnu1.0_NNU_012022-RA gnl_Phoda3.0_PDK_30s970271g001 88 gnl_Musac1.0_GSMUA_Achr8T31240_001 gnl_Musac1.0_GSMUA_Achr6T24850_001 0.1 gnl_Bradi1.2_Bradi3g31100.1 100 gnl_Orysa6.0_PACid_16885412 100 gnl_Orysa6.0_PACid_16885413 gnl_Orysa6.0_PACid_16885415 100 gnl_Orysa6.0_PACid_16885414 gnl_Orysa6.0_PACid_16866568 100 100 gnl_Bradi1.2_Bradi1g69880.1 76 gnl_Sorbi1.4_PACid_1954278 gnl_Sorbi1.4_PACid_1967640 gnl_Ambtr1.0.27_AmTr_v1.0_scaffold00209.4 gnl_Ambtr1.0.27_AmTr_v1.0_scaffold00209.2

Fig. S2.10 - orthogroup 2376 LaSa_1735 100HeAn_1761973 HeAn_1661968 HeAn_438 gnl_Solly2.3_Solyc07g055080.2.1 90 gnl_Soltu3.4_PGSC0003DMP400035506 gnl_Soltu3.4_PGSC0003DMP400018645 gnl_Solly2.3_Solyc10g008010.2.1 gnl_Utrgi_v4.1_Scf00206.g12537.t1 81 Gesneriaceae-DTNC-0010673-Sinningia_tuberosa Lamiaceae-EQDA-2054077-Salvia_spp. 93 gnl_Mimgu1.0_PACid_17671107 96 LiPhGnB2_1016.1 Striga_hermonthica_StHeBC3_3231.1 60Striga_gesneroides_StGe3_1180 Alectra_vogelii_AlVo2_16704 99 18 Alectra_vogelii_AlVo2_12447 65 Striga_gesneroides_StGe3_1398 Triphysaria_eriantha_TrEr3_1634 Triphysaria_eriantha_TrEr3_6099 V Triphysaria_pusilla_TrPu1_6476 83 Triphysaria_eriantha_TrEr3_1633 Orobanche_minor_OrMi2_6179 15 Orobanche_minor_OrMi2_4853 Orobanche_minor_OrMi2_1490 92 Orobanche_californica_OrCa3_17944 Phelipanche_aegyptiaca_PhAeBC5_3502.1 Phelipanche_ramosa_PhRa5_5002 16 Verbenaceae-GCFE-2001275-Verbena_hastata gnl_Mimgu1.0_PACid_17674865 Paulawniaceae-UMUL-2008327-Paulownia_fargesii Malus_domestica_ncbi_hit gnl_Glyma1.01_PACid_16300716 50 71gnl_Glyma1.01_PACid_16271520 gnl_Glyma1.01_PACid_16271522 gnl_Glyma1.01_PACid_16271518 gnl_Glyma1.01_PACid_16271521 gnl_Glyma1.01_PACid_16251836 9 gnl_Glyma1.01_PACid_16244469 gnl_Glyma1.01_PACid_16244470 75 gnl_Glyma1.01_PACid_16244471 gnl_Thepa2.0_Tp1g14620 43 gnl_Arath10_AT1G16470.1 100gnl_Arath10_AT1G79210.1 55 gnl_Thepa2.0_Tp5g34260 Jatropha_curcas_ncbi_hit 57 Ricinus_communis_ncbi_hit gnl_Carpa1.181_PACid_16426987 gnl_Theca1.0_Tc06_g012030 66 gnl_Poptr2.2_PACid_18233344 100 gnl_Poptr2.2_PACid_18229980 62 Prunus_persica_ncbi_hit 94 Fragaria_vesca_ncbi_hit 100 Phelipanche_ramosa_PhRa5_4383 Phelipanche_aegyptiaca_PhAeBC5_270.3 34 H Phelipanche_mutelii_PhMu2_6326 66 Phelipanche_ramosa_PhRa5_4382 Eucalyptus_grandis_ncbi_hit gnl_Vitvi12X_PACid_17823789 76 gnl_Aquco1.0_PACid_18158791 98 gnl_Nelnu1.0_NNU_016324-RA gnl_Musac1.0_GSMUA_Achr5T19360_001 99 gnl_Musac1.0_GSMUA_Achr8T13980_001 gnl_Sorbi1.4_PACid_1953163 100 gnl_Orysa6.0_PACid_16847356 68 gnl_Bradi1.2_Bradi3g49610.1 48 gnl_Sorbi1.4_PACid_1967357 gnl_Orysa6.0_PACid_16844271 gnl_Orysa6.0_PACid_16842056 gnl_Phoda3.0_PDK_30s6550949g006 gnl_Phoda3.0_PDK_30s1154301g003 0.1 Fig. S2.11 - orthogroup 3861 LaSa_42928 LaSa_42899 Lindenbergia_philippensis_LiPhGnB2_15157.1 Phelipanche_aegyptiaca_PhAeBC5_17480.2 100 V 99 Orobanche_californica_OrCa3_19474 66 gnl_Stras_v1.02_SGA1.0.scaffold171G00130 Lindenbergia_philippensis_LiPhGnB2_20423.1 47 96 Triphysaria_versicolor_TrVeBC3_15449.1 77 Phelipanche_ramosa_PhRa5_18614 100 100 V 66 Striga_hermonthica_StHeBC3_8657.3 93 Phelipanche_aegyptiaca_PhAeBC5_5452.1 gnl_Mimgu1.0_PACid_17688960 100 Sesamum_indicumSIN_1008614 66 gnl_Utrgi_v4.1_Scf00196.g12204.t1 gnl_Solly2.3_Solyc12g017920.1.1 100 gnl_Soltu3.4_PGSC0003DMP400051135 gnl_Soltu3.4_PGSC0003DMP400051136 gnl_Solly2.3_Solyc12g096610.1.1 gnl_Frave2.0_gene10159 gnl_Frave2.0_gene28028 Lindenbergia_philippensis_LiPhGnB2_6846.1 51 100 Phelipanche_mutelii_PhMu2_2110 H 90 Phelipanche_aegyptiaca_PhAeBC5_22555.2 100 99 gnl_Glyma1.01_PACid_16282271 gnl_Glyma1.01_PACid_16282272 62 Orobanche_minor_OrMi2_2808 Phelipanche_ramosa_PhRa5_12205 100 100 H Phelipanche_mutelii_PhMu2_12820 Phelipanche_mutelii_PhMu2_11052 85 Phelipanche_aegyptiaca_PhAeBC5_9914.1 42 gnl_Medtr3.5_Medtr3g029520.1 gnl_Poptr2.2_PACid_18242959 gnl_Poptr2.2_PACid_18209526 64 gnl_Carpa1.181_PACid_16428142 84 91 gnl_Arath10_AT2G31870.1 gnl_Thepa2.0_Tp4g14220 92 93 91 gnl_Thepa2.0_Tp2g03830 100 gnl_Arath10_AT2G31865.2 gnl_Theca1.0_Tc00_g035400 gnl_Vitvi12X_PACid_17842629 gnl_Aquco1.0_PACid_18146143 gnl_Nelnu1.0_NNU_020756-RA gnl_Sorbi1.4_PACid_1949469 100 gnl_Bradi1.2_Bradi1g01942.1 48 gnl_Bradi1.2_Bradi1g01980.1 100 gnl_Orysa6.0_PACid_16851025 gnl_Phoda3.0_PDK_30s1048841g004 69 gnl_Musac1.0_GSMUA_Achr3T21720_001 gnl_Ambtr1.0.27_AmTr_v1.0_scaffold00066.92 0.1

Fig. S2.12 - orthogroup 4067 gnl_Vitvi12X_PACid_17831143 gnl_Arath10_AT2G32320.2 100 gnl_Arath10_AT2G31580.1 37 gnl_Thepa2.0_Tp4g14010 gnl_Carpa1.181_PACid_16431986 34 gnl_Poptr2.2_PACid_18244294 gnl_Glyma1.01_PACid_16278099 57 gnl_Glyma1.01_PACid_16302163 gnl_Glyma1.01_PACid_16302546 100 77 gnl_Glyma1.01_PACid_16302712 gnl_Frave2.0_gene23761 100 gnl_Frave2.0_gene21135 100 Phelipanche_ramosa_PhRa5_10084 Phelipanche_aegyptiaca_PhAeBC5_9762.1 H Phelipanche_mutelii_PhMu2_15393 gnl_Solly2.3_Solyc04g051600.2.1 gnl_Soltu3.4_PGSC0003DMP400054342 gnl_Soltu3.4_PGSC0003DMP400054339 Phelipanche_ramosa_PhRa5_7628 45 Phelipanche_ramosa_PhRa5_55379 64 Phelipanche_mutelii_PhMu2_8074 V Phelipanche_mutelii_PhMu2_56057 Phelipanche_aegyptiaca_PhAeBC5_9480.1 93 Phelipanche_ramosa_PhRa5_33915 95 Sesamum_indicumSIN_1025118 Verbenaceae-GCFE-2057053-Verbena_hastata 26 60 gnl_Mimgu1.0_PACid_17678571 50 Lindenbergia_philippensis_LiPhGnB2_14111.5 Alectra_vogelii_AlVo2_21454 99 gnl_Stras_v1.02_SGA1.0.scaffold50G00130 54 Triphysaria_eriantha_TrEr3_23124 V 55 36 79 Triphysaria_pusilla_TrPu1_26273 Orobanchaceae-URZI-0076155-Epifagus_virginiana 33 LaSa_44805 gnl_kiwi_Achn042481 gnl_Nelnu1.0_NNU_014811-RA gnl_Nelnu1.0_NNU_014809-RA gnl_Aquco1.0_PACid_18146097 gnl_Aquco1.0_PACid_18144697 gnl_Aquco1.0_PACid_18140021 gnl_Phoda3.0_PDK_30s1172441g001 98 gnl_Musac1.0_GSMUA_Achr8T03920_001 100 gnl_Sorbi1.4_PACid_1981630 99 gnl_Bradi1.2_Bradi2g18677.2 gnl_Bradi1.2_Bradi3g06090.1 100 gnl_Orysa6.0_PACid_16862564 gnl_Orysa6.0_PACid_16862565 gnl_Ambtr1.0.27_AmTr_v1.0_scaffold00104.33 0.1

Fig. S2.13 - orthogroup 4572 gnl_Prupe_v1.0_ppa019924m 52 gnl_Betvu_v1.1_Bv9_217120_uykx.t1 43 gnl_kiwi_Achn050011 98 Gesneriaceae-DTNC-0066665-Sinningia_tuberosa 100 Sesamum_indicumSIN_1009534 78 Paulawniaceae-UMUL-2022595-Paulownia_fargesii 86 gnl_Stras_v1.02_SGA1.0.scaffold230G00040 49 100 Striga_gesneroides_StGe3_6504 100 gnl_Stras_v1.02_SGA1.0.scaffold244G00480 V 29 Phelipanche_ramosa_PhRa5_15411 100 Orobanche_californica_OrCa3_28524 gnl_kiwi_Achn141071 gnl_Vitvi_Genoscope.12X_GSVIVT01016843001 gnl_Vitvi_Genoscope.12X_GSVIVT01016837001 29 gnl_Poptr_v3.0_Potri.001G395000.1 12 gnl_Betvu_v1.1_Bv2_027460_hcyc.t1 22 gnl_kiwi_Achn198561 gnl_Stras_v1.02_SGA1.0.scaffold821G00010 100 Sesamum_indicumSIN_1010135 51 gnl_Stras_v1.02_SGA1.0.scaffold90G00440 100 100 gnl_Stras_v1.02_SGA1.0.scaffold90G00540 58 gnl_Stras_v1.02_SGA1.0.scaffold90G00530 100 gnl_Stras_v1.02_SGA1.0.scaffold90G00520 17 15 37 Triphysaria_pusilla_TrPu1_15 V 39 Phelipanche_ramosa_PhRa5_25490 100 Orobanche_minor_OrMi2_5957 gnl_Betvu_v1.1_Bv3u_068510_hfxt.t1 gnl_Poptr_v3.0_Potri.012G099400.1 72 29 Paulawniaceae-UMUL-2014906-Paulownia_fargesii gnl_Betvu_v1.1_Bv9_207570_uyyw.t1 100 93 gnl_Betvu_v1.1_Bv9_217220_muej.t1 gnl_Betvu_v1.1_Bv9_217200_jxww.t1 Sesamum_indicumSIN_1026648 4 Triphysaria_eriantha_TrEr3_4370 V Phelipanche_ramosa_PhRa5_7856 22 38 26 Sesamum_indicumSIN_1008324 gnl_Poptr_v3.0_Potri.013G146800.1 gnl_Prupe_v1.0_ppa017324m 86 100 gnl_Frave2.0_gene28576 gnl_Frave2.0_gene28575 100 gnl_Frave2.0_gene28262 100 gnl_Frave2.0_gene28565 97 gnl_Frave2.0_gene28567 76 Phelipanche_aegyptiaca_PhAeBC5_8623.1 100Phelipanche_ramosa_PhRa5_22472 H Orobanche_minor_OrMi2_36149 gnl_Frave2.0_gene28577 79 100 gnl_Frave2.0_gene12676 77 gnl_Frave2.0_gene28578 gnl_Frave2.0_gene28244 97 gnl_Frave2.0_gene06249 60 100 gnl_Frave2.0_gene04144 100 gnl_Frave2.0_gene04153 86 gnl_Frave2.0_gene19565 98 gnl_Frave2.0_gene19562 60 gnl_Frave2.0_gene19559 gnl_Frave2.0_gene18700 49 gnl_Prupe_v1.0_ppa017715m 44 gnl_Prupe_v1.0_ppa022705m 34 gnl_Frave2.0_gene18704 33 gnl_Frave2.0_gene23223 46 gnl_Frave2.0_gene23178 98 gnl_Frave2.0_gene28454 52 33 gnl_Frave2.0_gene23216 gnl_Frave2.0_gene04317 83 27 gnl_Prupe_v1.0_ppa023010m 100 gnl_Prupe_v1.0_ppa019433m 82 98 gnl_Prupe_v1.0_ppa016774m gnl_Prupe_v1.0_ppa022788m 63 gnl_Frave2.0_gene12971 68 gnl_Frave2.0_gene12935 100 gnl_Frave2.0_gene12937 gnl_Frave2.0_gene31616 22 100 gnl_Prupe_v1.0_ppa019570m gnl_Frave2.0_gene10265 89 gnl_Frave2.0_gene10267 gnl_Frave2.0_gene17268 97 65 gnl_Frave2.0_gene04055 gnl_Frave2.0_gene04468 gnl_Arath_TAIR10_AT5G22720.2 44 gnl_Orysa_v7.0_LOC_Os10g03780.1 24 gnl_Arath_TAIR10_AT2G42730.1 100 gnl_Arath_TAIR10_AT3G59200.1 68 gnl_Arath_TAIR10_AT3G58860.1 100 98 gnl_Arath_TAIR10_AT3G58880.1 gnl_Arath_TAIR10_AT3G59000.1 100 gnl_Stras_v1.02_SGA1.0.scaffold27G001030 V 41 36 gnl_Stras_v1.02_SGA1.0.scaffold27G001000 gnl_Nelnu_v1.0_NNU_024676-RA gnl_Aquco_v1.1_Aquca_003_00206.1 gnl_Aquco_v1.1_Aquca_003_00221.1 100 gnl_Aquco_v1.1_Aquca_003_00225.1 gnl_Aquco_v1.1_Aquca_022_00167.1 19 36 34 gnl_Aquco_v1.1_Aquca_022_00152.1 22 gnl_Aquco_v1.1_Aquca_054_00089.1 gnl_Nelnu_v1.0_NNU_014293-RA 0.1 100 52 gnl_Aquco_v1.1_Aquca_047_00117.1 65 gnl_Aquco_v1.1_Aquca_066_00033.1 93 gnl_Nelnu_v1.0_NNU_017532-RA gnl_Nelnu_v1.0_NNU_015070-RA gnl_Musac_v1.0_GSMUA_Achr2G13780_001 gnl_Nelnu_v1.0_NNU_015652-RA gnl_Ambtr_v1.0.27_AmTr_v1.0_scaffold00007.398

