Mitochondrial DNA suggests at least 11 origins of parasitism in angiosperms and reveals genomic chimerism in parasitic plants

Abstract

Background: Some of the most difficult phylogenetic questions in evolutionary biology involve identification of the free-living relatives of parasitic organisms, particularly those of parasitic flowering plants. Consequently, the number of origins of parasitism and the phylogenetic distribution of the heterotrophic lifestyle among angiosperm lineages is unclear.

Results: Here we report the results of a phylogenetic analysis of 102 species of seed plants designed to infer the position of all haustorial parasitic angiosperm lineages using three mitochondrial genes: atp1, coxI, and matR. Overall, the mtDNA phylogeny agrees with independent studies in terms of non-parasitic plant relationships and reveals at least 11 independent origins of parasitism in angiosperms, eight of which consist entirely of holoparasitic species that lack photosynthetic ability. From these results, it can be inferred that modern-day parasites have disproportionately evolved in certain lineages and that the endoparasitic habit has arisen by convergence in four clades. In addition, reduced taxon, single gene analyses revealed multiple horizontal transfers of atp1 from host to parasite lineage, suggesting that parasites may be important vectors of horizontal gene transfer in angiosperms. Furthermore, in Pilostyles we show evidence for a recent host-to-parasite atp1 transfer based on a chimeric gene sequence that indicates multiple historical xenologous gene acquisitions have occurred in this endoparasite. Finally, the phylogenetic relationships inferred for parasites indicate that the origins of parasitism in angiosperms are strongly correlated with horizontal acquisitions of the invasive coxI group I intron.

Conclusion: Collectively, these results indicate that the parasitic lifestyle has arisen repeatedly in angiosperm evolutionary history and results in increasing parasite genomic chimerism over time.

Figure 1

Phylogenetic tree estimated from 3 combined mt genes indicates at least 11 origins of parasitism in angiosperm phylogeny. Single best tree estimated from ML analysis of combined atp1 + coxI + matR mtDNA sequence data (-lnL = 43115.38). ML bootstrap support/Bayesian posterior probability values (BP/PP) are shown above all nodes with values >50/0.8. Green branches are non-parasitic, orange branches are mostly hemiparasitic, and red branches are holoparasitic lineages. A star next to a taxon represents the presence of the coxI intron in the sampled species. A filled circle next to an order or family represents the presence of the mitochondrial coxI intron based on literature reports [28].

Figure 2

Estimated branch lengths for combined 3 mt gene phylogenetic analysis. Single best tree from Fig. 1 with branch lengths estimated from ML analysis of combined atp1 + coxI + matR mtDNA sequence data (-lnL = 43115.38). Green branches are non-parasitic, orange branches are mostly hemiparasitic, and red branches are holoparasitic lineages. Some parasites have long estimated branch lengths for these 3 combined genes; however, even some non-parasites, like Scaevola, have long branch lengths as well. ML bootstrap support values (BP) are shown above all nodes with values >50.

Figure 3

Single gene analyses reveal potential cases of horizontal gene transfer of atp1 from host to parasite. Comparison of phylogenetic relationships, gene-specific branch lengths (drawn proportionally) and corrected pairwise divergences (K) for representatives of all rosid and asterid orders for the three mt genes, atp1, matR, and coxI. Endoparasites and their host lineages are shown in matching colors. BP/PP values are shown above all nodes with values >50/0.8. A. Single most likely tree from atp1-only analysis (-lnL = 5136.56). B. Single most likely tree from matR-only analysis (-lnL = 8443.48). C. Single most likely tree from coxI-only analysis (-lnL = 4426.50). D-F. Average pairwise divergences for the endoparasite taxa relative to all non-parasites in the atp1, matR, and coxI datasets, respectively. Calculations of pairwise divergences shown in D-F, were made by comparing each single endoparasite to all non-parasites and all non-parasites to each other.

Figure 4

Comparison of atp1 cDNA and DNA sequences in Rafflesia cantleyi. A. Agarose gel showing 1: RT-PCR results obtained using an initial reverse transcription step during thermalcycling, 2: RT-PCR results obtained without an initial reverse transcription step during thermalcycling, 3: RT-PCR results obtained without adding RNA to reaction but using an initial reverse transcription step during thermalcycling, and 4: 1 kb ladder. Results for lane 1 as compared to lane 2 indicate that cDNA was amplified from Rafflesia RNA. B. Comparisons of Rafflesia atp1 DNA and cDNA sequences. Position 931 appears to be RNA edited because a T was determined to be encoded in the cDNA while a C is encoded in the DNA.

Figure 5

Chimeric nature of atp1 in Pilostyles thurberi. A. Spatial delimitation of regions I and II of atp1 based on gene conversion analyses of sequence variation in Pilostyles and various Fabalean taxa. A region of 804 bp in Pilostyles (AZ) (region II) was inferred to be the result of gene conversion by an atp1 sequence from its host, Psorothamnus (P < 0.05). B. Single most likely tree obtained in phylogenetic analyses of regions I (-lnL = 883.42). C. Single most likely tree obtained in phylogenetic analyses of regions II (-lnL = 1383.82). Filled bar indicates caesalpinioid legumes and unfilled bar indicates faboid legumes. BP/PP values are shown above all nodes with values >50/0.8.

Figure 6

Ordinal level analysis of coxI intron presence among angiosperms. Character state tracing of coxI intron presence (shown in blue) among 45 orders of angiosperms. Numbers listed next to intron-negative orders show the number of families sampled for the coxI intron out of the total currently included within each order. Parasitism is inferred to have evolved 10 times on branches that also are inferred to have the intron (shown by numbers within circles). Only Krameriaceae and Cynomoriaceae do not appear to be associated with intron containing lineages. The probability of 10 origins of parasitism and zero losses of parasitism on branches that have the intron is < 0.001.

Figure 7

coxI intron phylogeny in flowering plants. Phylogenetic relationships among 49 angiosperm coxI intron DNA sequences inferred using ML (-lnL = 5938.60). Lineages shown in green are non-parasitic. Lineages in orange are mostly hemiparasitic, while those in red are holoparasitic. Bootstrap support values >50 and posterior probabilities > 0.8 are shown before and after the "/" respectively. Ordinal (or familial, if currently unplaced to order) classification is shown next to each taxon. Basal angiosperms and monocot orders are labeled in blue, rosid orders are labeled in purple, asterid orders are labeled in black, and Santalales are shown in yellow. General host plant preference for each parasitic plant lineage is shown next to each parasite (based on ref. # 3 and personal observations by TJB, JRM, and CWD). Overall phylogenetic relationships of the intron sequences are highly discordant with angiosperm phylogeny. Horizontal acquisition of the intron in parasites from their hosts does not seem likely because in no case is a highly supported relationship found between a parasite and any of its host lineages. Vertical acquisition of the intron in Cuscuta and Epifagus is supported by this tree because of the highly supported relationship found between these parasites and their close relatives.