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.

Molecular mechanism of different flower color formation of Cymbidium ensifolium.

Cymbidium ensifolium is one of the national orchids in China, which has high ornamental value with changeable flower colors. To understand the formation mechanism of different flower colors of C. ensifolium, this research conducted transcriptome and metabolome analyses on four different colored sepals of C. ensifolium. Metabolome analysis detected 204 flavonoid metabolites, including 17 polyphenols, 27 anthocyanins, 75 flavones, 34 flavonols, 25 flavonoids, 18 flavanones, and 8 isoflavones. Among them, purple-red and red sepals contain a lot of anthocyanins, including cyanidin, pelargonin, and paeoniflorin, while yellow-green and white sepals have less anthocyanins detected, and their metabolites are mainly flavonols, flavanones and flavonoids. Transcriptome sequencing analysis showed that the expression levels of the anthocyanin biosynthetic enzyme genes in red and purple-red sepals were significantly higher than those in white and yellow-green sepals of C. ensifolium. The experimental results showed that CeF3'H2, CeDFR, CeANS, CeF3H and CeUFGT1 may be the key genes involved in anthocyanin production in C. ensifolium sepals, and CeMYB104 has been proved to play an important role in the flower color formation of C. ensifolium. The results of transformation showed that the CeMYB104 is involved in the synthesis of anthocyanins and can form a purple-red color in the white perianth of Phalaenopsis. These findings provide a theoretical reference to understand the formation mechanism of flower color in C. ensifolium.

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.

Genome-Wide Identification and Characterization of the GRF Gene Family in Melastoma dodecandrum.

Growth-regulating factor (GRF) is a kind of transcription factor unique to plants, playing an important role in the flowering regulation, growth, and development of plants. Melastoma dodecandrum is an important member of Melastomataceae, with ornamental, medicinal, and edible benefits. The identification of the GRF gene family in M. dodecandrum can help to improve their character of flavor and continuous flowering. The members of the GRF gene family were identified from the M. dodecandrum genome, and their bioinformatics, selective pressure, and expression patterns were analyzed. The results showed that there were 20 GRF genes in M. dodecandrum. Phylogenetic analysis showed that the 71 GRF genes from M. dodecandrum, Arabidopsis thaliana, Camellia sinensis, and Oryza sativa can be divided into three clades and six subclades. The 20 GRF genes of M. dodecandrum were distributed in twelve chromosomes and one contig. Furthermore, the gene structure and motif analysis showed that the intron and motif within each clade were very similar, but there were great differences among different clades. The promoter contained cis-acting elements related to hormone induction, stress, and growth and development. Different transcriptomic expression of MdGRFs indicated that MdGRFs may be involved in regulating the growth and development of M. dodecandrum. The results laid a foundation for further study on the function and molecular mechanism of the M. dodecandrum GRF gene family.

Genome-Wide Identification and Analysis of bHLH Transcription Factors Related to Anthocyanin Biosynthesis in Cymbidium ensifolium.

The basic helix-loop-helix (bHLH) transcription factors are widely distributed across eukaryotic kingdoms and participate in various physiological processes. To date, the bHLH family has been identified and functionally analyzed in many plants. However, systematic identification of bHLH transcription factors has yet to be reported in orchids. Here, 94 bHLH transcription factors were identified from the Cymbidium ensifolium genome and divided into 18 subfamilies. Most CebHLHs contain numerous cis-acting elements associated with abiotic stress responses and phytohormone responses. A total of 19 pairs of duplicated genes were found in the CebHLHs, of which 13 pairs were segmentally duplicated genes and six pairs were tandemly duplicated genes. Expression pattern analysis based on transcriptome data revealed that 84 CebHLHs were differentially expressed in four different color sepals, especially CebHLH13 and CebHLH75 of the S7 subfamily. The expression profiles of CebHLH13 and CebHLH75 in sepals, which are considered potential genes regulating anthocyanin biosynthesis, were confirmed through the qRT-PCR technique. Furthermore, subcellular localization results showed that CebHLH13 and CebHLH75 were located in the nucleus. This research lays a foundation for further exploration of the mechanism of CebHLHs in flower color formation.