Fig. S2.14 - orthogroup 4598 other rosids 98 gi_920716850_gb_CM003380.1_Vigna_angularis_cultivar_Jingnong gi_593701027_gi_593701026_Phaseolus_vulgaris_hypothetical_protein gnl_Phavu_v1.0_Phvul.004G006800.1 gi_734391958_gb_KN653320.1_Glycine_soja_cultivar_W05 gi_571454424_gi_571454423_PREDICTED_Glycine_max_nuclear 100 gnl_Glyma1.01_PACid_16258742 Ljaponicus_lotus2g3v0932700.1 100 gi_828292947_gi_828292946_PREDICTED_Cicer_arietinum_nuclear 76 100 gi_922349752_gi_922349751_Medicago_truncatula_embryo_defective gnl_Medtr_Mt4.0v1_Medtr6g004840.1 100 Phelipanche_mutelii_PhMu2_15647 Phelipanche_aegyptiaca_PhAeBC5_13756.1 H Phelipanche_ramosa_PhRa5_33568 gnl_Eucgr_v1.1_Eucgr.J02245.1 gi_629086572_gb_KK198762.1_Eucalyptus_grandis_cultivar_BRASUZ1 gi_702481622_gi_702481621_PREDICTED_Eucalyptus_grandis_nuclear other rosids

other rosids basal asterids other asterids gi_747100634_gi_747100633_PREDICTED_Sesamum_indicum_nuclear Triphysaria_eriantha_TrEr3_12676 Triphysaria_pusilla_TrPu1_32798 Triphysaria_versicolor_TrVeBC3_10866.4 Triphysaria_versicolor_TrVeBC3_10866.3 V 100 100 Triphysaria_versicolor_TrVeBC3_10866.1 Triphysaria_versicolor_TrVeBC3_10866.2 Phelipanche_ramosa_PhRa5_20102 51 Phelipanche_mutelii_PhMu2_10392 gnl_Stras_v1.02_SGA1.0.scaffold108G00290 LiPhGnB2_13574.1 97 gi_604346328_gb_KI630210.1_Mimulus_guttatus_cultivar_IM62 gi_848854816_gi_848854815_PREDICTED_Erythranthe_guttatus_nuclear 100 gnl_Mimgu_v2.0_Migut.H00075.1 gnl_Mimgu_v2.0_Migut.J01523.1 gnl_Mimgu_v2.0_Migut.J01521.1 gnl_Utrgi_v4.1_Scf00266.g14164.t1 Lamiidae_asterid-EDEQ-2012677-Wrightia_natalensis 100 Coffea_Canephora_Cc07_g01650 0.1 gi_661893619_gi_661893603_Coffea_canephora_DH200=94_genomic asterid-BFJL-2017466-Cornus_floridana 92 gnl_kiwi_Achn389831 gnl_Nelnu_v1.0_NNU_002872-RA 42 gi_719976730_gi_719976729_PREDICTED_Nelumbo_nucifera_nuclear gnl_Aquco_v1.1_Aquca_011_00520.1

grasses

gnl_Ambtr_v1.0.27_AmTr_v1.0_scaffold00049.202

Fig. S2.15- orthogroup 5002 gnl_kiwi_Achn271061 94 gnl_kiwi_Achn321631 gnl_kiwi_Achn065441 Orobanche_californica_OrCa3_1090 V 53 Orobanche_californica_OrCa3_10227 58 gnl_kiwi_Achn218791 gnl_kiwi_Achn298471 21 gnl_kiwi_Achn315811 gnl_kiwi_Achn267851 gnl_kiwi_Achn307181 100 Phelipanche_mutelii_PhMu2_10848 Orobanche_californica_OrCa3_36975 V Phelipanche_mutelii_PhMu2_60948 Prunus_persica_ppa025773m 12 Prunus_persica_ppa015420m 100 Prunus_persica_ppa018881m Prunus_persica_ppa016227m Prunus_persica_ppa024297m Prunus_persica_ppa025795m Prunus_persica_ppa016010m 47 Prunus_persica_ppa024136m gnl_Frave2.0_gene09773 99 gnl_Frave2.0_gene02159 gnl_Frave2.0_gene31748 gnl_Frave2.0_gene26434 gnl_Frave2.0_gene31600 75 gnl_Frave2.0_gene30452 gnl_Frave2.0_gene07299 100 gnl_Frave2.0_gene29048 95 gnl_Frave2.0_gene03470 99 80 gnl_Frave2.0_gene20214 gnl_Frave2.0_gene12006 gnl_Frave2.0_gene06049 gnl_Frave2.0_gene23080 80 Prunus_persica_ppa016147m Prunus_persica_ppa019737m 100 Prunus_persica_ppa026150m Orobanche_californica_OrCa3_21790 99 70 H 20 Orobanche_californica_OrCa3_36984 Prunus_persica_ppa026428m PhAeBC5_9781.2 H gnl_Frave2.0_gene22366 84 Phelipanche_mutelii_PhMu2_4123 Alectra_vogelii_AlVo2_24277 H 65 Prunus_persica_ppa020338m Prunus_persica_ppa020692m Prunus_persica_ppa023823m 99 Prunus_persica_ppb018633m Prunus_persica_ppa003125m 58 Prunus_persica_ppa023051m 93 82 Prunus_persica_ppa023697m Prunus_persica_ppa018871m Prunus_persica_ppa025415m Orobanche_californica_OrCa3_675 H Orobanche_californica_OrCa3_28196 gnl_Frave2.0_gene12339 31 gnl_Frave2.0_gene00790 gnl_Frave2.0_gene03369 91 gnl_Frave2.0_gene12042 gnl_Frave2.0_gene12340 gnl_Frave2.0_gene03501 gnl_Theca1.0_Tc08_g013740 gnl_Theca1.0_Tc00_g053480 31 gnl_Theca1.0_Tc07_g007830 59 gnl_Theca1.0_Tc09_g026680 gnl_Theca1.0_Tc03_g005510 13 gnl_Theca1.0_Tc06_g003310 100 gnl_Theca1.0_Tc00_g060470 gnl_Theca1.0_Tc01_g022560 gnl_Theca1.0_Tc00_g028071 26 gnl_Theca1.0_Tc02_g023591 LaSa_54742 gnl_Stras_v1.02_SGA1.0.scaffold134G00240 V LaSa_4883 98 LaSa_12 LaSa_18962 Spolyrhiza_duckweed_Lemna28G0019700 BRUD-2007886-Typha_latifolia_monocot BRUD-2012376-Typha_latifolia_monocot 99 100 98 scaffold-OCWZ-2007249-Dioscorea_villosa 100 scaffold-OCWZ-2012665-Dioscorea_villosa MTII-2039925-Acorus_americanus_monocot MTII-2041638-Acorus_americanus_monocot 44 44 Spolyrhiza_duckweed_Lemna3G0068200 85 Spolyrhiza_duckweed_Lemna24G0000500 Spolyrhiza_duckweed_Lemna2G0037800 100 Spolyrhiza_duckweed_Lemna4G0087300 ICNN-2019470-Yucca_filamentosa_monocot 80 ICNN-2019471-Yucca_filamentosa_monocot ICNN-2019469-Yucca_filamentosa_monocot 27 87 100 BRUD-2061704-Typha_latifolia_monocot 71 BRUD-2013752-Typha_latifolia_monocot ICNN-2013337-Yucca_filamentosa_monocot MTII-2008402-Acorus_americanus_monocot ICNN-2148947-Yucca_filamentosa_monocot 44 98 20 ICNN-2149224-Yucca_filamentosa_monocot scaffold-OCWZ-2049833-Dioscorea_villosa 22 scaffold-OCWZ-2004081-Dioscorea_villosa ICNN-2032880-Yucca_filamentosa_monocot 15 1.0 BRUD-2000235-Typha_latifolia_monocot Fig. S2.16 - orthogroup 5896 HeAn_47681 LaSa_24621 97 HeAn_41638 gnl_Solly2.3_Solyc04g079320.2.1 Verbenaceae-GCFE-2013934-Verbena_hastata 95 100 gnl_Mimgu1.0_PACid_17691026 gnl_Mimgu1.0_PACid_17689222 Paulawniaceae-UMUL-2002659-Paulownia_fargesii Orobanchaceae-URZI-0014355-Epifagus_virginiana 93 Triphysaria_pusilla_TrPu1_5406 80 Triphysaria_eriantha_TrEr3_29871 Triphysaria_versicolor_TrVeBC3_14119.1 53 Triphysaria_pusilla_TrPu1_26889 Alectra_vogelii_AlVo2_29561 V 100 88 Alectra_vogelii_AlVo2_21473 91 100 gnl_Stras_v1.02_SGA1.0.scaffold123G00010 gnl_Stras_v1.02_SGA1.0.scaffold77G00150 43 gnl_Stras_v1.02_SGA1.0.scaffold1168G00030 58 Striga_hermonthica_StHeBC3_13163.3 Striga_gesneroides_StGe3_9700 97 Orobanche_minor_OrMi2_19523 Orobanche_californica_OrCa3_37752 80 Phelipanche_aegyptiaca_PhAeBC5_16415.7 Phelipanche_mutelii_PhMu2_32577 93 Lindenbergia_philippensis_LiPhGnB2_12184.1 55 gnl_Mimgu1.0_PACid_17678524 gnl_Mimgu1.0_PACid_17678523 Sesamum_indicumSIN_1011688 99 gnl_Theca1.0_Tc08_g000970 68 gnl_Carpa1.181_PACid_16424250 gnl_Arath10_AT2G26540.1 58 100 65 gnl_Thepa2.0_Tp4g07760 61 gnl_Poptr2.2_PACid_18222585 gnl_Medtr3.5_Medtr3g101350.1 54 100 gnl_Glyma1.01_PACid_16254781 gnl_Glyma1.01_PACid_16261644 89 Striga_hermonthica_StHeBC3_21126.2 Striga_gesneroides_StGe3_36184 H gnl_Vitvi12X_PACid_17821942 gnl_Phoda3.0_PDK_30s1210471g001 100 gnl_Phoda3.0_PDK_30s836921g002 62 gnl_Musac1.0_GSMUA_Achr2T07670_001 83 gnl_Bradi1.2_Bradi1g72180.2 gnl_Sorbi1.4_PACid_1954513 65 gnl_Orysa6.0_PACid_16845307 gnl_Aquco1.0_PACid_18152575 gnl_Aquco1.0_PACid_18152576 gnl_Aquco1.0_PACid_18152577 69 gnl_Aquco1.0_PACid_18152578 gnl_Nelnu1.0_NNU_001969-RA 0.1 gnl_Ambtr1.0.27_AmTr_v1.0_scaffold00123.1

Fig. S2.17 - orthogroup 8888 gnl_Solly2.3_Solyc11g018770.1.1 gnl_Solly2.3_Solyc11g018820.1.1 Sesamum_indicumSIN_1017177 Lindenbergia_philippensis_LiPhGnB2_4383.1 Orobanchaceae-URZI-0001495-Epifagus_virginiana Striga_gesneroides_StGe3_43233 43 6 gnl_Stras_v1.02_SGA1.0.scaffold6G00600 Striga_gesneroides_StGe3_9777 99 Alectra_vogelii_AlVo2_19276 Striga_gesneroides_StGe3_9722 Striga_hermonthica_StHeBC3_8636.1 100 gnl_Stras_v1.02_SGA1.0.scaffold20G00700 Orobanchaceae-URZI-0068114-Epifagus_virginiana Orobanche_minor_OrMi2_15141 65 Orobanche_minor_OrMi2_15929 Orobanche_californica_OrCa3_8565 V Phelipanche_mutelii_PhMu2_24461 7 Phelipanche_mutelii_PhMu2_24820 Phelipanche_mutelii_PhMu2_33283 57 Phelipanche_mutelii_PhMu2_24460 Phelipanche_mutelii_PhMu2_33284 78 Phelipanche_ramosa_PhRa5_17857 Phelipanche_ramosa_PhRa5_36857 Phelipanche_mutelii_PhMu2_36055 Orobanche_californica_OrCa3_21711 Triphysaria_eriantha_TrEr3_25033 Triphysaria_pusilla_TrPu1_21999 Triphysaria_eriantha_TrEr3_28457 Triphysaria_pusilla_TrPu1_35485 Triphysaria_eriantha_TrEr3_38325 gnl_Mimgu1.0_PACid_17680175 82 HeAn_49992 LaSa_19553 82 LaSa_34257 72 LaSa_18689 LaSa_29407 LaSa_29406 50 LaSa_51241 HeAn_41981 HeAn_50673 HeAn_50624 gnl_Betvu_v1.1_Bv1_001190_ugji.t1 gnl_Vitvi12X_PACid_17840176 90 gnl_Poptr2.2_PACid_18221628 72 gnl_Frave2.0_gene06236 100 Phelipanche_ramosa_PhRa5_6603 Phelipanche_mutelii_PhMu2_37370 H Phelipanche_ramosa_PhRa5_6605 Phelipanche_aegyptiaca_PhAeBC5_15086.1 99 Phelipanche_ramosa_PhRa5_6604 gnl_Glyma1.01_PACid_16317495 100 gnl_Glyma1.01_PACid_16281150 100 Orobanche_minor_OrMi2_43018 Orobanche_minor_OrMi2_9731 H gnl_Carpa1.181_PACid_16424591 74 gnl_Arath10_AT1G22660.1 40 gnl_Thepa2.0_Tp1g20170 gnl_Theca1.0_Tc08_g014540 gnl_Theca1.0_Tc10_g013720 gnl_Aquco1.0_PACid_18163162 gnl_Bradi1.2_Bradi4g41470.1 100gnl_Sorbi1.4_PACid_1977671 gnl_Sorbi1.4_PACid_1977672 100 gnl_Orysa6.0_PACid_16892943 gnl_Phoda3.0_PDK_30s671251g015 63 gnl_Musac1.0_GSMUA_Achr6T17850_001 gnl_Ambtr1.0.27_AmTr_v1.0_scaffold00017.26 1.0

Fig. S2.18 - orthogroup 9613 Gesneriaceae-DTNC-0011020-Sinningia_tuberosa Paulawniaceae-UMUL-2013986-Paulownia_fargesii Paulawniaceae-UMUL-2013987-Paulownia_fargesii 100 Lindenbergia_philippensis_LiPhGnB2_12551.1 Triphysaria_eriantha_TrEr3_30066 Triphysaria_pusilla_TrPu1_16839 100 Triphysaria_eriantha_TrEr3_28220 55 Triphysaria_eriantha_TrEr3_30067 Triphysaria_pusilla_TrPu1_26965 V 93 Triphysaria_pusilla_TrPu1_16838 91 Orobanche_minor_OrMi2_7956 80 Striga_hermonthica_StHeBC3_9019.1 Striga_gesneroides_StGe3_9589 75 Alectra_vogelii_AlVo2_8173 83 Orobanche_californica_OrCa3_6122 gnl_Mimgu1.0_PACid_17670015 gnl_Solly2.3_Solyc07g006190.2.1 65 gnl_Soltu3.4_PGSC0003DMP400019855 HeAn_33773 100 LaSa_13951 gnl_Vitvi12X_PACid_17832071 Glycine_soja_ncbi_hit Glycine_max_ncbi_hit gnl_Glyma1.01_PACid_16304786 100 gnl_Glyma1.01_PACid_16304787 Phaseolus_vulgaris_ncbi_hit 100Cicer_arietinum_ncbi_hit 97 Phelipanche_ramosa_PhRa5_14643 H Phelipanche_mutelii_PhMu2_11063 84 Phelipanche_aegyptiaca_PhAeBC5_16890.17 73 Medicago_truncatula_ncbi_hit 95 Citrus_sinensis_ncbi_hit 16 gnl_Theca1.0_Tc09_g015200 21 gnl_Arath10_AT2G23890.1 3640 gnl_Thepa2.0_Tp4g02790 gnl_Carpa1.181_PACid_16425939 gnl_Poptr2.2_PACid_18214284 16 gnl_Frave2.0_gene16847 50 gnl_Nelnu1.0_NNU_020849-RA gnl_Aquco1.0_PACid_18158606 gnl_Musac1.0_GSMUA_Achr5T24210_001 100 gnl_Phoda3.0_PDK_30s806571g005 80 gnl_Orysa6.0_PACid_16881654 gnl_Orysa6.0_PACid_16881655 49 gnl_Sorbi1.4_PACid_1957633 gnl_Bradi1.2_Bradi4g30660.1 gnl_Ambtr1.0.27_AmTr_v1.0_scaffold00019.105 0.1

