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Research Article
Danxiaorchis mangdangshanensis (Orchidaceae, Epidendroideae), a new species from central Fujian Province based on morphological and genomic data
expand article infoMiao Zhang‡, Xiao-Hui Zhang‡, Chang-Li Ge‡, Bing-Hua Chen‡
‡ Fujian Normal University, Fuzhou, China

Corresponding author: Bing-Hua Chen ( bhchen@fjnu.edu.cn )

Academic editor: Timothée Le Péchon
© 2022 Miao Zhang, Xiao-Hui Zhang, Chang-Li Ge, Bing-Hua Chen.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Zhang M, Zhang X-H, Ge C-L, Chen B-H (2022) Danxiaorchis mangdangshanensis (Orchidaceae, Epidendroideae), a new species from central Fujian Province based on morphological and genomic data. PhytoKeys 212: 37-55. https://doi.org/10.3897/phytokeys.212.91534
Open Access

Abstract

Danxiaorchis mangdangshanensis, a new mycoheterotrophic species from Fujian Province, China, is described and illustrated. The new species is morphologically similar to D. singchiana, but its callus of labellum is a less distinctive Y-shape with three auricles on the apex, four pollinia that are narrowly elliptic in shape and equal in size, and it lacks fine roots. The plastome of D. mangdangshanensis is highly degraded. Phylogenetic analyses distinguished D. mangdangshanensis from its congeners, D. singchiana and D. yangii, with strong support based on nrITS + matK and plastomes, respectively.

Keywords

Chloroplast genome, Epidendroideae, morphology, phylogeny, taxonomy

Introduction

The Orchidaceae, one of the largest families of angiosperms, were classified into five subfamilies based on their morphological and molecular characteristics, including Apostasioideae, Cypripedioideae, Vanilloideae, Orchidoideae, and Epidendroideae, with Epidendroideae being the largest (Chase et al. 2015). Identifying orchid species may be challenging, particularly during the vegetative stage when many orchid species plants exhibit very similar morphological characteristics. Moreover, many orchid species can crossbreed successfully across a wide range, giving rise to many intermediate types and natural variants. Therefore, phylogenetic analysis was more and more often employed to investigate the interrelationships among Orchidaceae species (Zhai et al. 2013; Lee et al. 2020; Li et al. 2020).

More than a few Epidendroideae species lack green leaves, resulting in reduced photosynthetic capacities and reliance on mycoheterotrophy for nourishment, i.e., indirectly exploiting other plants through mycorrhizal fungi (Brundrett 2009). Mycoheterotrophic plants, which are classified into two types, photosynthetic mycoheterotrophs and full mycoheterotrophs, are excellent examples of genomic modification due to relaxed selective constraints on photosynthetic function (Barrett et al. 2019). They possess distinct anatomical, physiological, and genomic features. One of these is a reduction in plastid genome size through loss or pseudogenization of photosynthesis-related genes. Early studies (Wolfe et al. 1992a; Wickett et al. 2008) suggested that the gene order of plastomes was conserved and that a large number of conserved genes were present; however, recent studies have revealed highly reduced (Delannoy et al. 2011; Wicke et al. 2013) and highly rearranged (Logacheva et al. 2014) plastomes. These findings indicate that mycoheterotrophic plants may have more diverse plastomes than previously thought.

Danxiaorchis (Calypsoinae, Epidendreae), a recently identified fully mycoheterotrophic orchid genus, was characterized by a distinct Y-shaped callus in its labellum. Only two species of Danxiaorchis have been documented, D. singchiana and D. yangii (Zhai et al. 2013; Yang et al. 2017). The plastid genome size of D. singchiana was found to have been dramatically reduced to 87, 910 dp (Li et al. 2020), however there is no plastome data available for its only congener, D. yangii.

In this paper, we describe a new orchid species found in Mangdang Mountain, Nanping City, in Fujian, China. The plant has a distinct morphology from the other known Danxiaorchis species. On the basis of morphological characteristics and molecular phylogenetic study, we propose a new species of Danxiaorchis and describe it below.

