Phylogenetic inference in Rafflesiales: the influence of rate heterogeneity and horizontal gene transfer

Abstract

Background: The phylogenetic relationships among the holoparasites of Rafflesiales have remained enigmatic for over a century. Recent molecular phylogenetic studies using the mitochondrial matR gene placed Rafflesia, Rhizanthes and Sapria (Rafflesiaceae s. str.) in the angiosperm order Malpighiales and Mitrastema (Mitrastemonaceae) in Ericales. These phylogenetic studies did not, however, sample two additional groups traditionally classified within Rafflesiales (Apodantheaceae and Cytinaceae). Here we provide molecular phylogenetic evidence using DNA sequence data from mitochondrial and nuclear genes for representatives of all genera in Rafflesiales.

Results: Our analyses indicate that the phylogenetic affinities of the large-flowered clade and Mitrastema, ascertained using mitochondrial matR, are congruent with results from nuclear SSU rDNA when these data are analyzed using maximum likelihood and Bayesian methods. The relationship of Cytinaceae to Malvales was recovered in all analyses. Relationships between Apodanthaceae and photosynthetic angiosperms varied depending upon the data partition: Malvales (3-gene), Cucurbitales (matR) or Fabales (atp1). The latter incongruencies suggest that horizontal gene transfer (HGT) may be affecting the mitochondrial gene topologies. The lack of association between Mitrastema and Ericales using atp1 is suggestive of HGT, but greater sampling within eudicots is needed to test this hypothesis further.

Conclusions: Rafflesiales are not monophyletic but composed of three or four independent lineages (families): Rafflesiaceae, Mitrastemonaceae, Apodanthaceae and Cytinaceae. Long-branch attraction appears to be misleading parsimony analyses of nuclear small-subunit rDNA data, but model-based methods (maximum likelihood and Bayesian analyses) recover a topology that is congruent with the mitochondrial matR gene tree, thus providing compelling evidence for organismal relationships. Horizontal gene transfer appears to be influencing only some taxa and some mitochondrial genes, thus indicating that the process is acting at the single gene (not whole genome) level.

Figure 1

ML strict consensus tree from mitochondrial matR. Strict consensus of two trees obtained from ML analysis of the 77-taxon mitochondrial matR matrix. Clades with Bayesian posterior probabilities between 0.9 and 1.0 are indicated by thick lines. Bootstrap percentages from MP analysis shown above lines. Rafflesiales taxa are shown in bold italics. Arrow represents a putative cases of horizontal gene transfer. The small phylogram is included to demonstrate branch length heterogeneity.

Figure 2

ML tree from mitochondrial atp1. Phylogram obtained from ML analysis of the 71-taxon mitochondrial atp1 matrix. Clades with Bayesian posterior probabilities between 0.9 and 1.0 are indicated by thick lines. Rafflesiales taxa are shown in bold italics. Note that the clade with Apodanthes and Polemonium (asterisk) is poorly supported with a posterior probability of 0.54.

Figure 3

Unconstrained MP tree from the 3-gene data matrix. Strict consensus of 12 trees obtained from an unconstrained maximum parsimony analysis of the 77-taxon "3-gene" matrix (nuclear SSU rDNA, rbcL, atpB). Bootstrap support is shown above the lines. Rafflesiales taxa are shown in bold italics.

Figure 4

Constrained ML tree from nuclear SSU rDNA. Tree resulting from the constrained ML analysis of the 77-taxon nuclear SSU rDNA matrix. Rafflesiales taxa are shown in bold italics.

Figure 5

Rafflesiales long branches mislead MP. Proportion of simulated data sets (replicates) for which incorrect "long-branch" clades are recovered in maximum parsimony (black bars, 77 taxa), maximum parsimony (grey bars, 20 taxa), and maximum likelihood (open bars, 20 taxa) analyses. Inset is the model tree used to generate the simulated data sets. M = Mitrastema, B = Bdallophyton + Cytinus, R = Rafflesia + Rhizanthes + Sapria, P = Pilostyles.