Fig. S2.19 - orthogroup 10050 LaSa_9736 LaSa_27458 LaSa_30143 29 HeAn_14324 34 LaSa_1078 LaSa_33441 HeAn_58251 HeAn_61694 96 gnl_Solly2.3_Solyc01g102800.2.1 gnl_Soltu3.4_PGSC0003DMP400031813 gnl_Mimgu1.0_PACid_17686933 100 Alectra_vogelii_AlVo2_3046 51 Striga_hermonthica_StHeBC3_2699.1 96 V 91 Striga_gesneroides_StGe3_2315 Phelipanche_aegyptiaca_PhAeBC5_5740.1 LiPhGnB2_3298.1 Acanthaceae-WEAC-0134883-Strobilanthes_dyerianus gnl_kiwi_Achn216951 gnl_kiwi_Achn212331 gnl_Theca1.0_Tc05_g019030 gnl_Carpa1.181_PACid_16409022 51 100 gnl_Arath10_AT3G02760.1 72 gnl_Thepa2.0_Tp3g01790 74 gnl_Poptr2.2_PACid_18218941 gnl_Glyma1.01_PACid_16260743 gnl_Glyma1.01_PACid_16269967 47 gnl_Frave2.0_gene31205 10062 100 Phelipanche_aegyptiaca_PhAeBC5_4284.2 Phelipanche_mutelii_PhMu2_5340 Phelipanche_ramosa_PhRa5_11783 H 60 Phelipanche_mutelii_PhMu2_31112 gnl_Vitvi12X_PACid_17841263 gnl_Vitvi12X_PACid_17841262 gnl_Aquco1.0_PACid_18142427 99 gnl_Nelnu1.0_NNU_009115-RA gnl_Sorbi1.4_PACid_1979785 gnl_Bradi1.2_Bradi2g35680.1 50 61 gnl_Orysa6.0_PACid_16858423 gnl_Phoda3.0_PDK_30s734041g002 68 gnl_Musac1.0_GSMUA_Achr5T14080_001 gnl_Phoda3.0_PDK_30s734041g001 gnl_Ambtr1.0.27_AmTr_v1.0_scaffold00071.77 1.0

Fig. S2.20 - orthogroup 10143 LaSa_44198 gnl_Soltu3.4_PGSC0003DMP400045030 gnl_Solly2.3_Solyc01g091080.2.1 gnl_Mimgu1.0_PACid_17694589 68 Alectra_vogelii_AlVo2_7693 99 gnl_Stras_v1.02_SGA1.0.scaffold12G002650 Striga_gesneroides_StGe3_19962 97 17 Striga_hermonthica_StHeBC3_6177.2 Orobanchaceae-URZI-0061697-Epifagus_virginiana 3 Orobanchaceae-URZI-0018854-Epifagus_virginiana Orobanche_minor_OrMi2_12581 Orobanche_minor_OrMi2_39726 8 Orobanchaceae-URZI-0082305-Epifagus_virginiana 100 7 Orobanchaceae-URZI-0013485-Epifagus_virginiana Orobanche_californica_OrCa3_15364 V 100 77 Phelipanche_ramosa_PhRa5_5131 Phelipanche_aegyptiaca_PhAeBC5_9156.1 37 29 Phelipanche_mutelii_PhMu2_5471 Orobanchaceae-URZI-0016678-Epifagus_virginiana Triphysaria_pusilla_TrPu1_17999 Triphysaria_eriantha_TrEr3_6767 44 Triphysaria_versicolor_TrVeBC3_10034.1 Triphysaria_pusilla_TrPu1_14279 100 Orobanche_minor_OrMi2_35729 Lindenbergia_philippensis_LiPhGnB2_8921.1 100 Orobanche_minor_OrMi2_28148 gnl_kiwi_Achn010621 gnl_Poptr2.2_PACid_18222357 83 gnl_Theca1.0_Tc01_g037890 gnl_Arath10_AT3G60740.1 49 100 100 gnl_Thepa2.0_Tp5g02510 96 gnl_Carpa1.181_PACid_16409070 95 gnl_Glyma1.01_PACid_16316674 gnl_Glyma1.01_PACid_16281882 87 Orobanche_minor_OrMi2_39134 H 52 Orobanche_minor_OrMi2_14052 100 Phelipanche_aegyptiaca_PhAeBC5_14056.1 gnl_Frave2.0_gene07454 gnl_Vitvi12X_PACid_17834998 gnl_Nelnu1.0_NNU_008901-RA 71 gnl_Aquco1.0_PACid_18164815 gnl_Musac1.0_GSMUA_Achr11T03730_001 100 gnl_Phoda3.0_PDK_30s862041g003 74 gnl_Orysa6.0_PACid_16886461 gnl_Bradi1.2_Bradi3g30790.1 81 gnl_Sorbi1.4_PACid_1951424 gnl_Ambtr1.0.27_AmTr_v1.0_scaffold00048.180 0.1

Fig. S2.21 - orthogroup 11437 gnl_Frave2.0_gene30996 gnl_Frave2.0_gene30988 85 gnl_Frave2.0_gene30980 gnl_Frave2.0_gene31008 50 gnl_Frave2.0_gene30982 Phelipanche_aegyptiaca_PhAeBC5_1584.1 H 64 gnl_Frave2.0_gene28737 90 gnl_Frave2.0_gene12227 gnl_Frave2.0_gene00418 90 82 gnl_Frave2.0_gene34410 gnl_Frave2.0_gene34407 100 gnl_Frave2.0_gene33872 gnl_Frave2.0_gene33871 51 gnl_Frave2.0_gene33873 gnl_Frave2.0_gene30876 gnl_Frave2.0_gene34406 PUT-163a-Fragaria_vesca-6216 gnl_Frave2.0_gene30906 gnl_Theca1.0_Tc00_g044010 15 gnl_Theca1.0_Tc09_g033670 14 gnl_Frave2.0_gene24429 Orobanche_californica_OrCa3_15790 H 18 45 HENI-2007991-Quercus_shumardii_oak Phelipanche_ramosa_PhRa5_6074 8 Phelipanche_mutelii_PhMu2_109 Phelipanche_aegyptiaca_PhAeBC5_13226.1 Phelipanche_mutelii_PhMu2_110 H 52 Phelipanche_mutelii_PhMu2_35871 gnl_Theca1.0_Tc06_g000820 gnl_Theca1.0_Tc06_g000840 Phelipanche_ramosa_PhRa5_232 12 Phelipanche_aegyptiaca_PhAeBC5_6799.1 Phelipanche_ramosa_PhRa5_1275 H 33 Phelipanche_mutelii_PhMu2_13252 gnl_Frave2.0_gene00429 5 25 gnl_Frave2.0_gene00428 80 gnl_Frave2.0_gene22425 gnl_Frave2.0_gene22426 4 gnl_Frave2.0_gene28027 gnl_Frave2.0_gene08529 gnl_Frave2.0_gene29907 gnl_Frave2.0_gene01672 97 gnl_Frave2.0_gene23583 90 gnl_Frave2.0_gene00385 50 gnl_Frave2.0_gene28402 gnl_Frave2.0_gene28418 100 gnl_Frave2.0_gene28417 97 gnl_Frave2.0_gene07738 99 gnl_Frave2.0_gene04277 63gnl_Frave2.0_gene20596 Prunus_persica_ppa005111m gnl_Frave2.0_gene13330 87 gnl_Frave2.0_gene09849 gnl_Frave2.0_gene09923 96 gnl_Frave2.0_gene26516 Phelipanche_aegyptiaca_PhAeBC5_3740.1 H 83 gnl_Frave2.0_gene19431 gnl_Frave2.0_gene19182 79 gnl_Frave2.0_gene19172 gnl_Frave2.0_gene13172 gnl_Frave2.0_gene13171 100 gnl_Frave2.0_gene13169 96 gnl_Frave2.0_gene13141 gnl_Glyma1.01_PACid_16317373 gnl_Glyma1.01_PACid_16281270 gnl_Medtr3.5_Medtr1g099370.1 97 gnl_Arath10_AT5G52910.1 86 gnl_Thepa2.0_Tp6g15090 gnl_Carpa1.181_PACid_16416435 90 gnl_Theca1.0_Tc03_g017820 gnl_Poptr2.2_PACid_18233697 100 gnl_Vitvi12X_PACid_17835783 LaSa_52885 gnl_Mimgu1.0_PACid_17674657 100 LiPhGnB2_19735.1 96 99 Phelipanche_aegyptiaca_PhAeBC5_12653.1 V gnl_Soltu3.4_PGSC0003DMP400011646 gnl_Soltu3.4_PGSC0003DMP400011645 gnl_Solly2.3_Solyc03g007800.2.1 gnl_Aquco1.0_PACid_18151553 73 gnl_Nelnu1.0_NNU_010368-RA gnl_Phoda3.0_PDK_30s1022831g001 gnl_Orysa6.0_PACid_16859082 32 100 gnl_Bradi1.2_Bradi2g15960.1 28 gnl_Sorbi1.4_PACid_1981906 gnl_Phoda3.0_PDK_30s1002881g001 gnl_Musac1.0_GSMUA_AchrUn_randomT23020_001 gnl_Ambtr1.0.27_AmTr_v1.0_scaffold00053.30 gnl_Selmo1.0_PACid_15404729 gnl_Selmo1.0_PACid_15416922 gnl_Phypa1.6_PACid_18054769 1.0 Fig. S2.22 - orthogroup 11841 gnl_Glyma1.01_PACid_16278523 86 gnl_Frave2.0_gene25610 100 gnl_Frave2.0_gene08069 100 Orobanche_californica_OrCa3_4009 Orobanche_minor_OrMi2_415 Phelipanche_mutelii_PhMu2_13703 H 55 Phelipanche_mutelii_PhMu2_6933 Phelipanche_aegyptiaca_PhAeBC5_3756.1 Phelipanche_ramosa_PhRa5_3602 31 gnl_Poptr2.2_PACid_18246017 40 gnl_Poptr2.2_PACid_18245040 gnl_Theca1.0_Tc00_g064570 LaSa_2850 90 LaSa_47034 Triphysaria_pusilla_TrPu1_38677 63 Striga_gesneroides_StGe3_44838 V Striga_gesneroides_StGe3_36009 65 13 gnl_Stras_v1.02_SGA1.0.scaffold83G00930 Verbenaceae-GCFE-2009106-Verbena_hastata 43 Gesneriaceae-DTNC-0013132-Sinningia_tuberosa 83 Sesamum_indicumSIN_1022333 gnl_kiwi_Achn381161 87 gnl_Soltu3.4_PGSC0003DMP400034797 gnl_Solly2.3_Solyc06g083910.2.1 gnl_Mimgu1.0_PACid_17679563 V 24 100 Triphysaria_eriantha_TrEr3_39852 45 Orobanchaceae-URZI-0074762-Epifagus_virginiana 79 Orobanchaceae-URZI-0067828-Epifagus_virginiana 100 49 Phelipanche_mutelii_PhMu2_43489 Phelipanche_ramosa_PhRa5_8967 Alectra_vogelii_AlVo2_1967 22 100 HeAn_61257 LaSa_24786 gnl_Vitvi12X_PACid_17835486 47 gnl_kiwi_Achn352341 55 gnl_kiwi_Achn359651 LaSa_11146 LaSa_11147 gnl_Frave2.0_gene08052 94 gnl_Frave2.0_gene08053 gnl_Frave2.0_gene08051 100 97 Sesamum_indicumSIN_1022335 gnl_kiwi_Achn359821 90 gnl_kiwi_Achn359831 gnl_kiwi_Achn357531 74 gnl_kiwi_Achn352791 54 gnl_kiwi_Achn359841 gnl_kiwi_Achn359811 gnl_Aquco1.0_PACid_18150554 gnl_Orysa6.0_PACid_16869706 gnl_Orysa6.0_PACid_16869707 gnl_Bradi1.2_Bradi1g56240.1 gnl_Sorbi1.4_PACid_1955730 0.1

Fig. S2.23 - orthogroup 12835 Phelipanche_aegyptiaca_PhAeBC5_24772.1 100 gnl_Stras_v1.02_SGA1.0.scaffold105G00190 gnl_Stras_v1.02_SGA1.0.scaffold147G00190 100 gnl_Stras_v1.02_SGA1.0.scaffold35G00460 40 gnl_Stras_v1.02_SGA1.0.scaffold81G00250 gnl_Stras_v1.02_SGA1.0.scaffold79G00260 V gnl_Stras_v1.02_SGA1.0.scaffold71G00860 gnl_Stras_v1.02_SGA1.0.scaffold80G00330 100 gnl_Stras_v1.02_SGA1.0.scaffold430G00030 LaSa_28322 LaSa_45833 94 LaSa_1379 97 LaSa_6044 LaSa_9873 69 LaSa_20530 82 LaSa_46312 49 HeAn_60209 100 76 HeAn_28195 LaSa_42718 LaSa_19173 95 LaSa_23853 79 LaSa_51707 gnl_Medtr3.5_Medtr7g061520.1 gnl_Medtr3.5_AC235757_20.1 48 gnl_Medtr3.5_Medtr4g116690.1 gnl_Medtr3.5_Medtr4g116510.1 gnl_Medtr3.5_Medtr2g061140.1 98 gnl_Medtr3.5_Medtr6g055350.1 gnl_Medtr3.5_Medtr6g079030.1 gnl_Glyma1.01_PACid_16289845 96 97 36 gnl_Glyma1.01_PACid_16290054 gnl_Medtr3.5_Medtr8g054330.1 Striga_hermonthica_StHeBC3_10075.1 H 100 LOC_Os01g60580.1_Orysa_sativa LOC_Os10g41350.1_Orysa_sativa LOC_Os10g31130.1_Orysa_sativa 68 76 gnl_Bradi1.2_Bradi3g24430.1 gnl_Bradi1.2_Bradi1g64655.1 92 gnl_Sorbi1.4_PACid_1970319 100 gnl_Sorbi1.4_PACid_1983153 gnl_Sorbi1.4_PACid_1969324 gnl_Bradi1.2_Bradi1g25296.1 100 gnl_Sorbi1.4_PACid_1958959 gnl_Bradi1.2_Bradi5g16030.1 74 gnl_Bradi1.2_Bradi2g07670.1 gnl_Bradi1.2_Bradi1g54870.1 99 gnl_Bradi1.2_Bradi3g03300.1 89 gnl_Bradi1.2_Bradi2g57400.1 gnl_Bradi1.2_Bradi1g38060.1 0.1

