Bulbophyllum is one of the largest genera and presents some of the most intricate taxonomic problems in the family Orchidaceae, including species of ornamental and medical importance. The lack of knowledge regarding the characterization of Bulbophyllum chloroplast (cp) genomes has imposed current limitations on our study. Here, we report the complete cp genomes of seven Bulbophyllum species, including B. ambrosia, B. crassipes, B. farreri, B. hamatum, B. shanicum, B. triste, and B. violaceolabellum, and compared with related taxa to provide a better understanding of their genomic information on taxonomy and phylogeny. A total of 28 Bulbophyllum cp genomes exhibit typical quadripartite structures with lengths ranging from 145,092 bp to 165,812 bp and a GC content of 36.60% to 38.04%. Each genome contained 125–132 genes, encompassing 74–86 protein-coding genes, 38 tRNA genes, and eight rRNA genes. The genome arrangements, gene contents, and length were similar, with differences observed in ndh gene composition. It is worth noting that there were exogenous fragment insertions in the IR regions of B. crassipes. A total of 18–49 long repeats and 38–80 simple sequence repeats (SSRs) were detected and the single nucleotide (A/T) was dominant in Bulbophyllum cp genomes, with an obvious A/T preference. An analysis of relative synonymous codon usage (RSCU) revealed that leucine (Leu) was the most frequently used codon, while cysteine (Cys) was the least used. Six highly variable regions (rpl32-trnL^UAG > trnT^UGU-trnL^UAA > trnF^GAA-ndhJ > rps15-ycf1 > rbcL-accD > psbI-trnS^GCU) and five coding sequences (ycf1 > rps12 > matK > psbK > rps15) were identified as potential DNA markers based on nucleotide diversity. Additionally, 31,641 molecular diagnostic characters (MDCs) were identified in complete cp genomes. A phylogenetic analysis based on the complete cp genome sequences and 68 protein-coding genes strongly supported that 28 Bulbophyllum species can be divided into four branches, sects. Brachyantha, Cirrhopetalum, and Leopardinae, defined by morphology, were non-monophyly. Our results enriched the genetic resources of Bulbophyllum, providing valuable information to illustrate the complicated taxonomy, phylogeny, and evolution process of the genus.

Gastrochilus pseudodresslerii (Aeridinae), a new species from Maguan County, Yunnan Province, China, is described and illustrated. Morphological comparisons indicated that this new species is similar to G. dresslerii, but differs by its longer and broadly lanceolate to oblong leaves, larger and elliptic dorsal sepal, longer and elliptic petals, subcupular hypochile, and narrowed toward apex of column. Phylogenetic analysis based on nuclear ribosomal ITS and four plastid sequences (atpI, matK, psbA and trnL-F) indicated that G. pseudodresslerii is also closely related to G. formosanus and G. raraensis, but can be easily distinguished from these two species based on its leaves, epichile and hypochile.

Epidendrum, one of the three largest genera of Orchidaceae, exhibits significant horticultural and ornamental value and serves as an important research model in conservation, ecology, and evolutionary biology. Given the ambiguous identification of germplasm and complex evolutionary relationships within the genus, the complete plastome of this genus (including five species) were firstly sequenced and assembled to explore their characterizations. The plastomes exhibited a typical quadripartite structure. The lengths of the plastomes ranged from 147,902 bp to 150,986 bp, with a GC content of 37.16% to 37.33%. Gene annotation revealed the presence of 78–82 protein-coding genes, 38 tRNAs, and 8 rRNAs. A total of 25–38 long repeats and 130–149 SSRs were detected. Analysis of relative synonymous codon usage (RSCU) indicated that leucine (Leu) was the most and cysteine (Cys) was the least. The consistent and robust phylogenetic relationships of Epidendrum and its closely related taxa were established using a total of 43 plastid genomes from the tribe Epidendreae. The genus Epidendrum was supported as a monophyletic group and as a sister to Cattleya. Meanwhile, four mutational hotspots (trnC^GCA–petN, trnD^GUC–trnY^GUA, trnS^GCU–trnG^UCC, and rpl32–trnL^UAG) were identified for further phylogenetic studies. Our analysis demonstrates the promising utility of plastomes in inferring the phylogenetic relationships of Epidendrum.

