Background: The taxonomy and infrageneric delimitation of Phalaenopsis Blume has been significantly disputed due to some overlapping morphological features between species related, which needed further evidence for clarification. The structural characterization of complete chloroplast genomes of P. storbatiana and P. wilsonii were analyzed and compared with those of related taxa to provide a better understanding of their genomic information on taxonomy and phylogeny. Results: It was shown that chloroplast genomes of Phalaenopsis storbatiana and P. wilsonii had a typical quadripartite structure with conserved genome arrangements and moderate divergence. The chloroplast genomes of P. storbatiana and P. wilsonii were 145,885 bp and 145,445 bp in length, respectively, and shared a similar GC content of 36.8%. Gene annotations of two species revealed 109 single-copy genes consistently. In addition, 20 genes duplicated in the inverted regions, 16 genes each possessed one or more introns, and five ndh (NA (D)H dehydrogenase) genes were observed in both. Comparative analysis of the total cp genomes of P. storbatiana and P. wilsonii with those of other six related Phalaenopsis species confirmed the stable sequence identity for coding and non-coding regions and higher sequence variation in SC regions than IR regions. Most of their protein-coding genes had a high degree of codon preference. Moreover, 45 genes were discovered with significantly positive selection. However, different amplifications in IR regions were observed in these eight species. Phylogenetic analysis based on CDS from 60 species representing main clades in Orchidaceae indicated that Phalaenopsis species including P. stobartiana and P. wilsonii formed a monophyletic clade with high bootstrap nested in tribe Vandeae of Epidendroideae, which was consistent with those from previous studies. Conclusions: The results could provide insight into understanding the plastome evolution and phylogenetic relationships of Phalaenopsis.

Bulbophyllum is the largest genus in Orchidaceae with a pantropical distribution. Due to highly significant diversifications, it is considered to be one of the most taxonomically and phylogenetically complex taxa. The diversification pattern and evolutionary adaptation of chloroplast genomes are poorly understood in this species-rich genus, and suitable molecular markers are necessary for species determination and phylogenetic analysis. A natural Asian section Macrocaulia was selected to estimate the interspecific divergence of chloroplast genomes in this study. Here, we sequenced the complete chloroplast genome of four Bulbophyllum species, including three species from section Macrocaulia. The four chloroplast genomes had a typical quadripartite structure with a genome size ranged from 156,182 to 158,524 bp. The chloroplast genomes included 113 unique genes encoding 79 proteins, 30 tRNAs and 4 rRNAs. Comparison of the four chloroplast genomes showed that the three species from section Macrocaulia had similar structure and gene contents, and shared a number of indels, which mainly contribute to its monophyly. In addition, interspecific divergence level was also great. Several exclusive indels and polymorphism SSR loci might be used for taxonomical identification and determining interspecific polymorphisms. A total of 20 intergenic regions and three coding genes of the most variable hotspot regions were proposed as candidate effective molecular markers for future phylogenetic relationships at different taxonomical levels and species divergence in Bulbophyllum. All of chloroplast genes in four Bulbophyllum species were under purifying selection, while 13 sites within six genes exhibited site-specific selection. A whole chloroplast genome phylogenetic analysis based on Maximum Likelihood, Bayesian and Parsimony methods all supported the monophyly of section Macrocaulia and the genus of Bulbophyllum. Our findings provide valuable molecular markers to use in accurately identifying species, clarifying taxonomy, and resolving the phylogeny and evolution of the genus Bulbophyllum. The molecular markers developed in this study will also contribute to further research of conservation of Bulbophyllum species.

Genome skimming has dramatically extended DNA barcoding from short DNA fragments to next generation barcodes in plants. However, conserved DNA barcoding markers, including complete plastid genome and nuclear ribosomal DNA (nrDNA) sequences, are inadequate for accurate species identification. Skmer, a recently proposed approach that estimates genetic distances among species based on unassembled genome skims, has been proposed to effectively improve species discrimination rate. In this study, we used Skmer to identify species based on genomic skims of 47 individuals representing 10 out of 13 species of Schima (Theaceae) from China. The unassembled reads identified six species, with a species identification rate of 60%, twice as high as previous efforts that used plastid genomes (27.27%). In addition, Skmer was able to identify Schima species with only 0.5× sequencing depth, as six species were well-supported with unassembled data sizes as small as 0.5 Gb. These findings demonstrate the potential for Skmer approach in species identification, where nuclear genomic data plays a crucial role. For taxonomically difficult taxa such as Schima, which have diverged recently and have low levels of genetic variation, Skmer is a promising alternative to next generation barcodes.

