The question on the patterns and limits of reduction of plastid genomes in nonphotosynthetic plants and the reasons of their conservation is one of the intriguing topics in plant genome evolution. Here, we report sequencing and analysis of plastid genome in nonphotosynthetic orchids Epipogium aphyllum and Epipogium roseum, which, with sizes of 31 and 19 kbp, respectively, represent the smallest plastid genomes characterized by now. Besides drastic reduction, which is expected, we found several unusual features of these “minimal” plastomes: Multiple rearrangements, highly biased nucleotide composition, and unprecedentedly high substitution rate. Only 27 and 29 genes remained intact in the plastomes of E. aphyllum and E. roseum—those encoding ribosomal components, transfer RNAs, and three additional housekeeping genes (infA, clpP, and accD). We found no signs of relaxed selection acting on these genes. We hypothesize that the main reason for retention of plastid genomes in Epipogium is the necessity to translate messenger RNAs (mRNAs) of accD and/or clpP proteins which are essential for cell metabolism. However, these genes are absent in plastomes of several plant species; their absence is compensated by the presence of a functional copy arisen by gene transfer from plastid to the nuclear genome. This suggests that there is no single set of plastid-encoded essential genes, but rather different sets for different species and that the retention of a gene in the plastome depends on the interaction between the nucleus and plastids.

Plastid genomes of nonphotosynthetic plants represent a perfect model for studying evolution under relaxed selection pressure. However, the information on their sequences is still limited. We sequenced and assembled plastid genome of Petrosavia stellaris, a rare mycoheterotrophic monocot plant. After orchids, Petrosavia represents only the second family of nonphotosynthetic monocots to have its plastid genome examined. Several unusual features were found: retention of the ATP synthase genes and rbcL gene; extensive gene order rearrangement despite a relative lack of repeat sequences; an unusually short inverted repeat region that excludes most of the rDNA operon; and a lack of evidence for accelerated sequence evolution. Plastome of photosynthetic relative of P. stellaris, Japonolirion osense, has standard gene order and does not have the predisposition to inversions. Thus, the rearrangements in the P. stellaris plastome are the most likely associated with transition to heterotrophic way of life.

Thismia is a genus of > 80 mycoheterotrophic species characterized by a peculiar appearance and complex floral morphology. A significant proportion of the species and morphological diversity of Thismia has only been uncovered in the past two decades, and new discoveries continue to be made. Given that many new data have recently become available, and the most comprehensive taxonomic revision of the genus from 1938 addresses less than half of the currently known species, previous hypotheses for species relationships and infrageneric taxonomic classification in Thismia was in need of review. Extensive molecular phylogenetic studies of Thismia at the genus level have never been presented. We investigate the phylogenetic relationships of 41 species (and one variety) of Thismia from the Old World. Our study comprises 68 specimens (for 28 of which the data were newly generated), including outgroup taxa broadly representing Thismiaceae (= Burmanniaceae p.p. sensuAPG IV, 2016), and is based on two nuclear and one mitochondrial marker. We use maximum likelihood and Bayesian inference to infer relationships among the taxa. We also constructed a morphological dataset of 12 mostly floral characters, comparing these characters to hypotheses based on molecular evidence to identify putative synapomorphies for major clades and to discuss hypotheses regarding the evolution of structural traits in the genus. Our analyses indicate that the majority of currently accepted infrageneric taxa of Thismia are polyphyletic. We find support for the monophyly of the Old World group, in which we recognize five well-supported lineages (clades); the only New World species studied appears to be related to the Neotropical genus Tiputinia. Ancestral state reconstructions demonstrate that the evolution of most morphological characters was homoplastic, but we identify characters that provide each of the five clades of Old World Thismia with a unique morphological description. The geographical distribution of the species under study is also shown to be consistent with the major clades. Our investigation provides a phylogenetic basis for the development of a novel sectional classification of Thismia reflecting morphological and geographical traits.

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Heterotrophic plants provide intriguing examples of reductive evolution. This is especially evident in the reduction of their plastid genomes, which can potentially proceed toward complete genome loss. Several milestones at the beginning of this path of degradation have been described; however, little is known about the latest stages of plastome reduction. Here we analyze a diversity of plastid genomes in a set of closely related non-photosynthetic plants. We demonstrate how a gradual loss of genes shapes the miniaturized plastomes of these plants. The subject of our study, the genus Thismia, represents the mycoheterotrophic monocot family Thismiaceae, a group that may have experienced a very ancient (60–80 mya) transition to heterotrophy. In all 18 species examined, the plastome is reduced to 14–18 kb and is highly AT-biased. The most complete observed gene set includes accD, seven ribosomal protein genes, three rRNA, and two tRNA genes. Different clades of Thismia have undergone further gene loss (complete absence or pseudogenization) compared to this set: in particular, we report two independent losses of rps2 and rps18.