Annals of Botany 118: 885–896, 2016 doi:10.1093/aob/mcw135, available online at www.aob.oxfordjournals.org Understanding the evolution of holoparasitic plants: the complete plastid genome of the holoparasite Cytinus hypocistis (Cytinaceae) Cristina Roquet1,2,*, Eric Coissac1,2, Corinne Cruaud3, Martı Boleda1,2, Fre´de´ric Boyer1,2, Adriana Alberti3, Ludovic Gielly1,2, Pierre Taberlet1,2, Wilfried Thuiller1,2,Je´re´mie Van Es4 and Se´bastien Lavergne1,2 1Laboratoire d’Ecologie Alpine, Universite´Grenoble Alpes, BP 53, FR-38000 Grenoble, France, 2Laboratoire d’Ecologie Alpine, CNRS, BP 53, FR-38000 Grenoble, France, 3CEA-Institut de Ge´nomique, Genoscope, Centre National de Se´quenc¸age, FR-91057 Evry Cedex, France and 4Conservatoire Botanique National Alpin, Domaine de Charance, FR-05000 Gap, France *For correspondence. E-mail [email protected] Received: 2 March 2016 Returned for revision: 9 April 2016 Accepted: 12 May 2016 Published electronically: 21 July 2016 Downloaded from Background and Aims Plant plastid genomes are highly conserved in size, gene content and structure; however, parasitic plants are a noticeable exception to this evolutionary stability. Although the evolution of parasites could help to better understand plastome evolution in general, complete plastomes of parasites have been sequenced only for some lineages so far. Here we contribute to filling this gap by providing and analysing the complete plastome se- quence of Cytinus hypocistis, the first parasite sequenced for Malvales and a species suspected to have an extremely http://aob.oxfordjournals.org/ small genome. Methods We sequenced and assembled de novo the plastid genome of Cytinus hypocistis using a shotgun ap- proach on genomic DNA. Phylogenomic analyses based on coding regions were performed on Malvidae. For each coding region present in Cytinus, we tested for relaxation or intensification of selective pressures in the Cytinus lin- eage compared with autotrophic Malvales. Key Results Cytinus hypocistis has an extremely divergent genome that is among the smallest sequenced to date (19Á4 kb), with only 23 genes and no inverted repeat regions. Phylogenomic analysis confirmed the position of Cytinus within Malvales. All coding regions of Cytinus plastome presented very high substitution rates compared with non-parasitic Malvales. Conclusions Some regions were inferred to be under relaxed negative selection in Cytinus, suggesting that further at INIST-CNRS on November 30, 2016 plastome reduction is occurring due to relaxed purifying selection associated with the loss of photosynthetic activ- ity. On the other hand, increased selection intensity and strong positive selection were detected for rpl22 in the Cytinus lineage, which might indicate an evolutionary role in the host–parasite arms race, a point that needs further research. Key words: Chloroplast genome, Cytinaceae, Cytinus hypocistis, Malvales, parasite, mycoheterotroph, plastome evolution, selective pressure. INTRODUCTION sequences have not been reported for some parasite lineages, and for others only one or few species have been studied. This The chloroplast genome (i.e. plastome) of seed plants is well gap is unfortunate, as it would permit a more general under- known for being remarkably conserved in size, structure and standing of the evolutionary forces that shape plastomes. gene content across the tree of life (Jansen and Ruhlman, 2012). The most striking exceptions to the evolutionary stability of the plastome have been described in parasite species (Westwood Evolutionary forces driving plastome reduction et al., 2010), which show a clear tendency towards plastome re- Since the main function of chloroplasts is photosynthesis, a duction (11–100 kb) in comparison with their autotrophic relatives (usually 120–170 kb; Ruhlman and Jansen, 2014). major question is why a plastid genome is maintained in non- This reduction is more pronounced in holoparasites, which are photosynthetic plants. Barbrook and colleagues (2006) pro- incapable of photosynthesis and thus completely dependent on posed the ‘essential tRNAs’ hypothesis, which is based on the their hosts for their nutrition. For instance, the plastome of the observation that the plastid trnE is involved in the synthesis of endoparasite Pilostyles aethiopica (Apodanthaceae) is the tetrapyrroles (i.e. molecules required for vital processes such as smallest reported to date for land plants, with 11 kb and five cellular respiration) and cannot be functionally replaced by any possibly functional genes (Bellot and Renner, 2016). In cytosolic tRNA for this function (Kumar et al.,1996). Orobanchaceae, the most studied plant parasite lineage to date, Therefore, even non-photosynthetic plants would need to retain some mechanisms of DNA retention have been suggested trnE (along with the machinery for its transcription), in order to (Wicke et al., 2013), but our understanding of plastome evolu- be able to synthesize essential tetrapyrroles such as the haem tion in parasites remains limited because complete plastome component of mitochondrial cytochromes and other essential VC The Author 2016. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: [email protected] 886 Roquet et al. — The plastid genome of the holoparasite Cytinus hypocistis (Cytinaceae) oxidative enzymes (Barbrook et al.,2006). However, two with those genes in operons. Regarding Santalales (a large recent studies based on high-throughput DNA sequencing af- order of plants consisting almost entirely of hemiparasites), firmed that plastid genomes had vanished at least in two non- only four species have had their plastome sequenced; the most photosynthetic organisms – the parasitic plant Rafflesia prominent plastome modifications were found in the species lagascae (Molina et al.,2014) and the colourless alga with the highest nutritional dependence on the host (Petersen Polytomella (Smith and Lee, 2014) – and a third study stated et al., 2015). Concerning mycoheterotrophs (i.e. species that that no functional tRNAs were found in Pilostyles (Bellot and parasitize other plants using fungi as vectors), several monocot Renner, 2016). These plastome losses would mean that the es- lineages have been studied, showing very different degrees of sential tRNAs hypothesis does not apply for these species, and plastome reduction, especially in orchids (Delannoy et al., that an alternative pathway exists for haem biosynthesis (Smith 2011; Logacheva et al.,2011, 2014; Barrett and Davis, 2012; and Asmail, 2014). Braukmann and Stefanovic, 2012; Barrett et al., 2014; Lam Another question related to plastome maintenance in para- et al., 2015; Schelkunov et al., 2015). In summary, all these sites is why different subsets of genes are retained in the plas- studies converge in showing that the plastome structure of tome of different parasitic species (Fig. 1), even within the parasitic plants is often radically altered in comparison with same genus (Supplementary Data Table S1). Within Cuscuta photosynthetic plants, with extensive gene losses and re- (Convolvulaceae), plastome differences of the four species arrangements in gene order. However, parasitism has inde- Downloaded from studied to date seem to be due to differences in their photosyn- pendently appeared at least 12 times in seed plants (Barkman thetic activity (Funk et al., 2007; McNeal et al., 2007). For et al., 2007; Su et al.,2015), and the fact that the entire plas- Orobanchaceae, the whole plastome has been reported for 16 tome is still not known for several parasite lineages limits our holoparasites and one hemiparasite (Wolfe et al.,1992; Li understanding of the macroevolutionary context of the emer- gence of parasitic lifestyles. et al., 2013; Wicke et al., 2013; Cusimano and Wicke, 2015), http://aob.oxfordjournals.org/ most of them showing notable differences in plastid gene con- tent and length and structural rearrangements. A recent study A new insightful case study in Cytinaceae (Wicke et al.,2013) indicated that the retention of plastid DNA fragments in this family could be affected by both the Our study focuses on Cytinus hypocistis, which belongs to proximity to genes under selection and their co-occurrence Cytinaceae. Cytinus was long considered to belong to Functional gene groups atp genes Ribosomal protein genes ndh genes RNA polymerase, maturase and initiation factor genes at INIST-CNRS on November 30, 2016 Other genes rRNAs Photosynthesis-related genes tRNAs 10 11 11 11 11 11 10 7 11 1 90 32 21 30 30 30 29 30 29 28 29 30 29 30 30 30 30 30 30 28 29 30 30 20 2 18 1 6 10 4 9 29 6 6 6 6 666 6 6 5 5 5 5 8 6 6 29 5 2 5 6 2 2 2 4 5 5 5 4 1 1 2 6 6 6 6 6 6 2 2 2 2 2 2 6 6 66 66 6 6 6 6 2 2 6 6 6 6 6 6 1 6 6 2 6 6 6 60 6 2 6 1 1 4 1 6 6 1 6 2 2 20 21 2 2 2 21 21 20 21 21 21 21 6 21 21 21 21 21 21 21 21 6 21 6 19 21 2 6 20 20 20 20 20 19 20 19 21 20 20 20 20 20 19 19 19 18 1 2 8 8 19 6 4 6 5 5 6 18 5 54 17 16 5 5 55554 54 21 5 5 4 5 3 4 4 44 4 4 16 4 4 3 4 4 Number of genes 1 15 30 5 4 1 1 4 3 3 1 5 15 4 4 4 15 30 31 31 14 30 15 30 30 30 30 30 30 30 29 14 29 29 29 29 29 29 29 29 28 30 29 30 28 29 28 29 26 28 27 28 28 29 27 29 29 15 4 24 21 22 22 2 17 19 18 17 2 2 1 3 6 7 9 3 6 6 0 444444 444444444444444444444444444444444444444444444 Trophic level Autotrophic Hemiparasite Vitis Osyrisvinifera alba Viscum album Cuscuta reflexa Holoparasite Aneura mirabilisHydnora visseri Cuscuta exaltata Piper cenocladum Neottia nidus-avis ViscumViscum crassulae