Plastome Evolution and Phylogeny of Orchidaceae, With 24 New Sequences

Abstract

In order to understand the evolution of the orchid plastome, we annotated and compared 124 complete plastomes of Orchidaceae representing all the major lineages in their structures, gene contents, gene rearrangements, and IR contractions/expansions. Forty-two of these plastomes were generated from the corresponding author's laboratory, and 24 plastomes-including nine genera (Amitostigma, Bulbophyllum, Dactylorhiza, Dipodium, Galearis, Gymnadenia, Hetaeria, Oreorchis, and Sedirea)-are new in this study. All orchid plastomes, except Aphyllorchis montana, Epipogium aphyllum, and Gastrodia elata, have a quadripartite structure consisting of a large single copy (LSC), two inverted repeats (IRs), and a small single copy (SSC) region. The IR region was completely lost in the A. montana and G. elata plastomes. The SSC is lost in the E. aphyllum plastome. The smallest plastome size was 19,047 bp, in E. roseum, and the largest plastome size was 178,131 bp, in Cypripedium formosanum. The small plastome sizes are primarily the result of gene losses associated with mycoheterotrophic habitats, while the large plastome sizes are due to the expansion of noncoding regions. The minimal number of common genes among orchid plastomes to maintain minimal plastome activity was 15, including the three subunits of rpl (14, 16, and 36), seven subunits of rps (2, 3, 4, 7, 8, 11, and 14), three subunits of rrn (5, 16, and 23), trnC-GCA, and clpP genes. Three stages of gene loss were observed among the orchid plastomes. The first was ndh gene loss, which is widespread in Apostasioideae, Vanilloideae, Cypripedioideae, and Epidendroideae, but rare in the Orchidoideae. The second stage was the loss of photosynthetic genes (atp, pet, psa, and psb) and rpo gene subunits, which are restricted to Aphyllorchis, Hetaeria, Hexalectris, and some species of Corallorhiza and Neottia. The third stage was gene loss related to prokaryotic gene expression (rpl, rps, trn, and others), which was observed in Epipogium, Gastrodia, Lecanorchis, and Rhizanthella. In addition, an intermediate stage between the second and third stage was observed in Cyrtosia (Vanilloideae). The majority of intron losses are associated with the loss of their corresponding genes. In some orchid taxa, however, introns have been lost in rpl16, rps16, and clpP(2) without their corresponding gene being lost. A total of 104 gene rearrangements were counted when comparing 116 orchid plastomes. Among them, many were concentrated near the IRa/b-SSC junction area. The plastome phylogeny of 124 orchid species confirmed the relationship of {Apostasioideae [Vanilloideae (Cypripedioideae (Orchidoideae, Epidendroideae))]} at the subfamily level and the phylogenetic relationships of 17 tribes were also established. Molecular clock analysis based on the whole plastome sequences suggested that Orchidaceae diverged from its sister family 99.2 mya, and the estimated divergence times of five subfamilies are as follows: Apostasioideae (79.91 mya), Vanilloideae (69.84 mya), Cypripedioideae (64.97 mya), Orchidoideae (59.16 mya), and Epidendroideae (59.16 mya). We also released the first nuclear ribosomal (nr) DNA unit (18S-ITS1-5.8S-ITS2-28S-NTS-ETS) sequences for the 42 species of Orchidaceae. Finally, the phylogenetic tree based on the nrDNA unit sequences is compared to the tree based on the 42 identical plastome sequences, and the differences between the two datasets are discussed in this paper.

Keywords: IR contraction/expansion; Orchidaceae; gene loss; genome rearrangement; plastome evolution.

Figure 1

Plastome length variation in 24 newly sequenced orchid species. The plastome of Gastrodia elata is 35,056 bp long and consists of only one single copy region. The species names in red indicate mycoheterotrophic species.

Figure 2

Diagram of nuclear ribosomal (nr) DNA repeat units consisting of 18S-ITS1-5.8S-ITS2-28S-NTS-ETS. The total length of the unit is approximately 10 kb, and it is arranged as a tandem repeat. The nrDNA repeat sequences of 42 orchid species were first reported in this paper.

