Comparative Chloroplast Genomes Analysis Provided Adaptive Evolution Insights in Medicago ruthenica.

A perennial leguminous forage, Medicago ruthenica has outstanding tolerance to abiotic stresses. The genome of Medicago ruthenica is large and has a complex genetic background, making it challenging to accurately determine genetic information. However, the chloroplast genome is widely used for researching issues related to evolution, genetic diversity, and other studies. To better understand its chloroplast characteristics and adaptive evolution, chloroplast genomes of 61 Medicago ruthenica were assembled (including 16 cultivated Medicago ruthenica germplasm and 45 wild Medicago ruthenica germplasm). These were used to construct the pan-chloroplast genome of Medicago ruthenica, and the chloroplast genomes of cultivated and wild Medicago ruthenica were compared and analyzed. Phylogenetic and haplotype analyses revealed two main clades of 61 Medicago ruthenica germplasm chloroplast genomes, distributed in eastern and western regions. Meanwhile, based on chloroplast variation information, 61 Medicago ruthenica germplasm can be divided into three genetic groups. Unlike the phylogenetic tree constructed from the chloroplast genome, a new intermediate group has been identified, mainly consisting of samples from the eastern region of Inner Mongolia, Shanxi Province, and Hebei Province. Transcriptomic analysis showed that 29 genes were upregulated and three genes were downregulated. The analysis of these genes mainly focuses on enhancing plant resilience and adapting adversity by stabilizing the photosystem structure and promoting protein synthesis. Additionally, in the analysis of adaptive evolution, the accD, clpP and ycf1 genes showed higher average Ka/Ks ratios and exhibited significant nucleotide diversity, indicating that these genes are strongly positively selected. The editing efficiency of the ycf1 and clpP genes significantly increases under abiotic stress, which may positively contribute to plant adaptation to the environment. In conclusion, the construction and comparative analysis of the complete chloroplast genomes of 61 Medicago ruthenica germplasm from different regions not only revealed new insights into the genetic variation and phylogenetic relationships of Medicago ruthenica germplasm, but also highlighted the importance of chloroplast transcriptome analysis in elucidating the model of chloroplast responses to abiotic stress. These provide valuable information for further research on the adaptive evolution of Medicago ruthenica.

Comparative Analysis and Phylogeny of the Complete Chloroplast Genomes of Nine Cynanchum (Apocynaceae) Species.

Cynanchum belongs to the Apocynaceae family and is a morphologically diverse genus that includes around 200 shrub or perennial herb species. Despite the utilization of CPGs, few molecular phylogenetic studies have endeavored to elucidate infrafamilial relationships within Cynanchum through extensive taxon sampling. In this research, we constructed a phylogeny and estimated divergence time based on the chloroplast genomes (CPGs) of nine Cynanchum species. We sequenced and annotated nine chloroplast (CP) genomes in this study. The comparative analysis of these genomes from these Cynanchum species revealed a typical quadripartite structure, with a total sequence length ranging from 158,283 to 161,241 base pairs (bp). The CP genome (CPG) was highly conserved and moderately differentiated. Through annotation, we identified a total of 129-132 genes. Analysis of the boundaries of inverted repeat (IR) regions showed consistent positioning: the rps19 gene was located in the IRb region, varying from 46 to 50 bp. IRb/SSC junctions were located between the trnN and ndhF genes. We did not detect major expansions or contractions in the IR region or rearrangements or insertions in the CPGs of the nine Cynanchum species. The results of SSR analysis revealed a variation in the number of SSRs, ranging from 112 to 150. In five types of SSRs, the largest number was mononucleotide repeats, and the smallest number was hexanucleotide repeats. The number of long repeats in the cp genomes of nine Cynanchum species was from 35 to 80. In nine species of Cynanchum, the GC3s values ranged from 26.80% to 27.00%, indicating a strong bias towards A/U-ending codons. Comparative analyses revealed four hotspot regions in the CPG, ndhA-ndhH, trnI-GAU-rrn16, psbI-trnS-GCU, and rps7-ndhB, which could potentially serve as molecular markers. In addition, phylogenetic tree construction based on the CPG indicated that the nine Cynanchum species formed a monophyletic group. Molecular dating suggested that Cynanchum diverged from its sister genus approximately 18.87 million years ago (Mya) and species diversification within the Cynanchum species primarily occurred during the recent Miocene epoch. The divergence time estimation presented in this study will facilitate future research on Cynanchum, aid in species differentiation, and facilitate diverse investigations into this economically and ecologically important genus.

