Comparative Plastid Genomes of Primula Species: Sequence Divergence and Phylogenetic Relationships.

Compared to traditional DNA markers, genome-scale datasets can provide mass information to effectively address historically difficult phylogenies. Primula is the largest genus in the family Primulaceae, with members distributed mainly throughout temperate and arctic areas of the Northern Hemisphere. The phylogenetic relationships among Primula taxa still maintain unresolved, mainly due to intra- and interspecific morphological variation, which was caused by frequent hybridization and introgression. In this study, we sequenced and assembled four complete plastid genomes (Primula handeliana, Primula woodwardii, Primula knuthiana, and Androsace laxa) by Illumina paired-end sequencing. A total of 10 Primula species (including 7 published plastid genomes) were analyzed to investigate the plastid genome sequence divergence and their inferences for the phylogeny of Primula. The 10 Primula plastid genomes were similar in terms of their gene content and order, GC content, and codon usage, but slightly different in the number of the repeat. Moderate sequence divergence was observed among Primula plastid genomes. Phylogenetic analysis strongly supported that Primula was monophyletic and more closely related to Androsace in the Primulaceae family. The phylogenetic relationships among the 10 Primula species showed that the placement of P. knuthiana-P. veris clade was uncertain in the phylogenetic tree. This study indicated that plastid genome data were highly effective to investigate the phylogeny.

Complete chloroplast genome sequence of Pinus tabuliformis var. henryi (Mast.) C.T.Kuan 1983 (Pinaceae).

Pinus tabuliformis var. henryi (Mast.) C.T.Kuan 1983 is an endemic and rare subtropical pine, mainly distributed in central China. In this study, we sequenced the complete chloroplast (cp) genome of P. tabuliformis var. henryi and reported it for the first time. The cp genome was 119,634 base pairs (bp) in total length, including two inverted repeats (IRs, 495 bp), separated by a large single-copy region (LSC, 65,600 bp) and a small single-copy region (SSC, 53,044 bp). There are 114 different genes in the cp genome of P. tabuliformis var. henryi, including 74 protein-coding genes, 36 transfer RNA genes, and four ribosomal RNA genes. The overall GC content of the cp genome was 38.5%. Our phylogenetic analysis of P. tabuliformis var. henryi demonstrated that it was closely related to P. tabuliformis and could be used to identify and analyze its genetic diversity, which was expected to provide new data for taxonomic and phylogenetic studies of Pinus.

Isolation and characterization of polymorphic microsatellite loci in the endangered plant Dipteronia sinensis (Sapindaceae).

PREMISE OF THE STUDY: Microsatellite markers were developed in Dipteronia sinensis to investigate the population genetics of this endangered plant. • METHODS AND RESULTS: Using the Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO) protocol, 19 microsatellite loci were developed in D. sinensis and evaluated for their variability in 29 samples from a natural population. For the 15 polymorphic loci, the number of alleles ranged from nine to 33, while the observed and expected heterozygosities ranged from 0.3793 to 0.9655 and from 0.6029 to 0.9609, respectively. Their cross-taxa transferability was also investigated in Acer miaotaiense, A. palmatum, and A. pictum subsp. mono, and 10 to 15 loci proved amplifiable in these species. • CONCLUSIONS: These microsatellite markers could be employed to investigate the population genetics of D. sinensis and may potentially be applicable to other related species.

Complete chloroplast genome sequence of Micranthes melanocentra (Saxifragaceae).

The phylogenetic relationships among Micranthes taxa remain unclear due to their diversification. Here, we report the complete chloroplast genome of Micranthes melanocentra obtained using high-throughput sequencing technology to provide genomic information for phylogenetic analyses. The plastome is 155,317 bp, with a large single-copy region (LSC) of 86,784 bp, a small single-copy region (SSC) of 18,007 bp, and a pair of 25,263 bp inverted repeat regions (IRs). The genome contains 132 genes, including 86 protein-coding genes, 37 tRNA genes, 8 rRNA genes, and 1 pseudogene. The GC content of the plastome is 37.9%; corresponding values in the LSC, SSC, and IR regions are 36.1%, 31.9%, and 43.3%, respectively. The phylogenetic tree supports the separation of Micranthes from Saxifraga s.l.

Complete chloroplast genome sequence of the carnivorous herb Pinguicula alpina (Lentibulariaceae).

We determined the complete chloroplast genome of Pinguicula alpina, a carnivorous plant using high-throughput sequencing technology. The deduced plastome is a closed circular molecule of 147,479 bp with a quadripartite structure, including a large single-copy region (LSC) of 81,937 bp, a small single-copy region (SSC) of 13,180 bp, and a pair of inverted repeat regions (IRs) of 26,181 bp. It contains 131 genes, comprising 82 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. The GC content of the plastome, LSC, SSC, and IR regions are 38.1%, 36.1%, 31.4% and 43.0%, respectively. Phylogenetic analysis revealed that P. alpina was related to P. ehlersiae.

Complete chloroplast genome sequence of Solanum erianthum: genome structure and genomic resources.

Solanum erianthum is known for its valuable medicinal properties. In this study, we report its complete chloroplast genome. The chloroplast genome size is 156,343 bp, including a LSC region of 86,855 bp, a SSC region of 18,608 bp and two inverted repeats (IR). The complete chloroplast genome includes a total of 128 unique genes with 83 protein-coding sequences, 37 tRNA and 8 rRNA genes. The results showed that S. erianthum was the most closely related to S. violaceum.

Strong phylogeographic pattern of cpDNA variation reveals multiple glacial refugia for Saruma henryi Oliv. (Aristolochiaceae), an endangered herb endemic to China.