Materials and methods

Morphological description

The morphological description of the new species was based on the study of specimens collected in a variety of spots in 2022. A stereoscopic zoom microscope (Carl Zeiss, Axio zoom. v.16, Germany), equipped with an attached digital camera (Axiocam), and a digital caliper were used to record the sizes of the morphological characters. Field observations provided habitats and phenology for the new species.

DNA extraction and sequencing

In this study, total DNA was extracted from freeze-dried gynostemium and the ovary of the new species using a DNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA). The phylogenetic position of the new species was determined by nrITS and plastid matK sequences. The nrITS (18S-ITS1-5.8S-ITS2-26S) was assembled using GetOrganelle v1.7.5, with -R of 7 and k-merset of “35, 85, 115”, the embplant_nr library was selected as the reference genome database, then annotated and visualized using Geneious v2021.2.2. The plastid matK was extracted from the genome sequence via Geneious v.2021.2.2.

Genome sequencing, assembly, annotation and analysis

Purified total DNA of the new species was fragmented, genome skimming was performed using next-generation sequencing technologies on the Illumina Novaseq 6000 platform with 150 bp paired-end reads and 480 bp insert size by Berry Genomics Co. Ltd. (Beijing, China), and 15.88 GB of reads was obtained.

The paired-end reads were filtered and assembled into complete plastome using a GetOrganelle v1.7.5.0 (Jin et al. 2020a) with appropriate parameters, with K-merset “21,45,65,85,105”, the word size is 0.6. Following previous studies, our workflow includes five key steps as well: 1. Mapping reads to seed and assembling seed-mapped reads for parameter estimation; 2. Recruiting more target-associated reads through extending iterations; 3. Conducting de novo assembly; 4. Roughly filtering fortarget-like contigs; 5. Identifying target contigs and exporting all configurations (Camacho et al. 2009; Bankevich et al. 2012; Langmead and Salzberg 2012; Jin et al. 2020). Graphs of the final assembly were visualized by Bandage (Wick et al. 2015) to assess their completeness. Gene annotation was performed using CPGAVAS2 (Shi et al. 2019) and PGA (Qu et al. 2019). The different annotations of protein coding sequences were confirmed using BLASTx. The tRNAs were checked with tRNAscan-SE v2.0.3. Final chloroplast genome maps were created using OGDRAW.

Phylogenetic analysis

The phylogenetic relationship was constructed using Maximum likelihood (ML) and Bayesian Inference (BI) analyses with the combined ITS and matK sequences. In total, 39 samples of Calypso, Changnienia, Chysis, Corallorhiza, Cremastra, Dactylostalix, Danxiaorchis, Ephippianthus, Govenia, Tipularia, Yoania and Yunorchis were included in our analysis. A species of Chysis was used as outgroup. Each individual locus was aligned using MAFFT 7.310 (Katoh and Standley 2013) with default settings. A concatenated supermatrix of the ITS sequences and matK was generated using PhyloSuitev.1.1.15 (Zhang et al. 2019) for the phylogenetic analysis. All missing data were treated as gaps. The best nucleotide substitution model according to the Bayesian Information Criterion (BIC) was K3Pu+F+R2, which was selected by ModelFinder (Kalyaanamoorthy et al. 2017) implemented in IQTREE v.1.6.8. Maximum likelihood phylogenies were inferred using IQ-TREE (Nguyen et al. 2015) under the model automatically selected by IQ-TREE (‘Auto’ option in IQ-TREE) for 2000 ultrafast (Minh et al. 2013) bootstraps. Bayesian Inference phylogenies were inferred using MrBayes 3.2.6 (Ronquist et al. 2012) under the GTR+F+G4 model (2 parallel runs, 2000000 generations). Phylograms were visualized in iTOLv.5 (iTOL: Interactive Tree Of Life (embl.de)).

