Supplementary Information
Ruling Out DNA Contamination or Mix-up
DNA contamination or misidentification/mix-up is always a serious concern in studies on
HGT, since one is looking for genes that are misplaced in molecular phylogenies or in
phylogenetic distribution, and misplaced DNA will create such effects. Indeed, certain published
claims for HGT in plants turn out to reflect contamination1,2. DNA contamination or mix-up can
be definitively ruled out in all fives cases of HGT reported in this study because the results are
entirely reproducible:

Sanguinaria rps11: We isolated (by direct sequencing of PCR products) the same,
chimeric rps11 gene, and only this gene, from DNA prepared from three different sources
of Sanguinaria material, with the third set of DNA preparations and PCR reactions
carried out in a different laboratory. As described in the main text, the near-identity of
Sanguinaria rps11 genomic and cDNA sequences, differing only by mitochondrialspecific RNA editing, further rules out artifact.

Amborella atp1: A eudicot-like atp1 gene of identical sequence was isolated by three
independent groups, each working from a different preparation of Amborella DNA (refs.
18 and 19 of main text and this study). Our unpublished data showing near-identity of
Amborella atp1 genomic and cDNA sequences, differing only by mitochondrial-specific
RNA editing, further rules out artifact.

Actinidia rps2: A monophyletic lineage of highly similar rps2 genes of monocot origin
was isolated from each of six species of Actinidia sampled.
1

Caprifoliaceae rps11: A monophyletic lineage of highly similar rps11 genes of
Ranunculalean origin was isolated from five genera of Caprifoliaceae (Fig. 2b of main
text).

