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Abstract
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
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
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
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
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
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
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
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
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,
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
Table 1. Taxa and corresponding genomic regions included in the study with their
Rafflesiaceae Rafflesia contig contig contig 4358 contig 19544 contig 975
(rbcL1),
contig
170975
(rbcL2)
Rafflesiaceae Rafflesia contig contig 78914 contig contig 283 contig 1054
philippensis
SRX209107 SRX209107
(rbcL1,
rbcL2*)
(non-host)
(R. leonardi
host)
(R.
philippensis
host)
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
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,
& 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
[26]. Purified PCR products were submitted to GENEWIZ, INC. for sequencing.
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
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
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
PhyML with 100 bootstrap replicates (BS) and aLRT SH-like branch support applying the GTR
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
(139.58.5 myr for eudicots+monocots [37]; 105.57 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:
6515 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
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
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].
In all genealogies (Fig 1), relationships among members of Vitaceae and among
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.
Rafflesia and/or Sapria embedded within Vitaceae with strong support. Sapria sequences include
sequences assembled from Genbank SRA SRX209107 (labeled), Genbank AY674768 (labeled),
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
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
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
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.
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.
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
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
Table 2. Bayes Factors (BF) of marginal likelihood estimates (MLE) obtained from
monophyletic
monophyletic
monophyletic
BF>3 indicates strong rejection of model with more negative MLE (Ronquist et al. 2011; McVay
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
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.
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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