To construct a phylogenetic tree based on plastome sequences, a total of 20 plastome sequences of Calypso, Corallorhiza, Cremastra, Danxiaorchis, Cattleya, Anathallis, Masdevallia, Neofinetia and Calanthe were included. Among them, Calypso, Corallorhiza, Cremastra and Danxiaorchis belong to Calypsoinae; Cattleya belongs to Laeliinae; and Anathallis and Masdevallia belong to Pleurothallidinae. Neofinetia falcata and Calanthe triplicata were used as outgroups. Each individual locus was aligned using MAFFT 7.310 (Katoh and Standley 2013) with default settings. The best nucleotide substitution model according to the Bayesian Information Criterion (BIC) was TVM+F+R4, which was selected by ModelFinder (Kalyaanamoorthy et al. 2017) implemented in IQTREE v.1.6.8. Maximum likelihood phylogenies were inferred using IQ-TREE (Nguyen et al. 2015) under the model automatically selected by IQ-TREE (‘Auto’ option in IQ-TREE) for 2000 ultrafast (Minh et al. 2013) bootstraps. Bayesian Inference phylogenies were inferred using MrBayes 3.2.6 (Ronquist et al. 2012) under the GTR+F+I+G4 model (2 parallel runs, 2000000 generations), in which the initial 25% of sampled data were discarded as burn-in. Phylograms were visualized in iTOLv.5 (iTOL: Interactive Tree Of Life (embl.de)).

Results

Comparative analysis of the plastomes

The plastome of Danxiaorchis mangdangshanensis was compared to those of the other 18 species in the subtribe Epidendreae. The plastome size of these species varied greatly from 85,273 bp in D. mangdangshanensis to 157,423 bp in Masdevallia coccinea (a photosynthetic orchid) (Table 1), with the new species being the smallest. The length of the IR region of D. mangdangshanensis was the shortest across all compared species, while the length of the LSC region was slightly longer than that of D. singchiana, but shorter than the remaining species studied. The SSC region of D. mangdangshanensis was intermediate in length compared to those of the other orchid species. The plastome size of mycoheterotrophic species showed high correlation with the size of both the SSC and IR.

Table 1.
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Statistics on the basic features of the plastid genomes of Danxiaorchis mangdangshanensis and related taxa.

Species Accession No. Voucher Number of Genes Length (bp) GC Content (%)
PCGs tRNA rRNA Total LSC SSC IR Total LSC SSC IR
Danxiaorchis mangdangshanensis OP122564 Huang &Chen 32 20 4 85,273 42,605 18,766 11,951 34.41 30.84 37.95 37.99
Danxiaorchis singchiana MN990438 Jin 29 22 4 87,910 42,494 17,890 13,763 34.55 31.12 39.01 36.97
Calypso bulbosa var. occidentalis MG874037 CFB 71 30 4 149,313 84,543 14,846 24,962 37.13 34.54 29.36 43.52
Corallorhiza bentleyi MG874035 Freudenstein 2550 52 31 4 124,482 64,420 10,722 24,670 36.60 32.62 25.81 42.94
Corallorhiza bulbosa KM390013 – 68 30 4 148,643 83,422 15,343 24,939 37.14 34.31 29.16 43.37
Corallorhiza macrantha KM390017 Salazar A 66 30 4 151,031 84,262 12,545 27,112 37.21 34.42 29.38 43.35
Corallorhiza mertensiana KM390018 Freudenstein 1999 54 30 4 147,941 81,109 13,774 26,529 36.78 33.92 28.10 43.41
Corallorhiza odontorhiza KM390021 – 67 30 4 147,317 82,259 13,508 25,775 36.99 34.24 28.28 43.66
Corallorhiza striata MG874034 CFB 47 29 4 141,202 75,701 13,319 26,091 36.34 33.12 27.33 43.27
Corallorhiza trifida MG874036 Freudenstein 2763a 67 30 4 149,376 83,685 15,285 25,203 37.21 34.55 28.99 43.75
Corallorhiza wisteriana KM390020 Freudenstein 2462 67 30 4 146,437 82,350 11,743 26,172 37.05 34.27 28.11 43.43
Cremastra appendiculata MG925366 – 73 30 4 155,320 87,098 15,478 26,372 37.19 34.55 30.41 43.54
Cattleya crispate KP168671 – 71 30 4 148,343 86,254 13,261 24,614 37.26 34.88 29.35 43.36
Cattleya liliputana KP202881 – 71 30 4 147,092 85,804 13,900 23,694 37.35 34.88 30.19 43.45
Anathallis obovata MH979332 UPCB:M.C. Santos 81 30 4 155,515 83,694 20,047 25,542 37.05 34.65 30.05 43.10
Masdevallia coccinea KP205432 – 79 30 4 157,423 84,957 18,448 27,009 36.81 34.42 29.44 43.10
Masdevallia picturata KJ566305 – 80 29 4 156,045 85,145 20,742 25,079 36.88 34.44 29.74 43.22
Neofinetia falcate KT726909 PDBK 67 30 4 156,045 84,948 18,029 26,534 36.64 34.44 29.74 43.22
Calanthe triplicata KF753635 – 80 30 4 132,271 87,263 18,476 26,510 36.74 34.40 29.73 43.03

