The complete chloroplast genome sequence of Isoetes baodongii (Isoetaceae).

Isoetes baodongii is a diploid species of Isoetaceae distributed in low altitude area, its megaspore ornamentation is similar to tetraploid species I. sinensis. We collected leaf material of I. baodongii and sequenced it for low depth whole genome sequence, then, a complete chloroplast genome of I. baodongii was assembled and annotated. This chloroplast genome has a circular structure of 145,494 bp in length with a GC content of 38.0%, comprising a large single copy (LSC) region of 91,860 bp, a pair of inverted repeat (IR) regions of 13,207 bp each, and a small single copy (SSC) region of 27,220 bp. 136 genes were annotated, including 84 protein-coding genes, 38 tRNA genes, and 8 rRNA genes. A maximum likelihood phylogeny tree was reconstructed after the sequences alignment, the result showed that I. baodongii formed a sister clade to the one clustered by I. sinensis, I. taiwanensis and I. orientalis. Although the chloroplast genome structure of I. baodongii is extremely similar to other species distributed in China, a well-supported phylogenetic relationship was reconstructed here, these results may provide new messages for further studies on phylogeny and evolution of vascular plant on the earth.

Genome-Wide Identification and Expression Pattern Analysis of TIFY Family Genes Reveal Their Potential Roles in Phalaenopsis aphrodite Flower Opening.

The TIFY gene family (formerly known as the zinc finger proteins expressed in inflorescence meristem (ZIM) family) not only functions in plant defense responses but also are widely involved in regulating plant growth and development. However, the identification and functional analysis of TIFY proteins remain unexplored in Orchidaceae. Here, we identified 19 putative TIFY genes in the Phalaenopsis aphrodite genome. The phylogenetic tree classified them into four subfamilies: 14 members from JAZ, 3 members from ZML, and 1 each from PPD and TIFY. Sequence analysis revealed that all Phalaenopsis TIFY proteins contained a TIFY domain. Exon-intron analysis showed that the intron number and length of Phalaenopsis TIFY genes varied, whereas the same subfamily and subgroup genes had similar exon or intron numbers and distributions. The most abundant cis-elements in the promoter regions of the 19 TIFY genes were associated with light responsiveness, followed by MeJA and ABA, indicating their potential regulation by light and phytohormones. The 13 candidate TIFY genes screened from the transcriptome data exhibited two types of expression trends, suggesting their different roles in cell proliferation and cell expansion of floral organ growth during Phalaenopsis flower opening. Overall, this study serves as a background for investigating the underlying roles of TIFY genes in floral organ growth in Phalaenopsis.

Phylogenomics and biogeographical diversification of Collabieae (Orchidaceae) and its implication in the reconstruction of the dynamic history of Asian evergreen broadleaved forests.

The tribe Collabieae (Epidendroideae, Orchidaceae) comprises approximately 500 species. Generic delimitation within Collabieae are confusing and phylogenetic interrelationships within the Collabieae have not been well resolved. Plastid genomes and nuclear internal transcribed spacer (ITS) sequences were used to estimate the phylogenetic relationships, ancestral ranges, and diversification rates of Collabieae. The results showed that Collabieae was subdivided into nine clades with high support. We proposed to combine Ancistrochilus and Pachystoma into Spathoglottis, merge Collabium and Chrysoglossum into Diglyphosa, and separate Pilophyllum and Hancockia as distinctive genera. The diversification of the nine clades of Collabieae might be associated with the uplift of the Himalayas during the Late Oligocene/Early Miocene. The enhanced East Asian summer monsoon in the Late Miocene may have promoted the rapid diversification of Collabieae at a sustained high diversification rate. The increased size of terrestrial pseudobulbs may be one of the drivers of Collabieae diversification. Our results suggest that the establishment and development of evergreen broadleaved forests facilitated the diversification of Collabieae.

Organelle Genomes of Epipogium roseum Provide Insight into the Evolution of Mycoheterotrophic Orchids.

