Analysis of the chloroplast genome and phylogenetic evolution of Bidens pilosa.

Chloroplast genomes for 3 Bidens plants endemic to China (Bidens bipinnata Linn., Bidens pilosa Linn., and Bidens alba var. radiata) have been sequenced, assembled and annotated in this study to distinguish their molecular characterization and phylogenetic relationships. The chloroplast genomes are in typical quadripartite structure with two inverted repeat regions separating a large single copy region and a small single copy region, and ranged from 151,599 to 154,478 bp in length. Similar number of SSRs and long repeats were found in Bidens, wherein mononucleotide repeats (A/T), forward and palindromic repeats were the most in abundance. Gene loss of clpP and psbD, IR expansion and contraction were detected in these Bidens plants. It seems that ndhE, ndhF, ndhG, and rpl32 from the Bidens plants were under positive selection while the majority of chloroplast genes were under purifying selection. Phylogenetic analysis revealed that 3 Bidens plants clustered together and further formed molophyletic clade with other Bidens species, indicating Bidens plants might be under radiation adaptive selection to the changing environment world-widely. Moreover, mutation hotspot analysis and in silico PCR analysis indicated that inter-genic regions of ndhD-ccsA, ndhI-ndhG, ndhF-rpl32, trnL_UAG-rpl32, ndhE-psaC, matK-rps16, rps2-atpI, cemA-petA, petN-psbM were candidate markers of molecular identification for Bidens plants. This study may provide useful information for genetic diversity analysis and molecular identification for Bidens species.

Chloroplast genome draft assembly of Falcataria moluccana using hybrid sequencing technology.

OBJECTIVES: Falcataria moluccana, known locally as Sengon, is a fast-growing legume tree that is commonly planted in community forests of Java Island, Indonesia. However, the plantations face attacks of Boktor stem borer (Xystrocera festiva) and gall-rust disease (Uromycladium falcatariae) as major threats to its productivity. To control those pest and disease, it is necessary to grow resistant sengon clones, which are developed through tree improvement program, of which needs genetic and genomic information. This dataset was created to construct draft of sengon chloroplast genome and to study the evolution of sengon based on matK and rbcL barcode genes. DATA DESCRIPTION: Genomic DNA was extracted from leaf samples of one individual healthy tree in a private plantation. The DNA was sequenced using Illumina Novaseq 6000 (Novogen AIT, Singapore) for short-reads data, and MinION of Nanopore following manufacture's protocols SQK-LSK110 for long-reads data. The 66,3 Gb short-reads and 12 Gb long-reads data were hybrid assembled and used to construct a 128.867 bp of F. moluccana chloroplast genome with a quadripartite structure, containing a pair of inverted repeats, a large single-copy and a small single-copy region. Phylogenetic tree constructed using matK and rbcL showed monophyletic origin of F. moluccana and other legume trees.

Complete chloroplast genome sequences of the medicinal plant Aconitum transsectum (Ranunculaceae): comparative analysis and phylogenetic relationships.

BACKGROUND: Aconitum transsectum Diels. (Ranunculaceae) is an important medicinal plant that is widely used in traditional Chinese medicine, but its morphological traits make it difficult to recognize from other Aconitum species. No research has sequenced the chloroplast genome of A.transsectum, despite the fact that phylogenetic analysis based on chloroplast genome sequences provides essential evidence for plant classification. RESULTS: In this study, the chloroplast (cp) genome of A. transsectum was sequenced, assembled, and annotated. A. transsectum cp genome is a 155,872 bp tetrameric structure including a large single copy (LSC, 87,671 bp) and a small single copy (SSC, 18,891 bp) section, as well as a pair of inverted repeat sequences (IRa and IRb, 25,894 bp each). 131 genes are encoded by the complete cp genome, comprising 86 protein-coding genes, 37 tRNAs, and 8 rRNAs. The most favored codon in the A. transsectum cp genome is AUG, and 46 repeats and 241 SSRs were also identified. The A. transsectum cp genome is similar in size, gene composition, and IR expansion and contraction to the cp genomes of seven Ranunculaceae species. Phylogenetic analysis of cp genomes of 28 plants from the Ranunculaceae family shows that A. transsectum is most closely related to A. vilmorinianum, A. episcopale, and A. forrestii of Subgen. Aconitum. CONCLUSIONS: Overall, this study provides complete cp genome resources for A. transsectum that will be beneficial for identifying potential.