Figure 3

Six representative plastomes of Orchidaceae. Cypripedium formosanum has the longest plastome (178.1 kb), while Epipogium roseum has the shortest. Gastrodia elata and Epipogium roseum hold 27 genes in their plastome, even though they have substantially different plastome sizes. The heavy vertical bars indicate inverted repeat (IR) regions and the broken lines among plastomes indicate the boundaries of IR and single copy regions.

Figure 4

Relationships between plastome lengths and gene numbers. (A): Terrestrial orchids show a wider range of variation than epiphytic orchids. (B): Mycoheterotrophic orchids show a wider range of variation than photosynthetic orchids. (C, D): Plastome lengths are more strongly correlated with LSC lengths than IR lengths in both mycoheterotrophic and photosynthetic orchids.

Figure 5

Distribution patterns of gene loss in Orchidaceae. The dark blue, light blue, and white blocks indicate presence, pseudogene, and absence of each gene, respectively. The non-functionalization of 11 ndh genes are distributed widely across all taxonomic groups of Orchidaceae. This frequently occurs in Epidendroideae and Vanilloideae. The non-functionalization of psa, psb, pet, and rpo gene classes are confined to mycoheterotrophic lineages. In addition, the loss of housekeeping genes such as the rps, rpl, or trn gene classes occur independently in four genera, Epipogium, Gastrodia, Lecanorchis, and Rhizanthella. The species names in red indicate mycoheterotrophic orchids.

Figure 6

Evolution of gene losses in the phylogenetic tree of Orchidaceae. A total of 129 taxa—124 Orchidaceae and five outgroup taxa—were the subject of tree reconstruction. The sequences of 83 protein coding genes were concatenated to a length of 87,399 bp. A maximum likelihood (ML) tree was constructed using RaxML-HPC2 with a GTR+G+I model (ML = −672774.637133 of ML optimization likelihood). All the genes were then plotted on the tree node using parsimony criteria under the condition of no parallel gains of the same gene. The species names in red indicate mycoheterotrophic orchids. (A): The basal portion of the tree showing the subfamilies Apostasioideae, Vanilloideae, Cypripedioideae, and Orchidoideae. (B): The upper portions of the tree showing the subfamily Epidendroideae.

Figure 7

Distribution patterns of the loss of 20 introns in Orchidaceae. The dark blue, light blue, and white blocks indicate intron presence, absence without corresponding gene loss, and absence with corresponding gene loss, respectively.

Figure 8

Distribution of gene blocks. A total of 134 gene blocks were recognized in the 124 orchid plastomes. Thirty were constant blocks. 104 blocks show both forward (dark blue) and reverse (red) orientations and confirmed the block rearrangements. Blocks that are non-applicable because of gene losses or shifts are indicated in gray. All large rearrangements longer than 15 kb are located on the LSC. The species names in red indicate mycoheterotrophic orchids. (A): Forward and reverse orientations of each gene block in the orchid species. (B): Locations, lengths, and frequencies of 134 gene blocks.

Figure 9

Comparison of a plastid tree and nrDNA tree for the same 42 orchid species. The 83 aligned protein coding genes were 80,798 bp long. A maximum likelihood (ML) tree was constructed using RaxML-HPC2 with a GTR+G+I model with 100 bootstrap replicates. The best plastid tree was obtained with ML = −332414.394814. The nrDNA unit (18S-ITS1-5.8S-ITS2-28S) was approximately 10 kb long. The tree reconstruction methods for nrDNA were identical to those of the plastid tree. The species names in red indicate mycoheterotrophic orchids. The lines between the two trees indicate the topological differences between them.

Figure 10

Fossil data showing the estimated divergence time of each node. Three fossil data were used to calibrate nodes (Asparagales—mean 105.3 mya, Dendrobium—23.2 mya, and Goodyera—15.0 mya). Orchidaceae diverged from its sister family at 99.20 mya, and then five subfamilies subsequently diverged in the order of Apostasioideae (79.91 mya), Vanilloideae (69.84 mya), Cypripedioideae (64.97 mya), Orchidoideae (59.16 mya), and Epidendroideae (59.16 mya). However, several specious subtribes within the Epidendroideae diverged in relatively short time periods (39.57–28.13 mya). The species names in red indicate mycoheterotrophic orchids.