Epipogium roseum, commonly known as one of the ghost orchids due to its rarity and almost transparent color, is a non-photosynthetic and fully mycoheterotrophic plant. Given its special nutritional strategies and evolutionary significance, the mitogenome was first characterized, and three plastomes sampled from Asia were assembled. The plastomes were found to be the smallest among Orchidaceae, with lengths ranging from 18,339 to 19,047 bp, and exhibited high sequence variety. For the mitogenome, a total of 414,552 bp in length, comprising 26 circular chromosomes, were identified. A total of 54 genes, including 38 protein-coding genes, 13 tRNA genes, and 3 rRNA genes, were annotated. Multiple repeat sequences spanning a length of 203,423 bp (45.47%) were discovered. Intriguingly, six plastid regions via intracellular gene transfer and four plastid regions via horizontal gene transfer to the mitogenome were observed. The phylogenomics, incorporating 90 plastomes and 56 mitogenomes, consistently revealed the sister relationship of Epipogium and Gastrodia, with a bootstrap percentage of 100%. These findings shed light on the organelle evolution of Orchidaceae and non-photosynthetic plants.

Comparative chloroplast genomics of 24 species shed light on the genome evolution and phylogeny of subtribe Coelogyninae (Orchidaceae).

BACKGROUND: The orchids of the subtribe Coelogyninae are among the most morphologically diverse and economically important groups within the subfamily Epidendroideae. Previous molecular studies have revealed that Coelogyninae is an unambiguously monophyletic group. However, intergeneric and infrageneric relationships within Coelogyninae are largely unresolved. There has been long controversy over the classification among the genera within the subtribe. RESULTS: The complete chloroplast (cp.) genomes of 15 species in the subtribe Coelogyninae were newly sequenced and assembled. Together with nine available cp. genomes in GenBank from representative clades of the subtribe, we compared and elucidated the characteristics of 24 Coelogyninae cp. genomes. The results showed that all cp. genomes shared highly conserved structure and contained 135 genes arranged in the same order, including 89 protein-coding genes, 38 tRNAs, and eight rRNAs. Nevertheless, structural variations in relation to particular genes at the IR/SC boundary regions were identified. The diversification pattern of the cp. genomes showed high consistency with the phylogenetic placement of Coelogyninae. The number of different types of SSRs and long repeats exhibited significant differences in the 24 Coelogyninae cp. genomes, wherein mononucleotide repeats (A/T), and palindromic repeats were the most abundant. Four mutation hotspot regions (ycf1a, ndhF-rp132, psaC-ndhE, and rp132-trnL) were determined, which could serve as effective molecular markers. Selection pressure analysis revealed that three genes (ycf1a, rpoC2 and ycf2 genes) might have experienced apparent positive selection during the evolution. Using the alignments of whole cp. genomes and protein-coding sequences, this study presents a well-resolved phylogenetic framework of Coelogyninae. CONCLUSION: The inclusion of 55 plastid genome data from a nearly complete generic-level sampling provide a comprehensive view of the phylogenetic relationships among genera and species in subtribe Coelogyninae and illustrate the diverse genetic variation patterns of plastid genomes in this species-rich plant group. The inferred relationships and informally recognized major clades within the subtribe are presented. The genetic markers identified here will facilitate future studies on the genetics and phylogeny of subtribe Coelogyninae.

Establishment of a Rapid and Effective Agrobacterium-Mediated Genetic Transformation System of Oxalis triangularis 'Purpurea'.

Oxalis triangularis 'Purpurea' has significant ornamental value in landscaping. There is a critical necessity to elucidate the gene functions of O. triangularis 'Purpurea' and dissect the molecular mechanisms governing key ornamental traits. However, a reliable genetic transformation method remains elusive. In this study, our investigation revealed that various transformation parameters, including recipient material (petioles), pre-culture time (2-5 days), acetosyringone (AS) concentration (100-400 µM), Agrobacterium concentrations (OD600 = 0.4-1.0), infection time (5-20 min), and co-culture time (2-5 days), significantly impacted the stable genetic transformation in O. triangular 'Purpurea'. Notably, the highest genetic transformation rate was achieved from the leaf discs pre-cultured for 3 days, treated with 200 µM AS infected with Agrobacterium for 11 min at OD600 of 0.6, and subsequently co-cultured for 3 days. This treatment resulted in a genetic transformation efficiency of 9.88%, and it only took 79 days to produce transgenic plants. Our transformation protocol offers advantages of speed, efficiency, and simplicity, which will greatly facilitate genetic transformation for O. triangular 'Purpurea' and gene function studies.

