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Origin of parasitism in Rafflesiaceae: insights from phylogenetic analyses of horizontally

transferred genes from its host family, Vitaceae

Jeanmaire Molina,* and Sharday Weaver

Department of Biology, Long Island University-Brooklyn, 1 University Plaza, Brooklyn, NY,

USA 11201

*corresponding author: jeanmaire.molina@liu.edu


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Abstract

Rafflesiaceae is an endoparasitic plant family that characteristically produces the largest

single flowers in the world. All three extant member genera (Sapria, Rafflesia, Rhizanthes) of the

family parasitize the genus Tetrastigma (Vitaceae). The parasitic nature of their relationship has

facilitated multiple genes to become horizontally transferred (HT) from the host plant to the

parasite. We aimed to understand the evolution of parasitism by reconstructing phylogenies

using genes that have been shown to be horizontally transferred between host and parasite

families but vertically transferred within Vitaceae and in other angiosperms. These HT genes

may give a minimum time approximation as to when parasitism may have evolved. We aimed

for markers that showed similar histories of HT among Rafflesia species but showed expected

phylogenetic relationships for non-Rafflesiaceae taxa. Five HT genes (rbcL, matK, rrn16, rrn23,

nad1) were analyzed phylogenetically using maximum likelihood and depicted congruent

topologies, except for rbcL, and were combined. The rbcL topology suggested ancient HT

predating Vitaceae or a plastid-like nuclear copy of rbcL (nupt) acquired by Raffesiaceae.

However, concatenation of the other 4 genes produced a phylogeny for non-Rafflesiaceae taxa

consistent with expected groupings. Placement of Sapria and Rafflesia in this combined

phylogeny may be taken as to when parasitism with Vitaceae originated. Dating analyses in

BEAST showed that Rafflesia and Sapria are not monophyletic but both are included within a

clade of Cayratia, Cyphostemma and Tetrastigma, estimating horizontal transfer as early as the

Eocene (53 myr), much later than the origin of Rafflesiaceae, which originated in late Cretaceous

(82 myr). Topology testing using Bayes Factor, however, did not support independent origins of

HT in Rafflesia and Sapria, at least for the genes sampled. Our study has shown that events of
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horizontal gene transfer analyzed within the context of Vitaceae phylogeny help elucidate the

evolutionary origin of parasitism in Rafflesiaceae.

Keywords: holoparasitism, Tetrastigma

Introduction

Throughout the world there are over 3,000 known species of parasitic plants [1], with

parasitism being shown to evolve independently at least 12 times [2]. Parasitic plants have

evolved from entirely autotrophic means of sustainment towards varying levels of dependence on

host plants [3]. Some parasitic plants depend entirely upon their host for both water and nutrients

establishing a holoparasitic relationship [4], while other parasitic plants maintain variable

degrees of photosynthetic activity and are classified as hemiparasites [3]. The connection

between the host and parasite is maintained by a strong graft union facilitated by haustoria

originating from the parasitic plant [5]. In plants, the haustorium is an absorptive structure with

multicellular organs and multiple cell types responsible for siphoning nutrients from the host

plant [3, 6].

The holoparasitic family Rafflesiaceae is known for producing the largest single flowers

in the world, up to 3 feet in some species. The family, which contains three genera: Sapria,

Rhizanthes, Rafflesia, is suggested to have originated in the Late Cretaceous based on molecular

dating (82 myr) [4]. Endemic to tropical Southeast Asia, these endoparasitic plants have giant,

transient flowers and have no vegetative parts and chlorophyll. They grow as strands of cells

embedded within the stem and root tissues of their sole host, the plant genus Tetrastigma [7].

Tetrastigma belongs to the grape family, Vitaceae, and its 95 species are distributed across the
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Asian tropics and subtropics extending into Australia. Tetrastigma has been dated to have

originated in the Eocene of Southeast Asia (~47.6 mya)[8]. Fourteen different species of

Tetrastigma have been reported as hosts of Rafflesiaceae [9].

Parasitic plant systems have provided strong evidence of horizontal gene transfers

(HGT), which is facilitated by the close physical relationship between host and parasite

[2,10,11]. Exchanged genes between remotely related organisms lead to mosaic genomes and a

gene phylogeny that might differ significantly from the organismal phylogeny [12]. A large

number of horizontal gene transfers between members of Rafflesiaceae and Vitaceae, involve

nuclear, plastid and mitochondrial genes and have been documented numerous times [13-16].