Comparative chloroplast genomes: insights into the identification and phylogeny of rapid radiation genus Rhodiola.

Rhodiola L. is a genus exhibiting rapid radiation and represents a typical case for studying plastid gene adaptation in species that spread from high altitudes to low altitudes. In this study, 23 samples of 18 Rhodiola species were collected from the Qinghai-Tibetan Plateau and five scattered alpine areas, and the plastid genomes (plastomes) of these species were sequenced, annotated, and compared between high-altitude and widely distributed groups. The plastomes of Rhodiola were found to be highly conserved in terms of gene size, content, and order but highly variable in several lineage-specific features, such as codon usage bias, IR boundary shifting, and distinct repeat sequence structures binding to SSRs. Codon usage in the genes of photosystem II exhibited an obvious preference, reflecting significant environmental adaptation pressures. In this study, three repeat regions compounded with trinucleotide and mononucleotide repeats were found for the first time in R. forrestii, R. himalensis, and R. yunnanensis. High-variability regions such as ndhF, ycf1, trnH-psbA, and rpoC1-rpoB were screened, laying the foundation for the precise identification of these species. The phylogenetic analysis revealed the occurrence of cyto-nuclear discordance, likely originating from the frequent interspecific hybridization events observed within Rhodiola species during rapid radiation. Dioecious and hermaphrodite species can be broadly categorized into two subclades, probably they have different environmental adaptation strategies in response to climate change. In addition, the phylogenetic tree supported the monophyly of R. forrestii and R. yunnanensis, which compose R. Sect. Pseudorhodiola. In conclusion, plastome data enrich the genetic information available for the Rhodiola genus and may provide insight into species migration events during climate change.

The complete chloroplast genome of Mussaenda pubescens and phylogenetic analysis.

The chloroplast (cp) genome sequence of Mussaenda pubescens, a promising resource that is used as a traditional medicine and drink, is important for understanding the phylogenetic relationships among the Mussaenda family and genetic improvement and reservation. This research represented the first comprehensive description of the morphological characteristics of M. pubescens, as well as an analysis of the complete cp genome and phylogenetic relationship. The results indicated a close relationship between M. pubescens and M. hirsutula based on the morphological characteristics of the flower and leaves. The cp was sequenced using the Illumina NovaSeq 6000 platform. The results indicated the cp genome of M. pubescens spanned a total length of 155,122 bp, including a pair of inverted repeats (IRA and IRB) with a length of 25,871 bp for each region, as well as a large single-copy (LSC) region and a small single-copy (SSC) region with lengths of 85,370 bp and 18,010 bp, respectively. The results of phylogenetic analyses demonstrated that species within the same genus displayed a tendency to group closely together. It was suggested that Antirhea, Cinchona, Mitragyna, Neolamarckia, and Uncaria might have experienced an early divergence. Furthermore, M. hirsutula showed a close genetic connection to M. pubescens, with the two species having partially overlapping distributions in China. This study presents crucial findings regarding the identification, evolution, and phylogenetic research on Mussaenda plants, specifically targeting M. pubescens.

Intraspecific differentiation of Lindera obtusiloba as revealed by comparative plastomic and evolutionary analyses.

Lindera obtusiloba Blume is the northernmost tree species in the family Lauraceae, and it is a key species in understanding the evolutionary history of this family. The species of L. obtusiloba in East Asia has diverged into the Northern and Southern populations, which are geographically separated by an arid belt. Though the morphological differences between populations have been observed and well documented, intraspecific variations at the plastomic level have not been systematically investigated to date. Here, ten chloroplast genomes of L. obtusiloba individuals were sequenced and analyzed along with three publicly available plastomes. Comparative plastomic analysis suggests that both the Northern and the Southern populations share similar overall structure, gene order, and GC content in their plastomes although the size of the plasome and the level of intraspecific variability do vary between the two populations. The Northern have relatively larger plastomes while the Southern population possesses higher intraspecific variability, which could be attributed to the complexity of the geological environments in the South. Phylogenomic analyses also support the split of the Northern and Southern clades among L. obtusiloba individuals. However, there is no obvious species boundary between var. obtusiloba and var. heterophylla in the Southern population, indicating that gene flow could still occur between these two varieties, and this could be used as a good example of reticulate evolution. It is also found that a few photosynthesis-related genes are under positive selection, which is mainly related to the geological and environmental differences between the Northern and the Southern regions. Our results provide a reference for phylogenetic analysis within species and suggest that phylogenomic analyses with a sufficient number of nuclear and chloroplast genomic target loci from widely distributed individuals could provide a deeper understanding of the population evolution of the widespread species.

