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

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 (trnNGUU-rpl32 > rpoB-trnCGCA > psbK-psbI > psaC-rps15 > trnEUUC-trnTGGU > 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.

Complete Chloroplast Genomes and Comparative Analyses of Three Paraphalaenopsis (Aeridinae, Orchidaceae) Species.

Paraphalaenopsis, a genus of perennial herbs from the family Orchidaceae, contains a number of ornamental species. However, there is no information on the chloroplast genomes of Paraphalaenopsis, which limits our studies of this genus. In this study, we reported the chloroplast genomes of three species of Paraphalaenopsis (P. labukensis, P. denevel, and P. laycockii 'Semi-alba') and performed comprehensive comparative analysis. These three chloroplast genomes showed a typical quadripartile structure. Their lengths ranged from 147,311 bp to 149,240 bp. Each genome contained 120 unique genes, including 74 protein-coding genes, 38 tRNA genes, and 8 rRNA genes. Comparative analysis revealed major differences in sequence divergence in the three chloroplast genomes. In addition, six hypervariable regions were identified (psbM-trnDGUC, psbB, ccsA, trnKUUU, trnSGCU-trnGUCC, rps16-trnQUUG) that can be used as DNA molecular markers. Phylogenetic relationships were determined using the chloroplast genomes of 28 species from 12 genera of Aeridinae. Results suggested that Paraphalaenopsis was a clade of Aeridinae that was sister to the Holcoglossum-Vanda clade, with 100% bootstrap support within Aeridinae. The findings of this study provided the foundation for future studies on the phylogenetic analysis of Aeridinae.

Comparative Phylogenetic Analysis for Aerides (Aeridinae, Orchidaceae) Based on Six Complete Plastid Genomes.

Aerides Lour. (Orchidaceae, Aeridinae) is a group of epiphytic orchids with high ornamental value, mainly distributed in tropical and subtropical forests, that comprises approximately 20 species. The species are of great value in floriculture and garden designing because of their beautiful flower shapes and colors. Although the morphological boundaries of Aerides are clearly defined, the relationship between Aerides and other closely related genera is still ambiguous in terms of phylogeny. To better understand their phylogenetic relationships, this study used next-generation sequencing technology to investigate the phylogeny and DNA barcoding of this taxonomic unit using genetic information from six Aerides plastid genomes. The quadripartite-structure plastomes ranged from 147,244 bp to 148,391 bp and included 120 genes. Among them, 74 were protein coding genes, 38 were tRNA genes and 8 were rRNA genes, while the ndh genes were pseudogenized or lost. Four non-coding mutational hotspots (rpl20-rpl33, psbM, petB, rpoB-trnCGCA, Pi > 0.06) were identified. A total of 71-77 SSRs and 19-46 long repeats (>30 bp) were recognized in Aerides plastomes, which were mostly located in the large single-copy region. Phylogenetic analysis indicated that Aerides was monophylic and sister to Renanthera. Moreover, our results confirmed that six Aerides species can be divided into three major clades. These findings provide assistance for species identification and DNA barcoding investigation in Aerides, as well as contributes to future research on the phylogenomics of Orchidaceae.

Leafless epiphytic orchids share Ceratobasidiaceae mycorrhizal fungi.

Some epiphytic orchids in the tribe Vandeae are characterized by extremely vestigial leaves (even leafless). Thus, their leaves provide only a small proportion of carbon required for their growth and development, while a large portion of carbon may need to be supplied by their roots and mycorrhizal fungi (MF). The MF richness and composition of leafless epiphytic orchids, which belong to numerous genera with diverse ecophysiologies and wide geographical ranges, remain poorly understood. In this study, we identified the MF communities of seven leafless epiphytic species from three orchid genera from up to 17 sites in China using high-throughput sequencing. Our analyses revealed that the leafless epiphytic orchids have a highly specialized association with Ceratobasidiaceae. Several fungal OTUs were found in three different orchid genera and have promoted germinations of Chiloschista and Phalaenopsis, which may have been caused by convergent evolution of leafless epiphytic orchids. Furthermore, the MF composition of Taeniophyllum glandulosum was significantly affected by collection site and host tree. Our study provides new insights into mycorrhizal associations of epiphytic orchids.

Plastome evolution and phylogeny of subtribe Aeridinae (Vandeae, Orchidaceae).