Due to its narrow host specialization, members of Rafflesiaceae provide a suitable model for the

study of the evolutionary origin of holoparasitism.

In the present study, we used DNA sequence data from mitochondrial and plastid markers

that have shown phylogenetic evidence of horizontal gene transfer between Rafflesiaceae and

Vitaceae [15,16], but vertically transferred within Vitaceae and angiosperm taxa outside

Rafflesiaceae. This provides the evolutionary context to understand the temporal and geographic

origin of the parasitic relationship or association between these two families. Though we do not

know the nature of the original symbiosis--parasitic, mutualistic or commensal [17], we assume

here that it started parasitic, as we do not have any other evidence otherwise. However, any of

these intimate associations may facilitate HGT, as in the case of Amborella, where HGT from

commensal epiphytes has been shown to occur [18]. Though HGT events can happen at

different times, molecular dating of these events can provide the minimum age for the

evolutionary origin of symbiotic relationships. The legume parasite Phelipnache , for example,
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was estiamted to had acquired a gene only known in legumes, about 16 mya, evolving new

functions in the parasite[19].

Given that the extant host of Rafflesiaceae is solely the genus Tetrastigma, it could be

expected that origin of parasitism and the onset of the oldest HGT should have occurred near the

time of the origin of the host genus. In this study we aim to test this by analyzing horizontally

transferred genes between Rafflesiaceae and Vitaceae within a calibrated phylogeny of vertically

transferred genes. This allows us to understand the biogeographical scenario that may have

facilitated the evolution of this unique host-parasite relationship.

Materials and Methods

Taxon sampling

A total of 23 taxa were sampled (see Table 1), including representative genera of

Rafflesiaceae (Rafflesia, Sapria), Vitaceae, and other angiosperms. Genera of Vitaceae were

selected to establish a “backbone” phylogeny for the host family (see Table 1) including genera

from the major clades within Vitaceae: Ampelopsis, Cayratia, Cissus, Cyphostemma, Rhoicissus,

Tetrastigma, Vitis [8]. Leea, sister to Vitaceae, was also included, and together they make up the

order Vitales. Additional sequences for homologous regions from rosid taxa (Arabidopsis,

Carica, Cucumis, Glycine, Phoenix, Populus, Ricinus, Zea) representative of different

angiosperm orders outside Vitales and Rafflesiaceae (see Table 1) were obtained from GenBank

[20] database using blastn [21] to serve as “control taxa”. Placement of these control rosid taxa

and backbone Vitaceae taxa in the resulting phylogenies should correspond with the expected
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relationships determined by the Angiosperm Phylogeny Group IV (APG IV) [22, 23],

respectively. The control taxa will provide support for the placement of Vitaceae within the

overall topology of APG IV, while the backbone taxa establish placement of Rafflesiaceae

within the host family.

Table 1. Taxa and corresponding genomic regions included in the study with their

GenBank accession numbers for sequences downloaded from GenBank.

Family Taxon matK rbcL rrn16 rrn23 nad1

Arecaceae Phoenix GU811709 GU811709 GU811709 GU811709 JN375330

Brassicaceae Arabidopsis AP000423 AP000423 AP000423 AP000423 JF729201

Caricaceae Carica EU431223 EU431223 EU431223 EU431223 EU431224

Cucurbitaceae Cucumis KF957866 KF957866 KF957866 KF957866 FJ007641

Euphorbiaceae Ricinus JF937588 JF937588 JF937588 JF937588 HQ874649

Fabaceae Glycine DQ317523 DQ317523 DQ317523 DQ317523 JX463295

Malvaceae Gossypium HQ325742 HQ325742 HQ325742 HQ325742 JX944505

Poaceae Zea KF241981 KF241981 KF241981 KF241981 DQ490951


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Rafflesiaceae Rafflesia contig contig contig 4358 contig 19544 contig 975

lagascae [24] 710409 127053

(rbcL1),

contig

170975

(rbcL2)

Rafflesiaceae Rafflesia contig contig 78914 contig contig 283 contig 1054

leonardi 224481 (rbcL2, 193797 (unpubl.) (unpubl.)

(unpubl.) unpubl.) (unpubl.)