Comparative analysis of complete Artemisia subgenus Seriphidium (Asteraceae: Anthemideae) chloroplast genomes: insights into structural divergence and phylogenetic relationships.

BACKGROUND: Artemisia subg. Seriphidium, one of the most species-diverse groups within Artemisia, grows mainly in arid or semi-arid regions in temperate climates. Some members have considerable medicinal, ecological, and economic value. Previous studies on this subgenus have been limited by a dearth of genetic information and inadequate sampling, hampering our understanding of their phylogenetics and evolutionary history. We therefore sequenced and compared the chloroplast genomes of this subgenus, and evaluated their phylogenetic relationships. RESULTS: We newly sequenced 18 chloroplast genomes of 16 subg. Seriphidium species and compared them with one previously published taxon. The chloroplast genomes, at 150,586-151,256 bp in length, comprised 133 genes, including 87 protein-coding genes, 37 tRNA genes, 8 rRNA genes, and one pseudogene, with GC content of 37.40-37.46%. Comparative analysis showed that genomic structures and gene order were relatively conserved, with only some variation in IR borders. A total of 2203 repeats (1385 SSRs and 818 LDRs) and 8 highly variable loci (trnK - rps16, trnE - ropB, trnT, ndhC - trnV, ndhF, rpl32 - trnL, ndhG - ndhI and ycf1) were detected in subg. Seriphidium chloroplast genomes. Phylogenetic analysis of the whole chloroplast genomes based on maximum likelihood and Bayesian inference analyses resolved subg. Seriphidium as polyphyletic, and segregated into two main clades, with the monospecific sect. Minchunensa embedded within sect. Seriphidium, suggesting that the whole chloroplast genomes can be used as molecular markers to infer the interspecific relationship of subg. Seriphidium taxa. CONCLUSION: Our findings reveal inconsistencies between the molecular phylogeny and traditional taxonomy of the subg. Seriphidium and provide new insights into the evolutionary development of this complex taxon. Meanwhile, the whole chloroplast genomes with sufficiently polymorphic can be used as superbarcodes to resolve interspecific relationships in subg. Seriphidium.

Characterization and phylogenetic analyses of ten complete plastomes of Spiraea species.

BACKGROUND: Spiraea is a genus of deciduous shrubs that contains 80-120 species, is mainly distributed in the Northern Hemisphere and has diversified in East Asia. Spiraea species are cultivated as ornamental plants and some are used in traditional herbal medicine. Based on morphological characteristics and genetic markers, phylogenetic classification exhibits low discriminatory power. RESULTS: In present study, we assembled and characterized the chloroplast (cp) genomes of ten Spiraea species and comparatively analysed with five reported cp genomes of this genus. The cp genomes of the fifteen Spiraea species, ranging from 155,904 to 158,637 bp in length, were very conserved and no structural rearrangements occurred. A total of 85 protein-coding genes (PCGs), 37 tRNAs and 8 rRNAs were annotated. We also examined 1,010 simple sequence repeat (SSR) loci, most of which had A/T base preference. Comparative analysis of cp genome demonstrated that single copy and non-coding regions were more divergent than the inverted repeats (IRs) and coding regions and six mutational hotspots were detected. Selection pressure analysis showed that all PCGs were under purifying selection. Phylogenetic analysis based on the complete cp genome data showed that Spiraea formed a monophyletic group and was further divided into two major clades. Infrageneric classification in each clade was supported with a high resolution value. Moreover, the phylogenetic trees based on each individual mutational hotspot segment and their combined dataset also consisted of two major clades, but most of the phylogenetic relationships of interspecies were not well supported. CONCLUSIONS: Although the cp genomes of Spiraea species exhibited high conservation in genome structure, gene content and order, a large number of polymorphism sites and several mutation hotspots were identified in whole cp genomes, which might be sufficiently used as molecular markers to distinguish Spiraea species. Phylogenetic analysis based on the complete cp genome indicated that infrageneric classification in two major clades was supported with high resolution values. Therefore, the cp genome data of the genus Spiraea will be effective in resolving the phylogeny in this genus.

Distinctive origin and evolution of endemic thistle of Korean volcanic island: Structural organization and phylogenetic relationships with complete chloroplast genome.