Phylogenetic analysis

Phylogenetic relationships were first reconstructed by Maximum likelihood (ML) and Bayesian Inference (BI) analyses using combined ITS and matK sequences, as well as the plastome data. The nrITS and matK tree (Fig. 1) clearly indicated the distinctiveness of Danxiaorchis mangdangshanensis from its two congeners, D. singchiana and D. yangii, with strong support (PP = 1, BS = 100), and the new species is closer to D. singchiana. Danxiaorchis is sister to Cremastra, which is consistent with previous studies (Freudenstein et al 2017; Li et al. 2019; 2020). In addition, the phylogenetic analysis based on entire plastomes also separates the new species from D. singchiana with strong support (PP = 1, BS = 100) (Fig. 2).

Figure 1. 

Phylogenetic tree of orchid species in the subtribe Epidendreae based on Bayesian Inference of nrITS and matK sequences. Numbers above and below branches indicate RAxML (left) bootstrap probabilities (BP) and Bayesian (right) posterior probabilities (PP), respectively.

Figure 2. 

Phylogenetic tree of orchid species in the subtribe Epidendreae based on Bayesian Inference of whole plastomes. Numbers above and below branches indicate RAxML (left) bootstrap probabilities (BP) and Bayesian (right) posterior probabilities (PP), respectively.

Taxonomic treatment

Danxiaorchis mangdangshanensis Q. S. Huang, Miao Zhang, B. Hua Chen & Wang Wu, sp. nov.

urn:lsid:ipni.org:names:77307479-1
Figs 3, 4, 5

Diagnosis

Danxiaorchis mangdangshanensis can be easily distinguished from D. singchiana by having no fine roots, fewer flowers in the raceme, the side lobes of the labellum are ivory-white rather than yellow, and it has only 3 colored strips rather than 4–5 pairs. Additionally, its callus is a less distinctive Y-shape and has three auricles, with a purple-red spot on each auricle at the front, and the callus has a remarkable striped appendage adaxially. Furthermore, there are narrow wings on the side of column, and the four pollinia are narrowly elliptic in shape and equal in size.

Figure 3. 

Danxiaorchis mangdangshanensis Q. S. Huang, Miao Zhang, B. Hua Chen & Wang Wu, sp. nov. A flowering and habitat (photographed by Wang Wu) B front view of a flower C-a dorsal sepal C-b lateral sepals C-c petals C-d labellum D gynostemium and labellum, front view, showing three purple-red spots (white arrows) on the Y-shaped callus (red arrows) E gynostemium and labellum, side view, showing three auricles(red arrows) F labellum, showing remarkable striped appendage G gynostemium, showing narrow wings on the both sides (red arrows) H cross section of labellum, showing indistinct Y-shaped callus (red arrows) I anther cap J pollinarium, front view, showing pollinia 4 in 2 pairs. Scale bars: 5 mm (B); 1 cm (C); 4 mm (D); 5 mm (E); 4 mm (F); 1 mm (G); 4 mm (H); 500 μm (I, J).