Genome-Wide Identification and Expression Analysis of WNK Kinase Gene Family in Acorus.

WNK (With No Lysine) kinases are members of serine/threonine protein kinase family, which lack conserved a catalytic lysine (K) residue in protein kinase subdomain II and this residue is replaced by either asparagine, serine, or glycine residues. They are involved in various physiological regulations of flowering time, circadian rhythms, and abiotic stresses in plants. In this study, we identified the WNK gene family in two species of Acorus, and analyzed their phylogenetic relationship, physiochemical properties, subcellular localization, collinearity, and cis-elements. The results showed twenty-two WNKs in two Acorus (seven in Ac. gramineus and fifteen in Ac. calamus) have been identified and clustered into five main clades phylogenetically. Gene structure analysis showed all WNKs possessed essential STKc_WNK or PKc_like superfamily domains, and the gene structures and conserved motifs of the same clade were similar. All the WNKs harbored a large number of light response elements, plant hormone signaling elements, and stress resistance elements. Through a collinearity analysis, two and fourteen segmental duplicated gene pairs were identified in the Ac. gramineus and Ac. calamus, respectively. Moreover, we observed tissue-specificity of WNKs in Acorus using transcriptomic data, and their expressions in response to salt stress and cold stress were analyzed by qRT-PCR. The results showed WNKs are involved in the regulation of abiotic stresses. There were significant differences in the expression levels of most of the WNKs in the leaves and roots of Acorus under salt stress and cold stress, among which two members in Ac. gramineus (AgWNK3 and AgWNK4) and two members in Ac. calamus (AcWNK8 and AcWNK12) were most sensitive to stress. In summary, this paper will significantly contribute to the understanding of WNKs in monocots and thus provide a set up for functional genomics studies of WNK protein kinases.

Origin and diversification of a Himalayan orchid genus Pleione.

Pleione is an orchid endemically distributed in high mountain areas across the Hengduan Mountains (HDM), Himalayas, Southeast Asia and South of China. The unique flower shapes, rich colors and immense medicinal importance of Pleione are valuable ornamental and economic resources. However, the phylogenetic relationships and evolutionary history of the genus have not yet been comprehensively resolved. Here, the evolutionary history of Pleione was investigated using single-copy gene single nucleotide polymorphisms and chloroplast genome datasets. The data revealed that Pleione could be divided into five clades. Discordance in topology between the two phylogenetic trees and network and D-statistic analyses indicated the occurrence of reticulate evolution in the genus. The evolution could be attributed to introgression and incomplete lineage sorting. Ancestral area reconstruction suggested that Pleione was originated from the HDM. Uplifting of the HDM drove rapid diversification by creating conditions favoring rapid speciation. This coincided with two periods of consolidation of the Asian monsoon climate, which caused the first rapid diversification of Pleione from 8.87 to 7.83 Mya, and a second rapid diversification started at around 4.05 Mya to Pleistocene. The interaction between Pleione and climate changes, especially the monsoons, led to the current distribution pattern and shaped the dormancy characteristic of the different clades. In addition to revealing the evolutionary relationship of Pleione with orogeny and climate changes, the findings of this study provide insights into the speciation and diversification mechanisms of plants in the East Asian flora.

Nuclear phylogeny and insights into whole-genome duplications and reproductive development of Solanaceae plants.