Unlike other Cirsium in Korea, Cirsium nipponicum (Island thistle) is distributed only on Ulleung Island, a volcanic island off the east coast of the Korean Peninsula, and a unique thistle with none or very small thorns. Although many researchers have questioned the origin and evolution of C. nipponicum, there is not much genomic information to estimate it. We thus assembled the complete chloroplast of C. nipponicum and reconstructed the phylogenetic relationships within the genus Cirsium. The chloroplast genome was 152,586 bp, encoding 133 genes consisting of 8 rRNA genes, 37 tRNA genes, and 88 protein-coding genes. We found 833 polymorphic sites and eight highly variable regions in chloroplast genomes of six Cirsium species by calculating nucleotide diversity, as well as 18 specific variable regions distinguished C. nipponicum from other Cirsium. As a result of phylogenetic analysis, C. nipponicum was closer to C. arvense and C. vulgare than native Cirsium in Korea: C. rhinoceros and C. japonicum. These results indicate that C. nipponicum is likely introduced through the north Eurasian root, not the mainland, and evolved independently in Ulleung Island. This study contributes to further understanding the evolutionary process and the biodiversity conservation of C. nipponicum on Ulleung Island.

Molecular characterizations of genes in chloroplast genomes of the genus Arachis L. (Fabaceae) based on the codon usage divergence.

Studies on the molecular characteristics of chloroplast genome are generally important for clarifying the evolutionary processes of plant species. The base composition, the effective number of codons, the relative synonymous codon usage, the codon bias index, and their correlation coefficients of a total of 41 genes in 21 chloroplast genomes of the genus Arachis were investigated to further perform the correspondence and clustering analyses, revealing significantly higher variations in genomes of wild species than those of the cultivated taxa. The codon usage patterns of all 41 genes in the genus Arachis were AT-rich, suggesting that the natural selection was the main factor affecting the evolutionary history of these genomes. Five genes (i.e., ndhC, petD, atpF, rpl14, and rps11) and five genes (i.e., atpE, psbD, psaB, ycf2, and rps12) showed higher and lower base usage divergences, respectively. This study provided novel insights into our understanding of the molecular evolution of chloroplast genomes in the genus Arachis.

Complete chloroplast genome sequences of Myristicaceae species with the comparative chloroplast genomics and phylogenetic relationships among them.

BACKGROUND: Myristicaceae was widly distributed from tropical Asia to Oceania, Africa, and tropical America. There are 3 genera and 10 species of Myristicaceae present in China, mainly distributed in the south of Yunnan Province. Most research on this family focuses on fatty acids, medicine, and morphology. Based on the morphology, fatty acid chemotaxonomy, and a few of molecular data, the phylogenetic position of Horsfieldia pandurifolia Hu was controversial. RESULTS: In this study, the chloroplast genomes of two Knema species, Knema globularia (Lam.) Warb. and Knema cinerea (Poir.) Warb., were characterized. Comparing the genome structure of these two species with those of other eight published species, including three Horsfieldia species, four Knema species, and one Myristica species, it was found that the chloroplast genomes of these species were relatively conserved, retaining the same gene order. Through sequence divergence analysis, there were 11 genes and 18 intergenic spacers were subject to positive selection, which can be used to analyze the population genetic structure of this family. Phylogenetic analysis showed that all Knema species were clustered in the same group and formed a sister clade with Myristica species support by both high maximum likelihood bootstrap values and Bayesian posterior probabilities; among Horsfieldia species, Horsfieldia amygdalina (Wall.) Warb., Horsfieldia kingii (Hook.f.) Warb., Horsfieldia hainanensis Merr. and Horsfieldia tetratepala C.Y.Wu. were grouped together, but H. pandurifolia formed a single group and formed a sister clade with genus Myristica and Knema. Through the phylogenetic analysis, we support de Wilde' view that the H. pandurifolia should be separated from Horsfieldia and placed in the genus Endocomia, namely Endocomia macrocoma subsp. prainii (King) W.J.de Wilde. CONCLUSION: The findings of this study provide a novel genetic resources for future research in Myristicaceae and provide a molecular evidence for the taxonomic classification of Myristicaceae.

Comparative chloroplast genomics of 34 species in subtribe Swertiinae (Gentianaceae) with implications for its phylogeny.

