Comparative and phylogenetic analysis of Chiloschista (Orchidaceae) species and DNA barcoding investigation based on plastid genomes

Abstract

Background: Chiloschista (Orchidaceae, Aeridinae) is an epiphytic leafless orchid that is mainly distributed in tropical or subtropical forest canopies. This rare and threatened orchid lacks molecular resources for phylogenetic and barcoding analysis. Therefore, we sequenced and assembled seven complete plastomes of Chiloschista to analyse the plastome characteristics and phylogenetic relationships and conduct a barcoding investigation.

Results: We are the first to publish seven Chiloschista plastomes, which possessed the typical quadripartite structure and ranged from 143,233 bp to 145,463 bp in size. The plastomes all contained 120 genes, consisting of 74 protein-coding genes, 38 tRNA genes and eight rRNA genes. The ndh genes were pseudogenes or lost in the genus, and the genes petG and psbF were under positive selection. The seven Chiloschista plastomes displayed stable plastome structures with no large inversions or rearrangements. A total of 14 small inversions (SIs) were identified in the seven Chiloschista plastomes but were all similar within the genus. Six noncoding mutational hotspots (trnN^GUU-rpl32 > rpoB-trnC^GCA > psbK-psbI > psaC-rps15 > trnE^UUC-trnT^GGU > accD-psaI) and five coding sequences (ycf1 > rps15 > matK > psbK > ccsA) were selected as potential barcodes based on nucleotide diversity and species discrimination analysis, which suggested that the potential barcode ycf1 was most suitable for species discrimination. A total of 47-56 SSRs and 11-14 long repeats (> 20 bp) were identified in Chiloschista plastomes, and they were mostly located in the large single copy intergenic region. Phylogenetic analysis indicated that Chiloschista was monophyletic. It was clustered with Phalaenopsis and formed the basic clade of the subtribe Aeridinae with a moderate support value. The results also showed that seven Chiloschista species were divided into three major clades with full support.

Conclusion: This study was the first to analyse the plastome characteristics of the genus Chiloschista in Orchidaceae, and the results showed that Chiloschista plastomes have conserved plastome structures. Based on the plastome hotspots of nucleotide diversity, several genes and noncoding regions are suitable for phylogenetic and population studies. Chiloschista may provide an ideal system to investigate the dynamics of plastome evolution and DNA barcoding investigation for orchid studies.

Keywords: Aeridinae; Chloroplast genome; DNA barcoding; Phylogenetic analysis; Small inversions.

Fig. 1

The annotation map of seven Chiloschista plastomes. The darker gray in the inner circle corresponds to the GC content. The IRA and IRB (two inverted repeating regions); LSC (large single-copy region); and SSC (Small single-copy region) are indicated outside of the GC content

Fig. 2

Comparison of junctions between the LSC, SSC, and IR regions among seven newly assembled Chiloschista plastomes and Phalaenopsis hygrochila

Fig. 3

Plastome comparison of seven species of Chiloschista and Phalaenopsis hygrochila using a progressive MAUVE algorithm

Fig. 4

Stem-loop structure of fourteen small inversions across seven Chiloschista species. Major types are represented. Details of free energy, sequences, loop length, and subtypes are described in Table S3

Fig. 5

Summary of simple sequence repeats (SSR) across the Chiloschista species. (A) Variation in repeat abundance and type in seven plastomes. (B) Number of SSRs for each Chiloschista species by SSR unit size, and number of SSRs for each Aeridinae species by location in IR, LSC, and SSC

Fig. 6

Global alignment of seven Chiloschista plastomes using mVISTA with Phalaenopsis hygrochila as reference. The y-axis shows the coordinates between the plastomes. The red boxes mean high variation regions in plastome sequence

Fig. 7

Sliding window test of nucleotide diversity (π) in the Chiloschista Plastomes. (A) The nucleotide diversity of complete plastome, six mutation hotspot regions (π > 0.06) were annotated. (B) The nucleotide diversity of 68 protein coding sequence, five mutation hotspot region (π > 0.03). The window size was set to 100 bp and the sliding windows size was 25 bp. X-axis, position of the midpoint of a window; Y-axis, π values of each window

Fig. 8

Phylogenetic tree of Chiloschista and other 15 Aeridinae species based on the complete plastome data. Numbers near the nodes are bootstrap percentages and Bayesian posterior probabilities (BS_ML left, BS_MP middle, and PP right). The nodes without values are 100% bootstrap or 1.00 posterior probability