Comparative Analyses of Complete Chloroplast Genomes of Microula sikkimensis and Related Species of Boraginaceae.

The present study provides a detailed analysis of the chloroplast genome of Microula sikkimensis. The genome consisted of a total of 149,428 bp and four distinct regions, including a large single-copy region (81,329 bp), a small single-copy region (17,261 bp), and an inverted repeat region (25,419 bp). The genome contained 112 genes, including 78 protein-coding genes, 30 tRNA genes, and 4 rRNA genes, and some exhibited duplication in the inverted repeat region. The chloroplast genome displayed different GC content across regions, with the inverted repeat region exhibiting the highest. Codon usage analysis and the identification of simple sequence repeats (SSRs) offer valuable genetic markers. Comparative analysis with other Boraginaceae species highlighted conservation and diversity in coding and noncoding regions. Phylogenetic analysis placed M. sikkimensis within the Boraginaceae family, revealing its distinct relationship with specific species.

Evolution of Cherries (Prunus Subgenus Cerasus) Based on Chloroplast Genomes.

Cherries (Prunus Subgenus Cerasus) have economic value and ecological significance, yet their phylogeny, geographic origin, timing, and dispersal patterns remain challenging to understand. To fill this gap, we conducted a comprehensive analysis of the complete chloroplast genomes of 54 subg. Cerasus individuals, along with 36 additional genomes from the NCBI database, resulting in a total of 90 genomes for comparative analysis. The chloroplast genomes of subg. Cerasus exhibited varying sizes and consisted of 129 genes, including protein-coding, transfer RNA, and ribosomIal RNA genes. Genomic variation was investigated through InDels and SNPs, showcasing distribution patterns and impact levels. A comparative analysis of chloroplast genome boundaries highlighted variations in inverted repeat (IR) regions among Cerasus and other Prunus species. Phylogeny based on whole-chloroplast genome sequences supported the division of Prunus into three subgenera, I subg. Padus, II subg. Prunus and III subg. Cerasus. The subg. Cerasus was subdivided into seven lineages (IIIa to IIIg), which matched roughly to taxonomic sections. The subg. Padus first diverged 51.42 Mya, followed by the separation of subg. Cerasus from subg. Prunus 39.27 Mya. The subg. Cerasus started diversification at 15.01 Mya, coinciding with geological and climatic changes, including the uplift of the Qinghai-Tibet Plateau and global cooling. The Himalayans were the refuge of cherries, from which a few species reached Europe through westward migration and another species reached North America through northeastward migration. The mainstage of cherry evolution was on the Qing-Tibet Plateau and later East China and Japan as well. These findings strengthen our understanding of the evolution of cherry and provide valuable insights into the conservation and sustainable utilization of cherry's genetic resources.

On Pattern-Cladistic Analyses Based on Complete Plastid Genome Sequences.

The fundamental Hennigian principle, grouping solely on synapomorphy, is seldom used in modern phylogenetics. In the submitted paper, we apply this principle in reanalyzing five datasets comprising 197 complete plastid genomes (plastomes). We focused on the latter because plastome-based DNA sequence data gained dramatic popularity in molecular systematics during the last decade. We show that pattern-cladistic analyses based on complete plastid genome sequences can successfully resolve affinities between plant taxa, simultaneously simplifying both the genomic and analytical frameworks of phylogenetic studies. We developed "Matrix to Newick" (M2N), a program to represent the standard molecular alignment of plastid genomes in the form of trees or relationships directly. Thus, massive plastome-based DNA sequence data can be successfully represented in a relational form rather than as a standard molecular alignment. Application of methods of median supertree construction (the Average Consensus method has been used as an example in this study) or Maximum Parsimony analysis to relational representations of plastome sequence data may help systematist to avoid the complicated assumption-based frameworks of Maximum Likelihood or Bayesian phylogenetics that are most used today in massive plastid sequence data analyses. We also found that significant amounts of pure genomic information that typically accommodate the majority of current plastid phylogenomic studies can be effectively dropped by systematists if they focus on the pattern-cladistics or relational analyses of plastome-based molecular data. The proposed pattern-cladistic approach is a powerful and straightforward heuristic alternative to modern plastome-based phylogenetics.