BACKGROUND: Subtribe Swertiinae, a medicinally significant and highly speciose Subtribe of family Gentianaceae. Despite previous extensive studies based on both morphology and molecular data, intergeneric and infrageneric relationships within subtribe Swertiinae remain controversial. METHODS: Here, we employed four newly generated Swertia chloroplast genomes with thirty other published genomes to elucidate their genomic characteristics. RESULTS: The 34 chloroplast genomes were small and ranged in size from 149,036 to 154,365 bp, each comprising two inverted repeat regions (size range 25,069-26,126 bp) that separated large single-copy (80,432-84,153 bp) and small single-copy (17,887-18,47 bp) regions, and all the chloroplast genomes showed similar gene orders, contents, and structures. These chloroplast genomes contained 129-134 genes each, including 84-89 protein-coding genes, 37 tRNAs, and 8 rRNAs. The chloroplast genomes of subtribe Swertiinae appeared to have lost some genes, such as rpl33, rpl2 and ycf15 genes. Comparative analyses revealed that two mutation hotspot regions (accD-psaI and ycf1) could serve as effective molecular markers for further phylogenetic analyses and species identification in subtribe Swertiinae. Positive selection analyses showed that two genes (ccsA and psbB) had high Ka/Ks ratios, indicating that chloroplast genes may have undergone positive selection in their evolutionary history. Phylogenetic analysis showed that the 34 subtribe Swertiinae species formed a monophyletic clade, with Veratrilla, Gentianopsis and Pterygocalyx located at the base of the phylogenetic tree. Some genera of this subtribe, however, were not monophyletic, including Swertia, Gentianopsis, Lomatogonium, Halenia, Veratrilla and Gentianopsis. In addition, our molecular phylogeny was consistent with taxonomic classification of subtribe Swertiinae in the Roate group and Tubular group. The results of molecular dating showed that the divergence between subtrib Gentianinae and subtrib Swertiinae was estimated to occur in 33.68 Ma. Roate group and Tubular group in subtribe Swertiinae approximately diverged in 25.17 Ma. CONCLUSION: Overall, our study highlighted the taxonomic utility of chloroplast genomes in subtribe Swertiinae, and the genetic markers identified here will facilitate future studies on the evolution, conservation, population genetics, and phylogeography of subtribe Swertiinae species.

A Comparative Analysis of the Chloroplast Genomes of Three Lonicera Medicinal Plants.

Both Lonicerae japonicae flos and Lonicerae similis flos are important components in traditional Chinese medicine (TCM) with precious medicinal value. However, the absence of studies on their chloroplast genomes and chromatography has considerably hindered the study of their evolutionary and phylogenetic relationships. In this study, the complete chloroplast (cp) genomes of Lonicera acuminata Wall. and Lonicera similis Hemsl. were sequenced using the Illumina sequencing platform and compared with that of Lonicera japonica Thunb., which has been previously reported. Furthermore, the chromatographic fingerprints of the three plants were constructed using HPLC and the content of quality marker (Q-Marker) was calculated. The annotation results showed that the two chloroplast genomes were typical quadripartite structures with lengths of 155,330 bp (L. acuminata) and 155,207 bp (L. similis). A total of 126 different genes were annotated, containing 82 protein-coding genes, 36 tRNA genes, and 8 rRNA genes. The expansion and contraction of the inverted repeat (IR) regions suggested that the boundary regions of IR/SC were comparatively conserved in the three species, and six regions (trnH-GUG-psbA, rps2-rpoC2, rbcL-psaI, trnN-GUU-ndhF, rps15-ycf1, and infA) with nucleotide diversity values (Pi) of variable sites higher than 1% were identified. Phylogenetic relation indicated that L. similis had a closer genetic relationship with L. japonica than L. acuminata. Additionally, the chromatographic fingerprints showed that the characteristic peaks of the three medicinal plants were similar, including Neochlorogenic acid, Chlorogenic acid, 4-Dicaffeoylquinic acid, Sweroside, Secoxyloganin, Luteoloside, Isochlorogenic acid A, Isochlorogenic acid B, and Isochlorogenic acid C. The content of chlorogenic acid and total phenolic acid in L. acuminata (7.4633 ± 0.4461%, 14.8953 ± 0.0728%) and L. similis (14.1055 ± 0.2566%, 21.9782 ± 0.1331%) was much higher than that of L. japonica (3.9729 ± 0.0928%, 6.0964 ± 0.1228%), respectively. This study provides appropriate information for species identification, phylogeny, quality assessment, and rational use of three medicinal plants of the genus Lonicera.