Solanaceae, the nightshade family, have ∼2700 species, including the important crops potato and tomato, ornamentals, and medicinal plants. Several sequenced Solanaceae genomes show evidence for whole-genome duplication (WGD), providing an excellent opportunity to investigate WGD and its impacts. Here, we generated 93 transcriptomes/genomes and combined them with 87 public datasets, for a total of 180 Solanaceae species representing all four subfamilies and 14 of 15 tribes. Nearly 1700 nuclear genes from these transcriptomic/genomic datasets were used to reconstruct a highly resolved Solanaceae phylogenetic tree with six major clades. The Solanaceae tree supports four previously recognized subfamilies (Goetzeioideae, Cestroideae, Nicotianoideae, and Solanoideae) and the designation of three other subfamilies (Schizanthoideae, Schwenckioideae, and Petunioideae), with the placement of several previously unassigned genera. We placed a Solanaceae-specific whole-genome triplication (WGT1) at ∼81 million years ago (mya), before the divergence of Schizanthoideae from other Solanaceae subfamilies at ∼73 mya. In addition, we detected two gene duplication bursts (GDBs) supporting proposed WGD events and four other GDBs. An investigation of the evolutionary histories of homologs of carpel and fruit developmental genes in 14 gene (sub)families revealed that 21 gene clades have retained gene duplicates. These were likely generated by the Solanaceae WGT1 and may have promoted fleshy fruit development. This study presents a well-resolved Solanaceae phylogeny and a new perspective on retained gene duplicates and carpel/fruit development, providing an improved understanding of Solanaceae evolution.

Two new bibenzyls from Pleione grandiflora (Rolfe) Rolfe and their antioxidant activity.

Two new bibenzyls (1 and 2) were isolated from the pseudobulbs of Pleione grandiflora (Rolfe) Rolfe along with six known compounds, including isoarundinin I (3), isoarundinin II (4), bulbocodin D (5), batatasin III (6), 5,3'-dihydroxy- 4-(p-hydroxybenzyl)-3-methoxybibenzyl (7) and shancigusin F (8). Their structures were established on the basis of spectroscopic methods. These compounds showed potent DPPH free radical scavenging effects with IC50 values ranging from 49.72 ± 0.35 µM to 65.41 ± 0.49 µM.

Population dynamics of Phaius flavus in southeast China: Reproductive strategies and plants conservation.

Because of species diversity and troubling conservation status in the wild, Orchidaceae has been one of the taxa with most concern in population ecological research for a long time. Although Orchidaceae is a group with high adaptability, they have become endangered for complex and various reasons such as the germination? difficulty and habitat loss, which makes it difficult to develop an accurate protection strategy. Phaius flavus is a terrestrial orchid which used to be widely distributed in central and southern Asia; however, large populations are difficult to find in the wild. Thus, the aim of this study was to provide a new perspective for conserving endangered P. flavus by investigating the mechanisms of its population decline; we established time-specific life and fertility tables, age pyramids, survival curves, and mortality curves for this plant and then conducted Leslie matrix model. We found that both of the populations from Wuhu Mount (WM) and Luohan Mount (LM) showed declining trends and exhibited pot-shaped age pyramids, low net reproductive rates, and negative intrinsic growth rates. The population from the Beikengding Mount (BM) showed a stable status with a bell-shaped age pyramid. However, it has a significant risk of decline because of the low net reproductive rate and intrinsic growth rate. This study use time-specific life and fertility tables, age pyramids, survival curves, and mortality curves, showed that the population decline of P. flavus could be attributed to 1) the shortage of seedlings caused by the low germination rate in the wild and 2) the loss of adult individuals caused by anthropogenic disturbances. To protect this species from extinction in these areas, we suggest that human activities in these habitats should be strictly forbidden and ex situ conservation of this plant in botanical gardens is also necessary.

Genomes of leafy and leafless Platanthera orchids illuminate the evolution of mycoheterotrophy.

