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Aroideae is an important subfamily of the Araceae family and contains many plants with medicinal and edible value. It is difficult to identify and classify Aroideae species accurately on the basis of morphology alone because of their polymorphic phenotypic traits. The chloroplast genome (CPG) is useful for studying on plant taxonomy and phylogeny, and the analysis of codon usage bias (CUB) in CPGs provides further insights into the intricate phylogenetic relationships among Aroideae. The results showed that the codon third position of the chloroplast genome coding sequence in Aroideae was rich in A and T, with a GC content of 37.91%. The ENC-plot and PR2-plot revealed that the codon usage bias of Aroideae was influenced by multiple factors, with natural selection as the dominant factor. Thirteen to twenty optimal codons ending in A/T were identified in 61 Aroideae species. Additionally, the comparative analysis of CPGs revealed that two single copy regions and non-coding regions were variable in Aroideae. Eight highly divergent regions (Pi > 0.064) were identified ( ndhF , rpl32 , ccsA, ndhE , ndhG , ndhF-rpl32 , ccsA-ndhD, and ndhE-ndhG ) , in which ndhE have the potential to serve as a reliable DNA marker to discriminate chloroplasts in Aroideae subfamily. Furthermore, the maximum likelihood-based phylogenetic trees constructed from complete chloroplast genomes and protein-coding sequences presented similar topologies. Principal component clustering analysis based on relative synonymous codon usage values (RSCUs) revealed that Calla was clearly deviated from Montrichardia and Anubias , and that Alocasia was closer to Colocasieae than to Arisaemateae. These findings suggest that the use of RSCU for clustering analysis could offer new theoretical support for species classification and evolution. Our research could provide a theoretical foundation for the chloroplast genetic engineering, taxonomy, and phylogenetic relationships of Aroideae chloroplasts.

Background Polystachya Hook. is a large pantropical orchid genus (c. 240 species) distributed in Africa, southern Asia and the Americas, with the center of diversity in Africa. Previous studies on species of this genus have not obtained the complete chloroplast genomes, structures and variations. Additionally, the phylogenetic position of the genus in the Orchidaceae is still controversial and uncertain. Therefore, in this study, we sequenced the complete plastomes of six Kenya Polystachya species based on genome skimming, subjected them to comparative genomic analysis, and reconstructed the phylogenetic relationships with other Orchidaceae species. Results The results exhibited that the chloroplast genomes had a typical quadripartite structure with conserved genome arrangement and moderate divergence. The plastomes of the six Polystachya species ranged from 145,484 bp to 149,274 bp in length and had an almost similar GC content of 36.9–37.0%. Gene annotation revealed 106–109 single-copy genes. In addition, 19 genes are duplicated in the inverted regions, and 16 genes each possessd one or more introns. Although no large structural variations were observed among the Polystachya plastomes, about 1 kb inversion was found in Polystachya modesta and all 11 ndh genes in the Polystachya plastomes were lost or pseudogenized. Comparative analysis of the overall sequence identity among six complete chloroplast genomes confirmed that for both coding and non-coding regions in Polystachya , SC regions exhibit higher sequence variation than IRs. Furthermore, there were various amplifications in the IR regions among the six Polystachya species. Most of the protein-coding genes of these species had a high degree of codon preference. We screened out SSRs and found seven relatively highly variable loci. Moreover, 13 genes were discovered with significant positive selection. Phylogenetic analysis showed that the six Polystachya species formed a monophyletic clade and were more closely related to the tribe Vandeae. Phylogenetic relationships of the family Orchidaceae inferred from the 85 chloroplast genome sequences were generally consistent with previous studies and robust. Conclusions Our study is the initial report of the complete chloroplast genomes of the six Polystachya species, elucidates the structural characteristics of the chloroplast genome of Polystachya , and filters out highly variable sequences that can contribute to the development of DNA markers for use in the study of genetic variability and evolutionary studies in Polystachya . In addition, the phylogenetic results strongly support that the genus of Polystachya is a part of the tribe Vandeae.