Comparative Analyses of Chloroplast Genomes for Parasitic Species of Santalales in the Light of Two Newly Sequenced Species, Taxillus nigrans and Scurrula parasitica.

When a flowering plant species changes its life history from self-supply to parasite, its chloroplast genomes may have experienced functional physical reduction, and gene loss. Most species of Santalales are hemiparasitic and few studies focus on comparing the chloroplast genomes of the species from this order. In this study, we collected and compared chloroplast genomes of 12 species of Santalales and sequenced the chloroplast genomes of Taxillus nigrans and Scurrula parasitica for the first time. The chloroplast genomes for these species showed typical quadripartite structural organization. Phylogenetic analysis suggested that these 12 species of Santalales clustered into three clades: Viscum (4 spp.) and Osyris (1 sp.) in the Santalaceae and Champereia (1 sp.) in the Opiliaceae formed one clade, while Taxillus (3 spp.) and Scurrula (1 sp.) in the Loranthaceae and Schoepfia (1 sp.) in the Schoepfiaceae formed another clade. Erythropalum (1 sp.), in the Erythropalaceae, appeared as a third, most distant, clade within the Santalales. In addition, both Viscum and Taxillus are monophyletic, and Scurrula is sister to Taxillus. A comparative analysis of the chloroplast genome showed differences in genome size and the loss of genes, such as the ndh genes, infA genes, partial ribosomal genes, and tRNA genes. The 12 species were classified into six categories by the loss, order, and structure of genes in the chloroplast genome. Each of the five genera (Viscum, Osyris, Champereia, Schoepfia, and Erythropalum) represented an independent category, while the three Taxillus species and Scurrula were classified into a sixth category. Although we found that different genes were lost in various categories, most genes related to photosynthesis were retained in the 12 species. Hence, the genetic information accorded with observations that they are hemiparasitic species. Our comparative genomic analyses can provide a new case for the chloroplast genome evolution of parasitic species.

Complete Chloroplast Genome of Four Thai Native Dioscorea Species: Structural, Comparative and Phylogenetic Analyses.

The chloroplast genomes of Dioscorea brevipetiolata, D. depauperata, D. glabra, and D. pyrifolia are 153,370-153,503 bp in size. A total of 113 genes were predicted, including 79 protein-coding sequences (CDS), 30 tRNA, and four rRNA genes. The overall GC content for all four species was 37%. Only mono-, di-, and trinucleotides were present in the genome. Genes adjacent to the junction borders were similar in all species analyzed. Eight distinct indel variations were detected in the chloroplast genome alignment of 24 Dioscorea species. At a cut-off point of Pi = 0.03, a sliding window analysis based on 25 chloroplast genome sequences of Dioscorea species revealed three highly variable regions, which included three CDS (trnC, ycf1, and rpl32), as well as an intergenic spacer region, ndhF-rpl32. A phylogenetic tree based on the complete chloroplast genome sequence displayed an almost fully resolved relationship in Dioscorea. However, D. brevipetiolata, D. depauperata, and D. glabra were clustered together with D. alata, while D. pyrifolia was closely related to D. aspersa. As Dioscorea is a diverse genus, genome data generated in this study may contribute to a better understanding of the genetic identity of these species, which would be useful for future taxonomic work of Dioscorea.

[Chloroplast genomic characterization and phylogenetic analysis of Castanopsis hystrix].

The structure and size of the chloroplast genome of Castanopsis hystrix was determined by Illumina HiSeq 2500 sequencing platform to understand the difference between C. hystrix and the chloroplast genome of the same genus, and the evolutionary position of C. hystrix in the genus, so as to facilitate species identification, genetic diversity analysis and resource conservation of the genus. Bioinformatics analysis was used to perform sequence assembly, annotation and characteristic analysis. R, Python, MISA, CodonW and MEGA 6 bioinformatics software were used to analyze the genome structure and number, codon bias, sequence repeats, simple sequence repeat (SSR) loci and phylogeny. The genome size of C. hystrix chloroplast was 153 754 bp, showing tetrad structure. A total of 130 genes were identified, including 85 coding genes, 37 tRNA genes and 8 rRNA genes. According to codon bias analysis, the average number of effective codons was 55.5, indicating that the codons were highly random and low in bias. Forty-five repeats and 111 SSR loci were detected by SSR and long repeat fragment analysis. Compared with the related species, chloroplast genome sequences were highly conserved, especially the protein coding sequences. Phylogenetic analysis showed that C. hystrix is closely related to the Hainanese cone. In summary, we obtained the basic information and phylogenetic position of the chloroplast genome of red cone, which will provide a preliminary basis for species identification, genetic diversity of natural populations and functional genomics research of C. hystrix.

