Unprecedented Parallel Photosynthetic Losses in a Heterotrophic Orchid Genus Craig F. Barrett,*,1 Brandon T. Sinn,1 and Aaron H. Kennedy2 1Department of Biology, West Virginia University, Morgantown, WV 2Mycology and Nematology Genetic Diversity and Biology Laboratory, USDA-APHIS, Beltsville, MD *Corresponding author: E-mail: [email protected]. Associate editor: Tal Pupko Abstract Heterotrophic plants are evolutionary experiments in genomic, morphological, and physiological change. Yet, genomic sampling gaps exist among independently derived heterotrophic lineages, leaving unanswered questions about the process of genome modification. Here, we have sequenced complete plastid genomes for all species of the leafless orchid genus Hexalectris, including multiple individuals for most, and leafy relatives Basiphyllaea and Bletia.Ourobjectivesare to determine the number of independent losses of photosynthesis and to test hypotheses on the process of genome degradation as a result of relaxed selection. We demonstrate four to five independent losses of photosynthesis in Hexalectris based on degradation of the photosynthetic apparatus, with all but two species displaying evidence of losses, and variation in gene loss extending below the species level. Degradation in the atp complex is advanced in Hexalectris warnockii, whereas only minimal degradation (i.e., physical loss) has occurred among some “housekeeping” genes. We find genomic rearrangements, shifts in Inverted Repeat boundaries including complete loss in one accession of H. arizonica, and correlations among substitutional and genomic attributes. Our unprecedented finding of multiple, independent transitions to a fully mycoheterotrophic lifestyle in a single genus reveals that the number of such transitions among land plants is likely underestimated. This study underscores the importance of dense taxon sampling, which is highly informative for advancing models of genome evolution in heterotrophs. Mycoheterotrophs such as Hexalectris provide forward-genetic opportunities to study the consequences of radical genome evolution beyond what is possible with mutational studies in model organisms alone. Key words: heterotroph, phylogenomics, plastome, pseudogene, selection, chloroplast, deletion, evolution. Introduction and contain the all four of the highly conserved, plastid- encoded ribosomal RNA species (e.g., Zhu et al. 2016). The Heterotrophic plants present prime examples of genomic sequencing of complete plastid genomes (plastomes) has be- Article modification due to relaxed selective constraints on photo- come more feasible because of high-throughput sequencing synthetic function. They are highly useful as “evolutionary technology and assembly algorithms, and thus plastomes experiments” in that they manage to survive and reproduce from throughout the tree of life are being published at a rapid under natural conditions despite drastic modifications, in pace (see Gitzendanner et al. 2018). contrast to many induced mutants in model systems which Plastid genome evolution in heterotrophic plants has re- would almost certainly go extinct in the wild. Thus, hetero- ceived much attention in recent years (e.g., reviewed by trophs can increase our knowledge on how whole-organismal Graham et al. [2017], Hadariova et al. [2018], and Wicke systems adjust to such radical changes. Plastid genomes are of and Naumann [2018]), yet only a handful of studies have particular interest because they are small, nonrecombining included taxon sampling of sufficient density to apply phylo- (i.e., in the sense of swapping material among homologous genetic, comparative approaches (Wicke et al. 2013, 2016; chromosomes as in nuclear DNA), and uniparentally inher- Barrett et al. 2014, 2018; Feng et al. 2016; Braukmann et al. ited; occur in relatively high copy number per cell compared 2017; Schneider et al. 2018). Many studies have focused on with nuclear and (typically) mitochondrial genomes; and are single plastomes, which may fail to capture several key aspects conserved in gene order, content, and, overall genome struc- of the process of plastid genome evolution, especially in cases ture, with a large and small single copy region bisected by a of rapidly accumulated genotypic and phenotypic change large Inverted Repeat (IR; typically 20–30 kb) across angio- due to relaxed selection and increased rates of substitution. sperms (see Ruhlman and Jansen 2014). The genes and Recent studies have begun to incorporate dense sampling of spacers of the IR are among the most conserved regions of