Betulaceae rps11: A monophyletic lineage of highly similar rps11 genes of unresolved
phylogenetic position but likely horizontal origin (see main text) was isolated from three
genera of Betulaceae (Fig. 2b of main text).
Mitochondrial Provenance of Horizontally-acquired Plant Genes
Three forms of evidence lead us to conclude that in all five cases of HGT reported in this
study the horizontally-acquired gene is located in the mitochondrial genome of the recipient
group. First, in all cases, these genes show the very low divergence typical of genes located in
angiosperm mitochondrial genomes and which have never existed in the much higher mutational
environment of angiosperm nuclear genomes (see main text, refs. 14-16 of main text, and
Supplemental Figure 1). Note that, as discussed in part in the main text, this same logic leads
one to conclude that in all five cases the donor genome was the mitochondrial genome and not
the nuclear genome. Second, in four of the five cases (all but Amborella atp1), Southern
hybridization experiments (ref. 13 of main text) show that at least one member of the relevant lineage
of plants contains a relevant sequence of the hybridization intensity expected for a mitochondrial
gene, as assayed under highly controlled conditions [note that all DNAs were made from green
leaves, which always contain significantly lower levels of mitochondrial DNA per cell (typically
hundreds of copies) than chloroplast DNA (thousands of copies), but higher levels than nuclear DNA
(two copies)3]. Third, that genomic and cDNA sequences for both Sanguinaria rps11 and
Amborella atp1 differ only by mitochondrial-specific RNA editing firmly establishes that both of
2
these genes are located in the mitochondrial genome (expression of the other three genes was not
assayed).
In terms of where these horizontally acquired genes were inserted into the mitochondrial
genome, the chimeric rps11 gene of Sanguinaria obviously reflects homologous integration of
horizontally acquired, monocot rps11 sequences into the endogenous rps11 locus. We have no
relevant information for the other four transfers, but predict, based on the loose organization of
angiosperm mitochondrial genomes (consisting almost entirely of single gene islands floating in
a large sea of intergenic spacers) and that virtually all known cases of gene duplicates in plant
mitochondrial genomes are unlinked4,5, that these foreign sequences were randomly inserted into
the genome.
The Tip of an Iceberg of Mitochondrial HGT in Plants
The five cases of plant mitochondrial HGT reported here must be the tip of a large
iceberg of mitochondrial HGT in plants considering 1) the discovery of relatively many cases
from such limited sampling (3 genes and a total of about 120 sequences), 2) the likelihood that
many cases of HGT will be missed owing to the relatively poor resolution of plant mitochondrial
single-gene trees (this reflects the very low mutation rate in plant mitochondrial genomes (refs.
14 and 15 of main text), as exacerbated by the short length of genes such as rps2 and rps11; see
Fig. 2 of main text), 3) the potential for chimeric HGT to further muddy the phylogenetic waters,
and 4) the inadequacy of Southern hybridization (used to detect recapture HGT) and PCR (used
to isolate mitochondrial genes) to detect long-distance HGT events, e.g., from a fungus.
3
Parametric Bootstrapping
To test further if the cases of inferred horizontal transfer can be explained under a vertical
transmission scenario, we performed parametric bootstrapping6 using 100 simulated sequence
replicates. The simulated sequences were generated with Seq-Gen7 using an HKY85 substitution
model with a gamma distribution and a constrained maximum likelihood input tree from PAUP8.
The model parameters were first estimated from the original data with Tree-Puzzle9. The
simulated data matrices were analyzed by parsimony in PAUP. Parametric bootstrapping
rejected (p<0.01) the vertical transmission hypotheses that group 1) rps2 from Actinidia with
Grevillea/Platanus, 2) rps11 from Caprifoliaceae and Betulaceae with Trochodendrales, 3) rps11
from Caprifoliaceae with Trochodendrales (with Betulaceae excluded from the analysis), 4) the
3’ half of rps11 from Sanguinaria with other Papaveraeceae and 5) the two Amborella atp1
sequences together. Parametric bootstrapping did not, however, reject the hypothesis that the
Betulaceae and Trochodendrales rps11 sequences form a monophyletic group.
4
Supplemental References:
1. Hudson, A. Fungal cytochrome c genes from plants. J. Mol. Evol. 41, 1170-1171 (1995).