Identification, Molecular Characteristics, and Evolution of YABBY Gene Family in Melastoma dodecandrum.

The YABBY gene family plays an important role in plant growth and development, such as response to abiotic stress and lateral organ development. YABBY TFs are well studied in numerous plant species, but no study has performed a genome-wide investigation of the YABBY gene family in Melastoma dodecandrum. Therefore, a genome-wide comparative analysis of the YABBY gene family was performed to study their sequence structures, cis-acting elements, phylogenetics, expression, chromosome locations, collinearity analysis, protein interaction, and subcellular localization analysis. A total of nine YABBY genes were found, and they were further divided into four subgroups based on the phylogenetic tree. The genes in the same clade of phylogenetic tree had the same structure. The cis-element analysis showed that MdYABBY genes were involved in various biological processes, such as cell cycle regulation, meristem expression, responses to low temperature, and hormone signaling. MdYABBYs were unevenly distributed on chromosomes. The transcriptomic data and real-time reverse transcription quantitative PCR (RT-qPCR) expression pattern analyses showed that MdYABBY genes were involved in organ development and differentiation of M. dodecandrum, and some MdYABBYs in the subfamily may have function differentiation. The RT-qPCR analysis showed high expression of flower bud and medium flower. Moreover, all MdYABBYs were localized in the nucleus. Therefore, this study provides a theoretical basis for the functional analysis of YABBY genes in M. dodecandrum.

Apostasia Mitochondrial Genome Analysis and Monocot Mitochondria Phylogenomics.

Apostasia shenzhenica belongs to the subfamily Apostasioideae and is a primitive group located at the base of the Orchidaceae phylogenetic tree. However, the A. shenzhenica mitochondrial genome (mitogenome) is still unexplored, and the phylogenetic relationships between monocots mitogenomes remain unexplored. In this study, we discussed the genetic diversity of A. shenzhenica and the phylogenetic relationships within its monocotyledon mitogenome. We sequenced and assembled the complete mitogenome of A. shenzhenica, resulting in a circular mitochondrial draft of 672,872 bp, with an average read coverage of 122× and a GC content of 44.4%. A. shenzhenica mitogenome contained 36 protein-coding genes, 16 tRNAs, two rRNAs, and two copies of nad4L. Repeat sequence analysis revealed a large number of medium and small repeats, accounting for 1.28% of the mitogenome sequence. Selection pressure analysis indicated high mitogenome conservation in related species. RNA editing identified 416 sites in the protein-coding region. Furthermore, we found 44 chloroplast genomic DNA fragments that were transferred from the chloroplast to the mitogenome of A. shenzhenica, with five plastid-derived genes remaining intact in the mitogenome. Finally, the phylogenetic analysis of the mitogenomes from A. shenzhenica and 28 other monocots showed that the evolution and classification of most monocots were well determined. These findings enrich the genetic resources of orchids and provide valuable information on the taxonomic classification and molecular evolution of monocots.

OrchidBase 5.0: updates of the orchid genome knowledgebase.

Containing the largest number of species, the orchid family provides not only materials for studying plant evolution and environmental adaptation, but economically and culturally important ornamental plants for human society. Previously, we collected genome and transcriptome information of Dendrobium catenatum, Phalaenopsis equestris, and Apostasia shenzhenica which belong to two different subfamilies of Orchidaceae, and developed user-friendly tools to explore the orchid genetic sequences in the OrchidBase 4.0. The OrchidBase 4.0 offers the opportunity for plant science community to compare orchid genomes and transcriptomes and retrieve orchid sequences for further study.In the year 2022, two whole-genome sequences of Orchidoideae species, Platanthera zijinensis and Platanthera guangdongensis, were de novo sequenced, assembled and analyzed. In addition, systemic transcriptomes from these two species were also established. Therefore, we included these datasets to develop the new version of OrchidBase 5.0. In addition, three new functions including synteny, gene order, and miRNA information were also developed for orchid genome comparisons and miRNA characterization.OrchidBase 5.0 extended the genetic information to three orchid subfamilies (including five orchid species) and provided new tools for orchid researchers to analyze orchid genomes and transcriptomes. The online resources can be accessed at https://cosbi.ee.ncku.edu.tw/orchidbase5/.