Complete Chloroplast Genome Sequence of the Long Blooming Cultivar Camellia 'Xiari Qixin': Genome Features, Comparative and Phylogenetic Analysis.

The camellia flower is a famous woody plant with a long-cultivated history and high ornamental value. It is extensively planted and utilized around the world and owns a massive germplasm resource. Camellia 'Xiari Qixin' belongs to one of the typical cultivars in the four seasons camellia hybrids series. Due to its long flowering period, this kind of cultivar is identified as a precious resource of camellia flowers. In this study, the complete chloroplast genome sequence of C. 'Xiari Qixin' was first reported. Its whole chloroplast genome is 157,039 bp in length with an overall GC content of 37.30%, composed of a large single copy region (LSC, 86,674 bp), a small single copy region (SSC, 18,281 bp), and a pair of inverted repeat regions (IRs, 26,042 bp each). A total of 134 genes were predicted in this genome, including 8 ribosomal RNA genes, 37 transfer RNA genes, and 89 protein-coding genes. In addition, 50 simple sequence repeats (SSRs) and 36 long repeat sequences were detected. By comparing C. 'Xiari Qixin' and seven Camellia species on the chloroplast genome, seven mutation hotspot regions were identified, including psbK, trnS (GCU)-trnG(GCC), trnG(GCC), petN-psbM, trnF(GAA)-ndhJ, trnP(UGG)-psaJ, and ycf1. Phylogenetic analysis of 30 chloroplast genomes showed that the genetic relationship between C. 'Xiari Qixin' and Camellia azalea is quite close in evolution. These results could not only provide a valuable database for determining the maternal origin of Camellia cultivars, but also contribute to the exploration of the phylogenetic relationship and utilization of germplasm resources for Camellia.

Comparative chloroplast genome analyses of diverse Phoebe (Lauraceae) species endemic to China provide insight into their phylogeographical origin.

The genus Phoebe (Lauraceae) includes about 90 evergreen tree species that are an ideal source of timber. Habitat destruction and deforestation have resulted in most of them being endemic to China. The accurate identification of endangered Phoebe species in China is necessary for their conservation. Chloroplast genome sequences can play an important role in species identification. In this study, comparative chloroplast genome analyses were conducted on diverse Phoebe species that are primarily distributed in China. Despite the conserved nature of chloroplast genomes, we detected some highly divergent intergenic regions (petA-psbE, ndhF-rpl32, and psbM-trnD-GUC) as well as three highly divergent genes (rbcL, ycf1, and ycf2) that have potential applications in phylogenetics and evolutionary analysis. The phylogenetic analysis indicated that various Phoebe species in China were divided into three clades. The complete chloroplast genome was better suited for phylogenetic analysis of Phoebe species. In addition, based on the phylogeographical analysis of Phoebe species in China, we inferred that the Phoebe species in China first originated in Yunnan and then spread to other southern areas of the Yangtze River. The results of this research will add to existing case studies on the phylogenetic analysis of Phoebe species and have the potential to contribute to the conservation of Phoebe species that are in danger of extinction.

Seven newly sequenced chloroplast genomes from the order Watanabeales (Trebouxiophyceae, Chlorophyta): Phylogenetic and comparative analysis.

The little-known order Watanabeales currently includes 10 genera with Chlorella-like species that reproduce by unequal-sized autospores and are predominantly solitary or terrestrial. The taxonomic scheme of Watanabeales has only been primarily inferred by short and less informative rDNA phylogenetic analysis. In the present study, seven newly sequenced genomes and one reported chloroplast genome representing the existing major branches of Watanabeales were harvested to phylogenetically reconstruct this order and to further understand its evolution. All chloroplast genomes of Watanabeales ranging from 133 to 274 kb were circular mapping and lacked a quadripartite structure. The chloroplast genome size, GC content, number of introns, and length of intergenic region proportion of the Watanabeales showed consistent trends, with Calidiella yingdensis D201 and Kalinella pachyderma 2601 having the lowest and highest values, respectively, echoing the positive correlation between organismal size and genome size. Phylogenetic analysis of Watanabeales based on 76 protein-coding genes coupled with the establishment of various complex analytical methods determined the unique robust taxonomic scheme which was incongruent with rDNA. Comparative genomic analyses revealed that the chloroplast genomes of Watanabeales accounted for numerous complex rearrangements and inversions which indicated high cryptic diversity. Substitution rate estimation indicated that the chloroplast genomes of Watanabeales were under purifying selection and similar evolutionary pressure and supported the view that genus Symbiochloris should be excluded from Watanabeales. Our results enrich the chloroplast genome resources of Watanabeales, clarify the phylogenetic status of species within this order, and provide more reference information for subsequent taxonomic and phylogenetic study.