Type

China. Fujian (福建) Province, Nanping (南平) City, Yanping (延平) District, Mangdangshan Mountain, Mangdangshan National Nature Reserve, forest margins, 26°41'N, 118°2'E, elevation 375 m, 5 May 2022, Q.S. Huang & B. Hua Chen CBH 04593 (Holotype, FNU, barcode FNU0041324; Isotypes, FNU, barcode FNU0041325).

Figure 4. 

Danxiaorchis mangdangshanensis Q. S. Huang, Miao Zhang, B. Hua Chen & Wang Wu, sp. nov. A fruit-bearing plant (photographed by Wang Wu) B infructescence and rhizome C longitudinal section of immature capsule D mature capsule, showing mature seeds (red arrow) E mature seeds. Scale bars: 1 cm (B, C); 1 mm (D, E).

Description

Plant erect, 10.6–22.2 cm tall, holomycotrophic. Rhizome tuberous, fleshy, cylindrical, 2.5–5.3 cm long, 7.0–11.2 mm thick, with short branches, 4.5–5.6 mm long, without roots. Scape terete, pale red-brown, 4.2–5.8 mm thick, 3-sheathed; sheaths cylindrical, clasping stem, membranous, 16.2–43.4 × 4.5–8.7 mm. Inflorescence racemose, 2.9–9.6 cm long, 4- to 10-flowered; floral bracts oblong-lanceolate, 10.5–29.8 × 3.0–11.1 mm, apex acuminate, pale yellow; pedicel and ovary bright yellow,13.8–22.9 mm long, glabrous; sepals yellow, obovate elliptic, dorsal sepals 13.5–17.2 × 4.8–6.5 mm, obtuse; lateral sepals 16.3–18.6 × 5.9–6.7 mm, obtuse; petals yellow, narrowly elliptic, 15.5–19.7 × 6.0–6.5 mm, acute; labellum 3-lobed, with 3 pairs of purple-red stripes on side lobes and purple-red spots on middle lobe; side lobes erect, ivory-white, slightly clasping the column, subsquare, 4.5–5.6 × 5.3–6.2 mm; mid-lobe oblong, 7.8–10.2 × 6.1–7.8 mm, apex acute to obtuse; labellum with two sacs at the base and a fleshy callus centrally, indistinctive Y-shaped (in the transition to “T-shape”), with 3 auricles on the apex, each of which has 1 purple-red spot at the front; callus extending from the base of disc to the base of mid-lobe, triangular at the base of mid-lobe, fleshy, ca. 3.1 mm wide, 0.25 m long, narrows into a raised band when extended, ca. 1.5 mm wide, 0.4 mm long, with sparse purple-red spots; column cream colored, straight, semi-cylindrical, narrow wings on the side, 4.9–6.3 mm long, 2.9 mm wide, footless; stigma concave, triangular, terminal; anther cap ellipsoid, ca. 1.3 mm in diameter; pollinia four, in two pairs, narrowly elliptic, granular-farinaceous, composed of friable massulae, each pair containing two pollinia equal in size with a thick caudicle attached to a common subsquare viscidium, ca. 0.5 mm in diameter. Capsule purple red, fusiform, 3 evident banded ridges, 37.3–46.8 mm long, 8.9–10.1 mm thick. Seeds light dark brown, cylindrical, 1.3 × 0.3 mm, fleshy, honeycombed stripes on the seed coat surface.

Figure 5. 

Danxiaorchis mangdangshanensis Q. S. Huang, Miao Zhang, B. Hua Chen & Wang Wu, sp. nov. A flowering plant B flower, front view C dissection of a flower, showing dorsal sepal, petal, lateral sepal D gynostemium and labellum, front view E gynostemium and labellum, side view F labellum G gynostemium H anther cap I pollinarium J immature capsule K mature seeds. Scale bars: 1.0 cm (A, B, C, J); 0.5 cm (D–F); 0.2 cm (G); 1.0 mm (H, I, K).

