Background/Objectives: Changnienia amoena is a rare and endangered terrestrial orchid endemic to China, valued for its ornamental and medicinal properties. However, limited genomic resources hinder its effective conservation strategies and sustainable utilization. This study aimed to generate comprehensive plastome resources and develop molecular markers to support the phylogenetics, identification, and conservation management of C. amoena. Methods: Genome skimming was employed to assemble and annotate the complete plastomes of seven geographically distinct C. amoena accessions. Comparative analyses were conducted to assess structural features and sequence divergence within C. amoena and across related species in the Calypsoinae subtribe. Phylogenetic relationships were inferred from protein-coding genes. Simple sequence repeats (SSRs), dispersed repeats, and hypervariable regions were identified from the plastomes, while nuclear SSRs were developed from assembled nuclear sequences. Results: All seven plastomes exhibited a conserved quadripartite structure with identical gene content and order, showing only minor variations in genome size. Sequence divergence was mainly confined to non-coding regions. Across Calypsoinae species, mycoheterotrophic taxa exhibited reduced plastomes. Phylogenetic analyses resolved four well-supported intergeneric clades within Calypsoinae and revealed a notable divergence between the HuNGZ accession and other C. amoena accessions, which otherwise showed low plastome-level differentiation. We also identified 69–74 plastome-derived SSRs, 22–25 dispersed repeats, and three hypervariable regions that may serve as informative molecular markers for C. amoena. Additionally, 16 polymorphic nuclear SSRs were developed from assembled nuclear sequences. Conclusions: These findings significantly expand the genomic resources available for C. amoena and provide essential insights for its phylogeny, molecular identification, conservation management, and future breeding efforts.

SUMMARY: Whole‐genome duplication (WGD, or polyploidization) is a major evolutionary force in plants, yet the genomic, transcriptional, and metabolic consequences of autopolyploidy remain less understood than those of allopolyploidy. Dioscorea nipponica , a medicinal species producing diosgenin‐type steroidal saponins, harbors both diploid and autopolyploid populations, providing a unique system to investigate these processes. Here, we present a haplotype‐resolved, chromosome‐scale genome assembly of autotetraploid D. nipponica (2 n = 4 x = 40). Comparative and population genomic analyses indicate a single, recent autotetraploid origin (<1.63 Ma), after the divergence of its diploid ancestors (~2.5–2.9 Ma), coinciding with Pliocene–Pleistocene climatic shifts that may have promoted population isolation and genome duplication. The four haplotypes exhibit high average nucleotide identity, extensive synteny and similar global gene expression, with pervasive allele loss (~32% of genes) and widespread allelic transcriptional suppression (~60% of expressed tetrads), together buffering dosage effects. Metabolomic profiling reveals a metabolic trade‐off in rhizomes, with autotetraploids accumulating higher flavonoids but lower diosgenin‐type steroidal saponins than diploids, potentially enhancing reproductive fitness and adaptation to marginal environments. This shift is supported by expanded flavonoid gene families (e.g., PAL, CHS), some exhibiting positive dosage effects, whereas diosgenin pathway genes (e.g., CYP90, CYP94) remain copy number stable and mostly show negative dosage effects in autotetraploids. Selective sweep analysis further identified genes linked to genome stability (e.g., AtTOP3α , AtDMC1 ) and abiotic stress response, crucial for polyploid establishment. Overall, these findings offer novel insights into how autopolyploidy shapes gene expression, metabolic evolution, and ecological adaptation, with implications for the conservation and utilization of this medicinally important species.