Complete chloroplast genome of eight Phaius (Orchidaceae) species from China: comparative analysis and phylogenetic relationship

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Keywords: Phaius; Chloroplast genome; Intergenetic delimitation; Orchidaceae; Phylogenetic relationship.

Fig. 1

The chloroplast genome maps of eight Phaius species. Genes drawn inside and outside of the circle are transcribed in clockwise and counterclockwise directions separately. Darker and lighter gray area in the inner circle corresponds to the GC and AT content, respectively. Genes belonging to different functional groups are color-coded in the outmost circle

Fig. 2

The total number of SSRs recorded in the cp genomes of eight Phaius species

Fig. 3

The frequency of classified repeat types in the cp genomes of eight Phaius species

Fig. 4

The total number of SSRs identified in the three genomic regions of eight Phaius cp genomes. (A) The total number of SSRs identified in the LSC regions. (B) The total number of SSRs identified in the SSC regions. (C) The total number of SSRs identified in the IRs regions

Fig. 5

Relative synonymous codon usage (RSCU) of eight Phaius species. Amino acid families are labelled on the x-axis. The histogram above each amino acid showing codon usage. Colors in the column graph reflecting codons in the same colors shown below the figure

Fig. 6

Comparison of the boundaries of LSC, SSC and IR regions among cp genomes of the eight Phaius species. The arrow indicated the number of bp representing genes that were distant from a particular region of the cp genome. JLB (LSC/IRb), JSB (IRb/SSC), JSA (SSC/IRa), and JLA (IRa/LSC) denoted the junction sites between each corresponding two regions on the cp genome

Fig. 7

Sequence alignment of complete cp genomes of the eight Phaius species with Phaius columnaris cp genome as a reference. The horizontal axis indicated the coordinates within the cp genome. Arrows up the alignments indicate the direction of the gene. Genome regions were color-coded as exon, rRNA gene, and conserved non-coding sequences (CNS)

Fig. 8

Sliding window analysis of cp genomes of eight Phaius species. (A) Comparison of the nucleotide diversity (Pi) among CDS regions. (B) Comparison of the nucleotide diversity among IGS regions. X-axis: position of the midpoint of a window; Y-axis: nucleotide diversity of each window. Highest variation hotspots for eight cp genomes are annotated on the graph. The colored lines at the bottom delineate these gene locations in different regions

Fig. 9

Phylogenetic tree reconstructed of Collabieae using the maximum likelihood (ML) method based on 71 single-copy CDS sequences from whole cp genomes of 49 Collabieae species as ingroups, with Eria corneri and E. lasiopetala as outgroups. Numbers at nodes indicate ML bootstrap values (BS; before the slash) and Bayesian posterior probabilities (PP; after the slash)

Fig. 10

Floral morphology of the eight Phaius species. A P. columnaris. B P. flavus. C P. mishmensis. D P. takeoi. E P. tancarvilleae. F P. tonkinensis. G P. wallichii. H P. wenshanensis