2. Doolittle, R. F. The case for gene transfers between very distantly related organisms, p. 311320. In M. Syvanen and C. I. Kado (ed.), Horizontal Gene Transfer. Chapman & Hall,
London, New York (1998).
3. Bendich, A. J. Why do chloroplasts and mitochondria contain so many copies of
their genome? Bioessays 6, 279-282 (1987).
4. Lonsdale, D. M. The plant mitochondrial genome. In Stumpf, P. K. & Conn, E. E. (eds.), The
Biochemistry of plants. Academic Press, San Diego, pp. 229-295 (1989).
5. Wolstenholme, D. R. & Fauron, C. M.-R. Mitochondrial genome organization. In Levings, C. S.,
III & Vasil, I. K. (eds.) The Molecular Biology of Plant Mitochondria, Kluwer, Netherlands pp.
1-59 (1995).
6. Huelsenbeck, J. P., Hillis, D. M., & Jones, R. Parametric bootstrapping in molecular
phylogenetics: Applications and performance. In Ferraris, J. D. & Palumbi, S. R. (eds.)
Molecular Zoology: Advances, strategies, and protocols, Wiley, New York pp 19-45 (1995).
7. Rambaut, A. & Grassly, N. C. Seq-Gen: An application for the Monte Carlo simulation of DNA
sequence evolution along phylogenetic trees. Comp. Appl. Biosci. 13, 235-238 (1997).
8. Strimmer, K. & von Haesler, A. Quartet puzzling: A quartet maximum likelihood method for
reconstructing tree topologies. Mol. Biol. Evol. 13, 964-969 (1996).
9. Swofford, D. L. PAUP*: Phylogenetic analysis using parsimony (*and other methods),
Sinauer Associates, Sunderland, Massachusetts (1988).
5
Table 1. PCR primers used in this study
Gene
rps2
rps2
rps2
rps2
rps2
rps2
rps2
rps11
rps11
rps11
rps11
rps11
rps11
rps11
rps11
rps11
rps11
rps11
atp1
atp1
atp1
atp1
atp1
atp1
Primer
INV.R
INV.F
rps2.f2
ub41
rps2.r3
rps2.r4
ub39
ub3
ub6
ub14
ub4
ub5
ub8
ub1
ub17
ub18
ub32
ub33
ub25
ub26
ub27
ub28
ub29
ub30
Sequence
ATAATAACTACACAATCTGGT
ATACCTATTGCATCTTCAGT
AAGACACTRATTTGTTTACGAA
ATGACAATCCWTTCTATDGT
AYGGGATAAGTKATTMKTTTAT
TCMAGAATSMCTGTTTTSRT
AACTGTATAGGATCATTC
GAGCGCGTAGAGCAACAAGT
ATGCCCCAGGAAAAAACAAC
ATGCCCCAGGAAAAAACGG
GGAAGTTGGGTCACATCGTGG
CTTTGGGAGRCGGCANCCATTATG
TCCGAGATGCTCTGTACGAAGTTCATG
CTTATTGTGGATCGGTGGTAAATG
CTAGCGCGCGTACTCTTCTTCTG
GTTATGACTCGATGACTAAG
CGAATCTACAGATCTMAA
AGAAGCGTTATGACTCGATGAC
TCGGTCGAGTGGTCTCAGTTG
GGAGATGGGATTGCACGTG
GAGAATGTAGGAAAAAAAG
TCGATACTTCTGTCAGCCTT
CAGCCTTGCACCTCTATTGA
AAGCCTAGCACCTCTATTTG
6
Orientation
R
F
F
F
R
R
R
F
F
F
R
R
R
R
F
F
F
F
F
F
F
R
R
R
Comment
Inverse PCR
Inverse PCR
For upstream analysis
For upstream analysis
For upstream analysis
For upstream analysis
For upstream analysis
For upstream analysis
HGT copy specific
HGT copy specific
HGT copy specific
Native copy specific
Supplemental Figure 1 Much greater divergence of nuclear than mitochondrial genes in plants.
Maximum likelihood trees of rps2 (a) and rps11 (b). Both scale bars correspond to 0.05 NT
substitutions per site. Trees contain the same set of mitochondrial sequences as in Figs. 2a and
2b of main text, respectively, with 5 or 8 nuclear sequences added to the analyses. In each case,
the nuclear sequences, all of which are from eudicots, form a monophyletic group, consistent
with the hypothesis that they result from a common mitochondrial-to-nucleus transfer event
occurring early in eudicot evolution (cf. timing of these genes’ loss from the mitochondrial
genome; see Fig. 1 of main text). However, in neither gene tree is the nuclear clade placed in the
position expected (marked with an asterisk) for a gene transfer event coincident in timing with
mitochondrial gene loss early in eudicot evolution. This is entirely unsurprisingly owing to the
highly divergent nature of the nuclear sequences relative to mitochondrial sequences and the
resulting potential for artifacts of long branch attraction. Note especially the position of the
rps11 nuclear clade: in the tree shown, it attaches to the long stem branch leading to monocots,
and under other analytical conditions it attaches to the long outgroup branch. The clade’s
alternative attachment, neither of which makes sense biologically, to either of the two longest
branches of mitochondrial sequences (also compare to Fig. 2b of main text) is almost certainly a
long-branch-attraction artifact.
Supplemental Figure 2 Nucleotide alignment used for the rps2 analysis shown in Fig. 2a of
main text.
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Supplemental Figure 3 Nucleotide alignment used for the rps11 analysis shown in Fig. 2b, 2d
and 2e of main text.
Supplemental Figure 4 Nucleotide alignment used for the atp1 analysis shown in Fig. 2f of
main text.
Supplemental Figure 5 Nucleotide alignment used for the rps11 upstream sequence analysis
shown in Fig. 2c of main text.
8