Phylogenetic analysis and character evolution of tribe Arethuseae (Orchidaceae) reveal a new genus Mengzia.

Delimitation of the tribe Arethuseae has varied considerably since it was first defined. The relationships within Arethuseae, particularly within the subtribe Arethusinae, remain poorly elucidated. In this study, we reconstructed the phylogeny of Arethuseae, using six plastid markers (matK, ycf1, rbcL rpoc1, rpl32-trnL and trnL-F) from 83 taxa. The ancestral state reconstruction of 11 selected morphological characters was also conducted to identify synapomorphies and assess potential evolutionary transitions. Morphological character comparision between the distinct species Bletilla foliosa and other species are conducted. Our results unequivocally supported the monophyly of Arethuseae, which included highly supported clades and a clear synapomorphy of non-trichome-like lamellae. Furthermore, B. foliosa formed a separate clade in the subtribe Arethusinae, instead of clustering with the other Bletilla species in the subtribe Coelogyninae. The morphological characters comparision further showed that the B. foliosa clade could be distinguished from other genera in Arethuseae by multiple characters, including presence of lateral inflorescence, three lamellae with trichome-like apex and four pollinia. In light of these molecular and morphological evidences, we propose Mengzia as a new genus to accommodate B. foliosa and accordingly provide descriptions of this new genus and combination.

OrchidBase 4.0: a database for orchid genomics and molecular biology.

BACKGROUND: The Orchid family is the largest families of the monocotyledons and an economically important ornamental plant worldwide. Given the pivotal role of this plant to humans, botanical researchers and breeding communities should have access to valuable genomic and transcriptomic information of this plant. Previously, we established OrchidBase, which contains expressed sequence tags (ESTs) from different tissues and developmental stages of Phalaenopsis as well as biotic and abiotic stress-treated Phalaenopsis. The database includes floral transcriptomic sequences from 10 orchid species across all the five subfamilies of Orchidaceae. DESCRIPTION: Recently, the whole-genome sequences of Apostasia shenzhenica, Dendrobium catenatum, and Phalaenopsis equestris were de novo assembled and analyzed. These datasets were used to develop OrchidBase 4.0, including genomic and transcriptomic data for these three orchid species. OrchidBase 4.0 offers information for gene annotation, gene expression with fragments per kilobase of transcript per millions mapped reads (FPKM), KEGG pathways and BLAST search. In addition, assembled genome sequences and location of genes and miRNAs could be visualized by the genome browser. The online resources in OrchidBase 4.0 can be accessed by browsing or using BLAST. Users can also download the assembled scaffold sequences and the predicted gene and protein sequences of these three orchid species. CONCLUSIONS: OrchidBase 4.0 is the first database that contain the whole-genome sequences and annotations of multiple orchid species. OrchidBase 4.0 is available at http://orchidbase.itps.ncku.edu.tw/.

Plastid phylogenomics improves resolution of phylogenetic relationship in the Cheirostylis and Goodyera clades of Goodyerinae (Orchidoideae, Orchidaceae).

Goodyerinae are one of phylogenetically unresolved groups of Orchidaceae. The lack of resolution achieved through the analyses of previous molecular sequences from one or a few markers has long confounded phylogenetic estimation and generic delimitation. Here, we present large-scale phylogenomic data to compare the plastome structure of the two main clades (Goodyera and Cheirostylis) in this subtribe and further adopt two strategies, combining plastid coding sequences and the whole plastome, to investigate phylogenetic relationships. A total of 46 species in 16 genera were sampled, including 39 species in 15 genera sequenced in this study. The plastomes of heterotrophic species are not drastically reduced in overall size, but display a pattern congruent with a loss of photosynthetic function. The plastomes of autotrophic species ranged from 147 to 165 kb and encoded from 132 to 137 genes. Three unusual structural features were detected: a 1.0-kb inversion in the large single-copy region of Goodyera schlechtendaliana; the loss and/or pseudogenization of ndh genes only in two species, Cheirostylis chinensis and C. montana; and the expansion of inverted repeat regions and contraction of small single-copy region in Hetaeria oblongifolia. Phylogenomic analyses provided improved resolution for phylogenetic relationships. All genera were recovered as monophyletic, except for Goodyera and Hetaeria, which were each recovered as non-monophyletic. Nomenclatural changes are needed until the broader sampling and biparental inherited markers. This study provides a phylogenetic framework of Goodyerinae and insight into plastome evolution of Orchidaceae.