Fig. S2.24 - orthogroup 13512 gnl_Frave2.0_gene13330 74 gnl_Theca1.0_Tc06_g000840 gnl_Theca1.0_Tc06_g000820 gnl_Frave2.0_gene22425 gnl_Frave2.0_gene08529 gnl_Frave2.0_gene28027 45 gnl_Frave2.0_gene22426 gnl_Frave2.0_gene00429 50 gnl_Frave2.0_gene24429 76 gnl_Frave2.0_gene00428 30 gnl_Frave2.0_gene29907 100 gnl_Frave2.0_gene01672 gnl_Frave2.0_gene00385 gnl_Frave2.0_gene23583 93 gnl_Frave2.0_gene28418 gnl_Frave2.0_gene28417 96 gnl_Frave2.0_gene07738 gnl_Frave2.0_gene20596 100 gnl_Frave2.0_gene04277 gnl_Frave2.0_gene28402 90 gnl_Frave2.0_gene12227 85 gnl_Frave2.0_gene30996 gnl_Frave2.0_gene30988 68 gnl_Frave2.0_gene30982 55 gnl_Frave2.0_gene31008 99 gnl_Frave2.0_gene30980 99 gnl_Frave2.0_gene28737 Orobanche_californica_OrCa3_3600 100 100 Phelipanche_aegyptiaca_PhAeBC5_1584.1 Phelipanche_ramosa_PhRa5_1522 H Phelipanche_mutelii_PhMu2_40952 56 gnl_Frave2.0_gene00418 82 gnl_Frave2.0_gene33872 99 gnl_Frave2.0_gene33871 gnl_Frave2.0_gene33873 62 82 gnl_Frave2.0_gene34406 gnl_Frave2.0_gene30876 gnl_Frave2.0_gene34407 45 77 gnl_Frave2.0_gene34410 gnl_Frave2.0_gene30906 gnl_Frave2.0_gene09849 gnl_Frave2.0_gene26516 100 gnl_Frave2.0_gene09923 Orobanche_californica_OrCa3_26054 H Phelipanche_ramosa_PhRa5_275 Phelipanche_aegyptiaca_PhAeBC5_3740.1 gnl_Frave2.0_gene19182 gnl_Frave2.0_gene19431 100gnl_Frave2.0_gene19172 97 gnl_Frave2.0_gene13169 gnl_Frave2.0_gene13172 gnl_Frave2.0_gene13171 LaSa_52885 gnl_Soltu3.4_PGSC0003DMP400011646 99 gnl_Solly2.3_Solyc03g007800.2.1 gnl_Soltu3.4_PGSC0003DMP400011645 100 Phelipanche_aegyptiaca_PhAeBC5_12653.1 V LiPhGnB2_19735.1 96 gnl_Mimgu1.0_PACid_17674657 gnl_Frave2.0_gene13141 95 gnl_Medtr3.5_Medtr1g099370.1 gnl_Glyma1.01_PACid_16281270 98 gnl_Glyma1.01_PACid_16317373 gnl_Poptr2.2_PACid_18233697 95 gnl_Carpa1.181_PACid_16416435 84 gnl_Thepa2.0_Tp6g15090 85 gnl_Arath10_AT5G52910.1 gnl_Theca1.0_Tc03_g017820 gnl_Vitvi12X_PACid_17835783 61 gnl_Aquco1.0_PACid_18151553 gnl_Nelnu1.0_NNU_010368-RA 31 gnl_Phoda3.0_PDK_30s1022831g001 gnl_Phoda3.0_PDK_30s1002881g001 5364 gnl_Sorbi1.4_PACid_1981906 gnl_Orysa6.0_PACid_16859082 33 gnl_Bradi1.2_Bradi2g15960.1 gnl_Musac1.0_GSMUA_AchrUn_randomT23020_001 gnl_Ambtr1.0.27_AmTr_v1.0_scaffold00053.30 gnl_Selmo1.0_PACid_15404729 gnl_Selmo1.0_PACid_15416922 gnl_Phypa1.6_PACid_18054769 1.0 Fig. S2.25 - orthogroup 13656 gnl_Phoda3.0_PDK_30s1094801g001 89 gnl_Phoda3.0_PDK_30s911091g001 gnl_Orysa6.0_PACid_16834607 68 gnl_Sorbi1.4_PACid_1963066 gnl_Orysa6.0_PACid_16830816 99 gnl_Orysa6.0_PACid_16830817 gnl_Sorbi1.4_PACid_1960032 gnl_Orysa6.0_PACid_16863947 42 100 gnl_Orysa6.0_PACid_16863945 54 gnl_Sorbi1.4_PACid_1982531 99 gnl_Sorbi1.4_PACid_1982530 gnl_Sorbi1.4_PACid_1982535 60 85 39 gnl_Orysa6.0_PACid_16863943 53 gnl_Orysa6.0_PACid_16863944 96 gnl_Sorbi1.4_PACid_1982529 gnl_Sorbi1.4_PACid_1982528 gnl_Bradi1.2_Bradi3g02450.1 gnl_Bradi1.2_Bradi3g02470.1 56 gnl_Bradi1.2_Bradi3g02480.1 18 gnl_Bradi1.2_Bradi3g02460.1 gnl_Sorbi1.4_PACid_1984153 gnl_Bradi1.2_Bradi1g37337.1 gnl_Orysa6.0_PACid_16844224 gnl_Orysa6.0_PACid_16838844 29 gnl_Orysa6.0_PACid_16838848 gnl_Orysa6.0_PACid_16838846 14 gnl_Orysa6.0_PACid_16838843 gnl_Orysa6.0_PACid_16838842 gnl_Orysa6.0_PACid_16844223 100 gnl_Orysa6.0_PACid_16838847 gnl_Orysa6.0_PACid_16838841 21 gnl_Orysa6.0_PACid_16838845 gnl_Orysa6.0_PACid_16867272 9 gnl_Orysa6.0_PACid_16868894 gnl_Sorbi1.4_PACid_1965806 62 Striga_hermonthica_StHeBC3_41710.1 H 18 gnl_Sorbi1.4_PACid_1965805 gnl_Sorbi1.4_PACid_1965804 gnl_Sorbi1.4_PACid_1980004 gnl_Sorbi1.4_PACid_1951525 82 gnl_Orysa6.0_PACid_16858777 gnl_Orysa6.0_PACid_16858781 95 gnl_Bradi1.2_Bradi2g05350.1 gnl_Bradi1.2_Bradi2g05340.1 gnl_Nelnu1.0_NNU_022856-RA gnl_Soltu3.4_PGSC0003DMP400028410 43 gnl_Poptr2.2_PACid_18210121 95 29 gnl_Frave2.0_gene01275 69 gnl_Frave2.0_gene01274 95 gnl_Glyma1.01_PACid_16310893 gnl_Theca1.0_Tc04_g008070 gnl_Ambtr1.0.27_AmTr_v1.0_scaffold00049.285 gnl_Ambtr1.0.27_AmTr_v1.0_scaffold00078.47 gnl_Ambtr1.0.27_AmTr_v1.0_scaffold00078.45 gnl_Ambtr1.0.27_AmTr_v1.0_scaffold00078.46 64 gnl_Ambtr1.0.27_AmTr_v1.0_scaffold00078.48 91 gnl_Ambtr1.0.27_AmTr_v1.0_scaffold00170.15 gnl_Ambtr1.0.27_AmTr_v1.0_scaffold00078.50 98 85 gnl_Ambtr1.0.27_AmTr_v1.0_scaffold00078.49 gnl_Ambtr1.0.27_AmTr_v1.0_scaffold00078.42 gnl_Ambtr1.0.27_AmTr_v1.0_scaffold00169.25 1.0 gnl_Ambtr1.0.27_AmTr_v1.0_scaffold00122.27

Fig. S2.26 - orthogroup 13892 gnl_Frave2.0_gene11100 gnl_Poptr2.2_PACid_18244173 38 77 gnl_Carpa1.181_PACid_16418392 89 gnl_Arath10_AT1G77750.1

52 gnl_Thepa2.0_Tp5g32920 gnl_Theca1.0_Tc08_g009990 gnl_Frave2.0_gene11101 26 gnl_Theca1.0_Tc08_g010000 92 gnl_Poptr2.2_PACid_18206356 gnl_Poptr2.2_PACid_18246298 95 gnl_Carpa1.181_PACid_16418413 Orobanche_minor_OrMi2_16170 Orobanche_californica_OrCa3_6702 H 88 Phelipanche_ramosa_PhRa5_28635 Phelipanche_aegyptiaca_PhAeBC5_4266.1 84 Phelipanche_mutelii_PhMu2_33390 gnl_Medtr3.5_Medtr4g081120.1 gnl_Medtr3.5_Medtr4g080830.2 90 gnl_Medtr3.5_Medtr4g080830.1 gnl_Glyma1.01_PACid_16247415 gnl_Glyma1.01_PACid_16247413 gnl_Glyma1.01_PACid_16247414 gnl_Carpa1.181_PACid_16415389 gnl_Solly2.3_Solyc11g056310.1.1 gnl_Aquco1.0_PACid_18166007 gnl_Selmo1.0_PACid_15411956 1.0

Fig. S2.27 - orthogroup 14230 Striga_gesneroides_StGe3_14832 Striga_hermonthica_StHeBC3_14884.1 100 gnl_Stras_v1.02_SGA1.0.scaffold625G00030 V gnl_Stras_v1.02_SGA1.0.scaffold532G00100 94 gnl_Stras_v1.02_SGA1.0.scaffold264G00090 Striga_gesneroides_StGe3_11086 Acanthaceae-WEAC-0134739-Strobilanthes_dyerianus Lamiaceae-EQDA-2058202-Salvia_spp. Triphysaria_eriantha_TrEr3_29072 94 Triphysaria_versicolor_TrVeBC3_14930.1 100 Triphysaria_pusilla_TrPu1_2075 100 Striga_hermonthica_StHeBC3_18662.1 V Striga_gesneroides_StGe3_14018 100 Triphysaria_versicolor_TrVeBC3_23121.1 100 Lindenbergia_philippensis_LiPhGnB2_21193.1 100 Lindenbergia_philippensis_LiPhGnB2_13982.2 Triphysaria_versicolor_TrVeBC3_16677.1 91 Triphysaria_eriantha_TrEr3_11128 V gnl_Poptr2.2_PACid_18215814 100 gnl_Poptr2.2_PACid_18250893 gnl_Poptr2.2_PACid_18250416 100 Graimondii_cotton_003G096100.2 90 Graimondii_cotton_003G096100.1 Csinensis.orange1.1g002062m_PAC_18100795 99 99 Prunus_persica_ppa020137m gnl_kiwi_Achn020651 Graimondii_cotton_006G037100.1 100 100 Mesculenta_cassava.15G155500.1 gnl_Utrgi_v4.1_Scf00249.g13750.t1 78 98 Orobanche_californica_OrCa3_24369 V Phelipanche_ramosa_PhRa5_17235 100 gnl_Betvu_v1.1_Bv9_222040_ujjq.t1 Graimondii_cotton_013G065300.1 46Prunus_persica_ppa001597m SIZE-2015853-Passiflora_caerulea 44 100 99 Mesculenta_cassava.15G114300.1 Mesculenta_cassava.15G114300.2 Egrandis.eucalyptus.I01547.1 Egrandis.eucalyptus.I01547.2 Egrandis.eucalyptus.B01589.1 Egrandis.eucalyptus.C01967.1 100 Egrandis.eucalyptus.B01259.1 Egrandis.eucalyptus.I00824.1 Egrandis.eucalyptus.E01219.1 68 Egrandis.eucalyptus.H01407.1 Egrandis.eucalyptus.B00042.1 87 Prunus_persica_ppa002389m 100 Prunus_persica_ppa018832m 100 gnl_Frave2.0_gene14932 Csinensis.orange1.1g007865m_PAC_18113773 Csinensis.orange1.1g008217m_PAC_18113774 84 Csinensis.orange1.1g005927m_PAC_18113772 Phelipanche_aegyptiaca_PhAeBC5_34854.1 H Phelipanche_ramosa_PhRa5_11242 73 Prunus_persica_ppa022622m Prunus_persica_ppa015375m gnl_Sorbi1.4_PACid_1977571 gnl_Bradi1.2_Bradi4g41960.1 gnl_Sorbi1.4_PACid_1966791 gnl_Sorbi1.4_PACid_1966792 gnl_Bradi1.2_Bradi3g45470.1 gnl_Bradi1.2_Bradi1g48350.1 gnl_Sorbi1.4_PACid_1958804 1.0 Fig. S2.28 - orthogroup 14233 gnl_Sorbi1.4_PACid_1960343 100 gnl_Orysa6.0_PACid_16844495 gnl_Sorbi1.4_PACid_1977009 gnl_Sorbi1.4_PACid_1977008 92 gnl_Bradi1.2_Bradi2g10470.1 99 gnl_Bradi1.2_Bradi2g10460.1 45 gnl_Orysa6.0_PACid_16857985 96 gnl_Orysa6.0_PACid_16891038 46 87 gnl_Orysa6.0_PACid_16891352 gnl_Orysa6.0_PACid_16830877 96 53 gnl_Bradi1.2_Bradi2g08360.1 gnl_Orysa6.0_PACid_16830878 gnl_Orysa6.0_PACid_16830843 100 gnl_Orysa6.0_PACid_16830837 gnl_Sorbi1.4_PACid_1960019 gnl_Sorbi1.4_PACid_1983565 19 100 gnl_Orysa6.0_PACid_16865527 gnl_Orysa6.0_PACid_16865528 gnl_Orysa6.0_PACid_16865529 gnl_Orysa6.0_PACid_16865530 gnl_Sorbi1.4_PACid_1960939 75 81 gnl_Sorbi1.4_PACid_1980105 gnl_Sorbi1.4_PACid_1980106 gnl_Sorbi1.4_PACid_1960110 gnl_Orysa6.0_PACid_16830882 100 97 gnl_Orysa6.0_PACid_16830891 gnl_Orysa6.0_PACid_16830892 100 gnl_Bradi1.2_Bradi2g08430.1 100gnl_Bradi1.2_Bradi2g08440.1 gnl_Bradi1.2_Bradi2g08420.1 91 gnl_Orysa6.0_PACid_16846824 86 gnl_Orysa6.0_PACid_16846823 88 100 gnl_Sorbi1.4_PACid_1975175 gnl_Sorbi1.4_PACid_1978774 95 gnl_Bradi1.2_Bradi3g18100.1 gnl_Orysa6.0_PACid_16861828 gnl_Orysa6.0_PACid_16861827 gnl_Orysa6.0_PACid_16861830 90 gnl_Orysa6.0_PACid_16861829 81 67 gnl_Orysa6.0_PACid_16861832 gnl_Orysa6.0_PACid_16861831 gnl_Orysa6.0_PACid_16879414 gnl_Orysa6.0_PACid_16892801 99 gnl_Orysa6.0_PACid_16861826 91 gnl_Bradi1.2_Bradi2g22710.1 gnl_Orysa6.0_PACid_16861823 100 gnl_Bradi1.2_Bradi2g22730.1 42 gnl_Orysa6.0_PACid_16859474 87 gnl_Orysa6.0_PACid_16859475 gnl_Orysa6.0_PACid_16859483 100 gnl_Orysa6.0_PACid_16859470 gnl_Orysa6.0_PACid_16859460 53 76 gnl_Orysa6.0_PACid_16859471 gnl_Sorbi1.4_PACid_1981200 gnl_Sorbi1.4_PACid_1981199 gnl_Bradi1.2_Bradi4g13490.1 67 gnl_Bradi1.2_Bradi5g23520.1 100 gnl_Bradi1.2_Bradi5g23530.1 gnl_Bradi1.2_Bradi1g13550.1 67 gnl_Bradi1.2_Bradi4g13380.1 100 gnl_Bradi1.2_Bradi4g13400.1 gnl_Bradi1.2_Bradi4g13420.1 gnl_Orysa6.0_PACid_16859740 gnl_Orysa6.0_PACid_16859741 gnl_Arath10_AT1G15680.1 100 gnl_Thepa2.0_Tp3g21990 gnl_Arath10_AT3G23950.1 gnl_Arath10_AT3G23970.1 gnl_Soltu3.4_PGSC0003DMP400043403 100 53 100 gnl_Soltu3.4_PGSC0003DMP400004741 Striga_gesneroides_StGe3_20526 Striga_gesneroides_StGe3_22413 88 Striga_gesneroides_StGe3_25459 Striga_gesneroides_StGe3_26212 V 100 Striga_gesneroides_StGe3_34926 100 Striga_gesneroides_StGe3_26383 Phelipanche_mutelii_PhMu2_50622 86 Phelipanche_ramosa_PhRa5_51185 Phelipanche_mutelii_PhMu2_38171 Phelipanche_ramosa_PhRa5_40789 gnl_Mimgu1.0_PACid_17673589 Striga_gesneroides_StGe3_521 100 Striga_gesneroides_StGe3_4270 H gnl_Stras_v1.02_SGA1.0.scaffold994G00050 82 gnl_Orysa6.0_PACid_16847332 gnl_Orysa6.0_PACid_16847337 gnl_Orysa6.0_PACid_16847329 gnl_Orysa6.0_PACid_16879603 73 100 gnl_Orysa6.0_PACid_16833223 gnl_Sorbi1.4_PACid_1976426 gnl_Sorbi1.4_PACid_1956529 gnl_Sorbi1.4_PACid_1977208 100 38 100 gnl_Bradi1.2_Bradi1g68200.1 35 gnl_Bradi1.2_Bradi1g70240.1 gnl_Orysa6.0_PACid_16845944 93 Striga_gesneroides_StGe3_10627 H Striga_hermonthica_StHeBC3_16619.1 99 gnl_Sorbi1.4_PACid_1965357 gnl_Orysa6.0_PACid_16829622 58 gnl_Orysa6.0_PACid_16829623 100 gnl_Orysa6.0_PACid_16829656 gnl_Orysa6.0_PACid_16830249 1.0 gnl_Orysa6.0_PACid_16830250

Fig. S2.29- orthogroup 14624 100 Striga_hermonthica_StHeGnB1_80049 H Zea_mays_speckle-type_POZ_protein_gi_226499726 72 100 Sorghum_bicolor_10g026740_gi_242096658 Oryza_Os08g0198300_gi_937929319 65 Oryza_gi_125602495 gnl_Orysa6.0_PACid_16864927 58 Oryza_BTB_POZ_and_MATH_domain-containing_protein gnl_Orysa6.0_PACid_16864926 gnl_Orysa6.0_PACid_16868116 100 94 gnl_Sorbi1.4_PACid_1956568 gnl_Bradi1.2_Bradi1g32230.1 84 61 gnl_Bradi1.2_Bradi1g32247.1 gnl_Bradi1.2_Bradi1g32260.1 gnl_Sorbi1.4_PACid_1984655 90 gnl_Sorbi1.4_PACid_1984652 gnl_Sorbi1.4_PACid_1984651 0.1