Rafflesiaceae Rafflesia none X (rbcL2) X none X

philippensis

Rafflesiaceae Sapria SRX209107* X (rbcL1); SRX209107 X; AY674768

SRX209107 SRX209107

(rbcL1,

rbcL2*)

Salicaceae Populus KJ664927 KJ664927 KJ664927 KJ664927 AY674757

Vitaceae Ampelopsis KP088956 AJ419722 none X none

Vitaceae Cayratia HG004950 X X X none


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Vitaceae Cissus JX517840 X X X X

Vitaceae Cyphostemma KR735040 AJ419719 X X none

Vitaceae Leea AM396497 FJ976143 HQ664562 HQ664562 AY674724

Vitaceae Rhoicissus JX518018 AJ419724 X none X

Vitaceae Tetrastigma HG004976 AJ419717 X HQ664626 AY674775

(non-host)

Vitaceae Tetrastigma none X X X X

(R. leonardi

host)

Vitaceae Tetrastigma none none none X X

(R.

philippensis

host)

Vitaceae Vitis DQ424856 DQ424856 DQ424856 DQ424856 GQ220325

Cells marked “X” correspond to PCR sequences generated in the present study. Some sequences

for Rafflesia were obtained from previously assembled contigs [16] and unpublished data for R.

leonardi. Some sequences for Sapria were obtained from reassembly of Illumina reads published

in Genbank (SRA SRX209711), and are indicated as such. Non-overlapping fragments for rbcL

(rbcL1 and rbcL2) were obtained from Rafflesia and Sapria. Poor-quality sequences (<150 bp
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with many ambiguous bases) for matK and rbcL2 were assembled from Sapria SRX209107 and

were excluded in the analyses (*).

DNA extraction, amplification, and sequencing

Genomic DNA for Rafflesia and Sapria was provided by [16]. Leaf material for

Cyphostemma was provided by the US Botanic Garden, leaf material for Cayratia was obtained

from Betsy Jackes (James Cook University, Australia), and leaf material for Cissus, Rhoicissus,

and Tetrastigma (non-host) was provided by University of Connecticut’s Biodiversity Education

& Research Greenhouses. In addition, leaf material for Rafflesia-infected Tetrastigma, host of R.

philippensis [24], was collected by JM, while host material for R. leonardi [25] was kindly

provided by Pieter Pelser (University of Canterbury, New Zealand). Inclusion of host and non-

host Tetrastigma allows determination of whether the genes were transferred horizontally in real-

time or were ancient transfers. We were not able to obtain plant material for Rhizanthes, which is

Rafflesia’s evolutionary sister [4], but the more basal Sapria, included here, should allow us to

determine horizontal transfer (HT) events that occurred earlier prior to Rafflesia’s.

DNA extraction was performed using Qiagen DNeasy Plant Mini Kit (cat# 69104). PCR

primer sequences were designed by JM (available upon request) for the mitochondrial (mt) gene

region nad1 and plastid (cp) gene regions rbcL (composed of non-overlapping fragments rbcL1,

rbcL2), rrn16, rrn23, and matK, which were demonstrated to show phylogenetic evidence of

horizontal transfer between Rafflesiaceae and Vitaceae [15, 16]. However, these are probably

plastid DNA insertions into the nucleus of Rafflesia (nupt), [16]. We aimed for markers that

showed similar histories of HT among Rafflesia species but showed expected phylogenetic
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relationships based on APGIV [22] for non-Rafflesiaceae taxa. Polymerase chain reaction (PCR)

amplification reactions for all genes followed the protocol in [26], but different PCR profiles for

the different genomic regions were implemented: nad1 followed the PCR program in [27], while

matK, rbcL1, rbcL2, rrn16 and rrn23 were run using PCR conditions in [28]. Agarose gel

electrophoresis of PCR products and enzymatic purification of amplicons were performed as in

[26]. Purified PCR products were submitted to GENEWIZ, INC. for sequencing.

Sequence alignment and phylogenetic analyses

Sequence contig assembly and editing of sequenced PCR products were performed using

Geneious version (R7) (http://www.geneious.com) [29]. Sequences generated from this study

have been deposited to Genbank (XXXXX). Other sequences were obtained from previously

assembled contigs [16], (see Table 1) and from GenBank as described in “Taxon Sampling”. We

also assembled the plastid genome of Sapria himalayana from published Illumina data (Genbank

SRA SRX209107) in Geneious R7 [29] using suggested parameters, with Vitis vinifera complete

chloroplast genome (Genbank accession DQ424856) as reference to obtain Sapria plastid

sequences for rrn16, rrn23, matK in addition to the PCR-amplified sequences.