To improve our understanding of the origin and evolution of mycoheterotrophic plants, we here present the chromosome-scale genome assemblies of two sibling orchid species: partially mycoheterotrophic Platanthera zijinensis and holomycoheterotrophic Platanthera guangdongensis. Comparative analysis shows that mycoheterotrophy is associated with increased substitution rates and gene loss, and the deletion of most photoreceptor genes and auxin transporter genes might be linked to the unique phenotypes of fully mycoheterotrophic orchids. Conversely, trehalase genes that catalyse the conversion of trehalose into glucose have expanded in most sequenced orchids, in line with the fact that the germination of orchid non-endosperm seeds needs carbohydrates from fungi during the protocorm stage. We further show that the mature plant of P. guangdongensis, different from photosynthetic orchids, keeps expressing trehalase genes to hijack trehalose from fungi. Therefore, we propose that mycoheterotrophy in mature orchids is a continuation of the protocorm stage by sustaining the expression of trehalase genes. Our results shed light on the molecular mechanism underlying initial, partial and full mycoheterotrophy.

Integration of the metabolome and transcriptome reveals indigo biosynthesis in Phaius flavus flowers under freezing treatment.

Background: Indigo-containing plant tissues change blue after a freezing treatment, which is accompanied by changes in indigo and its related compounds. Phaius flavus is one of the few monocot plants containing indigo. The change to blue after freezing was described to explore the biosynthesis of indigo in P. flavus. Methods: In this study, we surveyed the dynamic change of P. flavus flower metabolomics and transcriptomics. Results: The non-targeted metabolomics and targeted metabolomics results revealed a total of 98 different metabolites, the contents of indole, indican, indigo, and indirubin were significantly different after the change to blue from the freezing treatment. A transcriptome analysis screened ten different genes related to indigo upstream biosynthesis, including three anthranilate synthase genes, two phosphoribosyl-anthranilate isomerase genes, one indole-3-glycerolphosphate synthase gene, five tryptophan synthase genes. In addition, we further candidate 37 cytochrome P450 enzyme genes, one uridine diphosphate glucosyltransferase gene, and 24 ß-D-glucosidase genes were screened that may have participated in the downstream biosynthesis of indigo. This study explained the changes of indigo-related compounds at the metabolic level and gene expression level during the process of P. flavus under freezing and provided new insights for increasing the production of indigo-related compounds in P. flavus. In addition, transcriptome sequencing provides the basis for functional verification of the indigo biosynthesis key genes in P. flavus.

Genome-Wide Identification and Expression Profiles of bZIP Genes in Cannabis sativa L.

Background: The bZIP gene family plays roles in biotic and abiotic stress, secondary metabolism, and other aspects in plants. They have been reported in Arabidopsis thaliana, Oryza sativa, Artemisia annua, and other plants, but their roles in Cannabis sativa have not been determined. Materials and Methods: In this study, we analyzed the genome-wide identification and expression profile of the bZIP gene family in C. sativa. Results: A total of 51 members of the bZIP gene family were identified based on the C. sativa genome and numbered in order from CsbZIP1 to CsbZIP51. Their phylogenetic relationships, cis-elements in promoter region, gene structures and motif compositions, physicochemical properties, chromosome locations, and expression profiles, were analyzed. The results showed that the 51 CsbZIPs were unevenly distributed on 10 chromosomes and could be clustered into 11 subfamilies. Furthermore, CsbZIPs located in the same subfamilies presented similar intron/exon organization and motif composition. The expression levels of CsbZIPs in various tissues (flowers, bracts, vegetative leaves, stems, and seeds) were determined using reverse transcription quantitative polymerase chain reaction. The expression levels of CsbZIPs were higher in flowers and bracts. The 51 CsbZIPs were explored, and their structure, evolution, and expression pattern in different tissues of C. sativa were characterized synthetically. The findings indicated that CsbZIPs are essential for the growth and development of C. sativa. Conclusions: These results provide a theoretical basis for subsequent research on hemp bZIP transcription factors and the cultivation of high-cannabidiol and low-tetrahydrocannabinol high-quality cannabis varieties.

Genome-wide identification and expression profile of YABBY genes in Averrhoa carambola.