Fig. S2.30 - orthogroup 14675 Orobanche_californica_OrCa3_9383 Phelipanche_mutelii_PhMu2_51910 H Phelipanche_ramosa_PhRa5_17244 Phelipanche_aegyptiaca_PhAeBC5_12620.5 83 gnl_Frave2.0_gene21241 74 gnl_Frave2.0_gene21231 98 gnl_Frave2.0_gene21233 gnl_Frave2.0_gene21245 gnl_Frave2.0_gene19118 80 97 gnl_Frave2.0_gene21232 71 gnl_Frave2.0_gene21242 gnl_Frave2.0_gene21238 gnl_Frave2.0_gene21186 gnl_Frave2.0_gene14595 98 gnl_Frave2.0_gene14597 gnl_Frave2.0_gene14586 63 gnl_Frave2.0_gene18557 gnl_Frave2.0_gene31811 64 gnl_Frave2.0_gene19461 gnl_Frave2.0_gene09533 gnl_Medtr3.5_Medtr5g075990.1 35 gnl_Medtr3.5_Medtr4g049350.1 94 gnl_Medtr3.5_Medtr3g069650.1 gnl_Medtr3.5_Medtr5g075950.1 gnl_Phoda3.0_PDK_30s865971g001 gnl_Phoda3.0_PDK_30s800451g001 39 100 gnl_Sorbi1.4_PACid_1953483 67 gnl_Bradi1.2_Bradi1g63420.1 gnl_Orysa6.0_PACid_16846802 gnl_Solly2.3_Solyc09g055660.1.1 gnl_Soltu3.4_PGSC0003DMP400034416 59 Phelipanche_mutelii_PhMu2_32856 V 8 gnl_Mimgu1.0_PACid_17677845 66 gnl_Theca1.0_Tc04_g015730 gnl_Carpa1.181_PACid_16403961 14 gnl_Frave2.0_gene13596 22 gnl_Medtr3.5_Medtr8g005100.1 18 gnl_Medtr3.5_Medtr8g005340.1 7 gnl_Poptr2.2_PACid_18250931 gnl_Poptr2.2_PACid_18212145 21 gnl_Vitvi12X_PACid_17836522 gnl_Vitvi12X_PACid_17832708 gnl_Carpa1.181_PACid_16412084 gnl_Theca1.0_Tc10_g016830 gnl_Aquco1.0_PACid_18147454 gnl_Ambtr1.0.27_AmTr_v1.0_scaffold00077.184 1.0

Fig. S2.31 - orthogroup 15149 Prunus_persica_ppa023353m Prunus_persica_ppa025795m 98 Prunus_persica_ppa016010m Prunus_persica_ppa024136m Prunus_persica_ppa015420m 100 Prunus_persica_ppa016227m gnl_Frave2.0_gene19468 Phelipanche_ramosa_PhRa5_15804 H Phelipanche_aegyptiaca_PhAeBC5_8832.4 100 Phelipanche_mutelii_PhMu2_63448 gnl_Frave2.0_gene03891 78 gnl_Frave2.0_gene27661 73 gnl_Frave2.0_gene15631 gnl_Frave2.0_gene33503 95gnl_Frave2.0_gene04826 gnl_Frave2.0_gene30312 80 gnl_Frave2.0_gene19093 gnl_Frave2.0_gene02768 gnl_kiwi_Achn271061 100 gnl_kiwi_Achn065441 1.0 gnl_kiwi_Achn321631

Fig. S2.32 - orthogroup 15246 gnl_Medtr3.5_Medtr1g019650.1 74 gnl_Medtr3.5_Medtr6g036620.1 45 gnl_Medtr3.5_Medtr4g026590.1 32 gnl_Medtr3.5_Medtr3g067430.1 100 gnl_Medtr3.5_Medtr3g067280.1 70 gnl_Medtr3.5_Medtr3g067270.1 gnl_Medtr3.5_Medtr7g044920.1 100 gnl_Medtr3.5_Medtr7g044980.1 77 gnl_Medtr3.5_Medtr8g056800.1 53 Alectra_vogelii_AlVo2_35769 Phelipanche_aegyptiaca_PhAeBC5_2525.1 56 98 Triphysaria_eriantha_TrEr3_39363 H Phelipanche_ramosa_PhRa5_530 Orobanche_californica_OrCa3_47561 Alectra_vogelii_AlVo2_19886 gnl_Glyma1.01_PACid_16291630 gnl_Glyma1.01_PACid_16291629 gnl_Medtr3.5_AC233140_23.1 83 gnl_Medtr3.5_Medtr8g022420.1 34 gnl_Medtr3.5_Medtr8g022400.1 gnl_Medtr3.5_Medtr8g022430.1 gnl_Medtr3.5_AC233140_86.1 0.1

Fig. S2.33 - orthogroup 16703 gnl_Orysa6.0_PACid_16872953 64 gnl_Orysa6.0_PACid_16890481 gnl_Orysa6.0_PACid_16840413 81 gnl_Orysa6.0_PACid_16839434 gnl_Orysa6.0_PACid_16877750 95 gnl_Orysa6.0_PACid_16848970 gnl_Orysa6.0_PACid_16862048 4 gnl_Orysa6.0_PACid_16877419 gnl_Orysa6.0_PACid_16886673 29 gnl_Orysa6.0_PACid_16858440 gnl_Orysa6.0_PACid_16876064 gnl_Orysa6.0_PACid_16832742 7 gnl_Orysa6.0_PACid_16880542 7 gnl_Bradi1.2_Bradi3g30580.1 gnl_Bradi1.2_Bradi4g17300.1 97 gnl_Bradi1.2_Bradi5g07330.1 gnl_Bradi1.2_Bradi2g41420.1 gnl_Orysa6.0_PACid_16871978 gnl_Orysa6.0_PACid_16871969 100 gnl_Orysa6.0_PACid_16867378 gnl_Orysa6.0_PACid_16860437 57 gnl_Orysa6.0_PACid_16847486 25 gnl_Orysa6.0_PACid_16844619 gnl_Orysa6.0_PACid_16839791 Striga_hermonthica_StHeBC3_11327.15 H gnl_Sorbi1.4_PACid_1951824 51 gnl_Orysa6.0_PACid_16832894 gnl_Sorbi1.4_PACid_1980074 gnl_Sorbi1.4_PACid_1954917 100 100 gnl_Sorbi1.4_PACid_1972982 22 gnl_Sorbi1.4_PACid_1956882 gnl_Sorbi1.4_PACid_1955602 gnl_Sorbi1.4_PACid_1980472 gnl_Sorbi1.4_PACid_1974761 97 gnl_Bradi1.2_Bradi5g08880.1 gnl_Bradi1.2_Bradi4g22540.1 90 gnl_Bradi1.2_Bradi1g47680.1 gnl_Bradi1.2_Bradi5g10560.1 gnl_Selmo1.0_PACid_15410349 1.0 gnl_Selmo1.0_PACid_15406368

Fig. S2.34 - orthogroup 18354 gnl_Frave2.0_gene07387 gnl_Frave2.0_gene08314 51 gnl_Frave2.0_gene14494 90 gnl_Frave2.0_gene07552 gnl_Frave2.0_gene07551 88 gnl_Frave2.0_gene07550 gnl_Frave2.0_gene18321 99 Phelipanche_ramosa_PhRa5_19568 80 73 Phelipanche_mutelii_PhMu2_4060 H Phelipanche_mutelii_PhMu2_19778 Phelipanche_aegyptiaca_PhAeBC5_5786.1 61 gnl_Frave2.0_gene13262 gnl_Frave2.0_gene13239 82 gnl_Frave2.0_gene13179 Orobanche_minor_OrMi2_14186 H Orobanche_minor_OrMi2_14157 gnl_Frave2.0_gene13181 gnl_Frave2.0_gene13154 1.0

Fig. S2.35 - orthogroup 18709 HeAn_55438 100 HeAn_33799 100 LaSa_49220 46 LaSa_55647 gnl_Solly2.3_Solyc02g014310.2.1 16 gnl_Soltu3.4_PGSC0003DMP400002176 71 LiPhGnB2_889.1 Striga_hermonthica_StHeBC3_685.1 100 Phelipanche_aegyptiaca_PhAeBC5_998.1 V Triphysaria_versicolor_TrVeBC3_1542.1 16 92 gnl_Mimgu1.0_PACid_17678048 gnl_Frave2.0_gene00819 40 gnl_Aquco1.0_PACid_18159464 gnl_Aquco1.0_PACid_18159465 gnl_Vitvi12X_PACid_17823768 gnl_Arath10_AT3G18610.1 gnl_Arath10_AT1G48920.1 36 97 gnl_Thepa2.0_Tp1g35830 gnl_Arath10_AT1G73490.2 100 84 gnl_Thepa2.0_Tp7g36960 gnl_Arath10_AT5G05430.1 63 84 gnl_Arath10_AT3G09160.1 77 Phelipanche_ramosa_PhRa5_6642 H Phelipanche_aegyptiaca_PhAeBC5_654.1 34 100 gnl_Arath10_AT5G03495.1 100 gnl_Arath10_AT5G03480.1 gnl_Arath10_AT5G53700.1 54 gnl_Poptr2.2_PACid_18243531 gnl_Poptr2.2_PACid_18243532 gnl_Poptr2.2_PACid_18206110 gnl_Medtr3.5_Medtr4g074930.2 23 gnl_Medtr3.5_Medtr4g074930.1 100 gnl_Medtr3.5_Medtr4g074800.1 35 gnl_Glyma1.01_PACid_16286191 21 gnl_Glyma1.01_PACid_16283407 gnl_Theca1.0_Tc02_g013990 59 gnl_Carpa1.181_PACid_16411620 gnl_Nelnu1.0_NNU_000420-RA gnl_Phoda3.0_PDK_30s975021g004 75 gnl_Musac1.0_GSMUA_Achr11T14880_001 35 gnl_Musac1.0_GSMUA_Achr10T09980_001 gnl_Bradi1.2_Bradi5g22117.1 100 gnl_Sorbi1.4_PACid_1951695 62gnl_Orysa6.0_PACid_16856787 100 Spolyrhiza_duckweed_Lemna13G0045800 gnl_Ambtr1.0.27_AmTr_v1.0_scaffold00092.50 100 gnl_Selmo1.0_PACid_15402332 gnl_Phypa1.6_PACid_18069145 gnl_Phypa1.6_PACid_18069144 gnl_Phypa1.6_PACid_18068370 gnl_Phypa1.6_PACid_18068369 1.0

Fig. S2.36 - orthogroup 18774 gnl_Bradi1.2_Bradi1g73680.1 48 gnl_Bradi1.2_Bradi1g73690.1 gnl_Orysa6.0_PACid_16845224 gnl_Sorbi1.4_PACid_1968103 100 gnl_Orysa6.0_PACid_16883029 gnl_Orysa6.0_PACid_16873470 gnl_Orysa6.0_PACid_16873469 gnl_Bradi1.2_Bradi2g25790.1 58 10099 gnl_Bradi1.2_Bradi3g55500.1 63 gnl_Orysa6.0_PACid_16895713 H 28 Striga_gesneroides_StGe3_521 33 Striga_hermonthica_StHeBC3_4423.1 100Setaria_italica_gi_514767976_ref_XP_004966374.1 Setaria_italica_gi_835961998_ref_XP_012700825.1 Setaria_italica_gi_835966179_ref_XP_004966375.2 gnl_Bradi1.2_Bradi4g04240.1 gnl_Bradi1.2_Bradi4g04227.1 gnl_Orysa6.0_PACid_16868700 gnl_Orysa6.0_PACid_16868699 gnl_Orysa6.0_PACid_16868698 gnl_Orysa6.0_PACid_16868697 gnl_Orysa6.0_PACid_16868706 gnl_Orysa6.0_PACid_16868707 100 gnl_Orysa6.0_PACid_16868719 gnl_Orysa6.0_PACid_16868722 100 gnl_Orysa6.0_PACid_16868733 100gnl_Orysa6.0_PACid_16868726 gnl_Orysa6.0_PACid_16869007 gnl_Orysa6.0_PACid_16868716 100 gnl_Orysa6.0_PACid_16868730 100 gnl_Orysa6.0_PACid_16868691 gnl_Bradi1.2_Bradi3g10796.1 gnl_Orysa6.0_PACid_16885981 100 gnl_Sorbi1.4_PACid_1964039 gnl_Sorbi1.4_PACid_1964040 gnl_Sorbi1.4_PACid_1964041 82 100 gnl_Orysa6.0_PACid_16835231 gnl_Orysa6.0_PACid_16835230 gnl_Bradi1.2_Bradi1g00410.1 95 94 gnl_Sorbi1.4_PACid_1949182 97 gnl_Bradi1.2_Bradi2g36840.1 Striga_gesneroides_StGe3_4270 H gnl_Stras_v1.02_SGA1.0.scaffold994G00050 gnl_Sorbi1.4_PACid_1979586 79 100gnl_Orysa6.0_PACid_16837691 gnl_Orysa6.0_PACid_16837687 gnl_Orysa6.0_PACid_16837690 gnl_Bradi1.2_Bradi2g02830.1 56 gnl_Bradi1.2_Bradi2g02850.1 94 100 100 gnl_Bradi1.2_Bradi2g02860.1 gnl_Orysa6.0_PACid_16845205 gnl_Orysa6.0_PACid_16845228 gnl_Bradi1.2_Bradi1g72580.1 gnl_Bradi1.2_Bradi1g73060.1 gnl_Orysa6.0_PACid_16845182 gnl_Orysa6.0_PACid_16845183 gnl_Orysa6.0_PACid_16878767 gnl_Sorbi1.4_PACid_1967154 1.0 gnl_Sorbi1.4_PACid_1960730

gnl_Orysa6.0_PACid_16883029 Fig. S2.37 - orthogroup 19297 100 gnl_Sorbi1.4_PACid_1968103 61 gnl_Bradi1.2_Bradi4g04240.1 gnl_Bradi1.2_Bradi4g04227.1 42 gnl_Orysa6.0_PACid_16873469 gnl_Orysa6.0_PACid_16873470 gnl_Bradi1.2_Bradi2g25790.1 100 97 gnl_Bradi1.2_Bradi3g55500.1 87 Striga_hermonthica_StHeBC3_4423.1 H 58 Striga_gesneroides_StGe3_521 gnl_Orysa6.0_PACid_16895713 gnl_Sorbi1.4_PACid_1979586 100 100 gnl_Orysa6.0_PACid_16837691 gnl_Orysa6.0_PACid_16837690 gnl_Orysa6.0_PACid_16837687 gnl_Orysa6.0_PACid_16885981 gnl_Bradi1.2_Bradi3g10796.1 gnl_Orysa6.0_PACid_16869007 100 gnl_Orysa6.0_PACid_16868700 100 gnl_Orysa6.0_PACid_16868697 100 gnl_Orysa6.0_PACid_16868699 gnl_Orysa6.0_PACid_16868698 gnl_Orysa6.0_PACid_16868706 gnl_Orysa6.0_PACid_16868707 100 gnl_Orysa6.0_PACid_16868733 gnl_Orysa6.0_PACid_16868726 gnl_Orysa6.0_PACid_16868722 100 gnl_Orysa6.0_PACid_16868719 62 gnl_Orysa6.0_PACid_16868691 gnl_Orysa6.0_PACid_16868716 gnl_Orysa6.0_PACid_16868730 gnl_Orysa6.0_PACid_16835230 gnl_Orysa6.0_PACid_16835231 100 gnl_Bradi1.2_Bradi1g00410.1 95 gnl_Sorbi1.4_PACid_1949182 ShContig9483_satoko_gi_295389393_hypothetical_protein 100 gnl_Stras_v1.02_SGA1.0.scaffold994G00050 H 100 Striga_gesneroides_StGe3_4270 79 89 gnl_Bradi1.2_Bradi2g36840.1 gnl_Sorbi1.4_PACid_1964040 gnl_Sorbi1.4_PACid_1964039 gnl_Sorbi1.4_PACid_1964041 gnl_Bradi1.2_Bradi2g02850.1 86 100 gnl_Bradi1.2_Bradi2g02830.1 100 gnl_Bradi1.2_Bradi2g02860.1 gnl_Orysa6.0_PACid_16845205 100 69 gnl_Orysa6.0_PACid_16845228 gnl_Bradi1.2_Bradi1g72580.1 gnl_Bradi1.2_Bradi1g73060.1 99 gnl_Bradi1.2_Bradi1g73680.1 100 gnl_Bradi1.2_Bradi1g73690.1 97 gnl_Orysa6.0_PACid_16845224 60 gnl_Orysa6.0_PACid_16845183 gnl_Orysa6.0_PACid_16845182 gnl_Orysa6.0_PACid_16878767 gnl_Sorbi1.4_PACid_1967154 gnl_Sorbi1.4_PACid_1960730 0.1 Fig. S2.38 - orthogroup 19696 Mulberry_Morus_natabilis002841 Csinensis.orange1.1g020018m_PAC_18122451 54 Csinensis.orange1.1g022637m_PAC_18122452 28 gnl_Frave2.0_gene15801 97 Prunus_persica_ppa015729m Prunus_persica_ppa021784m 46 Prunus_persica_ppa022824m Mesculenta_cassava.10G138200.1 Mesculenta_cassava.10G137600.1 42 Phelipanche_aegyptiaca_PhAeBC5_17034.1 H 100 gnl_Poptr2.2_PACid_18222057 gnl_Poptr2.2_PACid_18210472 gnl_Poptr2.2_PACid_18222056 gnl_Poptr2.2_PACid_18210463 Lamiaceae-EQDA-2005970-Salvia_spp. Paulawniaceae-UMUL-2012754-Paulownia_fargesii Paulawniaceae-UMUL-2012753-Paulownia_fargesii Gesneriaceae-DTNC-0004440-Sinningia_tuberosa gnl_Stras_v1.02_SGA1.0.scaffold113G00290 Orobanche_minor_OrMi2_14035 V Acanthaceae-WEAC-0003066-Strobilanthes_dyerianus 43Verbenaceae-GCFE-2009960-Verbena_hastata 6 Alectra_vogelii_AlVo2_18365 Striga_gesneroides_StGe3_13331 gnl_Stras_v1.02_SGA1.0.scaffold72G00170 V Striga_gesneroides_StGe3_3882 7 gnl_Stras_v1.02_SGA1.0.scaffold137G00180 gnl_Betvu_v1.1_Bv_21630_omdg.t1 40 gnl_Utrgi_v4.1_Scf00101.g8492.t1 gnl_Utrgi_v4.1_Scf00400.g17019.t1 Verbenaceae-GCFE-2009467-Verbena_hastata 1.0 Bignoniaceae-TKEK-0028546-Mansoa_alliacea Fig. S2.39 - orthogroup 20188 gnl_Frave2.0_gene16595 gnl_Frave2.0_gene16593 99 Phelipanche_ramosa_PhRa5_172 H 89 Phelipanche_aegyptiaca_PhAeBC5_1574.1 gnl_Frave2.0_gene24560 gnl_Frave2.0_gene12583 gnl_Frave2.0_gene00411 1.0