Sequencing and Analysis of Complete Chloroplast Genomes Provide Insight into the Evolution and Phylogeny of Chinese Kale (Brassica oleracea var. alboglabra).

Chinese kale is a widely cultivated plant in the genus Brassica in the family Brassicaceae. The origin of Brassica has been studied extensively, but the origin of Chinese kale remains unclear. In contrast to Brassica oleracea, which originated in the Mediterranean region, Chinese kale originated in southern China. The chloroplast genome is often used for phylogenetic analysis because of its high conservatism. Fifteen pairs of universal primers were used to amplify the chloroplast genomes of white-flower Chinese kale (Brassica oleracea var. alboglabra cv. Sijicutiao (SJCT)) and yellow-flower Chinese kale (Brassica oleracea var. alboglabra cv. Fuzhouhuanghua (FZHH)) via PCR. The lengths of the chloroplast genomes were 153,365 bp (SJCT) and 153,420 bp (FZHH) and both contained 87 protein-coding genes and eight rRNA genes. There were 36 tRNA genes in SJCT and 35 tRNA genes in FZHH. The chloroplast genomes of both Chinese kale varieties, along with eight other Brassicaceae, were analyzed. Simple sequence repeats, long repeats, and variable regions of DNA barcodes were identified. An analysis of inverted repeat boundaries, relative synonymous codon usage, and synteny revealed high similarity among the ten species, albeit the slight differences that were observed. The Ka/Ks ratios and phylogenetic analysis suggest that Chinese kale is a variant of B. oleracea. The phylogenetic tree shows that both Chinese kale varieties and B. oleracea var. oleracea were clustered in a single group. The results of this study suggest that white and yellow flower Chinese kale comprise a monophyletic group and that their differences in flower color arose late in the process of artificial cultivation. Our results also provide data that will aid future research on genetics, evolution, and germplasm resources of Brassicaceae.

Habitat-related plastome evolution in the mycoheterotrophic Neottia listeroides complex (Orchidaceae, Neottieae).

BACKGROUND: Mycoheterotrophs, acquiring organic carbon and other nutrients from mycorrhizal fungi, have evolved repeatedly with substantial plastid genome (plastome) variations. To date, the fine-scale evolution of mycoheterotrophic plastomes at the intraspecific level is not well-characterized. A few studies have revealed unexpected plastome divergence among species complex members, possibly driven by various biotic/abiotic factors. To illustrate evolutionary mechanisms underlying such divergence, we analyzed plastome features and molecular evolution of 15 plastomes of Neottia listeroides complex from different forest habitats. RESULTS: These 15 samples of Neottia listeroides complex split into three clades according to their habitats approximately 6 million years ago: Pine Clade, including ten samples from pine-broadleaf mixed forests, Fir Clade, including four samples from alpine fir forests and Fir-willow Clade with one sample. Compared with those of Pine Clade members, plastomes of Fir Clade members show smaller size and higher substitution rates. Plastome size, substitution rates, loss and retention of plastid-encoded genes are clade-specific. We propose to recognized six species in N. listeroides complex and slightly modify the path of plastome degradation. CONCLUSIONS: Our results provide insight into the evolutionary dynamics and discrepancy of closely related mycoheterotrophic orchid lineages at a high phylogenetic resolution.

Comparative analyses of five complete chloroplast genomes from the endemic genus Cremanthodium (Asteraceae) in Himalayan and adjacent areas.

