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Botany What are the genomic consequences for plastids in a mixotrophic orchid (Epipactis helleborine)? Journal: Botany Manuscript ID cjb-2020-0054.R1 Manuscript Type: Article Date Submitted by the 06-Aug-2020 Author: Complete List of Authors: Valencia-D., Janice; Southern Illinois University Carbondale, School of Biological Science Whitten, William; Florida Museum of Natural History Neubig, Kurt; Southern Illinois University Carbondale, School of Biological ScienceDraft Albinism, <i>Epipactis helleborine</i>, mixotrophy, mycoheterotrophy, Keyword: plastome Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? : © The Author(s) or their Institution(s) Page 1 of 33 Botany What are the genomic consequences for plastids in a mixotrophic orchid (Epipactis helleborine)? Janice Valencia-D.¹,3, W. Mark Whitten²†, Kurt Neubig¹ ¹School of Biological Sciences, Southern Illinois University, 1125 Lincoln Dr., Carbondale, Illinois, 62901, USA; [email protected], [email protected] ²Florida Museum of Natural History, University of Florida, 1659 Museum Rd., Gainesville, FL 32611, USA 3Corresponding author †Author deceased Corresponding author: Draft Janice Valencia-D.1 1125 Lincoln Dr, Carbondale, Illinois, 62901-6509, USA Phone: 618-203-2724, Fax: 618-453-3441 Email address: [email protected] © The Author(s) or their Institution(s) Botany Page 2 of 33 Abstract The chloroplast (plastid) controls carbon uptake, so its DNA sequence and function are highly conserved throughout the land plants. But for those that have alternative carbon supplies, the plastid genome is susceptible to suffer mutations in the photosynthetic genes and overall size reduction. Fully mycoheterotrophic plants receive organic carbon from their fungi partner, do not photosynthesize and also do not exhibit green coloration (or produce substantial quantities of chlorophyll). Epipactis helleborine (L.) Crantz exhibits all trophic modes from autotrophy to full mycoheterotrophy. Albinism is a stable condition in individuals of this species and does not prevent them from producing flowers and fruits. Here we assemble and compare the plastid genome of green and albino individuals. Our results show that there is still strong selective pressure in the plastid genome. Therefore, the few punctual differences among them, to ourDraft knowledge, do not affect any normal photosynthetic capability in the albino plant. These findings suggest that mutations or other genetically controlled processes in other genomes, or environmental conditions, are responsible for the phenotype. Albinism, Epipactis helleborine, mixotrophy, mycoheterotrophy, plastome © The Author(s) or their Institution(s) Page 3 of 33 Botany Introduction About 23,000 species of land plants derive their resources from fungi at least during some part of their lives, and the vast majority of them are orchids (Merckx 2013). This way of living, known as mycoheterotrophism, provides part or all the carbon supplies that the plants need (Leake 1994; Freudenstein and Barrett 2010; Merckx 2013; Selosse et al. 2016; Lallemand 2018). Based on the dependency of plants to the fungi, they can be ‘initially mycoheterotrophic’ if they only have a strong association during early developmental stages, ‘partially mycoheterotrophic’ if they retain their relationship with the fungi and develop photosynthetic capabilities (also called mixotrophic, Gebauer and Meyer 2003; Selosse and Roy 2009), or ‘fully mycoheterotrophic’ if they only rely on the organic carbon provided by the fungus (Merckx 2013; Wicke and Naumann 2018; Jacquemyn and Merckx 2019). During the initial stages of seed germination andDraft protocorm development, orchids depend on fungal partners for nutrition. Later on, they become self-sufficient, but retain a close relationship with their mycorrhizal partner (reviewed in Dearnaley et al. 2012). Scattered through the family, fully mycoheterotrophic taxa have arisen at least 35 times in the Orchidaceae family based on phylogenetic reconstructions (Freudenstein and Barrett 2010; Barrett et al. 2019) and they comprise around 235 species in 43 genera (Merckx 2013). Fully mycoheterotrophic taxa lose their photosynthetic capabilities in a process that is correlated with the reduction of the plastid (cp) genome (e.g., Delannoy et al. 2011; Wicke et al. 2013; Feng et al. 2016; Lam et al. 2018). The common steps in the cp degradation across parasitic and heterotrophic land plants have been described in a model consisting of five stages (Barrett and Davis 2012; Barrett et al. 2014; Wicke et al. 2016); slightly modified by (Wicke and Naumann 2018). The first stage is the loss or degradation of ndh genes, which is a recurrent event observed in orchids and is not always related