Multi-omics integration provides insights into the symbiotic evolution of the mycoheterotrophic medicinal orchid Gastrodia elata

Mycoheterotrophy represents an extreme evolutionary strategy in which plants abandon photosynthesis and become obligately dependent on fungal partners for carbon and nutrients. Gastrodia elata, a medicinal orchid forming long-term symbioses with Armillaria and Mycena, provides an ideal system to investigate the genomic basis of obligate plant-fungus symbiosis. Here, we generate a chromosome-level genome assembly (1.09 Gb) of a dark red G. elata accession and construct a pan-gene set from 11 G. elata accessions plus G. menghaiensis as an outgroup; within the sampled cultivated G. elata accessions, SNP-based clustering was more concordant with tuber32 shape grouping than with stem-color labels. Comparative genomic analyses reveal a pronounced degeneration of photosynthetic capacity. We did not detect intact nuclear-encoded 33 rbcS loci in the dark red G. elata assembly, consistent with loss of Rubisco-dependent Calvin-Benson carbon fixation. Notably, a subset of photosynthesis-related genes is retained and transcriptionally active, consistent with possible non-photosynthetic or plastid-associated roles that require functional validation. To systematically dissect horizontal gene transfer (HGT) events during symbiotic adaptation in orchids, we conducted a genome-wide screen and phylogenomic validation of HGT across 13 orchid genomes and identified a total of 44 putative HGT-acquired genes with donor lineages spanning Bacteria, Metazoa, and Viruses, clustered into 20 distinct gene families. Notably, despite millions of years of close symbiotic interaction between G. elata and its symbiotic fungi, under this multi-step phylogenomic validation pipeline, no detectable fungal-derived HGT was found in the G. elata genome. Together with the candidate transporter/metabolic-gene evidence, these findings are consistent with a model in which metabolic interactions, rather than detectable stable fungal-to-plant gene integration, may contribute to G. elata-fungus symbiosis. This is supported by a specialized nutrient-acquisition framework adapted to obligate heterotrophy: genes associated with trehalose hydrolysis and expanded sugar transporter families suggest potential routes for hexose carbon acquisition, while retained amino acid/oligopeptide transporter and urease-related genes suggest potential organic nitrogen and ammonium assimilation routes. We further identify the GAFP gene family as a candidate molecular innovation potentially contributing to symbiotic homeostasis. The recently derived Class 1 GAFP lineage exhibits distinct domain architecture, tandem expansion, and elevated expression, making it a priority candidate for testing roles in limiting fungal overgrowth and maintaining symbiotic homeostasis. Taken together, these results establish a pan-gene framework/resource that provides new insights into the symbiotic adaptation and reductive genome evolution in G. elata.