Plastid phylogenomic data yield new and robust insights into the phylogeny of Cleisostoma-Gastrochilus clades (Orchidaceae, Aeridinae).

The Cleisostoma-Gastrochilus clades are among the most speciose and diverse groups of Asian orchids and are a taxonomically problematic group. Phylogenetic relationships among the genera of these clades have remained unresolved with traditional sequences from one or a few markers. We present large-scale phylogenomic data sets, incorporating complete chloroplast genome sequences from 53 species (including 41 species sequenced in this study), to compare plastome structure and to resolve the phylogenetic relationships of these clades. The plastomes of Cleisostoma-Gastrochilus clades possessed the quadripartite structure and plastome genes of typical angiosperms with sizes ranging from 142 to 149 kb and encoding a set of 118-120 genes. Unusual structural features were detected in the plastome of Uncifera acuminata, including the presence of a large 17-kb inversion (19 genes) in the Large Single-Copy region and the loss of the rpl32 gene in Cleisostoma fuerstenbergianum. The pseudogenization of ndh genes was widespread in these clades. Phylogenomic analyses, including 68 plastid protein-coding genes, showed that these clades can be subdivided into three major groupings and six subgroupings: Vandopsis undulata, the Gastrochilus clade (including the Trichoglottis and Gastrochilus subclades) and the Cleisostoma clade (including the Vandopsis, Diploprora, Cleisostoma and Schoenorchis subclades). Two genera, Vandopsis and Cleisostoma, were not monophyletic. A new genus, Cymbilabia, was proposed to avoid non-monophyly of Vandopsis. Our results demonstrate the power of plastid phylogenomics to improve the phylogenetic relationships of intricate groups and provide new insight into plastome evolution in Orchidaceae.

Molecular systematics of Goodyerinae (Cranichideae, Orchidoideae, Orchidaceae) based on multiple nuclear and plastid regions.

Goodyerinae are one of the most species-rich and widespread subtribes of Orchidaceae but notorious for their taxonomic difficulty. Here, a comprehensive molecular phylogenetic study of the subtribe is presented based on two nuclear (ITS, Xdh) and five plastid (matK, psaB, rbcL, trnL, trnL-F) regions. A total of 119 species were included representing all clades recovered by previous phylogenetic analyses as well as seven outgroups. Maximum parsimony, maximum likelihood and Bayesian inference methods were used to infer the phylogenetic relationships. The results show that the Goodyerinae subdivided into three major subdivisions and six groupings: Pachyplectron, Goodyera clade (including Goodyera procera, Microchilus subclade and Goodyera subclade) and Cheirostylis clade (including Gonatostylis, Cheirostylis subclade and Ludisia subclade). Four genera, Erythrodes, Goodyera, Myrmechis and Odontochilus, are not monophyletic. The results support Odontochilus s. l. to include Myrmechis and Kuhlhasseltia. The systematic positions of Goodyera procera and two isolated genera, Herpysma and Orchipedum, are difficult to determine.

A perspective on crassulacean acid metabolism photosynthesis evolution of orchids on different continents: Dendrobium as a case study.

