0.004 * * * * * * 95 * 97 * * * 93 * * * 68 * * * * * 98 * Epipactis microphylla Neottia ovata Neottia pinetorum Tropidia polystachya Neottia cordata Palmorchis trilobulata Epipactis purpurata Epipactis palustris Epipactis mairei1 Palmorchis pabstii Cephalanthera damasonium Neottia japonica Epipactis helleborine Russia Cephalanthera longifolia Diceratostele gabonensis Epipactis mairei Epipactis helleborine- White Epipactis gigantea Epipactis atrorubens Cephalanthera longibracteata Neottia suzukii Epipactis albensis Neottia fugongensis Cephalanthera rubra Epipactis veratrifolia Limodorum abortivum Epipactis helleborine-Green Neottia japonica1 E. helleborine species complex Mycoparasitism does not correlate with changes in the plastid genome in the orchid Epipactis helleborine Janice Valencia-D.¹, Kierstin Lipe¹, Kurt Neubig¹, W. Mark Whitten² ¹Plant Biology Department, Southern Illinois University, Carbondale, Illinois, 62901 ²Florida Museum of Natural History, University of Florida, P.O. Box 117800, Gainesville, FL 32611 Introduction Epipactis helleborine L.(Crantz) is a mixotrophic orchid that can obtain its carbon supplies in different ways that range from autotrophism to mycoheterotrophism (Fig. 1). Full mycoheterotrophism (=mycoparasitism) is associated with a drastic reduction in the chloroplast (plastid) genome due to the loss of genes related to photosynthetic pathways. However, this has not been shown in mixotrophic species. To determine if achlorophyllous individuals of Epipactis helleborine have alterations in their photosynthesis-related genes, we sequenced and assembled plastid genomes for green and albino plants. Figure 1. Jacquemyn & Merckx´s continuum trophic model (2019). A. E. helleborine albino form, a full mycoheterotroph (Photo: Damon Tighe). B. E. helleborine green form, a partial mycoheterotroph (Photo: Roh Curtis) 1. Green and albino forms of E. helleborine were collected in Virginia, Giles County, Mountain Lake Biological Station. DNA was extracted using a CTAB method (Doyle & Doyle, 1987), followed by a silica purification column step (Neubig et al., 2014). Sequencing was performed on an Illumina HiSeqX. The poorly assembled region at trnC- ACA intron was improved by adding sequences from PCR. 2. Plastid genomes were assembled in Geneious 10.2.3. Annotations were made using CHLOROBOX and were manually curated. 3. A Maximum Likelihood analysis of the Neottieae tribe, to which Epipactis belongs, was performed using coding regions and rRNAs from our two samples, three unpublished orchid plastid genomes, and 23 more downloaded from GenBank. For the E. helleborine species complex, the full plastid genome was compared to assess substitutions per site and hotspots. • The plastid genome of both forms of E. helleborine consisted of a single, circular DNA sequence, in the typical quadripartite structure (Fig. 2). • The plastid genome of E. helleborine contains all gene regions expected in an autotrophic orchid, including complete and functional ndh genes, which are the first group of genes that tend to be lost in taxa that exhibit some transition to partial or full mycoheterotrophy. • There was no evidence of mutations in coding regions in the plastid genome of the albino plant, which suggests that this condition is regulated by nuclear or mitochondrial genes in this species. • Among the widespread E. helleborine, numerous varieties, subspecies and forms have been described due to the high plasticity in vegetative morphology and floral size and color. A broad taxonomic revision is needed to fully understand the morphological breadth of the species complex, considering the genetic variation. • We identify 4 hotspots in the plastid genome and 141 sites that can be useful to identify phylogenetic groups inside the E. helleborine complex. These results are relevant for population genetic studies and for determining evolutionary dynamics like foundational effect in the North American individuals, inbreeding and fluct