plastomes within clades, for example, at or near the genus the plastome in terms of gene order and substitution rates and species level (Wicke et al. 2013; Barrett et al. 2014, 2018; and contribute to overall structural stability of the chromo- Feng et al. 2016; Braukmann et al. 2017; Schneider et al. 2018). some, multimeric structure among plastid chromosomes, This approach is important in that it allows plastome ß The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] Open Access 1884 Mol. Biol. Evol. 36(9):1884–1901 doi:10.1093/molbev/msz111 Advance Access publication May 6, 2019 Parallel Photosynthetic Losses in a Heterotrophic Orchid Genus . doi:10.1093/molbev/msz111 MBE evolution to be interpreted on a finer scale—and more im- Transitions to full mycoheterotrophy are most likely facili- portantly, from a phylogenetic-comparative perspective— tated by the condition of “initial mycoheterotrophy” in revealing details on the types of changes that occur and their orchids, all of which begin their lives as nonphotosynthetic, mechanistic causes, not merely the result after several millions mycoparasitic seedlings (protocorms) that eventually be- of years of evolution on a single plastome. come photosynthetic with the development of leaves or Species-level sampling in the leafless orchid genus other organs capable of photosynthesis (Rasmussen 1995; Corallorhiza (plastome sequencing) and parasitic vine Bidartondo 2005; Leake 2005). Full mycoheterotrophy can Cuscuta (family Convolvulaceae; DNA probe blotting) has be interpreted as a pedomorphic shift that has occurred revealed multiple independent shifts to full heterotrophy many times in separate orchid lineages, and initial mycohe- within single genera, and thus it is likely that the number of terotrophy can be viewed as a preadaptation to such shifts, hypothesized transitions to complete heterotrophy and loss for example, with the characteristic mycotrophic coralloid of photosynthesis have been underestimated in other groups rhizome structure found in heterotrophic species represent- (Revill et al. 2005; Funk et al. 2007; McNeal, Arumugunathan, ing a protracted, juvenile protocorm stage (Rasmussen 1995; et al. 2007; McNeal, Kuehl, et al. 2007; Braukmann et al. 2013; Freudenstein and Barrett 2010; Merckx, Freudenstein, et al. Barrett et al. 2014, 2018). Further, it remains to be determined 2013). These changes are often accompanied by fungal host if photosynthetic loss and subsequent plastome degradation shifts from “typical” orchid mycorrhizal fungal partners— can be detected below the species level. saprotrophic taxa termed rhizoctonias (teleomorphic What types of genomic changes occur in heterotrophic families Ceratobasidiaceae, Tulasnellaceae, and plants? Much attention has been given to the evolutionary Sebacinaceae)—to ectomycorrhizal or even arbuscular my- fates of plastid-encoded gene systems coinciding with or fol- corrhizal fungi, though some of the former are known to lowing the evolution of heterotrophy (e.g., dePamphilis and form ectomycorrhizas (Taylor and Bruns 1997; Taylor et al. Palmer 1990; Wolfe et al. 1992; Barbrook et al. 2006; Krause 2003; Barrett et al. 2010; Kennedy et al. 2011). 2008, 2011, 2012; Wicke et al. 2011, 2013). Barrett and Davis Mycoheterotrophs in clades throughout the orchids ex- (2012) and subsequently Barrett et al. (2014) proposed a gen- hibit several convergent morphoanatomical characteristics eral conceptual model, in which plastid-encoded gene sys- during the transition from autotrophy to heterotrophy. tems are lost in a more-or-less linear, irreversible pattern (but These changes have resulted in modifications to several allowing exceptions), following the general sequence of 1) organs, including reduction or loss of roots and leaves, prev- ndh genes, encoding a chloroplast NAD(P)H dehydrogenase; alence of cleistogamy (closed-flowers), autonomous self- 2) psa, psb, rbcL, ccsA, cemA, ycf3,andycf4, encoding various pollination, reduction or loss of stomata, and reduction in complexes involved directly in photosynthesis (e.g., photo- vascular tissues (Leake 1994; Leake et al. 2008; Merckx and systems I and II, RuBisCO); 3) rpo, a plastid-encoded RNA Freudenstein 2010). polymerase; 4) atp, encoding a thylakoid ATP synthase; and 5) The leafless orchid genus Hexalectris is composed of nine “housekeeping” genes involved in basic organellar functions species, all of which have been presumed to be full mycohe- such as intron splicing and translation (e.g., matK, rpl, rps, rrn, terotrophs, distributed throughout Western