Conserved chloroplast genome sequences of the genus Clerodendrum Linn. (Lamiaceae) as a super-barcode.

BACKGROUND: The plants of the genus Clerodendrum L. have great potential for development as an ornamental and important herbal resource. There is no significant morphological difference among many species of the genus Clerodendrum, which will lead to confusion among the herbs of this genus and ultimately affect the quality of the herbs. The chloroplast genome will contribute to the development of new markers used for the identification and classification of species. METHODS AND RESULTS: Here, we obtained the complete chloroplast genome sequences of Clerodendrum chinense (Osbeck) Mabberley and Clerodendrum thomsoniae Balf.f. using the next generation DNA sequencing technology. The chloroplast genomes of the two species all encode a total of 112 unique genes, including 80 protein-coding, 28 tRNA, and four rRNA genes. A total of 44-42 simple sequence repeats, 19-16 tandem repeats and 44-44 scattered repetitive sequences were identified. Phylogenetic analyses showed that the nine Clerodendrum species were classified into two clades and together formed a monophyletic group. Selective pressure analyses of 77 protein-coding genes showed that there was no gene under positive selection in the Clerodendrum branch. Analyses of sequence divergence found two intergenic regions: trnH-GUG-psbA, nhdD-psaC, exhibiting a high degree of variations. Meanwhile, there was no hypervariable region identified in protein coding genes. However, the sequence identities of these two intergenic spacers (IGSs) are greater than 99% among some species, which will result in the two IGSs not being used to distinguish Clerodendrum species. Analysis of the structure at the LSC (Large single copy) /IR (Inverted repeat) and SSC (Small single copy)/IR boundary regions showed dynamic changes. The above results showed that the complete chloroplast genomes can be used as a super-barcode to identify these Clerodendrum species. The study lay the foundation for the understanding of the evolutionary process of the genus Clerodendrum.

Evolutionary Patterns of the Chloroplast Genome in Vanilloid Orchids (Vanilloideae, Orchidaceae).

The Vanilloideae (vanilloids) is one of five subfamilies of Orchidaceae and is composed of fourteen genera and approximately 245 species. In this study, the six new chloroplast genomes (plastomes) of vanilloids (two Lecanorchis, two Pogonia, and two Vanilla species) were decoded, and then the evolutionary patterns of plastomes were compared to all available vanilloid plastomes. Pogonia japonica has the longest plastome, with 158,200 bp in genome size. In contrast, Lecanorchis japonica has the shortest plastome with 70,498 bp in genome size. The vanilloid plastomes have regular quadripartite structures, but the small single copy (SSC) region was drastically reduced. Two different tribes of Vanilloideae (Pogonieae and Vanilleae) showed different levels of SSC reductions. In addition, various gene losses were observed among the vanilloid plastomes. The photosynthetic vanilloids (Pogonia and Vanilla) showed signs of stage 1 degradation and had lost most of their ndh genes. The other three species (one Cyrotsia and two Lecanorchis), however, had stage 3 or stage 4 degradation and had lost almost all the genes in their plastomes, except for some housekeeping genes. The Vanilloideae were located between the Apostasioideae and Cypripedioideae in the maximum likelihood tree. A total of ten rearrangements were found among ten Vanilloideae plastomes when compared to the basal Apostasioideae plastomes. The four sub-regions of the single copy (SC) region shifted into an inverted repeat (IR) region, and the other four sub-regions of the IR region shifted into the SC regions. Both the synonymous (dS) and nonsynonymous (dN) substitution rates of IR in-cooperated SC sub-regions were decelerated, while the substitution rates of SC in-cooperated IR sub-regions were accelerated. A total of 20 protein-coding genes remained in mycoheterotrophic vanilloids. Almost all these protein genes show accelerated base substitution rates compared to the photosynthetic vanilloids. Two of the twenty genes in the mycoheterotrophic species faced strong "relaxed selection" pressure (p-value < 0.05).