BACKGROUND: Members of the plant-specific YABBY gene family are thought to play an important role in the development of leaf, flower, and fruit. The YABBY genes have been characterized and regarded as vital contributors to fruit development in Arabidopsis thaliana and tomato, in contrast to that in the important tropical economic fruit star fruit (Averrhoa carambola), even though its genome is available. METHODS: In the present study, a total of eight YABBY family genes (named from AcYABBY1 to AcYABBY8) were identified from the genome of star fruit, and their phylogenetic relationships, functional domains and motif compositions, physicochemical properties, chromosome locations, gene structures, protomer elements, collinear analysis, selective pressure, and expression profiles were further analyzed. RESULTS: Eight AcYABBY genes (AcYABBYs) were clustered into five clades and were distributed on five chromosomes, and all of them had undergone negative selection. Tandem and fragment duplications rather than WGD contributed to YABBY gene number in the star fruit. Expression profiles of AcYABBYs from different organs and developmental stages of fleshy fruit indicated that AcYABBY4 may play a specific role in regulating fruit size. These results emphasize the need for further studies on the functions of AcYABBYs in fruit development.

The Melastoma dodecandrum genome and the evolution of Myrtales.

Melastomataceae has abundant morphological diversity with high economic and ornamental merit in Myrtales. The phylogenetic position of Myrtales is still contested. Here, we report the chromosome-level genome assembly of Melastoma dodecandrum in Melastomataceae. The assembled genome size is 299.81 Mb with a contig N50 value of 3.00 Mb. Genome evolution analysis indicated that M. dodecandrum, Eucalyptus grandis, and Punica granatum were clustered into a clade of Myrtales and formed a sister group with the ancestor of fabids and malvids. We found that M. dodecandrum experienced four whole-genome polyploidization events: the ancient event was shared with most eudicots, one event was shared with Myrtales, and the other two events were unique to M. dodecandrum. Moreover, we identified MADS-box genes and found that the AP1-like genes expanded, and AP3-like genes might have undergone subfunctionalization. The SUAR63-like genes and AG-like genes showed different expression patterns in stamens, which may be associated with heteranthery. In addition, we found that LAZY1-like genes were involved in the negative regulation of stem branching development, which may be related to its creeping features. Our study sheds new light on the evolution of Melastomataceae and Myrtales, which provides a comprehensive genetic resource for future research.

Comparative analysis of plastomes in Oxalidaceae: Phylogenetic relationships and potential molecular markers.

The wood sorrel family, Oxalidaceae, is mainly composed of annual or perennial herbs, a few shrubs, and trees distributed from temperate to tropical zones. Members of Oxalidaceae are of high medicinal, ornamental, and economic value. Despite the rich diversity and value of Oxalidaceae, few molecular markers or plastomes are available for phylogenetic analysis of the family. Here, we reported four new whole plastomes of Oxalidaceae and compared them with plastomes of three species in the family, as well as the plastome of Rourea microphylla in the closely related family Connaraceae. The eight plastomes ranged in length from 150,673 bp (Biophytum sensitivum) to 156,609 bp (R. microphylla). Genome annotations revealed a total of 129-131 genes, including 83-84 protein-coding genes, eight rRNA genes, 37 tRNA genes, and two to three pseudogenes. Comparative analyses showed that the plastomes of these species have minor variations at the gene level. The smaller plastomes of herbs B. sensitivum and three Oxalis species are associated with variations in IR region sizes, intergenic region variation, and gene or intron loss. We identified sequences with high variation that may serve as molecular markers in taxonomic studies of Oxalidaceae. The phylogenetic trees of selected superrosid representatives based on 76 protein-coding genes corroborated the Oxalidaceae position in Oxalidales and supported it as a sister to Connaraceae. Our research also supported the monophyly of the COM (Celastrales, Oxalidales, and Malpighiales) clade.

Phylogenetic estimation and morphological evolution of Alsineae (Caryophyllaceae) shed new insight into the taxonomic status of the genus Pseudocerastium.