Sequence alignment for each gene (nad1, rbcL1, rbcL2, matK, rrn16, rrn23) was

conducted in MAFFT [30] using default parameters. Each gene was first analyzed

phylogenetically using PhyML [31] with 100 bootstrap replicates (BS) and aLRT (approximate

likelihood ratio test) Shimodaira-Hasegawa-like (SH-like) branch support applying the general

time reversible (GTR) DNA model [32]. SH-like aLRT has been shown to outperform BS when
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applied to polytomies [33] and is less prone to false positives [34]. SH-like support greater than

0.9 is considered significant [35].

Sequences were concatenated if the individual gene phylogenies showed topological

congruence and agreed with expected genus-level phylogenetic relationships [23, 22], which was

the case for matK, rrn16, rrn23 and nad1. rbcL1 and rbcL2 showed a different placement for

Rafflesia and Sapria (i.e. “sister” to Vitaceae instead of being embedded in it, which was

observed for the other HT markers), and were not concatenated with the other genes. However,

rbcl1 and rbcl2 were concatenated except for the Rafflesiaceae taxa. Genealogies for

concatenated alignments (rbcL1+rbcL2, matK+rrn16+rrn23+nad1) were also generated in

PhyML with 100 bootstrap replicates (BS) and aLRT SH-like branch support applying the GTR

DNA model [32].

Dating of horizontal transfer

To estimate the time of horizontal transfer, matk, rrn16, rrn23 and nad1 were analyzed in

BEAST v1.8.0 [36] where the plastid regions (matK, rrn16, rrn23) were conatenated and

allowed its own set of parameters (unlinked substitution and clock models) independent of the mt

nad1. The GTR+G substitution model, empirical base frequencies, lognormal relaxed clock

model, and a Yule tree prior were selected in the BEAST analysis. The tree model root height

was set to normal distribution, with nodes variously calibrated based on literature values

(139.58.5 myr for eudicots+monocots [37]; 105.57 myr for Vitales+rosids [38]). All members

of Vitales and Rafflesiaceae were forced to belong to one clade based on the results of the

maximum likelihood analyses with the node of this clade calibrated with normal distribution:

6515 myr to span 35.6-94.4 myr, the latter as maximum age of Vitaceae based on calibration by
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[39], assuming the horizontal transfer cannot be older than the host family. The node for

Tetrastigma was calibrated to span 36.4-59.4 myr based on [8] estimation of the Tetrastigma

crown group. Markov Chain Monte Carlo (MCMC) was ran for 20 million generations sampling

every 2000 generations. The log file was inspected in Tracer v1. 5 [40] to check if the effective

sampling size (ESS) for relevant parameters was greater than 200. TreeAnnotator v1. 8.1

(included in the BEAST package) was used to generate the maximum clade credibility tree from

the BEAST tree files after removing 25% of the trees as burnin. FigTree v1. 4. 2 [41] was used to

visualize the maximum clade credibility tree.

Topology testing for the single origin of horizontal transfer in Rafflesiaceae

Since the different genealogies showed Rafflesia and Sapria not monophyletic, albeit

with weak support, we decided to test the hypothesis of single-origin of horizontal transfer

(positive constraint), occurring in the Rafflesiaceae common ancestor against the hypothesis of

independent HT origins (negative constraint), using the concatenated matk+rrn16+rrn23+nad1

data. We constrained Rafflesia spp. and Sapria to be monophyletic, and performed a stepping

stone run of 1,000,000 generations (using the default of 50 steps) to obtain the marginal

likelihood estimate (MLE), that was then compared with the MLE produced from the analysis of

the negative constraint. A difference of at least 3 log likelihood units between the two models

(i.e. Bayes Factor) implies that model with higher MLE is significantly better [42,43].

Results and Discussion


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Phylogenies of horizontally transferred genes

In all genealogies (Fig 1), relationships among members of Vitaceae and among

angiosperms outside Rafflesiaceae conformed generally with phylogenetic expectations [23,22],

suggesting that the markers used in this study were vertically transferred among the non-

Rafflesiaceae taxa, as desired, to preclude effects of incomplete lineage sorting and other

homoplasious events.

