The Evolution of Mitochondrial Genomes between Two Cymbidium Sister Species: Dozens of Circular Chromosomes and the Maintenance and Deterioration of Genome Synteny.

Plant mitochondrial genomes (mitogenomes) exhibit fluid genome architectures, which could lead to the rapid erosion of genome synteny over a short evolutionary time scale. Among the species-rich orchid family, the leafy Cymbidium lancifolium and leafless Cymbidium macrorhizon are sister species with remarkable differences in morphology and nutritional physiology. Although our understanding of the evolution of mitochondria is incomplete, these sister taxa are ideal for examining this subject. In this study, the complete mitogenomes of C. lancifolium and C. macrorhizon, totaling 704,244 bp and 650,751 bp, respectively, were assembled. In the 2 mitogenomes, 38 protein-coding genes, 18 cis- and 6 trans-spliced introns, and approximately 611 Kb of homologous sequences are identical; overall, they have 99.4% genome-wide similarity. Slight variations in the mitogenomes of C. lancifolium and C. macrorhizon in repeat content (21.0 Kb and 21.6 Kb, respectively) and mitochondrial DNA of plastid origin (MIPT; 38.2 Kb and 37.5 Kb, respectively) were observed. The mitogenome architectures of C. lancifolium and C. macrorhizon are complex and comprise 23 and 22 mini-circular chromosomes, respectively. Pairwise comparisons indicate that the two mitogenomes are largely syntenic, and the disparity in chromosome numbers is likely due to repeat-mediated rearrangements among different chromosomes. Notably, approximately 93.2 Kb C. lancifolium mitochondrial sequences lack any homology in the C. macrorhizon mitogenome, indicating frequent DNA gains and losses, which accounts mainly for the size variation. Our findings provide unique insights into mitogenome evolution in leafy and leafless plants of sister species and shed light on mitogenome dynamics during the transition from mixotrophy to mycoheterotrophy.

High-quality Cymbidium mannii genome and multifaceted regulation of crassulacean acid metabolism in epiphytes.

Epiphytes with crassulacean acid metabolism (CAM) photosynthesis are widespread among vascular plants, and repeated evolution of CAM photosynthesis is a key innovation for micro-ecosystem adaptation. However, we lack a complete understanding of the molecular regulation of CAM photosynthesis in epiphytes. Here, we report a high-quality chromosome-level genome assembly of a CAM epiphyte, Cymbidium mannii (Orchidaceae). The 2.88-Gb orchid genome with a contig N50 of 22.7 Mb and 27 192 annotated genes was organized into 20 pseudochromosomes, 82.8% of which consisted of repetitive elements. Recent expansions of long terminal repeat retrotransposon families have made a major contribution to the evolution of genome size in Cymbidium orchids. We reveal a holistic scenario of molecular regulation of metabolic physiology using high-resolution transcriptomics, proteomics, and metabolomics data collected across a CAM diel cycle. Patterns of rhythmically oscillating metabolites, especially CAM-related products, reveal circadian rhythmicity in metabolite accumulation in epiphytes. Genome-wide analysis of transcript and protein level regulation revealed phase shifts during the multifaceted regulation of circadian metabolism. Notably, we observed diurnal expression of several core CAM genes (especially ßCA and PPC) that may be involved in temporal fixation of carbon sources. Our study provides a valuable resource for investigating post-transcription and translation scenarios in C. mannii, an Orchidaceae model for understanding the evolution of innovative traits in epiphytes.

DNA barcoding of Cymbidium by genome skimming: Call for next-generation nuclear barcodes.

Cymbidium is an orchid genus that has undergone rapid radiation and has high ornamental, economic, ecological and cultural importance, but its classification based on morphology is controversial. The plastid genome (plastome), as an extension of plant standard DNA barcodes, has been widely used as a potential molecular marker for identifying recently diverged species or complicated plant groups. In this study, we newly generated 237 plastomes of 50 species (at least two individuals per species) by genome skimming, covering 71.4% of members of the genus Cymbidium. Sequence-based analyses (barcoding gaps and automatic barcode gap discovery) and tree-based analyses (maximum likelihood, Bayesian inference and multirate Poisson tree processes model) were conducted for species identification of Cymbidium. Our work provides a comprehensive DNA barcode reference library for Cymbidium species identification. The results show that compared with standard DNA barcodes (rbcL + matK) as well as the plastid trnH-psbA, the species identification rate of the plastome increased moderately from 58% to 68%. At the same time, we propose an optimized identification strategy for Cymbidium species. The plastome cannot completely resolve the species identification of Cymbidium, the main reasons being incomplete lineage sorting, artificial cultivation, natural hybridization and chloroplast capture. To further explore the potential use of nuclear data in identifying species, the Skmer method was adopted and the identification rate increased to 72%. It appears that nuclear genome data have a vital role in species identification and are expected to be used as next-generation nuclear barcodes.

Plastid RNA editing reduction accompanied with genetic variations in Cymbidium, a genus with diverse lifestyle modes.

Recent sequencing efforts have broadly uncovered the evolutionary trajectory of plastid genomes (plastomes) of flowering plants in diverse habitats, yet our knowledge of the evolution of plastid posttranscriptional modifications is limited. In this study, we generated 11 complete plastomes and performed ultra-deep transcriptome sequencing to investigate the co-evolution of plastid RNA editing and genetic variation in Cymbidium, a genus with diverse trophic lifestyles. Genome size and gene content is reduced in terrestrial and green mycoheterotrophic orchids relative to their epiphytic relatives. This could be partly due to extensive losses and pseudogenization of ndh genes for the plastid NADH dehydrogenase-like complex, but independent pseudogenization of ndh genes has also occurred in the epiphyte C. mannii, which was reported to use strong crassulacean acid metabolism photosynthesis. RNA editing sites are abundant but variable in number among Cymbidium plastomes. The nearly twofold variation in editing abundance is mainly due to extensive reduction of ancestral editing sites in ndh transcripts of terrestrial, mycoheterotrophic, and C. mannii plastomes. The co-occurrence of editing reduction and pseudogenization in ndh genes suggests functional constraints on editing machinery may be relaxed, leading to nonrandom loss of ancestral edited sites via reduced editing efficiency. This study represents the first systematic examination of RNA editing evolution linked to plastid genome variation in a single genus. We also propose an explanation for how genomic and posttranscriptional variations might be affected by lifestyle-associated ecological adaptation strategies in Cymbidium.