Fig. S2.40 - orthogroup 20190 gnl_Frave2.0_gene28307

gnl_Frave2.0_gene02726

83 gnl_Frave2.0_gene24697

100 gnl_Frave2.0_gene19495 100 Phelipanche_aegyptiaca_PhAeBC5_2529.1 H

gnl_Frave2.0_gene00529 1.0

Fig. S2.41 - orthogroup 23343 Orobanche_californica_OrCa3_16494 100 Phelipanche_ramosa_PhRa5_6015 H Phelipanche_mutelii_PhMu2_13013 100 Phelipanche_aegyptiaca_PhAeBC5_4800.1 100 Ricinus_communis_gi_255565992_ref_XP_002523984.1

46 Jatropha_curcas_gi_802585223_ref_XP_012070310.1 gnl_Theca1.0_Tc05_g023030 100 Graimondii_cotton_008G193500.2 84 Graimondii_cotton_008G193500.1 Csinensis.orange1.1g000983m_PAC_18110905 78 Csinensis.orange1.1g000964m_PAC_18110904 Csinensis.orange1.1g000962m_PAC_18110903 100 gnl_Betvu_v1.1_Bv1_007370_pjms.t1 gnl_Frave2.0_gene05887 80 gnl_kiwi_Achn378361 gnl_Vitvi12X_PACid_17828991 gnl_Vitvi12X_PACid_17828992 0.1 Fig. S2.42 - orthogroup 23480 gnl_Frave2.0_gene13130 84 gnl_Frave2.0_gene13131 53 Phelipanche_mutelii_PhMu2_5714 86 H 49 Phelipanche_aegyptiaca_PhAeBC5_5395.1

gnl_Frave2.0_gene13129

gnl_Frave2.0_gene13128

Cricetulus_griseus_hamster 1.0

Figure S2. RAxML-based maximum likelihood trees for 42 HGT orthogroups. Yellow highlighting and “H” symbol refer to the HGT clade, green highlighting and “V” symbol refer to the vertical clade. The species are represented by "gnl_" followed by the first three letters of the genus and first two letters of the species name, which can also be referred to in Fig. S1.

1 HGT VGT 8

2 6 4 depth of coverage 2 0 −2

3.0 3.5 4.0 4.5 contig length

Figure S3. A stoichiometry plot showing the coverage of HGT genomic contigs and vertically transmitted genomic contigs. The X-axis is the log10 transformed contig length (to remove noise, only contigs longer than 1kb were included), and the Y-axis is the log2 transformed read deapth of coverage. This analysis includes 56 HGT-derived genomic contigs, and 493 vertically transmitted gneomic contigs from all three parasitic plants. Two high-coverage genomic contigs are labeled to show outliers: 1 is a mitochondrial genomic contig with much greater sequence depth than nuclear contigs, 2 is a highly-repetitive sequence. Acanthaceae-WEAC-0128093-Strobilanthes_dyerianus (A) (B) 37 gnl_Utrgi_v4.1_Scf00040.g4681.t1 TrVeBC3_3854.9 TrVeBC3_3854.9 64 Orobanche_californica_OrCa3_31544 21 Alectra_vogelii_AlVo2_5753 75 gnl_Poptr2.2_PACid_18223259 88 38 5 Striga_gesneroides_StGe3_9626 gnl_Poptr2.2_PACid_18223260 99 Sesamum_indicumSIN_1023962 gnl_Poptr2.2_PACid_18223258 25 Bignoniaceae-TKEK-0013373-Mansoa_alliacea gnl_Poptr2.2_PACid_18244408 Paulawniaceae-UMUL-2082842-Paulownia_fargesii gnl_Poptr2.2_PACid_18223257 Verbenaceae-GCFE-2009486-Verbena_hastata 57 Verbenaceae-GCFE-2009487-Verbena_hastata 77 Lamiaceae-EQDA-2006479-Salvia_spp. Gesneriaceae-DTNC-0055568-Sinningia_tuberosa 89 Plantaginaceae-EBOL-0066383-Anthirrhinum_majus-B 47 Oleaceae-TORX-0127693-Olea_europaea 29 Nicotiana_sylvestris_gi_698472814_ref_XP_009784338.1 57 Citrus_clementina_gi_567882157_ref_XP_006433637.1 Gossypium_raimondii_gi_823133574_ref_XP_012466627.1 Eucalyptus_grandis_gi_702371196_ref_XP_010061674.1 94 50 Arabidopsis_lyrata_subsp_lyrata_gi_297797395_ref_XP_002866582.1 Phaseolus_vulgaris_gi_593795754_ref_XP_007160915.1 87 gnl_Medtr3.5_Medtr1g018020.1 gnl_Medtr3.5_Medtr1g018230.1 88 Ricinus_communis_gi_255538054_ref_XP_002510092.1 gnl_Poptr2.2_PACid_18223257 gnl_Poptr2.2_PACid_18244408 gnl_Poptr2.2_PACid_18223258 gnl_Poptr2.2_PACid_18223260 gnl_Poptr2.2_PACid_18223259 0.1

HeAn_38808 (C) TrVeBC3_14358.1 80 LiPhGnB2_10428.1 73 StHeBC3_12704.1 gnl_Mimgu1.0_PACid_17676823 71 gnl_Mimgu1.0_PACid_17694128 17 gnl_Solly2.3_Solyc08g007380.2.1 gnl_Frave2.0_gene08950 11 27 gnl_Glyma1.01_PACid_16282551 99 gnl_Glyma1.01_PACid_16282550 21 TrVeBC3_27563.1 40 17 gnl_Poptr2.2_PACid_18218220 18 gnl_Thepa2.0_Tp7g15180 100 25 gnl_Arath10_AT4G16566.1 gnl_Theca1.0_Tc03_g025070 gnl_Vitvi12X_PACid_17817529 0.1 27 gnl_Nelnu1.0_NNU_008844-RA gnl_Aquco1.0_PACid_18156842 gnl_Bradi1.2_Bradi5g16890.1 100 gnl_Sorbi1.4_PACid_1973290 70 gnl_Orysa6.0_PACid_16855908 gnl_Orysa6.0_PACid_16855906 76 gnl_Orysa6.0_PACid_16855907 gnl_Orysa6.0_PACid_16855909 gnl_Musac1.0_GSMUA_Achr5T20890_001 gnl_Ambtr1.0.27_AmTr_v1.0_scaffold00106.76

(D) gnl_Medtr3.5_Medtr1g068580.1 (E) StHeBC3_5017.3_corrected gnl_Medtr3.5_Medtr2g005070.1 67 PhAeBC5_2757.2 gnl_Medtr3.5_Medtr2g048470.2 59 TrVeBC3_10819.9 gnl_Medtr3.5_Medtr2g048470.1 68TrVeBC3_10819.6 100 StHeBC3_5017.3 gnl_Mimgu1.0_PACid_17684638 100 32 97 gnl_Poptr2.2_PACid_18207162 gnl_Solly2.3_Solyc01g067820.2.1 gnl_Glyma1.01_PACid_16278625 gnl_Soltu3.4_PGSC0003DMP400039608 75 32 gnl_Glyma1.01_PACid_16290845 gnl_Arath10_AT3G62450.1 gnl_Glyma1.01_PACid_16290844 28 gnl_Thepa2.0_Tp5g00970 gnl_Carpa1.181_PACid_16410134 54 gnl_Theca1.0_Tc04_g027250 68 gnl_Theca1.0_Tc04_g027250 gnl_Carpa1.181_PACid_16410134 gnl_Arath10_AT3G62450.1 gnl_Medtr3.5_Medtr2g048470.2 gnl_Thepa2.0_Tp5g00970 gnl_Medtr3.5_Medtr2g048470.1_corrected 73 gnl_Solly2.3_Solyc01g067820.2.1 gnl_Medtr3.5_Medtr1g068580.1 100 gnl_Soltu3.4_PGSC0003DMP400039608 gnl_Medtr3.5_Medtr2g005070.1 PhAeBC5_2757.2 gnl_Glyma1.01_PACid_16278625 71 63 TrVeBC3_10819.6 gnl_Glyma1.01_PACid_16290845 46TrVeBC3_10819.9 89 gnl_Glyma1.01_PACid_16290844 74 gnl_Mimgu1.0_PACid_17684638 gnl_Poptr2.2_PACid_18207162_corrected gnl_Orysa6.0_PACid_16887483 gnl_Orysa6.0_PACid_16887483 gnl_Bradi1.2_Bradi4g26776.1 52gnl_Bradi1.2_Bradi4g26776.1 gnl_Bradi1.2_Bradi4g44832.2 gnl_Bradi1.2_Bradi4g44832.2 gnl_Sorbi1.4_PACid_1977171 gnl_Sorbi1.4_PACid_1968853 gnl_Sorbi1.4_PACid_1968853 1.0 gnl_Sorbi1.4_PACid_1977171 1.0

Figure S4. HGT artifacts due to insufficient taxon sampling (A-B), host plant contamination (C), and frame-shift errors (D-E). RAxML-based maximum likelihood tree for orthogroup 20348 from the initial automated pipeline (A) shows the placement of a Triphysaria gene (purple arrow; TrVeBC3_3854.9) as a member of a rosid clade composed of many Populus sequences (Poptr), indicative of a potential HGT. (B) Increase of taxon sampling in the Lamiales order (green non-shaded) and the rosid groups (red) identifies the Triphysaria sequence (TrVeBC3_3854.9) as a vertically transmitted gene which groups with its closely related parasitic taxa (green foreground, grey shading) in Lamiales. (C) Discovery of an unannotated host plant gene, rather than HGT (orthogroup 8224), explains the placement of the Triphysaria gene (TrVeBC3_27563.1) with Glycine max (Glyma) sequences, a close relative of its experimental host – Medicago truncatula. (D-E) show an HGT artifact due to frame-shift errors in orthogroup 20348. (D) Purple branch shows a clade with long branch composed of three sequences – StHeBC3_5017.3, gnl_Medtr3.5_Medtr2g048470.1, and gnl_Poptr2.2_PACid_18207162. Striga sequence was identified in preliminary screening as a potential HGT sequence because of strong support for placement with a rosid donor. Careful examination of the alignment revealed frame-shift errors in all these three sequences, and correction of this error resolved the Striga gene as a vertical gene (E). The two rosid sequences from Poplar and Medicago also went to their expected positions within the rosid clades without long branches (E).

Figure S5. The conserved 5’-UTR intron shared between the donor represented by Sorghum bicolor gene Sb10g026740 and the HGT Striga hermonthica gene StHeGnB1_80049 (orthogroup 14624). Geneious alignment view of the sequence alignment between the donor and recipient gene for 5’-UTR region (A), 5’- UTR intron (B), CDS (C), and 3’-UTR (D). The 5’-UTR intron was revealed by read mapping data.

Figure S6. The conserved 3’-UTR intron shared between the donor represented by Sorghum bicolor gene Sb01g013250 and the HGT Striga hermonthica gene ShContig9483 (orthogroup 19297). Geneious alignment view of the sequence alignment between the donor and recipient gene for 5’-UTR region (A), CDS (B), 3’-UTR (C), and 3’-UTR intron (D). A shortened intron revealed in the parasitic Striga HGT gene, as shown in (C) where the N-terminal intron of the host gene resulted in unspliced transcribed mRNA in the 3’-UTR region. The 3’-UTR intron was revealed by read mapping data.

(A1)

D

H

V

VR

pos 333 515 591 741 819 983 1347 1587 1796 2202 2457 2967 3074 3177 3335 (A2) (A3) gnl_Theca1.0_Tc09_g018620 D Conserved intron sequence btw D & H 97 PhAeBC5_4239_scaffold_HGT H 41 Conserved intron sequence btw V & VR gnl_Mimgu1.0_PACid_17693905 83 VR 96 PhAeBC5_4810.1_VGT V pos Intron positions in coding sequence alignment gnl_Mimgu1.0_PACid_17678221

(B1) Intron loss

D

H

V

VR

pos 379 524 678 846 1077 1221 1312 1419 1654 1980 2298

(B2) (C) gnl_Bradi1.2_Bradi1g69880.1 gnl_Sorbi1.4_PACid_1954278 orthogroup annotation # of CIPs 58 gnl_Sorbi1.4_PACid_1967640 806 valyl-tRNA 15 gnl_Orysa6.0_PACid_16885413 synthetase 7 50 gnl_Orysa6.0_PACid_16885412 gnl_Bradi1.2_Bradi3g31100.1 2270 methionyl-tRNA 11 10 gnl_Soltu3.4_PGSC0003DMP400050083 synthetase 34 gnl_Theca1.0_Tc04_g020790 96 D 4067 tRNAHis guanylyl- 9 OrMiGB1_4206_HGT 17 19 transferase 82 StHeBC3_2868.1_HGT H 100 gnl_Glyma1.01_PACid_16290193 gnl_Glyma1.01_PACid_16286058 PhRaGB1_464_VGT V 36 gnl_Mimgu1.0_PACid_17695503 VR Figure S7. Intron phylogeny and intron-positions for three HGT orthogroups that encode tRNA synthetase/transferase. (A) and (B) show the conserved intron positions and intron sequence comparisons between two pairs of sequences: H (the HGT gene) and D ( the represented donor), V (the vertical gene) and VR (the vertical sequence relative) (Mimulus sequence) (corresponding coding sequence phylogeny are shown in Fig. S1. (A1) (orthogroup 806) and (B1) (orthogroup 2270) show coding sequence alignment with introns marked with boxes. (A3) and (B2) show the intron sequence phylogenies supporting strong sequence similarity of two pairs, the HGT gene and its donor, the vertical gene and its vertical relative in Mimulus. Intron sequence phylogeny were made by the conserved introns colored in red and blue of (A1) and (B1). The intron origins match the coding sequence origin, supporting genomic transfers. (C) Annotation of three HGT orthogroups, all encoding tRNA synthetase/tranferases involved in the attachment of the codon and tRNA anticodon. Also, the number of conserved intron positions (CIPs) in the forced CDS alignment were also shown, 4067 is the orthogroup in main Figure 3 that also encodes tRNAHis transferase.