Fig 1. Phylogenies/genealogies generated with maximum likelihood. All markers show

Rafflesia and/or Sapria embedded within Vitaceae with strong support. Sapria sequences include

sequences assembled from Genbank SRA SRX209107 (labeled), Genbank AY674768 (labeled),

and PCR-generated sequences (not indicated). Rafflesia sequences include PCR-generated

sequences and previously assembled sequences from [16]. Tetrastigma sequences include

Genbank sequences and PCR-generated sequences (hosts of R. phil=R. philippensis and R. leon=

R. leonardi). Branches in bold represent SH-like support >90% and bootstrap support >50%. A.

rbcL (rbcL1, rbcL2; Sapria only has rbcL1), B. matK, C. rrn16, D. rrn23, E. nad1, F. phylogeny

from concatenated matk-rrn16-rrn23-nad1 alignment.

Rafflesia and Sapria were found strongly associated with Vitaceae suggesting HT

between host and parasite, but did not form a monophyletic group, except in rbcL (Fig 1A;

alignment 884 bp long). All three species of Rafflesia included here, however, were always

monophyletic, implying that the markers used represent more ancient horizontal transfers that

occurred prior to the speciation of these Rafflesia species. matK (Fig 1B; 406 bp), rrn16 (Fig 1C;
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1488 bp), rrn23 (Fig 1D; 538 bp), and nad1 (Fig 1E; 1145 bp), presented Rafflesia and Sapria,

interspersed within Vitaceae and are non-monophyletic, but their topologies were not strongly

discordant with each other.

Interestingly in the rbcL genealogy (Fig 1A), Rafflesia and Sapria were monophyletic

and “sister” to Vitaceae. This may reflect that the physical relationship between Rafflesiaceae

and Vitaceae evolved once and early prior to the diversification of both families, and that the

other HT events occured later and independently in Rafflesia and Sapria. It is also possible that

the rbcL copies in Rafflesia and Sapria represent nuclear plastid-like sequences (nupt), and that

this nupt was acquired early in the common ancestor of Rafflesiaceae.

Excluding rbcL, as its position for Rafflesia and Sapria strongly contradict the other

genes, we combined matk, rrn16, rrn23 and nad1 (for a total of 3577 bp), resulting in the

combined phylogeny of Fig 1F and S1 Fig. In S1 Fig, only the genes for non-Rafflesiaceae taxa

were concatenated, and those for Rafflesia and Sapria were not, so as to accurately determine

their placement within the combined phylogeny of vertically transfered genes. Rafflesia and

Sapria appeared in different positions, but there was no strong discordance, as would be

indicated by high BS (>50%) and SH-like (>90%) support for conflicting clades, motivating us

to further concatenate the different genes within accessions of Rafflesia and Sapria, resulting in

the combined phylogeny in Fig 1F. The concatenation of sequence data is still an “important

part of the phylogeneticist’s toolbox” because recent studies have shown concatenated data to be

robust with the effects of sampling error, homoplasy, and missing data [44]. The combined

phylogeny (Fig 1F) still reflected the expected groupings among the non-Rafflesiaceae taxa,

which suggests that concatenation of weakly discordant gene trees did not affect the topology of

the underlying species tree. Though Rafflesia and Sapria are not monophyletic, they are both
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found strongly embedded within the CCT clade (Cyphostemma, Cayratia, Tetrastigma; >50%

BS and >90% SH in Fig 1F). The placement of Sapria and Rafflesia in this topology may be

used to estimate as to when they evolved physical association with the grape family.

Single origin of parasitism sometime in the Eocene?

We estimated timing of events in BEAST using the concatenated data of the 4 genes.

Sapria and Rafflesia were nested inside CCT, with strong support (100% pp, Fig 2) which

suggests that the physical relationship between Vitaceae and Rafflesiaceae may have evolved

sometime during or after the evolution of the CCT common ancestor, which according to our

results, goes back to early Eocene (53.0 myr, 39.5-66.2 myr 95% HPD), coincidentally during

the Global Thermal Maximum, when megathermal closed canopy forests were widespread [45].

This date is younger than the estimated age of Rafflesiaceae itself (82 myr) [4], and may suggest

that parasitism in Rafflesiaceae, as estimated by HT genes included here, could have evolved

much later.

Fig 2. Calibrated phylogeny from BEAST analysis of concatenated matk-rrn16-rrn23-nad1

alignment. Solid circles represent calibration points (see Materials and Methods). Rafflesia and

Sapria are not monophyletic but both are nested within the Cyphostemma-Cayratia-Tetrastigma

(CCT) clade (100% pp), which originated in Early Eocene (53.0 myr).

