Chloroplast genomic diversity in Bulbophyllum section Macrocaulia (Orchidaceae, Epidendroideae, Malaxideae): Insights into species divergence and adaptive evolution

Abstract

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.

Keywords: Bulbophyllum; Chloroplast genome; DNA markers; Orchidaceae; SSR; Sequence divergence.

Fig. 1

Chloroplast genome map for four Bulbophyllum species. Genes located outside the outer rim are transcribed in a counterclockwise direction, whereas genes inside the outer rim are transcribed in a clockwise direction. The colored bars indicate known different functional groups. The dashed gray area in the inner circle shows the percentage GC contents of the corresponding genes. LSC, SSC, and IR denote large single copy, small single copy, and inverted repeat, respectively.

Fig. 2

Simple sequence repeats (SSRs) in chloroplast genomes of four Bulbophyllum species. (A) Frequency distribution of different types of SSRs in the cp genomes. (B) Frequency of SSRs identified in the coding regions (CDS), intergenic spacers (IGS) and introns of the cp genomes.

Fig. 3

Alignment of the cp genome sequences of four Bulbophyllum species, with B. gedangense as a reference. The X-axis corresponds to coordinates within the cp genome. The Y-axis shows the percentage identity in the range 50%–100%.

Fig. 4

Comparison of the borders between neighboring genes and junctions of LSC, SSC, and IR regions in the chloroplast genomes in four Bulbophyllum species. Boxes above or below the main line indicate genes adjacent to borders.

Fig. 5

Comparative analysis of nucleotide diversity (Pi) values among the cp genome sequences of four Bulbophyllum species. (A) Nucleotide diversity (Pi) values of coding genes in the LSC, SSC, and IR regions; (B) Nucleotide diversity (Pi) values of intergenic regions in the LSC, SSC, and IR regions.

Fig. 6

Non-synonymous (dN), synonymous (dS) and dN/dS average ratios of four Bulbophyllum species.

Fig. 7

Phylogenetic tree conducted using Maximum Likelihood (ML), Maximum Parsimonious (MP) and Bayesian Inference (BI) methods based on 68 protein-coding chloroplast genes from different species. The numbers above branches represent bootstrap percentage (BP) of ML/BP of MP/posterior probability (PP) of BI.