Distribution and habitat

Danxiaorchis mangdangshanensis is only found in Mangdangshan National Nature Reserve, Fujian, China (Fig. 6), where it grows at the margin of mid-subtropical evergreen broad-leaved forest, beside a canal near a Musa balbisiana forest. Many other plants grow in the surrounding habitat, whose tree layer includes Castanopsis fargesii Franch. (Fagaceae), C. fissa (Champion ex Bentham) Rehder et E. H. Wilson (Fagaceae), and Vernicia montana Lour. (Euphorbiaceae); the shrub layer includes Ficus erecta Thunb. (Moraceae), F. hirta Vahl (Moraceae), Maesa japonica (Thunb.) Moritzi. ex Zoll. (Primulaceae), Callicarpa kochiana Makino (Lamiaceae), and Aucuba chinensis Benth. (Garryaceae); the vegetation layer includes Angiopteris fokiensis Hieron. (Marattiaceae), Viola diffusa Ging. (Violaceae), Mazus fukienensis Tsoong (Mazaceae), Gynostemma pentaphyllum (Thunb.) Makino (Cucurbitaceae), Iris japonica Thunb. (Iridaceae), Musa balbisiana Colla (Musaceae), and Miscanthus floridulus (Lab.) Warb. ex Schum et Laut. (Poaceae); the interlayer plants include Fissistigma oldhamii (Hemsl.) Merr. (Annonaceae), and Stauntonia obovatifoliola Hayata subsp. urophylla (Hand.-Mazz.) H.N.Qin (Lardizabalaceae).

Figure 6. 

Distribution of Danxiaorchis mangdangshanensis, D. singchiana, D. yangii in China. Legend: (red star) D. mangdangshanensis, (black circle) D. singchiana, (black triangle) D. yangii.

Phenology

Flowering was observed from mid-April to early May, and fruiting from mid-May to mid-June.

Etymology

The Mang dang shan dang xia lang (茫荡山丹霞兰).The epithet mangdangshanensis (茫荡山) refers to Mangdangshan Mountain, Mangdangshan National Nature Reserve, Fujian Province where this new species was found.

Conservation status

During our fieldwork in 2022, three populations of about 14 plants of the new species were found in Mangdangshan National Nature Reserve, Fujian Province, China. And hence, we suggest its placement in the Data Deficient category of IUCN (2022). According to the Updated List of National Key Protected Wild Plants (Decree No. 15) by the country’s State Forestry and Grassland Administration and the Ministry of Agriculture and Rural Affairs, Danxiaorchis are classified in the national secondary protection list. The new recorded genus should also be included on the national secondary protection list during the upcoming revision process.

Characteristics of the Danxiaorchis mangdangshanensis plastome

The highly reduced plastid genome of Danxiaorchis mangdangshanensis still has a quadripartite structure and is 85,273 bp with a large single-copy (LSC) region of 42,605 bp separated from a small single-copy (SSC) region of 18,766 bp by two inverted repeat regions (IRs), each of 11,951 bp (Fig. 7). A total of 56 unique genes were identified in the plastome and it contains 32 protein-coding genes, 20 tRNAs, and four rRNAs. A total of seven genes were duplicated in the IR regions, including rpl22, rps19, trnH-GUG, rpl2, rpl23, trnI-CAU, ycf2 (Table 2). The total GC content of the plastome is 34.40%. Two inversions were detected in the plastome of D. mangdangshanensis (Suppl. material 1: Fig. S1), which are also reported for D. singchiana (Li et al. 2020). The annotated plastome was deposited in GenBank (accession number OP122564).

Table 2.
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Gene contents in the plastid genome of Danxiaorchis mangdangshanensis.