Figure S8. Phylogeny of orthogroup 11841 and predicted 3D structure. The HGT genes identified are PhAeBC5_3756.1 in Phelipanche aegyptiaca and StHeBC3_55745.1 in Striga hermonthica. For this particular orthogroup, the HGT gene was also identified in Orobanche fasciculata (OrFa_1kp_VYDM_130285) from the 1KP database. Additional Orobanchaceae orthologs were also found through shallow transcriptome sequencing of additional species, including Myzorrhiza californica (OrCaGB1_4009), Phelipanche ramosa (PhRaGB1_3602) and Phelipanche mutelii (PhMuGB1_6933). HGT and vertical clade was labeled with “H” and “V” respectively on the phylogeny. (A). phylogeny of orthogroup 11841 supporting HGT; (B). Protein 3D structure of Anthocyanidin synthase from Arabidopsis thaliana (PDB ID: 1GP4); (C). Predicted protein 3D structure of PhAeBC5_3756.1. Color scheme: alpha helixes shown in red; beta sheets shown in yellow; loops shown in green.

(A) Color Key

−2 0 2 after HIF interface Value before HIF after HIF before HIF imbibed germinatedseed germinated seedling seedling underground shoots above groundabove shoots ground germinatedgerminated seedling seedlingearly stage late haustoria stage haustoria undergroundunderground shoots underground shoots underground roots roots floral buds StHeBC3_4423.1 StHeBC3_10075.1 StHeBC3_21126.3 StHeBC3_21126.1 StHeBC3_21126.2 StHeBC3_16619.1 StHeBC3_32425.1 StHeBC3_11327.15 StHeBC3_41710.1 StHeBC3_2868.1

StHe0G StHe2G StHe3G StHe4G StHe1G2StHe1G3 StHe51G StHe52G StHe61G StHe62G StHe2G2 StHe51G2 StHe51G3 StHe52G2 (B) StHeIntSorbiG

Color Key

−2 0 1 2 after HIF before HIF tissues Value imbibed seed tissues attached above germinated seedlinggerminated seedlingearly-stage haustoria Zea interface ground shoots roots - unattached late-stage haustoriaMedicago interface ground shootsunattached aboveattached floralunattached floral roots - attached TrVeBC3_24500.1

TrVeBC3_29569.1

TrVe0G TrVe1G TrVe2G TrVe61G TrVe62G TrVe63G TrVe3G2TrVe41G TrVe61Gu TrVe62Gu TrVe63Gu TrVeIntMedtrGTrVeIntZeamaG Figure S9. Heatmaps of HGT transgenes in Striga hermonthica (A), and Triphysaria versicolor (B). These genes were identified from the 42 HGT orthogroups. Heatmap intensity represents the FPKM-transformed z-score.

Table S1. Scoring scheme used to score each phylogenetic tree based on bootstrap support, depth of donor clades, and long branches. Criterion1 Criterion2 Criterion3 Scoring Bootstrap score Sampling depth score Branch length score Summed confidence support score x=100 10 rich sampling in the 5 branch length 3 x≤9 low 90≤x<100 8 donor clade and not long strong support 80≤x<90 6 rich sampling in the 3 branch length 2 10≤x≤14 medium 70≤x<80 4 donor clade and low < 2 times of 60≤x<70 3 support the average 50≤x<60 2 mixed samples in 1 branch length 1 15≤x≤18 high x<50 1 the ancestral node >=2 times

Table S2. SH test to evaluate number of transfers in HGT trees by constraining multiple HGT genes to one monophyletic clade. orthogroup logL (original) logL (constrained) Significant worse # of transfers (0.01)? 218 -106049.78 -106490.58 Y ≥ 2 1021 -40178.73 -40281.56 Y ≥ 2 3861 -42295.88 -42296.95 N 1 5002 -95429.05 -95694.82 Y ≥ 2 8888 -35555.79 -35813.86 Y ≥ 2 11437 -121967.75 -122293.12 Y ≥ 2 13512 113878.47 -114045.44 Y ≥ 2 14233 -88970.83 -89052.98 Y ≥ 2 18354 -11889.45 -11941.91 Y ≥ 2 18774 -48200.19 -48661.65 Y ≥ 2 19297 -48200.19 -48661.65 Y ≥ 2 logL: log likelihood value, Y means the constrained tree is significant worse than the original tree, N means the constrained tree is not significant worse than the original tree.

Table S3. Verification of HGT sequences by multiple lines of evidences including RT-PCR (RT), genomic PCR (GP), covered by genomic sequencing (GS), present in transcriptomes of multiple parasitic species (MP), or by existing studies. Orthogroup (genes) Verification Orthogroup Verification 1886 (PhAeBC5_15496.1) RT, GP, GS 12835 (StHeBC3_10075.1) GS 14624 (StHeGnB1_80049) GP, GS 14230 (PhAeBC5_34854.1) GS 1685 (PhAeBC5_6791.2/9731.1/15355.1) RT, GP 17 (PhAeBC5_8480.1) GS 10143 (PhAeBC5_14056.1) RT 226 (PhAeBC5_14888.1) GS 8888 (2) (PhAeBC5_15086.1) RT 9613 (PhAeBC5_16890.17) GS 10050 (PhAeBC5_4284.2) RT, GP (I) 19696 (PhAeBC5_17034.1) GS 13656 (StHeBC3_41710.1) RT 16703 (StHeBC3_11327.15) GS 14233 (2) (StHeBC3_16619.1) RT 18709 (PhAeBC5_654.1) GS 18774 (2) (StHeBC3_4423.1) RT 13892 (PhAeBC5_4266.1) GS 1021 (2) GS 20190 (PhAeBC5_2529.1) RT (PhAeBC5_17559.1) 2376 (PhAeBC5_270.1 5002 (2) GS StHeBC3_48088.1) RT, GS (PhAeBC5_9781.2) RT, GS, GP GS 11841 (PhAeBC5_3756.1 (I- StHeBC3_55745.1) Pa_3756.1) 15149 (PhAeBC5_8832.4) 1226 (PhAeBC5_11914.4) RT, GS 20188 (PhAeBC5_1574.1) GS RT, GS, GP 3861 (PhAeBC5_ 9914.1 /22555.2) (I) 4572 (PhAeBC5_8623.1) MP (3) 5896 (StHeBC3_21126.1) RT, GS 4598 (PhAeBC5_13756.1) MP(3) RT, GS, GP 11437 (2) 806 (PhAeBC5_4239.1) (I) (PhAeBC5_13226.1/6799.1) MP (4) 13512 (2) 2270 (StHeBC3_2868.1) RT, GS (PhAeBC5_1584.1/3740.1) MP (4) 4067 (PhAeBC5_9762.1) RT, GS 14675 (PhAeBC5_12620.5) MP (4) 18354 (2) 218 (2) (PhAeBC5_20413.1) RT, GS (PhAeBC5_5786.1) MP (4) Reference – Yoshida et 19297 (2) (StGe3_4270) al. 2010 (26) 23343 (PhAeBC5_4800.1) MP(4) Reference – Zhang et al. 15246 (PhAeBC5_2525.1) 2013 (28) 23480 (PhAeBC5_5395.1) MP (2) Number in the parenthesis following orthogroup and transcriptome stands for the number of events for that orthogroup, and the number of parasite transcriptomes containing the HGT gene, respectively. “I” followed by GP (genomic PCR) represents introns in the sequenced genomic PCR product.

Table S4. An example of orthogroup 806 showing conserved intron positions in coding sequence (CDS) multiple sequence alignment (MSA). Sequence_id pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos pos gnl_Ambtr1.0.27_AmTr 333 515 591 657 741 819 903 983 1347 1587 1796 2202 2457 2967 3074 3177 3335 _v1.0_scaffold00038.43 gnl_Sorbi1.4_PACid_19 333 515 591 657 741 819 903 983 1347 1587 1796 2202 2457 2967 3074 3177 3335 75126 gnl_Orysa6.0_PACid_16 333 515 591 657 741 819 903 983 1347 1587 1796 2510 846845 gnl_Orysa6.0_PACid_16 333 515 591 657 741 819 903 983 1347 1587 1796 2202 2457 2967 3074 3177 844493 gnl_Orysa6.0_PACid_16 333 515 591 657 741 819 903 983 1347 1587 1796 2202 2457 2967 3074 3177 844494 gnl_Orysa6.0_PACid_16 333 515 591 657 741 819 903 983 1347 1587 1796 2202 2457 2967 3074 3177 3335 849430 gnl_Orysa6.0_PACid_16 333 515 591 657 741 819 903 983 1347 1587 1796 2202 2457 2967 3074 3177 3335 849429 gnl_Bradi1.2_Bradi2g0 333 515 591 657 741 819 903 983 1347 1587 1796 2202 2457 2967 3074 3177 3335 4960.1 gnl_Bradi1.2_Bradi3g3 333 504 657 741 819 903 987 1347 1587 1796 2457 2967 3074 3177 3335 0350.1 gnl_Orysa6.0_PACid_16 333 515 591 657 741 819 903 983 1347 1545 1796 2202 2457 2967 3074 3177 3335 886435 gnl_Aquco1.0_PACid_1 333 515 591 657 741 819 903 983 1347 1587 1796 2202 2457 2967 3074 3177 3335 8164810 gnl_Aquco1.0_PACid_1 333 515 591 657 741 819 903 983 1347 1587 1796 2202 2457 2967 3074 3177 3335 8157961 gnl_Aquco1.0_PACid_1 333 515 591 657 741 819 903 983 1347 1587 1796 2202 2457 2967 3074 3177 3335 8144325 gnl_Poptr2.2_PACid_18 741 819 903 983 1347 1587 1796 2202 2457 2967 3074 3177 3335 207203 gnl_Poptr2.2_PACid_18 515 591 657 741 819 903 983 1347 1587 1796 2202 2457 2967 3074 3177 3335 244025 gnl_Arath10_AT1G146 333 515 591 657 741 819 903 983 1347 1587 1796 2202 2457 2967 3074 3177 3335 10.1 gnl_Thepa2.0_Tp1g129 333 515 591 657 741 819 903 983 1347 1587 1796 2202 2457 2967 3074 3177 70 gnl_Theca1.0_Tc09_g0 333 515 591 657 741 819 903 983 1347 1587 1796 2202 2457 2967 3074 3177 3335 18620 (HGT donor) PhAeBC5_4239.1 (HGT 333 515 591 657 741 819 903 983 1347 1587 1796 2202 2457 2967 3074 3177 3335 gene) gnl_Medtr3.5_Medtr1g 515 591 657 741 819 903 983 1347 1587 1796 2202 2457 2967 3074 3177 3335 101620.1 gnl_Glyma1.01_PACid_ 515 591 657 741 819 903 983 1347 1587 1796 2202 2457 2967 3074 3177 3335 16281403 gnl_Glyma1.01_PACid_ 515 591 657 741 819 903 983 1347 1587 1796 2202 2457 2967 3074 3177 3335 16281404 gnl_Vitvi12X_PACid_17 333 515 591 657 741 819 903 983 1347 1587 1796 2202 2457 2967 3074 3177 3335 822742 gnl_Solly2.3_Solyc09g0 333 515 591 657 741 819 903 983 1347 1587 1796 2202 2457 2967 3074 3177 3335 07540.2.1 gnl_Mimgu1.0_PACid_1 333 515 591 657 741 819 903 983 1347 1587 1796 2202 2457 2967 3074 3177 7678221 (Vertical relative) gnl_Mimgu1.0_PACid_1 333 515 591 657 741 819 903 983 1347 1587 1796 2202 2457 2967 3074 3177 3335 7684099 (Vertical relative) gnl_Mimgu1.0_PACid_1 333 515 591 657 741 819 903 983 1347 1587 1796 2202 2457 2967 3074 3177 7693905 (Vertical relative) PhAeBC5_4810.1 333 515 591 - 741 819 - 983 1347 1587 1796 2202 2457 2967 3074 3177 3335 (vertical gene) pos: intron position in MSA; “-“ means not available due to low coverage of genomic DNA sequencing data.

Table S5. Summary of conserved intron positions (CIPs) in HGT clade and the overall MSA. orthogroup annotation HGT recipient Donor # of CIPs in scorable # of # of genus representative HGT clade CIPs in HGT CIPs clade in MSA 2376 proteasome subunit P, S Poptr 8 8 10 11841 Oxidoreductase, 2OG- P, S, O Frave 1 3 3 Fe oxygenase 4067 tRNAHis P Frave 8 9 12 guanylytransferase 8888 tRNA P Frave 7 7 14 adenylyltransferase activity 10050 tRNA synthetase class P Frave 4 4 6 II core domain containing protein 806 Valyl-tRNA P, O Theca 15 15 19 synthetase 2270 methionyl-tRNA P, S, O Theca 11 13 16 synthetase 1685 Gnk2-homologous P Medtr, Glyma 7 7 7 domain 1886 Ankyrin repeat family P Frave 0 2 2 protein 9613 HAD-superfamily P Glyma 2 3 12 hydrolase 3861 Poly (ADP-ribose) P, L Glyma 7 7 9 glycohydrolase (PARG) 5896 uroporphyrinogen-III S Glyma 8 8 8 synthase 18774 Domain of unknown S Orysa 0 0 0 function Total 78 79 118 HGT recipient genus: P – Phelipanche, S – Striga, O – Orobanche, L – Lindenbergia. Nomenclature of the donor follows the five-letter rule (The first three letters of the genus, and first two letters of the species).

Table S6. PAML analyses with branch test on codon alignment of 42 HGT orthogroups, testing mode of selection in HGT sequences (foreground) compared to VGT sequences (background).

Ortho ID Dn Ds Foreground ω Background ω P-value Significant at 0.05 Selection 806 0.09 0.33 0.27 0.14 < 0.00001 S RP 1886 0.48 0.82 0.58 0.16 < 0.00001 S RP 2270 0.09 0.28 0.31 0.14 < 0.00001 S RP 2376 0.03 0.15 0.18 0.04 0.000108 S RP 4067 0.10 0.24 0.43 0.20 0.000187 S RP 8888 0.12 0.28 0.43 0.23 0.001553 S RP 9613 0.51 1.03 0.50 0.14 < 0.00001 S RP 10050 0.13 0.26 0.51 0.20 < 0.00001 S RP 10143 0.10 0.21 0.46 0.20 < 0.00001 S RP 1226 0.22 0.41 0.55 0.27 0.002847 S RP 4598 0.22 0.56 0.39 0.27 0.016327 S RP 11437 0.40 0.53 0.74 0.36 < 0.00001 S RP 13512 0.29 0.17 1.65 0.34 < 0.00001 S POS 3861 0.14 0.10 1.48 0.28 < 0.00001 S POS 18709 0.19 0.17 1.16 0.22 0.000495 S POS 19696 0.56 73.08 0.01 0.29 0.022212 S SP 11841 0.03 0.20 0.14 0.26 0.03422 S SP 13656 0.01 3.08 0.00 0.30 < 0.00001 S SP 14233 0.32 1.16 0.27 0.48 0.005556 S SP 5896 0.06 0.21 0.29 0.30 0.943628 NS P 1685 0.06 0.21 0.29 0.27 0.811464 NS P 17 0.21 0.78 0.27 0.23 0.054126 NS P 218 0.07 0.54 0.12 0.20 0.431345 NS P 226 0.12 0.51 0.24 0.27 0.449545 NS P 1021 0.13 0.72 0.18 0.19 0.943886 NS P 4572 0.13 0.42 0.31 0.47 0.054707 NS P 5002 0.17 1.00 0.17 0.23 0.172164 NS P 12835 0.11 0.68 0.16 0.18 0.731762 NS P 13892 0.02 0.09 0.26 0.24 0.917998 NS P 14230 0.28 2.22 0.13 0.16 0.331354 NS P 14624 0.22 1.02 0.22 0.26 0.420936 NS P 14675 0.49 1.22 0.40 0.47 0.705675 NS P 15149 0.31 1.24 0.25 0.28 0.734189 NS P 15246 0.06 0.10 0.65 0.24 0.139996 NS P 16703 0.40 70.39 0.01 0.33 0.09305 NS P 18354 0.54 1.01 0.54 0.62 0.908563 NS P 18774 0.20 0.32 0.61 0.51 0.26167 NS P 19297 0.08 0.14 0.61 0.51 0.26167 NS P 20188 0.55 0.66 0.83 0.82 0.971238 NS P 20190 0.19 0.22 0.88 0.91 0.946516 NS P 23343 0.08 0.32 0.26 0.34 0.05642 NS P 23480 0.22 0.26 0.56 0.84 0.218423 NS P Significant branch tests (at p<0.05) reveal relaxed constraint (RP), positive selection (POS), or stronger purifying selection (SP) in the HGT foreground compared to background. Non-significant branch tests show a common level of purifying selection (P) in the foreground HGT sequences compared to the VGT background.