Subtribe Aeridinae (Vandeae, Epidendroideae, Orchidaceae) consists of 83 genera and 2,345 species. The present study completely decoded the plastomes and nuclear ribosomal (nr) RNA gene clusters of seven species of Aeridinae belonging to Gastrochilus, Neofinetia, Pelatantheria, and Thrixspermum and compared them with existing data to investigate their genome evolution and phylogeny. Although no large structural variations were observed among the Aeridinae plastomes, 14 small inversions (SI) were found in Orchidaceae for the first time. Therefore, the evolutionary trends and usefulness of SI as molecular identification markers were evaluated. Since all 11 ndh genes in the Aeridinae plastome were lost or pseudogenized, the evolutionary trends of ndh genes are discussed at the tribe and family levels. In the maximum likelihood tree reconstructed from 83 plastome genes, the five Orchidaceae subfamilies were shown to have diverged in the following order: Apostasioideae, Vanilloideae, Cypripedioideae, Orchioideae, Epidendroideaeae. Divergence times for major lineages were found to be more recent, 5-10 Mya, than previous studies, which only used two or three genes. Vandeae, which includes Aeridinae, formed a sister group with Cymbidieae and Epidendreae. The Vandeae, Cymbidieae, and Epidendreae lineages were inferred to have diverged at 25.31 Mya; thus, numerous speciation events within Aeridineae occurred since then. Furthermore, the present study reconstructed a phylogenetic tree from 422 nrITS sequences belonging to Aerdinae and allied taxa and uses it to discuss the phylogenetic positions and species identities of five endangered species.

A molecular phylogeny of Aeridinae (Orchidaceae: Epidendroideae) inferred from multiple nuclear and chloroplast regions.

The subtribe Aeridinae, which contains approximately 90 genera, is one of the most diverse and taxonomically puzzling groups in Orchidaceae. In the present study, the phylogenetic relationships of Aeridinae were reconstructed utilizing five DNA sequences (ITS, atpI-H, matK, psbA-trnH, and trnL-F) from 211 taxa in 74 genera. The results of the phylogenetic analyses indicate that Aeridinae is monophyletic and that the subtribe can primarily be grouped into 10 clades: (1) Saccolabium clade, (2) Chiloschista clade, (3) Phalaenopsis clade, (4) Thrixspermum clade, (5) Vanda clade, (6) Aerides clade, (7) Trichoglottis clade, (8) Abdominea clade, (9) Gastrochilus clade, and (10) Cleisostoma clade. In our examination, most genera of Aeridinae were well-supported as monophyletic, and several genera, namely, Pteroceras, Cleisostoma, Vandopsis, Diploprora, Malleola, and Robiquetia, were found to be polyphyletic as currently circumscribed. In addition, several classifications of intra-genera, such as the subgenus Codonosepalum of Taeniophyllum and the section Gastrochilus of Gastrochilus, were also revealed to be paraphyletic. Due to the many questions raised by our phylogenies, the present study may serve as a reference for future taxonomic studies of Aeridinae.

Comparative Analysis of Luisia (Aeridinae, Orchidaceae) Plastomes Shed Light on Plastomes Evolution and Barcodes Investigation.

Luisia, a genus of the subtribe Aeridinae of Orchidaceae, comprises ca. 40 species. Members of Luisia exhibit unique morphological characteristics and represent a valuable ornamental orchid genus. However, due to the scarcity of distinct morphological characters, species identification within this genus is ambiguous and controversial. In the present study, next-generation sequencing (NGS) methods were used to assemble the plastomes of five Luisia species and compare them with one publicly available Luisia plastid genome data. The plastomes of Luisia possessed a quadripartite structure, with sizes ranging from 146,243 bp to 147,430 bp. The plastomes of six Luisia species contained a total of 120 genes, comprising 74 protein-coding genes, 38 tRNA genes and eight rRNA genes. Notably, all ndh genes were pseudogenized or lost. An analysis of codon usage bias showed that leucine (Leu) exhibited the highest frequency, while cysteine (Cys) exhibited the lowest frequency. A total of 57 to 64 SSRs and 42 to 49 long repeats were identified. Five regions and five coding sequences were identified for DNA barcodes, based on the nucleotide diversity (Pi) analysis. The species of Luisia constituted a monophyletic group and were sister to Paraphalaenopsis with strong support. Our study deepens the understanding of species identification, plastome evolution and the phylogenetic positions of Luisia.

The complete plastid genome of Thrixspermum centipeda (Orchidaceae, Aeridinae).

The complete plastid genome of the type species of Thrixspermum, Th. centipeda, was determined and analyzed in this work. The plastome was 147,888 bp in length with 85,899 bp of the large single-copy (LSC) region, 11,055 bp of the small single-copy (SSC) region and 25,467 bp of the invert repeats (IR) regions. The genome contained 120 genes, including 74 protein-coding genes, 38 tRNA genes, and 8 rRNA genes. Phylogenetic analysis divided 18 Aeridinae plastomes into four groups, and Th. centipeda was sister to Th. tsii.