with the transition to © The Author(s) or their Institution(s) Botany Page 4 of 33 mycoheterotrophic conditions (e.g., Kim et al. 2015, 2018; Wicke and Naumann 2018). The second is the degradation of primary photosynthetic genes (subunits of pet, psa, psb genes) and the plastid-encoded polymerase gene (rpo). Once the plant becomes fully heterotrophic, during the third step, it loses the atp and rbcL genes and then housekeeping genes (tRNAs, rRNAs and plastid ribosomal proteins). During the fourth stage, genes associated with other metabolic functions (accD, clpP, ycf1, ycf2) are lost. Finally, there can be a complete loss of the cp-genome as exemplified in Rafflesia (Molina et al. 2014). Mutations in the cp- genome can lead to an achlorophyllous phenotype. Although some fully mycoheterotrophic plants produce small amounts of chlorophyll (Cummings and Welschmeyer 1998; Barrett et al. 2014), most exhibit a variety of colors such as red, purple, brown, or white. Achlorophyllous plantsDraft can also be produced artificially in the lab by altering plastid genes, as has been done in tobacco (i.e., Santis‐Maciossek et al. 1999; Fleischmann et al. 2011), wheat (Xia et al. 2012) and several other taxa (Kumari et al. 2009 and references therein). In these cases, alterations of photosynthesis-related genes cause an albino phenotype in normally green (i.e., autotrophic) species. Among Neottieae, albinism has been best described in members of Cephalanthera (Renner 1938; Salmia 1989; Julou et al. 2005; Abadie et al. 2006; Roy et al. 2013) and Epipactis (Salmia 1989; Selosse et al. 2004; Jakubska and Schmidt 2005; Lallemand 2018). In Epipactis helleborine, albino individuals are not abundant in natural populations, but individuals produce white shoots every growing season which make the albinism a permanent condition (Salmia 1989). To date, no studies have addressed the question of if the albino phenotype is heritable and linked to cp-genome modifications. To determine if © The Author(s) or their Institution(s) Page 5 of 33 Botany achlorophyllous individuals of E. helleborine have alterations in their photosynthesis-related genes, we sequenced and assembled cp-genomes for green and albino plants. Epipactis helleborine belongs taxonomically to Epipactis section Epipactis, a clade in which the species are difficult to recognize taxonomically and genetically (Tyteca and Dufrene 1994; Squirrell et al. 2001; Ehlers et al. 2002; Brzosko et al. 2004; Sramkó et al. 2019). Most of the species belonging to the E. helleborine alliance originate from a few populations that are restricted to small regions (with the exception of a paraphyletic group forming E. helleborine) (Sramkó et al. 2019). To understand the variation in the cp-genome composition across these closely related species, we compared our findings with other Epipactis species for which the plastid genome was available through GenBank. Draft Materials and Methods Collection, extraction, sequencing Green and albino forms of E. helleborine (W. Mark Whitten 4891 and 4890, respectively) were freshly collected in Virginia, at the Mountain Lake Biological Station in an Appalachian montane Quercus-Tsuga forest. Vouchers were deposited at the University of Florida Herbarium (FLAS) and leaf tissues were preserved in silica-gel. Total DNA was extracted from each sample using a CTAB method (Doyle and Doyle 1987), followed by a silica purification column step (Neubig et al. 2014). The samples were quantified using Qubit dsDNA BR assay (Thermo Fisher, Waltham, MA, USA) and concentrations adjusted approximately to the same value for both (Whitten 4890: 57.4ng/µL, Whitten 4891: 57.6ng/µL). Library preparation, barcoding and sequencing were conducted at © The Author(s) or their Institution(s) Botany Page 6 of 33 Rapid Genomics, LLC (Gainesville, FL, USA). Pooled fragments of 350-450 bp were sequenced on an Illumina HiSeq X platform to get 150 bp paired-end reads. Plastid genome assembly The paired-end reads were trimmed for quality at 0.05 probability and assembled in Geneious 10.2.3 (https://www.geneious.com) with a combination of reference and de novo assemblies. Epipactis mairei (KU551264) and E. veratrifolia (KU551267) produced by Feng et al. (2016) were used as references. Polymerase Chain Reaction (PCR) Primers were designed for the amplification of the 32,000-32,400 bp region in the trnC(CAC) intron, which could not be confidentlyDraft recovered from the Illumina data. We designed two primers for each flank in selected regions with good Illumina data coverage (depth >30). Then the four possible combinations of the four primers were amplified per sample in order to ensure complete length coverage. Designed forward primers were 5’- CTTCCGCCTTGACAGGGCGG-3’ and 5’-CTTCCGCCTTGACAGGGCGG, and the