Table S7. PAML analyses with the branch-site model on codon alignment of 15 HGT orthogroups with greater Dn/Ds on HGT genes compared to the background, identifying the presence of sites under positive selection. HGT transgene IDs Orthogroup ID Sites identified having positive selection (P<=0.05) PhAeBC5_4239.1 OrMi2_4015 806* 556 D; PhRa5_26371 PhMu2_16115 PhAeBC5_11914.4 PhRa5_49596 1226* 135 E; 250 V; PhRa5_27280 PhMu2_10869 PhMu2_12766 PhAeBC5_15496.1 PhRa5_103413 1886* 1 M; 39 E; 83 F; 84 S; 86 E; 92 K; 146 L; PhRa5_58721 153 S; 170 T; 173 D; 176 C; 210 T; 214 L; 230 T; 236 T; 247 R; 249 Q; 251 C; 254 D; 256 V; 317 G; 318 K; 322 E; 330 P; 347 S; 363 A; 403 G; 404 F ; 406 S; 416 C; 421 G; 426 F; 428 G; 429 P; 438 C; 444 W; 472 A; 485 A; 488 V; 493 A; 500 G; 530 S; 533 E; 535 V; 581 D; 582 L; StHeBC3_2868.1 PhAeBC5_3356.1 2270 NA PhRa5_6157 PhMu2_12619 PhMu2_44156 PhMu2_20937 PhMu2_5516 PhMu2_14031 OrMi2_4206 StGe3_7730 StGe3_20099 PhAeBC5_270.3 StHeBC3_48088.1 2376* 20 V; PhRa5_4383 PhMu2_6326 PhRa5_4382 PhAeBC5_9914.1OrMi2_2808 3861* 23 S; 45 E; 68 A; 71 D; 93 L; 96 D; 97 D; PhRa5_12205 PhMu2_12820 254 D; 289 K; 301 V; 486 C; 543 W; 568 PhMu2_11052 P; PhAeBC5_9762.1 PhRa5_10084 4067* 218 K; PhMu2_15393 PhAeBC5_13756.1 PhMu2_15647 4598 31 R; 32 P; 156 S; 159 G; 160 L; 169 K; PhRa5_33568 187 L; 188 S; 233 Q; 235 K; 240 K; 244 T; 247 E; 248 A; 249 M; 251 L; 253 P; 282 L; 284 S; 379 K; 481 N; 495 E; 500 G; 535 A; 536 T; 539 V; 540 A; 543 C; 545 P; 547 N; Phelipanche_ramosa_PhRa5_6603 8888* 209 Q; 538 D; 559 P; Phelipanche_mutelii_PhMu2_37370 Phelipanche_ramosa_PhRa5_6605 PhAeBC5_15086.1 Phelipanche_ramosa_PhRa5_6604 PhAeBC5_16890.17 PhRa5_14643 9613* 3 P; 4 S; 22 S; 26 R; 33 F; 42 K; 50 K; 53 PhRa5_14643 T; 55 N; 73 L; 74 P; 77 D; 78 A; 81 I; 82 G; 85 L; 86 Q; 87 I; 90 E; 95 V; 96 E; 99 F; 100 V; 101 H; 102 L; 104 F; 106 C; 107 E; 109 K; 110 P; 112 H; 114 V; 116 S; 121 S; 122 K; 123 P; 126 K; 127 F; 162 T; PhAeBC5_4284.2 PhMu2_5340 10050* 472 S; 666 S; 709 G; 710 S; 763 F; PhRa5_11783 PhMu2_31112 PhAeBC5_14056.1 OrMi2_39134 10143* 521 H; 599 G; OrMi2_14052 PhAeBC5_1584.1 PhAeBC5_3740.1 11437* 1201 Y; 1242 E; 1287 T; 1417 A; 1637 G; PhAeBC5_1584.1 OrCa3_3600 13512* 367 R; 479 C; 480 R; 497 S; 520 S; 533 PhRa5_1522 PhMu2_40952 F; 536 E; 547 L; 768 G; 831 N; 844 N; 886 E; 1005 P; 1107 Y; 1129 L; 1174 K; 1391 S; PhAeBC5_654.1 PhRa5_6642 18709* 664 C; 676 Y; 710 K; 715 S; 724 R; NA indicates no adaptive sites were identified. “*” after each orthogroup indicates presence of adaptive sites were identified only in parasitic lineages, not in a nonparasitic genome, such as Mimulus.

Table S8. RELAX analyses on codon alignment of 42 HGT orthogroups testing stronger selection or relaxed constraint in HGT sequences compared to the background. Ortho ID K P-value Significant at 0.05 Selection: HGT vs background 806 1.01 0.838 NS same 9613 0.75 0.090 NS same 14230 0.66 0.064 NS same 10050 1.01 0.970 NS same 11841 1.35 0.237 NS same 19696 0.65 0.193 NS same 18709 0.85 0.987 NS same 11437 1.03 0.747 NS same 13656 0.78 0.584 NS same 17 1.03 0.720 NS same 1021 1.14 0.325 NS same 5002 1.05 0.538 NS same 12835 1.78 0.103 NS same 14624 1.02 0.914 NS same 15246 1 0.995 NS same 16703 1.02 0.914 NS same 18354 1 0.970 NS same 18774 0.92 0.436 NS same 20188 0 0.067 NS same 20190 0.73 0.294 NS same 23343 1.21 0.227 NS same 23480 1.02 0.960 NS same 4572 50 0.004 S stronger 5896 50 0.026 S stronger 19297 49.44 0.013 S stronger 1886 49.42 0.001 S stronger 4598 35.1 0.000 S stronger 226 2.22 0.000 S stronger 14233 2.16 0.000 S stronger 4067 1.99 0.000 S stronger 218 1.93 0.000 S stronger 13512 1.9 0.000 S stronger 1226 1.61 0.000 S stronger 14675 1.14 0.004 S stronger 1685 0.82 0.004 S relaxed 2270 0.68 0.000 S relaxed 15149 0.66 0.038 S relaxed 8888 0.61 0.013 S relaxed 3861 0.55 0.000 S relaxed 13892 0.54 0.030 S relaxed 2376 0.21 0.000 S relaxed 10143 0.08 0.001 S relaxed Significant RELAX results (at p<0.05) reveal stronger selection (stronger), relaxed constraint (relaxed). Non-significant RELAX results show the same level of selection in the foreground HGT sequences compared to background (same).

Table S9. Inferred donor lineage and recipient taxa from the gene trees for the final 42 HGT orthogroups. orthogroup ID recipient taxa donor family taxa or common name 17 Phe Amaranthaceae Beta vulgaris (beet) 218 Tri, Ale, Phe, Str Rosaceae Prunus (peach) 226 Phe Euphorbiaceae Morchella (Cassava) 806 Phe, Oro Malvaceae Theobroma cacao (cocoa) 1021 Phe, Tri Rosaceae Fragaria (strawberry), Malus (apple) 1226 Phe Euphorbiaceae Ricinus communis (castor bean) 1685 Phe Fabaceae Medicago, Glycine 1886 Phe Rosaceae Fragaria vesca (strawberry) 2270 Phe, Oro, Str Malvaceae Theobroma cacao (cocoa) 2376 Phe, Str Rosaceae Fragaria vesca (strawberry) 3861 LiPh, Phe, Oro Fabaceae Glycine (soybean) 4067 Phe Rosaceae Fragaria vesca (strawberry) 4572 Phe, Oro Rosaceae Fragaria vesca (strawberry) 4598 Phe Fabaceae Medicago (alfalfa, barrel millet) 5002 Phe, Oro, Ale Fabaceae Fragaria (strawberry), Prunus (peach) 5896 Str Fabaceae Glycine (sobyean) 8888 Phe, Oro Rosaceae, Fragaria (strawberry), Glycine Fabaceae (soybean) 9613 Phe Fabaceae Medicago (alfalfa, barrel millet) 10050 Phe Rosaceae Fragaria vesca (strawberry) 10143 Phe, Oro Rosaceae Fragaria vesca (strawberry) 11437 Phe Rosaceae Fragaria vesca (strawberry) 11841 Phe, Oro Rosaceae Fragaria vesca (strawberry) 12835 Str Poaceae Sorghum bicolor 13512 Phe, Oro Rosaceae Fragaria vesca (strawberry) 13656 Str Poaceae Sorghum bicolor 13892 Phe, Oro Fabaceae Medicago (alfalfa, barrel millet) 14230 Phe Rosaceae Prunus (peach) 14233 Str Poaceae Oryza sativa (rice) 14624 Str Poaceae Sorghum bicolor, 14675 Phe, Oro Rosaceae Fragaria vesca (strawberry) 15149 Phe, Oro Rosaceae Fragaria vesca (strawberry) 15246 Phe, Oro, Ale, Tri Fabaceae Medicago (alfalfa, barrel millet) 16703 Str Poaceae Sorghum bicolor 18354 Phe, Oro Rosaceae Fragaria vesca (strawberry) 18709 Phe Brassicaceae Arabidopsis thaliana (thale cress) 18774 Str Poaceae Setaria italica (foxtail millet) 19297 Str Poaceae Sorghum bicolor 19696 Phe Salicaceae Populus trichocarpa (poplar) 20188 Phe, Str Rosaceae Fragaria vesca (strawberry) 20190 Phe Rosaceae Fragaria vesca (strawberry) 23343 Phe, Oro Euphorbiaceae Ricinus communis (castor bean) 23480 Phe Rosaceae Fragaria vesca (strawberry) Phe: Phelipanche, Oro: Orobanche, Str: Striga, Tri: Triphysaria, Ale: Alectra, Lin: Lindebergia

Table S10. Inferred PFam domains for the HGT genes in the 42 orthogroups. orthogroup HGT unigene Pfam domain 226 PhAeBC5_14888.1 [Cytochrome P450(31-483)] [Bacteriophage coat protein B(47-66), ABC1 family(109-154), Beta- amyloid peptide (beta-APP)(154-162), Phosphotransferase enzyme PhAeBC5_15353.2 family(215-253)] [Salt stress response/antifungal(62-151), Protein of PhAeBC5_9731.1 unknown function (DUF2970)(177-195), ABC1 family(238-281)] [Salt 1685 PhAeBC5_6791.2 stress response/antifungal(148-244), ABC1 family(327-376)] 2376 PhAeBC5_270.3 [Proteasome subunit A N-terminal signature(142-147)] 14624 StHeGnB1_80049 [BTB/POZ domain(272-300)] 23343 PhAeBC5_4800.1 [NB-ARC domain(174-451), CENP-Q, a CENPA-CAD centromere complex subunit(703-758), AAA ATPase domain(761-805), Leucine rich repeat(862-872)] [non-haem dioxygenase in morphine synthesis N-terminal(39-150), 11841 PhAeBC5_3756.1 2OG-Fe(II) oxygenase superfamily(200-294)] 1886 PhAeBC5_15496.1 [PUL domain(281-350)] PhAeBC5_13226.1 [Kelch motif(207-212), Kelch motif(228-244)] [Kelch motif(155-190), 11437 PhAeBC5_6799.1 Kelch motif(257-283)] [Poly A polymerase head domain(38-175), Probable RNA and SrmB- 8888 PhAeBC5_15086.1 binding site of polymerase A(221-262)] 18709 PhAeBC5_654.1 [Nup53/35/40-type RNA recognition motif(116-150)] [Leucyl-tRNA synthetase, Domain 2(347-405), tRNA synthetases class I (C) catalytic domain(632-673), Anticodon-binding domain of 806 PhAeBC5_4239.1 tRNA(774-922), KRI1-like family(995-1025)] [Adenylate kinase, active site lid(171-198)] [tRNA synthetases class I PhAeBC5_3356.1 (I, L, M and V)(435-474), Anticodon-binding domain of tRNA(537- 2270 StHeBC3_2868.1 621)] 4067 PhAeBC5_9762.1 [tRNAHis guanylyltransferase(265-391)] [Histidyl-tRNA synthetase(109-430), Anticodon binding domain(462- 10050 PhAeBC5_4284.2 538)] 13892 PhAeBC5_4266.1 [Ribosomal protein S13/S18(60-132)] [ABC transporter transmembrane region(725-970), AAA 17 PhAeBC5_8480.1 domain(1076-1091), RNA helicase(1247-1282)] [3-beta hydroxysteroid dehydrogenase/isomerase family(10-73), 9613 PhAeBC5_16890.17 Dimerisation domain of Zinc Transporter(333-357)] 15246 PhAeBC5_2525.1 [Albumin I chain a(71-112)] [DNA polymerase III beta subunit, central domain(12-79), BAAT / 1226 PhAeBC5_11914.4 Acyl-CoA thioester hydrolase C terminal(135-162)] [HEAT-like repeat(383-391), Tubulin folding cofactor D C 10143 PhAeBC5_14056.1 terminal(427-614), HEAT repeat(640-657)] PhAeBC5_22555.2 [Poly (ADP-ribose) glycohydrolase (PARG)(614-632)] [Poly (ADP- 3861 PhAeBC5_9914.1 ribose) glycohydrolase (PARG)(78-432)] 4598 PhAeBC5_13756.1 [Colicin D(303-330)] 19696 PhAeBC5_17034.1 [Peptidase C39 family(300-311)] 16703 StHeBC3_11327.15 [GRF zinc finger(56-86)] [Leucine Rich Repeat(182-192), Leucine Rich repeat(206-214), 4572 PhAeBC5_8623.1 Leucine Rich repeats (2 copies)(316-325)] [Proline-rich nuclear receptor coactivator(17-65), Uncharacterised 5896 StHeBC3_21126.2 protein family (UPF0180)(206-237), PCI domain(299-377)] [Protein of unknown function (DUF 659)(16-145), Domain of unknown function (DUF4413)(278-387), hAT family C-terminal dimerisation region(456-535)] [BED zinc finger(57-115), Protein of PhAeBC5_31016.1 unknown function (DUF 659)(199-334), Domain of unknown function StHeBC3_32425.1 (DUF4413)(492-590)] [Protein of unknown function (DUF 659)(371- PhAeBC5_20413.1 392)] [Domain of unknown function (DUF4413)(250-355), Protein of 218 TrVeBC3_29569.1 unknown function (DUF 659)(393-413)] [DDE superfamily endonuclease(66-145), hAT family C-terminal PhAeBC5_17559.1 dimerisation region(419-489)] [BED zinc finger(69-113), Protein of 1021 TrVeBC3_24500.1 unknown function (DUF 659)(247-393)] [BED zinc finger(316-368), Domain of unknown function (DUF4413)(414-512), hAT family C-terminal dimerisation region(568- 5002 PhAeBC5_9781.2 650)] 12835 StHeBC3_10075.1 [Plant transposon protein(167-274)] [RhoGEF domain(297-379), MULE transposase domain(433-464), 14230 PhAeBC5_34854.1 Domain of Unknown Function with PDB structure (DUF3862)(495- 547)] 15149 PhAeBC5_8832.4 [hAT family C-terminal dimerisation region(142-224)] [Kelch motif(200-213), Domain of unknown function (DUF4585)(252- PhAeBC5_1584.1 270), Galactose oxidase, central domain(279-310)] [Kelch motif(144- 13512 PhAeBC5_3740.1 157), Protein of unknown function (DUF1668)(222-244)] 14233 StHeBC3_16619.1 [Kelch motif(339-357)] 14675 PhAeBC5_12620.5 [Kelch motif(198-213)] 18354 PhAeBC5_5786.1 [Zinc-finger of C2H2 type(255-261), C2H2-type zinc finger(311-314)] 20190 PhAeBC5_2529.1 [DNA polymerase phi(10-104)] 23480 PhAeBC5_5395.1 [GAG-polyprotein viral zinc-finger(137-151)] 18774 StHeBC3_4423.1 [Protein of unknown function (DUF1618)(174-337)] 13656 StHeBC3_41710.1 NO HITS ShContig9483_gi29 19297 5389393_19297 NO HITS 20188 PhAeBC5_1574.1 NO HITS Genes from each orthogroup were annotated with a Pfam domain, with bracket separating the information for each gene. Domains for a gene are separated by comma, and each domain is followed by the corresponding region in the encoded protein sequence.