Category, group of Genes Gene names
Photosynthesis:
Subunits of photosystem I psaC, psaI
Subunits of photosystem II –
Subunits of NADH dehydrogenase –
Subunits of cytochrome b/f complex petG, petL, petN
Subunits of ATP synthase –
Large subunit of rubisco –
Subunits photochlorophyllide reductase –
Self-replication:
Proteins of large ribosomal subunit rpl14, rpl16*, rpl2*(2), rpl20, rpl22(2), rpl23(2), rpl32, rpl33, rpl36
Proteins of small ribosomal subunit rps11, rps12**, rps14, rps16*, rps18, rps19 (2), rps2, rps3, rps4, rps7, rps8
Subunits of RNA polymerase –
Ribosomal RNAs rrn16S, rrn23S, rrn4.5S, rrn5S
Transfer RNAs trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnG-GCC, trnH-GUG (2), trnI-CAU (2), trnL-UAA*, trnL-UAG, trnM-CAU, trnN-GUU, trnP-UGG, trnQ-UUG, trnR-ACG, trnS-GGA, trnT-UGU, trnV-GAC, trnW-CCA, trnY-GUA, trnfM-CAU
Other genes:
Maturase matK
Protease clpP**
Envelope membrane protein –
Acetyl-CoA carboxylase accD
c-type cytochrome synthesis gene –
Translation initiation factor infA
Genes of unknown function:
Conserved hypothetical chloroplast ORF ycf1, ycf15, ycf2(2)

Notes: *gene with one introns; **gene with two introns; #Pseudo gene; Gene (2): Number of copies of multi-copy genes.
Figure 7. 

Circular gene map of the plastid genome of Danxiaorchis mangdangshanensis. Genes inside the circle are transcribed clockwise, while those drawn outside are transcribed counterclockwise. Genes are color-coded according to their functional groups. The circle inside the GC content graph marks the 50% threshold.

Discussion

The characteristic Y-shaped callus on its labellum clearly indicates the new species Danxiaorchis mangdangshanensis belongs to the genus Danxiaorchis, and this conclusion was strongly supported by phylogenetic analyses based on combined datasets of ITS and matK, as well as the whole plastome. On the basis of a comprehensive morphological comparison, the new species can be distinguished from its two congeners, D. singchiana and D. yangii (Table 3). It was noticeable that the callus of D. mangdangshanensis had a less distinctive Y-shape, with three auricles on the apex, and a purple-red spot on each auricle at the front. Adaxially, the callus features a unique striped appendage. The Y-shaped callus of D. yangii was remarkably large and had an obovoid appendage at its base adaxially (Yang et al. 2017). Among the mycoheterotrophic taxa of Epidendroideae, Danxiaorchis have four sectile pollinia that are granular-farinaceous, with distinct caudicles and viscidium (Zhai et al. 2013; Yang et al. 2017). This configuration is unique in Epidendreae (i.e., Yoania; Chen et al. 2009), and its possible taxonomic significance awaits further study.

Table 3.
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Morphological and distribution altitude differences between Danxiaorchis mangdangshanensis, D. singchiana and D. yangii.

Characteristics D. mangdangshanensis D. singchiana D. yangii
Roots Branches, no fine roots Fine roots and branches Fine branches, no fine roots
Flowers in the raceme 4–10 6–18 5–30
Color of side lobes of labellum Ivory-white Yellow Yellow
Number of stripes on the side lobes 3 4–5 3
callus Indistinctive Y-shaped, three auricles at the front Distinctive Y-shaped Y-shaped, remarkable large
Front view of the callus 3 distinct purple-red spots None None
Callus adaxially bearing A remarkable striped appendage An obovoid appendage A remarkable obovoid appendage
Size of four pollinia Equal in size Different in size Equal in size
Narrow wings on the side of the stamen column Yes No No
Distribution altitude/m ca. 370 ca.130 ca. 360

The plastome of Danxiaorchis mangdangshanensis was compared to those of the other 18 species in the subtribe Epidendreae. Although the genome sizes of the investigated species varied greatly, they all possessed typical quadripartite structures. This variance in genome size was mostly caused by variations in the length of the IR and SSC regions. The plastome of Danxiaorchis is more “degraded” than those of the other orchid species in the tribe Epidendreae examined, which is mostly due to gene losses associated with mycoheterotrophic habitats. However, the 15 essential genes among orchid plastomes to maintain minimal plastome activity (Kim et al. 2020) were all present in the plastome of D. mangdangshanensis, 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 (5s, 16s, and 23s), trnC-GCA, and clpP genes. The IR region of D. mangdangshanensis was half that of most orchid species studied, and even smaller than its congener, D. singchiana (Li et al. 2020). The IR region plays a role in the structural stability of plastomes and its expansion or contraction due to changes in the amount of repeated DNA and/or changes in sequence complexity (Palmer and Thompson 1982).