Incarvillea Juss. is an herbaceous genus comprising only 16 species of the Bignoniaceae family. Here, we investigated Incarvillea potaninii Batalin in Mongolia, using conservation assessments, distribution modeling, taxonomic treatment, and comparative plastome analysis. Although previously found in Mongolia and China, we confirmed that it currently occurs only in Mongolia. Globally, I. potaninii is considered vulnerable. Its plastome is 154,003 bp long, containing a large single-copy (LSC) region of 81,790 bp and a small single-copy (SSC) region of 10,037 bp, separated by a pair of inverted repeats (IRs) of 31,088 bp. The genome comprises 111 unique genes, including 77 protein-coding genes, 30 tRNA genes, and four rRNA genes, and the ndh A gene was lost. Compared to typical angiosperm plastomes, Incarvillea shows several large-scale inversions, translocations, acc D gene loss in genus, IR expansion, and SSC contraction. The IR region, integrated with one to thirteen genes of the SSC region, showed great variation in size, order, and content. Phylogenetic analysis revealed that I. potaninii is closely related to I. sinensis and I. semiretschenskia . We determined the current and future distributions of I. potaninii using MaxEnt. Under current climatic conditions, the potential habitat of I. potaninii spans approximately 19,655.54 km², with highly suitable areas concentrated in southern Mongolia. Although the total suitable habitat area is projected to remain stable, the area of extreme suitability will decline markedly in future. Under the SSP126 scenario, extreme suitability areas may decrease by 67.83% by 2070. Key predictors of distribution include temperature seasonality, precipitation during the driest quarter, and soil pH. We provide descriptions, taxonomic notes, and distribution of I. potaninii .

Background Pinus is the largest genus of Pinaceae and the most primitive group of modern genera. Pines have become the focus of many molecular evolution studies because of their wide use and ecological significance. However, due to the lack of complete chloroplast genome data, the evolutionary relationship and classification of pines are still controversial. With the development of new generation sequencing technology, sequence data of pines are becoming abundant. Here, we systematically analyzed and summarized the chloroplast genomes of 33 published pine species. Results Generally, pines chloroplast genome structure showed strong conservation and high similarity. The chloroplast genome length ranged from 114,082 to 121,530 bp with similar positions and arrangements of all genes, while the GC content ranged from 38.45 to 39.00%. Reverse repeats showed a shrinking evolutionary trend, with IRa/IRb length ranging from 267 to 495 bp. A total of 3,205 microsatellite sequences and 5,436 repeats were detected in the studied species chloroplasts. Additionally, two hypervariable regions were assessed, providing potential molecular markers for future phylogenetic studies and population genetics. Through the phylogenetic analysis of complete chloroplast genomes, we offered novel opinions on the genus traditional evolutionary theory and classification. Conclusion We compared and analyzed the chloroplast genomes of 33 pine species, verified the traditional evolutionary theory and classification, and reclassified some controversial species classification. This study is helpful in analyzing the evolution, genetic structure, and the development of chloroplast DNA markers in Pinus .

Complete chloroplast genome sequences are widely used in the analyses of phylogenetic relationships among angiosperms. As a species-rich genus, species diversity centers of Saxifraga L. include mountainous regions of Eurasia, such as the Alps and the Qinghai–Tibetan Plateau (QTP) sensu lato. However, to date, datasets of chloroplast genomes of Saxifraga have been concentrated on the QTP species; those from European Alps are largely unavailable, which hinders comprehensively comparative and evolutionary analyses of chloroplast genomes in this genus. Here, complete chloroplast genomes of 19 Saxifraga species were de novo sequenced, assembled and annotated, and of these 15 species from Alps were reported for the first time. Subsequent comparative analysis and phylogenetic reconstruction were also conducted. Chloroplast genome length of the 19 Saxifraga species range from 149,217 bp to 152,282 bp with a typical quadripartite structure. All individual chloroplast genome included in this study contains 113 unique genes, including 79 protein-coding genes, four rRNAs and 30 tRNAs. The IR boundaries keep relatively conserved with minor expansion in S. consanguinea. mVISTA analysis and identification of polymorphic loci for molecular markers shows that six intergenic regions (ndhC-trnV, psbE-petL, rpl32-trnL, rps16-trnQ, trnF-ndhJ, trnS-trnG) can be selected as the potential DNA barcodes. A total of 1204 SSRs, 433 tandem repeats and 534 Large sequence repeats were identified in the 19 Saxifraga chloroplast genomes. The codon usage analysis revealed that Saxifraga chloroplast genome codon prefers to end in A/T. Phylogenetic reconstruction of 33 species (31 Saxifraga species included) based on 75 common protein coding genes received high bootstrap support values for nearly all identified nodes, and revealed a tree topology similar to previous studies.