Chloroplast genomic comparison provides insights into the evolution of seagrasses.

BACKGROUND: Seagrasses are a polyphyletic group of monocotyledonous angiosperms that have evolved to live entirely submerged in marine waters. Thus, these species are ideal for studying plant adaptation to marine environments. Herein, we sequenced the chloroplast (cp) genomes of two seagrass species (Zostera muelleri and Halophila ovalis) and performed a comparative analysis of them with 10 previously published seagrasses, resulting in various novel findings. RESULTS: The cp genomes of the seagrasses ranged in size from 143,877 bp (Zostera marina) to 178,261 bp (Thalassia hemprichii), and also varied in size among different families in the following order: Hydrocharitaceae > Cymodoceaceae > Ruppiaceae > Zosteraceae. The length differences between families were mainly related to the expansion and contraction of the IR region. In addition, we screened out 2,751 simple sequence repeats and 1,757 long repeat sequence types in the cp genome sequences of the 12 seagrass species, ultimately finding seven hot spots in coding regions. Interestingly, we found nine genes with positive selection sites, including two ATP subunit genes (atpA and atpF), three ribosome subunit genes (rps4, rps7, and rpl20), one photosystem subunit gene (psbH), and the ycf2, accD, and rbcL genes. These gene regions may have played critical roles in the adaptation of seagrasses to diverse environments. In addition, phylogenetic analysis strongly supported the division of the 12 seagrass species into four previously recognized major clades. Finally, the divergence time of the seagrasses inferred from the cp genome sequences was generally consistent with previous studies. CONCLUSIONS: In this study, we compared chloroplast genomes from 12 seagrass species, covering the main phylogenetic clades. Our findings will provide valuable genetic data for research into the taxonomy, phylogeny, and species evolution of seagrasses.

Comparison of plastid genomes and ITS of two sister species in Gentiana and a discussion on potential threats for the endangered species from hybridization.

BACKGROUND: Gentiana rigescens Franchet is an endangered medicinal herb from the family Gentianaceae with medicinal values. Gentiana cephalantha Franchet is a sister species to G. rigescens possessing similar morphology and wider distribution. To explore the phylogeny of the two species and reveal potential hybridization, we adopted next-generation sequencing technology to acquire their complete chloroplast genomes from sympatric and allopatric distributions, as along with Sanger sequencing to produce the nrDNA ITS sequences. RESULTS: The plastid genomes were highly similar between G. rigescens and G. cephalantha. The lengths of the genomes ranged from 146,795 to 147,001 bp in G. rigescens and from 146,856 to 147,016 bp in G. cephalantha. All genomes consisted of 116 genes, including 78 protein-coding genes, 30 tRNA genes, four rRNA genes and four pseudogenes. The total length of the ITS sequence was 626 bp, including six informative sites. Heterozygotes occurred intensively in individuals from sympatric distribution. Phylogenetic analysis was performed based on chloroplast genomes, coding sequences (CDS), hypervariable sequences (HVR), and nrDNA ITS. Analysis based on all the datasets showed that G. rigescens and G. cephalantha formed a monophyly. The two species were well separated in phylogenetic trees using ITS, except for potential hybrids, but were mixed based on plastid genomes. This study supports that G. rigescens and G. cephalantha are closely related, but independent species. However, hybridization was confirmed to occur frequently between G. rigescens and G. cephalantha in sympatric distribution owing to the lack of stable reproductive barriers. Asymmetric introgression, along with hybridization and backcrossing, may probably lead to genetic swamping and even extinction of G. rigescens. CONCLUSION: G. rigescens and G. cephalantha are recently diverged species which might not have undergone stable post-zygotic isolation. Though plastid genome shows obvious advantage in exploring phylogenetic relationships of some complicated genera, the intrinsic phylogeny was not revealed because of matrilineal inheritance here; nuclear genomes or regions are hence crucial for uncovering the truth. As an endangered species, G. rigescens faces serious threats from both natural hybridization and human activities; therefore, a balance between conservation and utilization of the species is extremely critical in formulating conservation strategies.