Phosphoenolpyruvate carboxylase (PEPC) gene family plays a crucial role in both plant growth and response to abiotic stress. Approximately half of the Orchidaceae species are estimated to perform CAM pathway, and the availability of sequenced orchid genomes makes them ideal subjects for investigating the PEPC gene family in CAM plants. In this study, a total of 33 PEPC genes were identified across 15 orchids. Specifically, one PEPC gene was found in Cymbidium goeringii and Platanthera guangdongensis; two in Apostasia shenzhenica, Dendrobium chrysotoxum, D. huoshanense, Gastrodia elata, G. menghaiensis, Phalaenopsis aphrodite, Ph. equestris, and Pl. zijinensis; three in C. ensifolium, C. sinense, D. catenatum, D. nobile, and Vanilla planifolia. These PEPC genes were categorized into four subgroups, namely PEPC-i, PEPC-ii, and PEPC-iii (PTPC), and PEPC-iv (BTPC), supported by the comprehensive analyses of their physicochemical properties, motif, and gene structures. Remarkably, PEPC-iv contained a heretofore unreported orchid PEPC gene, identified as VpPEPC4. Differences in the number of PEPC homolog genes among these species were attributed to segmental duplication, whole-genome duplication (WGD), or gene loss events. Cis-elements identified in promoter regions were predominantly associated with light responsiveness, and circadian-related elements were observed in each PEPC-i and PEPC-ii gene. The expression levels of recruited BTPC, VpPEPC4, exhibited a lower expression level than other VpPEPCs in the tested tissues. The expression analyses and RT-qPCR results revealed diverse expression patterns in orchid PEPC genes. Duplicated genes exhibited distinct expression patterns, suggesting functional divergence. This study offered a comprehensive analysis to unveil the evolution and function of PEPC genes in Orchidaceae.

Apostasioideae, the early divergent subfamily of Orchidaceae, comprises Apostasia and Neuwiedia genera with approximately 20 species. Despite extensive research on Apostasioideae, previous studies have struggled to resolve taxonomic issues, particularly concerning the position of species within this subfamily. Here, we sequenced and annotated plastomes of Apostasia fujianica and Neuwiedia malipoensis, unveiling their phylogenetic relationships and shared plastome features with the other five published plastomes. We identified and analyzed the length, GC content, repeat sequences, and RSCU values of the chloroplast genomes. It is noteworthy that the chloroplast genome of N. malipoensis stands out as the largest among all known chloroplast genomes within the Apostasioideae subfamily, primarily due to contributions from both the LSC and SSC regions. Furthermore, our analysis revealed three unique structural rearrangements located approximately 10k–47k bp (ycf3–trnS-GCU) and 58k–59k bp(accD) in the LSC region and 118k–119k (ndhI) bp in the SSC region of the chloroplast genomes across all five species within the Apostasia genus, which presents a potential avenue for identifying distinctive chloroplast genetic markers, setting them apart from other orchid plants. And a total of four mutational hotspots (rpoC2, atpH, rps4, ndhK, and clpP) were identified. Moreover, our study suggested that Apostasia and Neuwiedia formed a monophyletic group, with Apostasia being sister to Neuwiedia. Within the Apostasia genus, five species were classified into two major clades, represented as follows: (A. odorata (A. shenzhenica and A. fujianica) (A. ramifera and A. wallichii)). These findings hold significance in developing DNA barcoding of Apostasioideae and contribute to the further phylogenetic understanding of Apostasioideae species.

Dendrobium tetrapterum, a cryptic species in the D. lindleyi alliance, is described and illustrated. Detailed morphological comparisons indicate that it is similar to D. lindleyi and D. jenkinsii but can be distinguished by four morphological characters, namely petals without (vs. with) claw, slit (vs. entire) transverse septum at lip base, anther cap with (vs. without) indentation on each side and a rhombic (vs. pentagonal–octagonal or oblong) pseudobulb cross-section. Combined with the geographical distribution and whole plastid genome data, we confirm that D. tetrapterum is not a natural hybrid of the other two species, but a separate species.