However, Rafflesia and Sapria were not monophyletic within CCT, which may suggest

independent origins of HT events (and/or parasitism). Evidence for independent origins of


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parasitism among Rafflesia species, offers conceivable support that parasitism independently

originated in Sapria and in Rafflesia [9]. However, topology testing yielded a BF value (4.51,

see Table 2) strongly rejecting the hypothesis of independent origin of HT in Rafflesia and

Sapria (M2) vs. the single-origin model (M1) suggesting that HT of the concerned genes may

have occurred only once in the common ancestor of Rafflesia and Sapria sometime in the last 50

myr. Yet, if Rafflesia and Sapria acquired the genes from Tetrastigma itself, which can be

implemented by enforcing monophyly of Tetrastigma with Rafflesia and Sapria (M3), is another

question. M3 resulted in an MLE (and BF) 2.41 log likelihood units higher compared to M1,

below the suggested BF=3 for significant rejection of opposing model, but possibly suggests that

Tetrastigma itself potentially could have been the source of the HT genes. The Eocene date that

we obtained for the HT also overlaps with Tetrastigma’s evolutionary origin (~47.6 mya) [8],

which may imply that evolution of parasitism in Rafflesiaceae probably originated close to the

time of origin of Tetrastigma. Nonetheless, this single origin may not hold for other HT genes

we did not analyze here, as they could have been horizontally transferred at different times

independently among members of Rafflesiaceae.

Table 2. Bayes Factors (BF) of marginal likelihood estimates (MLE) obtained from

topology testing in MrBayes.

Constraints applied MLE

M1: Rafflesia+Sapria monophyletic -13586.79

M2: Raff+Sapria not monophyletic -13591.3


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BF 4.51 (M2 strongly rejected)

M3: Rafflesia+Sapria+Tetrastigma -13584.38

monophyletic

M4: Rafflesia+Sapria+Tetrastigma not -13591.97

monophyletic

BF 7.59 (M4 strongly rejected)

M1: Rafflesia+Sapria monophyletic -13586.79

M3: Rafflesia+Sapria+Tetrastigma -13584.38

monophyletic

BF 2.41 (M3 rejected)

BF>3 indicates strong rejection of model with more negative MLE (Ronquist et al. 2011; McVay

and Carstens 2013).

As our result did not conclusively identify Tetrastigma as the source of HT genes, it is

also possible that Sapria and/or Rafflesia may have experienced host switching throughout their

evolution. Other members of Vitaceae, perhaps now extinct, may have served as these

holoparasites’ hosts prior to Tetrastigma, as Sapria is reported to parasitize not only Tetrastigma,

but Vitis as well [46]. Thus, it could be that host preference in Rafflesiaceae is a labile trait that

contributes to an increase in species survival over time in the event of host extinction [47].

Multiple origins of parasitism, independent HT events, and host switching may have all

been at play during the evolution of Rafflesiaceae. Only after detailed investigation of the
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ecological and molecular genetics of the parasitic association that we can fully appreciate the

processes that led to the evolution of these large-flowered holoparasites. It remains to be seen

how the results of the present study will change upon inclusion of other HT genes, as well as

more accessions of Vitaceae and Rafflesiaceae. Similar studies in other plant parasite systems

may help decipher their ancestral origins and shed light on the evolutionary history of parasitic

relationships.

Acknowledgments

We thank Julie Barcelona, Daniel Nickrent, and Pieter Pelser for helping JM collect

Rafflesia. We are grateful to the US Botanic Garden, University of Connecticut Research

Greenhouse, Pieter Pelser, Daniel Nickrent, Betsy Jackes for providing plant materials for DNA

extraction. Craig Manbauman and Claire-Iphanise Michel provided initial technical support. We

are also indebted to Joseph Morin, Jonathan Flowers, Michael Purugganan, Giselle Concepcion,

José Tello, Carole Griffiths, Danielle Dirienzo, and SW’s family for various forms of support.

Some sequencing was funded by the Professional Development Fund JM received from LIU.

This paper is dedicated to the memory of Leonardo Co.

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Supporting Information

S1 Fig. Phylogeny from concatenated matk-rrn16-rrn23-nad1 alignment. Only the genes for

non-Rafflesiaceae taxa were concatenated, and those for Rafflesia and Sapria were not. Rafflesia

and Sapria appeared in different positions, but there was no strong discordance, as would be

indicated by high BS (>50%) and SH-like (>90%) support for conflicting clades. This motivated

us to further concatenate the different genes within accessions of Rafflesia and Sapria, resulting

in the combined phylogeny in Fig 1f.


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