Cremanthodium Benth. is an endemic genus in the Himalayas and adjacent areas. Some plants of the genus are traditional medicinal plants in Tibetan medicine. In this study, the chloroplast genomes of five species (Cremanthodium arnicoides (DC. ex Royle) Good, Cremanthodium brunneopilosum S. W. Liu, Cremanthodium ellisii (Hook. f.) Kitam., Cremanthodium nervosum S. W. Liu, and Cremanthodium rhodocephalum Diels) were collected for sequencing. The sequencing results showed that the size of the chloroplast genome ranged from 150,985 to 151,284 bp and possessed a typical quadripartite structure containing one large single copy (LSC) region (83,326-83,369 bp), one small single copy (SSC) region (17,956-18,201 bp), and a pair of inverted repeats (IR) regions (24,830-24,855 bp) in C. arnicoides, C. brunneopilosum, C. ellisii, C. nervosum, and C. rhodocephalum. The chloroplast genomes encoded an equal number of genes, of which 88 were protein-coding genes, 37 were transfer ribonucleic acid genes, and eight were ribosomal ribonucleic acid genes, and were highly similar in overall size, genome structure, gene content, and order. In comparison with other species in the Asteraceae family, their chloroplast genomes share similarities but show some structural variations. There was no obvious expansion or contraction in the LSC, SSC or IR regions among the five species, indicating that the chloroplast gene structure of the genus was highly conserved. Collinearity analysis showed that there was no gene rearrangement. The results of the phylogenetic tree showed that the whole chloroplast genomes of the five species were closely related, and the plants of this genus were grouped into one large cluster with Ligularia Cass. and Farfugium Lindl.

The Complete Chloroplast Genomes of Nine Smilacaceae Species from Hong Kong: Inferring Infra- and Inter-Familial Phylogeny.

The Smilacaceae is a cosmopolitan family consisting of 200-370 described species. The family includes two widely accepted genera, namely Smilax and Heterosmilax. Among them, the taxonomical status of Heterosmilax has been continuously challenged. Seven Smilax and two Heterosmilax species can be found in Hong Kong, with most of them having medicinal importance. This study aims to revisit the infra-familial and inter-familial relationships of the Smilacaceae using complete chloroplast genomes. The chloroplast genomes of the nine Smilacaceae species from Hong Kong were assembled and annotated, which had sizes of 157,885 bp to 159,007 bp; each of them was identically annotated for 132 genes, including 86 protein-coding genes, 38 transfer RNA genes, and 8 ribosomal RNA genes. The generic status of Heterosmilax was not supported because it was nested within the Smilax clade in the phylogenetic trees, echoing previous molecular and morphological studies. We suggest delimitating the genus Heterosmilax as a section under the genus Smilax. The results of phylogenomic analysis support the monophyly of Smilacaceae and the exclusion of Ripogonum from the family. This study contributes to the systematics and taxonomy of monocotyledons, authentication of medicinal Smilacaceae, and conservation of plant diversity.

Codon usage bias and genetic diversity in chloroplast genomes of Elaeagnus species (Myrtiflorae: Elaeagnaceae).

Codon usage bias (CUB) reveals the characteristics of species and can be utilized to understand their evolutionary relationship, increase the target genes' expression in the heterologous receptor plants, and further provide theoretic assistance for correlative study on molecular biology and genetic breeding. The chief aim of this work was to analyze the CUB in chloroplast (cp.) genes in nine Elaeagnus species to provide references for subsequent studies. The codons of Elaeagnus cp. genes preferred to end with A/T bases rather than with G/C bases. Most of the cp. genes were prone to mutation, while the rps7 genes were identical in sequences. Natural selection was inferred to have a powerful impact on the CUB in Elaeagnus cp. genomes, and their CUB was extremely strong. In addition, the optimal codons were identified in the nine cp. genomes based on the relative synonymous codon usage (RSCU) values, and the optimal codon numbers were between 15 and 19. The clustering analyses based on RSCU were contrasted with the maximum likelihood (ML)-based phylogenetic tree derived from coding sequences, suggesting that the t-distributed Stochastic Neighbor Embedding clustering method was more appropriate for evolutionary relationship analysis than the complete linkage method. Moreover, the ML-based phylogenetic tree based on the conservative matK genes and the whole cp. genomes had visible differences, indicating that the sequences of specific cp. genes were profoundly affected by their surroundings. Following the clustering analysis, Arabidopsis thaliana was considered the optimal heterologous expression receptor plant for the Elaeagnus cp. genes. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01289-6.

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.

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.

Complete Chloroplast Genomes of Pterodon emarginatus Vogel and Pterodon pubescens Benth: Comparative and Phylogenetic Analyses.