Pseudocerastium is a monotypic genus in Caryophyllaceae endemic to China. The genus has been widely accepted since it was described in 1998, however its phylogenetic position within Caryophyllaceae has never been studied. In the present study, the whole plastid genome and nuclear ribosomal internal transcribed spacer (ITS) sequences of Pseudocerastium stellarioides was obtained through genome skimming, and the phylogenetic position of the species was studied for the first time. Plastid phylogenomic analysis of Caryophyllaceae revealed that Pseudocerastium is clustered within the tribe Alsineae with strong support. Phylogenetic analyses based on an enlarged taxon sampling of Alsineae using five DNA regions (matK, rbcL, rps16 intron, trnL-F and ITS) revealed that P. stellarioides was nested deeply within Cerastium with strong support. Analyses of morphological character evolution suggest that the ancestral states in Alsineae include three styles and a six-lobed capsule at the apex, while both Cerastium and Pseudocerastium have five styles and ten lobes at the apex of the capsule, further supporting their close relationship. The species Pseudocerastium stellarioides is similar to Cerastium wilsonii in morphology, but differs in having villous indumentum on the lower part of the filaments and compressed globose seeds. Therefore, based on the present molecular and morphological evidence, the generic name Pseudocerastium is reduced here as a new synonym of Cerastium and the species P. stellarioides is transferred to Cerastium as C. jiuhuashanense.

Phylogenetic incongruence in Cymbidium orchids.

Cymbidium, which includes approximately 80 species, is one of the most ornamental and cultivated orchid genera. However, a lack of markers and sparse sampling have posed great challenges to resolving the phylogenetic relationships within the genus. In the present study, we reconstructed the phylogenetic relationships by utilizing one nuclear DNA (nrITS) and seven plastid genes (rbcL, trnS, trnG, matK, trnL, psbA, and atpI) from 70 species (varieties) in Cymbidium. We also examined the occurrence of phylogenetic conflict between nuclear (nrITS) and plastid loci and investigated how phylogenetic conflict bears on taxonomic classification within the genus. We found that phylogenetic conflict and low support values may be explained by hybridization and a lack of informative characteristics. Our results do not support previous classification of the subgenera and sections within Cymbidium. Discordance between gene trees and network analysis indicate that reticulate evolution occurred in the genus Cymbidium. Overall, our study indicates that Cymbidium has undergone a complex evolution.

Plastome structure and adaptive evolution of Calanthe s.l. species.

Calanthe s.l. is the most diverse group in the tribe Collabieae (Orchidaceae), which are pantropical in distribution. Illumina sequencing followed by de novo assembly was used in this study, and the plastid genetic information of Calanthe s.l. was used to investigate the adaptive evolution of this taxon. Herein, the complete plastome of five Calanthe s.l. species (Calanthe davidii, Styloglossum lyroglossa, Preptanthe rubens, Cephalantheropsis obcordata, and Phaius tankervilliae) were determined, and the two other published plastome sequences of Calanthe s.l. were added for comparative analyses to examine the evolutionary pattern of the plastome in the alliance. The seven plastomes ranged from 150,181 bp (C. delavayi) to 159,014 bp (C. davidii) in length and were all mapped as circular structures. Except for the three ndh genes (ndhC, ndhF, and ndhK) lost in C. delavayi, the remaining six species contain identical gene orders and numbers (115 gene). Nucleotide diversity was detected across the plastomes, and we screened 14 mutational hotspot regions, including 12 non-coding regions and two gene regions. For the adaptive evolution investigation, three species showed positive selected genes compared with others, C. obcordata (cemA), S. lyroglossa (infA, ycf1 and ycf2) and C. delavayi (nad6 and ndhB). Six genes were under site-specific positive selection in Calanthe s.l., namely, accD, ndhB, ndhD, rpoC2, ycf1, and ycf2, most of which are involved in photosynthesis. These results, including the new plastomes, provide resources for the comparative plastome, breeding, and plastid genetic engineering of orchids and flowering plants.