Background Dendrobium is the second-largest genus in the Orchidaceae family. However, research on codon usage bias (CUB) in Dendrobium genus remains relatively limited. This knowledge gap impedes progress in genetic engineering and the breeding of superior varieties, making it challenging to meet the growing demand for higher yield and quality of Dendrobium . Results The cp genomes of Dendrobium species exhibited codon composition biased toward A/T bases, with GC content below 50% (GC1 > GC2 > GC3). Eleven to nineteen optimal codons were identified across these species, with the third base of most codons predominantly ending with A/U. Parity rule 2-bias plots, effective number of codons plots, and neutrality plot analysis further revealed that natural selection serves as the primary driver of CUB variation, while mutation pressure played a secondary role. Notably, the positions of some species were different in the phylogenetic topologies from cp genomes and cp CDS. Conclusions CUB is an effective tool for investigating the phylogenetic evolution of Dendrobium. The CUB in Dendrobium species is primarily driven by natural selection, while mutation pressure plays a secondary role.

Background Scutellaria , a sub-cosmopolitan genus, stands as one of the Lamiaceae family’s largest genera, encompassing approximately 500 species found in both temperate and tropical montane regions. Recognized for its significant medicinal properties, this genus has garnered attention as a research focus, showcasing anti-cancer, anti-inflammatory, antioxidant, and hepatoprotective qualities. Additionally, it finds application in agriculture and horticulture. Comprehending Scutellaria ’s taxonomy is pivotal for its effective utilization and conservation. However, the current taxonomic frameworks, primarily based on morphological characteristics, are inadequate. Despite several phylogenetic studies, the species relationships and delimitations remain ambiguous, leaving the genus without a stable and reliable classification system. Results This study analyzed 234 complete chloroplast genomes, comprising 220 new and 14 previously published sequences across 206 species, subspecies, and varieties worldwide. Phylogenetic analysis was conducted using six data matrices through Maximum Likelihood and Bayesian Inference, resulting in a robustly supported phylogenetic framework for Scutellaria . We propose three subgenera, recommending the elevation of Section Anaspis to subgeneric rank and the merging of Sections Lupulinaria and Apeltanthus . The circumscription of Subgenus Apeltanthus and Section Perilomia needs to be reconsidered. Comparative analysis of chloroplast genomes highlighted the IR/SC boundary feature as a significant taxonomic indicator. We identified a total of 758 SSRs, 558 longer repetitive sequences, and ten highly variable regions, including trnK–rps16 , trnC–petN , petN–psbM , accD–psaI , petA–psbJ , rpl32–trnL , ccsA–ndhD , rps15–ycf1 , ndhF , and ycf1 . These findings serve as valuable references for future research on species identification, phylogeny, and population genetics. Conclusions The phylogeny of Scutellaria , based on the most comprehensive sample collection to date and complete chloroplast genome analysis, has significantly enhanced our understanding of its infrageneric relationships. The extensive examination of chloroplast genome characteristics establishes a solid foundation for the future development and utilization of Scutellaria , an important medicinal plant globally.