Members of the Orchidaceae, one of the largest families of flowering plants, evolved the crassulacean acid metabolism (CAM) photosynthesis strategy. It is thought that CAM triggers adaptive radiation into new niche spaces, yet very little is known about its origin and diversification on different continents. Here, we assess the prevalence of CAM in Dendrobium, which is one of the largest genera of flowering plants and found in a wide range of environments, from the high altitudes of the Himalayas to relatively arid habitats in Australia. Based on phylogenetic time trees, we estimated that CAM, as determined by δ 13C values less negative than -20.0‰, evolved independently at least eight times in Dendrobium. The oldest lineage appeared in the Asian clade during the middle Miocene, indicating the origin of CAM was associated with a pronounced climatic cooling that followed a period of aridity. Divergence of the four CAM lineages in the Asian clade appeared to be earlier than divergence of those in the Australasian clade. However, CAM species in the Asian clade are much less diverse (25.6%) than those in the Australasian clade (57.9%). These findings shed new light on CAM evolutionary history and the aridity levels of the paleoclimate on different continents.

A new phylogenetic analysis sheds new light on the relationships in the Calanthe alliance (Orchidaceae) in China.

The taxonomy of the Calanthe alliance (Epidendroideae, Orchidaceae), consisting of Calanthe, Cephalantheropsis, and Phaius, has been difficult for orchidologists to understand because of the presence of common morphological features. In this study, in addition to morphological and geographical analyses, maximum parsimony and Bayesian inference analyses were performed based on nucleotide sequences of the nuclear internal transcribed spacer and cpDNA genes of 88 taxa representing the major clades of the Calanthe alliance in China. The results indicated that Cephalantheropsis is monophyletic, while both Phaius and Calanthe are polyphyletic. In Phaius, a total of three species, P. flavus, P. columnaris, and P. takeoi, were segregated to form a new genus, Paraphaius. In Calanthe, subgenus Preptanthe and sect. Styloglossum were both categorized as distinct genera from Calanthe. Our results also confirm that Calanthe delavayi and C. calanthoides are members of Calanthe. Previous studies assigned C. delavayi to Phaius and C. calanthoides to Ghiesbrechtia. Five sections, namely, Alpinocalanthe, Puberula, Ghiesbrechtia, Tricarinata, and Calanthe, three of which are new taxa, were recognized in Calanthe. Therefore, we propose that the Calanthe alliance is composed of six genera: Calanthe, Cephalantheropsis, Paraphaius, Phaius, Preptanthe and Styloglossum.

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.

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.

The complete chloroplast genome sequence of Alniphyllum fortunei (Styracaceae) from Fujian, China.

Alniphyllum fortunei is a subtropical tree species, a large deciduous tree with a tall and straight trunk, which is an excellent fast-growing and broad-leaved tree species with a wide range of uses we resequenced complete chloroplast (cp) genome of A. fortunei from Fujian, China. The whole genome was 154,166 bp in length, consisting of a pair of inverted repeats (IR 26,658 bp), a large single-copy region (LSC 82,438 bp), and a small single-copy region (SSC 18,367 bp). The complete genome contained 139 genes, including 89 protein-coding genes, 40 tRNA, and 8 rRNA genes. The phylogenetic analyses based on the complete chloroplast genome sequence provided solid evidence that A. fortunei has a close relationship with A. pterospermum and Bruinsmia polysperma.

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.

Frequent germplasm exchanges drive the high genetic diversity of Chinese-cultivated common apricot germplasm.

The genetic diversity of germplasm is critical for exploring genetic and phenotypic resources and has important implications for crop-breeding sustainability and improvement. However, little is known about the factors that shape and maintain genetic diversity. Here, we assembled a high-quality chromosome-level reference of the Chinese common apricot 'Yinxiangbai', and we resequenced 180 apricot accessions that cover four major ecogeographical groups in China and other accessions from occidental countries. We concluded that Chinese-cultivated common apricot germplasms possessed much higher genetic diversity than those cultivated in Western countries. We also detected seven migration events among different apricot groups, where 27% of the genome was identified as being introgressed. Remarkably, we demonstrated that these introgressed regions drove the current high level of germplasm diversity in Chinese-cultivated common apricots by introducing different genes related to distinct phenotypes from different cultivated groups. Our results highlight the consideration that introgressed regions may provide an important reservoir of genetic resources that can be used to sustain modern breeding programs.