Background: The disputed phylogenetic position of Aerides flabellata Rolfe ex Downie, due to morphological overlaps with related species, was investigated based on evidence of complete chloroplast (cp) genomes. The structural characterization of complete cp genomes of A. flabellata and A. rosea Lodd. ex Lindl. & Paxton were analyzed and compared with those of six related species in “Vanda-Aerides alliance” to provide genomic information on taxonomy and phylogeny. Results: The cp genomes of A. flabellata and A. rosea exhibited conserved quadripartite structures, 148,145 bp and 147,925 bp in length, with similar GC content (36.7 ~ 36.8%). Gene annotations revealed 110 single-copy genes, 18 duplicated in inverted regions, and ten with introns. Comparative analysis across related species confirmed stable sequence identity and higher variation in single-copy regions. However, there are notable differences in the IR regions between two Aerides Lour. species and the other six related species. The phylogenetic analysis based on CDS from complete cp genomes indicated that Aerides species except A. flabellata formed a monophyletic clade nested in the subtribe Aeridinae, being a sister group to Renanthera Lour., consistent with previous studies. Meanwhile, a separate clade consisted of A. flabellata and six Vanda R. Br. species was formed, as a sister taxon to Holcoglossum Schltr. Conclusions: This research was the first report on the complete cp genomes of A. flabellata. The results provided insights into understanding of plastome evolution and phylogenetic relationships of Aerides. The phylogenetic analysis based on complete cp genomes showed that A. flabellata should be placed in Vanda rather than in Aerides.

Phaius Lour. (Collabieae, Orchidaceae) is a small genus consisting of about 45 species, with highly ornamental and medicinal values. However, the phylogenetic relationship of Phaius among Calanthe s. l. has been highly debated based on morphological and molecular data. The complete chloroplast (cp) genome has been widely used as a useful molecular marker for resolving phylogenetic problems, while few genomic data on Phaius was available. Therefore, complete cp genomes of eight Phaius species were sequenced and characterized in detail to provide a better understanding of its phylogenetics in Calanthe s. l. The cp genomes of eight species investigated exhibited conserved quadripartite structures with varied lengths ranging between 157,997 bp to 158,735 bp. The overall GC content of these genomes ranged between 36.82 and 36.97%. Gene annotation revealed 136 genes in all eight genomes, of which 21 were duplicated in inverted regions and 15 with introns. Comparative analysis of eight cp genomes revealed stable sequence identity with greater variation in the single-copy regions, alongside notable differences in the genes at the LSC/IRb and IRb/SSC junctions, as well as in the number of SSRs. The phylogenetic analysis based on CDS from 49 complete cp genomes of Collabieae indicated that the eight Phaius species, together with other two species P. philippinensis and P. hainanensis, were clustered into a monophyletic clade among Calanthe s. l. and divided into two subclades with strong supports. Additionally, it was also supported that Calanthe s. l. should be divided into five genera with strong supports, including Calanthe s. s., Cephalantheropsis, Styloglossum, Phaius, and Preptanthe. It was the first report on the complete cp genome of six Phaius species (P. columnaris, P. mishmensis, P. takeoi, P. tonkinensis, P. wallichii and P. wenshanensis) and has been comparatively analyzed in detail with P. flavus and P. tancarvilleae. It provided a comprehensive investigation of various cp genomic features for phylogenetic implications, including overall genome structure, codon usage, repeat sequences, IR boundaries, DNA polymorphisms, and phylogenetic reconstruction. It was suggested that Phaius and Calanthe s. s. should be treated as two independent genera. The concept of new genus Paraphaius was not confirmed by complete cp genomic data here. The intergeneric relationship of Phaius and its alliance in Calanthe group could be understood better by more cp genomic data.

Dendrobium wattii is a rare and ornamental orchid that is placed in Sect. Formosae of Dendrobium. The complete chloroplast (cp) genome sequence and the genome features of D. wattii were reported for new data on the phylogeny of Dendrobium. The complete cp genome sequence of D. wattii is 159,366 bp in length, including a large single-copy region (LSC, 87,192 bp), a small single-copy region (SSC, 18,422 bp), and two inverted repeat sequences (IRs, 26,876 bp, each). The total content of GC is 37.2%. The cp genome contains 133 genes, consisting of 131 unique genes (79 protein-coding genes, 30 tRNAs, and 4 rRNAs). The phylogenetic analysis suggested that D. wattii be closely related to other species of Dendrobium.