Walnuts ( of the Juglandaceae) are well-known economically important resource plants for the edible nuts, high-quality wood, and medicinal use, with a distribution from tropical to temperate zones and from Asia to Europe and Americas. There are about 21 species in . Classification of at section level is problematic, because the phylogenetic position of is disputable. Lacking morphological and DNA markers severely inhibited the development of related researches. In this study, the complete chloroplast genomes and two nuclear DNA regions (the internal transcribed spacer and ubiquitin ligase gene) of 10 representative taxa of were used for comparative genomic analyses in order to deepen the understanding on the application value of genetic information for inferring the phylogenetic relationship of the genus. The chloroplast genomes possessed the typical quadripartite structure of angiosperms, consisting of a pair of inverted repeat regions separated by a large single-copy region and a small single-copy region. All the 10 chloroplast genomes possessed 112 unique genes arranged in the same order, including 78 protein-coding, 30 tRNA, and 4 rRNA genes. A combined sequence data set from two nuclear DNA regions revealed that plants could be classified into three branches: (1) section , (2) section including which is closer to , and (3) section . However, three branches with a different phylogenetic topology were recognized in using the complete chloroplast genome sequences: (1) section , (2) section , and (3) section plus . The molecular taxonomy of is almost compatible to the morphological taxonomy except (section ). Based on the complete chloroplast genome sequence data, the divergence time between section and section was 44.77 Mya, while section diverged from other sections in the genus was 47.61 Mya. Eleven of the 12 small inversions in the chloroplast genomes provided valuable phylogenetic information for classification of walnut plants at section and species levels. Our results are valuable for future studies on genetic diversity and will enhance the understanding on the phylogenetic evolution of Juglandaceae.

Nekemias grossedentata ( N. grossedentata ) is a medicinal and edible plant. The young leaves and tender stems are specifically utilized to manufacture vine tea, which is traditionally employed in the treatment of conditions such as the common cold fever, sore throat, jaundice hepatitis, and other ailments. The morphologically of N. grossedentata similar to Nekemias cantoniensis ( N. cantoniensis ) and Nekemias megalophylla ( N. megalophylla ), which lead to a chaotic market supply. Numerious studies have confirmed that chloroplast genomes and chromatography play important role in plant classification. Here, the whole chloroplast (cp) genomes of the three Nekemias species were sequenced in Illumina sequencing platform. Meanwhile, their chromatographic fingerprints have constructed using high-performance liquid chromatography (HPLC). The annotation results demonstrated that the three chloroplast genomes were typical quadripartite structures, with lengths of 162,147 bp ( N. grossedentata ), 161,981 bp ( N. megalophylla ), and 162,500 bp ( N. cantoniensis ), respectively. A total of 89 ( N. grossedentata ) /86 ( N. megalophylla and N. cantoniensis ) protein-coding genes, 37 tRNA gene and 8 rRNA genes were annotated. The IR/SC boundary regions were relatively conserved across the three species, although three regions ( rps19-rpl2, rpl32-trnL-UAG, ccsA-ndhD ) exhibited nucleotide diversity values (Pi) of variable sites higher than 1%. Phylogenetic analysis indicated that N. grossedentata had a closer genetic relationship with N. megalophylla than that of N. cantoniensis . Moreover, the chromatographic fingerprints revealed that the main functional components and genetic relatedness of three species were highly similar with their morphological results. In conclusion, N. grossedentata and N. megalophylla can be consider as the origin plants of vine tea. This study provides appropriate information for species identification, phylogeny, quality assessment of three medicinal plants of the genus Nekemias and will contribute to the standardization of vine tea raw materials.

Background Limited access to genetic information has greatly hindered our understanding of the molecular evolution, phylogeny, and differentiation time of subg. Amygdalus . This study reported complete chloroplast (cp) genome sequences of subg. Amygdalus , which further enriched the available valuable resources of complete cp genomes of higher plants and deepened our understanding of the divergence time and phylogenetic relationships of subg. Amygdalus . Results The results showed that subg. Amygdalus species exhibited a tetrad structure with sizes ranging from 157,736 bp ( P. kansuensis ) to 158,971 bp ( P. davidiana ), a pair of inverted repeat regions (IRa/IRb) that ranged from 26,137–26,467 bp, a large single-copy region that ranged from 85,757–86,608 bp, and a small single-copy region that ranged from 19,020–19,133 bp. The average GC content of the complete cp genomes in the 12 species was 36.80%. We found that the structure of the subg. Amygdalus complete cp genomes was highly conserved, and the 12 subg. Amygdalus species had an rps 19 pseudogene. There was not rearrangement of the complete cp genome in the 12 subg. Amygdalus species. All 12 subg. Amygdalus species clustered into one clade based on both Bayesian inference and maximum likelihood. The divergence time analyses based on the complete cp genome sequences showed that subg. Amygdalus species diverged approximately 15.65 Mya. Conclusion Our results provide data on the genomic structure of subg. Amygdalus and elucidates their phylogenetic relationships and divergence time.