Background: The species Pterodon emarginatus and P. pubescens, popularly known as white sucupira or faveira, are native to the Cerrado biome and have the potential for medicinal use and reforestation. They are sister species with evolutionary proximity. Objective: Considering that the chloroplast genome exhibits a conserved structure and genes, the analysis of its sequences can contribute to the understanding of evolutionary, phylogenetic, and diversity issues. Methods: The chloroplast genomes of P. emarginatus and P. pubescens were sequenced on the Illumina MiSeq platform. The genomes were assembled based on the de novo strategy. We performed the annotation of the genes and the repetitive regions of the genomes. The nucleotide diversity and phylogenetic relationships were analyzed using the gene sequences of these species and others of the Leguminosae family, whose genomes are available in databases. Results: The complete chloroplast genome of P. emarginatus is 159,877 bp, and that of P. pubescens is 159,873 bp. The genomes of both species have circular and quadripartite structures. A total of 127 genes were predicted in both species, including 110 single-copy genes and 17 duplicated genes in the inverted regions. 141 microsatellite regions were identified in P. emarginatus and 140 in P. pubescens. The nucleotide diversity estimates of the gene regions in twenty-one species of the Leguminosae family were 0.062 in LSC, 0.086 in SSC, and 0.036 in IR. The phylogenetic analysis demonstrated the proximity between the genera Pterodon and Dipteryx, both from the clade Dipterygeae. Ten pairs of primers with potential for the development of molecular markers were designed. Conclusion: The genetic information obtained on the chloroplast genomes of P. emarginatus and P. pubescens presented here reinforces the similarity and evolutionary proximity between these species, with a similarity percentage of 99.8%.

Codon usage bias in chloroplast genes implicate adaptive evolution of four ginger species.

Codon usage bias (CUB) refers to different codons exhibiting varying frequencies of usage in the genome. Studying CUB is crucial for understanding genome structure, function, and evolutionary processes. Herein, we investigated the codon usage patterns and influencing factors of protein-coding genes in the chloroplast genomes of four sister genera (monophyletic Roscoea and Cautleya, and monophyletic Pommereschea and Rhynchanthus) from the Zingiberaceae family with contrasting habitats in southwestern China. These genera exhibit distinct habitats, providing a unique opportunity to explore the adaptive evolution of codon usage. We conducted a comprehensive analysis of nucleotide composition and codon usage on protein-coding genes in the chloroplast genomes. The study focused on understanding the relationship between codon usage and environmental adaptation, with a particular emphasis on genes associated with photosynthesis. Nucleotide composition analysis revealed that the overall G/C content of the coding genes was ˂ 48%, indicating an enrichment of A/T bases. Additionally, synonymous and optimal codons were biased toward ending with A/U bases. Natural selection is the primary factor influencing CUB characteristics, particularly photosynthesis-associated genes. We observed differential gene expressions related to light adaptation among sister genera inhabiting different environments. Certain codons were favored under specific conditions, possibly contributing to gene expression regulation in particular environments. This study provides insights into the adaptive evolution of these sister genera by analyzing CUB and offers theoretical assistance for understanding gene expression and regulation. In addition, the data support the relationship between RNA editing and CUB, and the findings shed light on potential research directions for investigating adaptive evolution.

Complete Chloroplast Genome Sequence of the Endemic and Endangered Plant Dendropanax oligodontus: Genome Structure, Comparative and Phylogenetic Analysis.