The loss of the plastid genes within heterotrophic lineages occurred in a general order. The first was the loss of the NADH dehydrogenase-like (ndh) complex, which may frequently trigger irreversible evolutionary cascade losses of photosynthetic genes (atp, psa/psb, pet, rbcL, ycf3, 4) and a plastid-encoded RNA polymerase (rpo). Followed by the loss of housekeeping genes involved in basic organellar functions such as intron splicing and translation (rpl, rps, rrn, trn, accD, clpP, matK, ycf1, 2) (Barrett and Davis 2012; Kim et al. 2020). In Danxiaorchis mangdangshanensis, the ndh genes have completely disappeared, which is common in mycoheterotrophic orchids. This is interesting because they are also lost or become pseudogenized in photosynthetic orchids, such as Oncidium (Wu et al. 2010) and Phalaenopsis (Chang et al. 2006), raising questions about their significance to photosynthetic chloroplasts. In addition, nearly all photosynthetic genes and the rpo gene were lost in D. mangdangshanensis, representing whole-organismal loss of photosynthetic functions, which thus is a major transitory event in both physiology and genome evolution of the plant. Ycf3 and ycf4, which are crucial to photosystem polypeptide function, were lost in D. mangdangshanensis, although the former was present but has become pseudogene in D. singchiana (Li et al. 2020). Furthermore, several housekeeping genes, including rps15 and some trn genes, were lost in D. mangdangshanensis, which might be due to the increasing dependence on external carbon. It has been hypothesized that perhaps only a few loci, such as tRNA-Glu, tRNA-fMet, are absolutely essential in heterotrophic plants (Barbrook et al. 2006).

Plastid genome evolution in mycoheterotrophic lineages should be of concern in relation to the conservation of these plants, as many of them are rare or endangered (Leake 1994; Merckx and Freudenstein 2010). The mycoheterotrophs represent replicated evolutionary experiments in the loss of photosynthetic function, and its effect on genome evolution. It is evident that photosynthesis-related genes are the first to become pseudogenes or to be deleted in heterotrophic plants (Wolfe et al. 1992b; Wickett et al. 2008; Delannoy et al. 2011; Barrett and Davis 2012; Logacheva et al. 2014; Li et al. 2020). In spite of this, a number of questions remain unanswered regarding the evolution of heterotrophic plastomes, and the current study provides new information on these issues.

Acknowledgements

We are grateful to Ms. D.L. Cai for the illustration, Mr. Wang Wu for the finding and the preceding observation of the new species and Mr. Q.S. Huang for his kind help during our fieldwork. This work was financially supported by the biodiversity investigation programme of Fujian Mangdangshan National Nature Reserve (2022–2025), Special Project of Orchid Survey of National Forestry and Grassland Administration (contract no. 2020-07), the Sub-project VI of National Program on Key Basic Research Project (Grant No. 2015FY110200), the National Special Fund for Chinese medicine resources Research in the Public Interest of China (Grant No.2019-39), the Natural Science Foundation of Fujian Province (2020J05037 to MZ), the Foundation of Fujian Educational Committee (JAT190089 to MZ), and the scientific research innovation program “Xiyuanjiang River Scholarship” of College of Life Sciences, Fujian Normal University (22FSSK018).

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Supplementary material

Supplementary material 1 

Appendix S1

Miao Zhang, Xiao-Hui Zhang, Chang-Li Ge, Bing-Hua Chen

Data type: Docx file.

Explanation note: Figure S1. The two inversions in the plastome of Danxiaorchis mangdangshanensis. Table S1. GenBank information for the taxa used in the present study (matK and nrITS). Table S2. GenBank information for the taxa used in the present study (plastid genome).

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
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