Dendropanax oligodontus, which belongs to the family Araliaceae, is an endemic and endangered species of Hainan Island, China. It has potential economic and medicinal value owing to the presence of phenylpropanoids, flavonoids, triterpenoids, etc. The analysis of the structure and characteristics of the D. oligodontus chloroplast genome (cpDNA) is crucial for understanding the genetic and phylogenetic evolution of this species. In this study, the cpDNA of D. oligodontus was sequenced for the first time using next-generation sequencing methods, assembled, and annotated. We observed a circular quadripartite structure comprising a large single-copy region (86,440 bp), a small single-copy region (18,075 bp), and a pair of inverted repeat regions (25,944 bp). The total length of the cpDNA was 156,403 bp, and the GC% was 37.99%. We found that the D. oligodontus chloroplast genome comprised 131 genes, with 86 protein-coding genes, 8 rRNA genes, and 37 tRNAs. Furthermore, we identified 26,514 codons, 13 repetitive sequences, and 43 simple sequence repeat sites in the D. oligodontus cpDNA. The most common amino acid encoded was leucine, with a strong A/T preference at the third position of the codon. The prediction of RNA editing sites in the protein-coding genes indicated that RNA editing was observed in 19 genes with a total of 54 editing sites, all of which involved C-to-T transitions. Finally, the cpDNA of 11 species of the family Araliaceae were selected for comparative analysis. The sequences of the untranslated regions and coding regions among 11 species were highly conserved, and minor differences were observed in the length of the inverted repeat regions; therefore, the cpDNAs were relatively stable and consistent among these 11 species. The variable hotspots in the genome included clpP, ycf1, rnK-rps16, rps16-trnQ, atpH-atpI, trnE-trnT, psbM-trnD, ycf3-trnS, and rpl32-trnL, providing valuable molecular markers for species authentication and regions for inferring phylogenetic relationships among them, as well as for evolutionary studies. Evolutionary selection pressure analysis indicated that the atpF gene was strongly subjected to positive environmental selection. Phylogenetic analysis indicated that D. oligodontus and Dendropanax dentiger were the most closely related species within the genus, and D. oligodontus was closely related to the genera Kalopanax and Metapanax in the Araliaceae family. Overall, the cp genomes reported in this study will provide resources for studying the genetic diversity and conservation of the endangered plant D. oligodontus, as well as resolving phylogenetic relationships within the family.

Complete chloroplast genome sequences of Acanthocalyx alba and Acanthocalyx nepalensis subsp. delavayi (Caprifoliaceae).

In this study, the chloroplast genomes of Acanthocalyx alba (Hand.-Mazz., 1925) and Acanthocalyx nepalensis subsp. delavayi (Franchet, 1885) were sequenced, and their total lengths were 148,720 bp and 149,253 bp, respectively. The A. alba genome contained two inverted repeat regions (IRs) of 21,849 bp, a large single-copy region (LSC) of 89,084 bp, and a small single-copy region (SSC) of 15,938 bp, whereas A. nepalensis subsp. delavayi contained two IRs of 21,736 bp, one LSC of 89,034 bp, and one SSC of 16,747 bp. The chloroplast genomes of both A. alba and A. nepalensis subsp. delavayi contained 109 genes, including 72 mRNA, 33 tRNA, and four rRNA genes. Phylogenetic analysis suggested that A. alba is in a clade with A. nepalensis subsp. delavayi. This study provides useful data for further phylogenetic studies of A. alba and A. nepalensis subsp. delavayi.

The complete chloroplast genome sequences of three lilies: genome structure, comparative genomic and phylogenetic analyses.

We sequenced and analyzed the complete chloroplast genomes of Lilium amoenum, Lilium souliei, and Nomocharis forrestii in detail, including the first sequence and structural comparison of Nomocharis forrestii. We found that the lengths and nucleotide composition of the three chloroplast genes showed little variation. The chloroplast genomes of the three Lilium species contain 87 protein coding genes (PCGs), 38 tRNAs, and 8 rRNA genes. The only difference is that Nomocharis forrestii had an additional infA pseudogene. In the sequence analysis of the Lilium chloroplast genomes, 216 SSRs, 143 pairs of long repeats, 571 SNPs, and 202 indels were detected. In addition, we identified seven hypervariable regions that can be used as potential molecular markers and DNA barcodes of Lilium through complete sequence alignment. The phylogenetic tree was constructed from the three chloroplast genome sequences of Lilium obtained here and 40 chloroplast genome sequences from the NCBI database (including 35 Lilium species, 4 Fritillaria species, and one species of Smilax). The analysis showed that the species clustering of the genus Lilium essentially conformed to the classical morphological classification system of Comber, but differences in the classification of individual species remained. In our report, we support the reclassification of Lilium henryi and Lilium rosthorniiy in the genus Lilium. In general, this study not only provides genome data for three Lilium species, but also provides a comparative analysis of the Lilium chloroplast genomes. These advances will help to identify Lilium species, clarify the phylogenetic analysis of the Lilium genus, and help to solve and improve the disputes and deficiencies in the traditional morphological classification.