Saruma henryi Oliv. (Aristolochiaceae) is an endangered herb endemic to China. In this study, chloroplast microsatellites (cpSSRs) and sequences of the atpB-rbcL intergenic spacers were employed to reveal its genetic diversity and phylogeographic patterns. We detected high within-species genetic diversity (H(T)=0.939 for cpSSR; H(T)=0.862 for atpB-rbcL) and pronounced among-population genetic differentiation (H(S)=0.182, G(ST)=0.811, R(ST)=0.9, F(ST)=0.93 for cpSSR; H(S)=0.238, G(ST)=0.724, N(ST)=0.758, F(ST)=0.79 for atpB-rbcL) with a strong phylogeographic pattern (R(ST)>G(ST), P<0.01 for cpSSR; N(ST)>G(ST), U=0.25 for atpB-rbcL). Eleven haplotypes were distinguished by cpSSRs and atpB-rbcL intergenic spacers, respectively. The molecular phylogenetic data, together with the geographic distribution of the haplotypes, suggested the existence of multiple localized glacial refugia in Mts. Qinling, eastern Mts. Bashan and the southeastern edge of Yunnan-Guizhou Plateau. Nested clade analysis (NCA) and population genetic analyses supported the limited gene flow (caused by low dispersal capacity and complex topography of its habitats) as the major factor responsible for the strong population differentiation and phylogeographic pattern. Past fragmentation and allopatric fragmentation were inferred as important processes responsible for the modern phylogeograhpic pattern. In addition, contiguous range expansions occurred in western Mts. Qinling and eastern Mts. Bashan.

The complete chloroplast genome sequence of Picea schrenkiana (Pinaceae).

Picea is a phylogenetically complicate genus with great economic and ecological values. Here, we determined the whole complete chloroplast genome of Picea schrenkiana to provide genomic information for phylogenetic analysis of the genus. The plastome of P. schrenkiana is 124,060 bp in size and contains 114 genes, including 74 protein-coding genes, 36 tRNA genes, and four rRNA genes. The overall GC content is 38.7%. Unlike the typical plastome with a conserved quadripartite structure, loss of inverted repeat regions is found in the chloroplast genome. The phylogenetic tree shows that monophyly of P. schrenkiana is well supported.

The complete chloroplast genome sequence of Limonium sinense (Plumbaginaceae).

Limonium is a species-rich genus in Plumbaginaceae. In this study, we determined the complete chloroplast genome of Limonium sinense for potential phylogenomic analysis in the future. The plastome is 174,033 bp in length, with a large single-copy region (LSC) of 96,128 bp, a small single-copy region (SSC) of 13,517 bp and a pair of inverted repeat regions (IRs) of 32,194 bp. It contains 132 genes, including 84 protein-coding genes, 37 tRNA genes, 8 rRNA genes, and 3 pseudogenes. The overall GC content is 36.7%, while the corresponding values for the LSC, SSC, and IR region are 34.9, 31.1, and 40.6%, respectively. The phylogenetic analysis supported that L. sinense was clustered together with Plumbago auriculata in family Plumbaginaceae.

Transit From Autotrophism to Heterotrophism: Sequence Variation and Evolution of Chloroplast Genomes in Orobanchaceae Species.

The family Orobanchaceae including autotrophic, hemiparasitic, and holoparasitic species, is becoming a key taxa to study the evolution of chloroplast genomes in different lifestyles. But the early evolutionary trajectory in the transit from autotrophism to hemiparasitism still maintains unclear for the inadequate sampling. In this study, we compared 50 complete chloroplast genomes in Orobanchaceae, containing four newly sequenced plastomes from hemiparasitic Pedicularis, to elucidate the sequence variation patterns in the evolution of plastomes. Contrasted to the sequence and structural hypervariabilities in holoparasites, hemiparasitic plastomes exhibited high similarity to those of autotrophs in gene and GC contents. They are generally characterized with functional or physical loss of ndh/tRNA genes and the inverted small-single-copy region. Gene losses in Orobanchaceae were lineage-specific and convergent, possibly related to structural reconfiguration and expansion/contraction of the inverted region. Pseudogenization of ndh genes was unique in hemiparasites. At least in Pedicularis, the ndhF gene might be most sensitive to the environmental factors and easily pseudogenized when autotrophs transit to hemiparasites. And the changes in gene contents and structural variation potentially deeply rely on the feeding type. Selective pressure, together with mutational bias, was the dominant factor of shaping the codon usage patterns. The relaxed selective constraint, potentially with genome-based GC conversion (gBGC) and preferential codon usage, drive the fluctuation of GC contents among taxa with different lifestyles. Phylogenetic analysis in Orobanchaceae supported that parasitic species were single-originated while holoparasites were multiple-originated. Overall, the comparison of plastomes provided a good opportunity to understand the evolution process in Orobanchaceae with different lifestyles.

Characteristics of Organellar Genomes and Nuclear Internal Transcribed Spacers in the Tertiary Relict Genus Dipelta and Their Phylogenomic Implications.

Dipelta (Caprifoliaceae) is a Tertiary relict genus endemic to China, comprising three species with horticultural and medicinal values. For the lack of genomic information, interspecific relationships and divergence times in the genus remain unresolved. In the present study, we assembled and characterized the complete plastomes, the partial mitogenomes, and nuclear internal transcribed spacer (ITS) fragments from genome skimming datasets of 14 Dipelta individuals. The plastomes were conserved in genomic structure, gene order, and gene content, but with highly variable repeat sequences. Three genes (rpl23, ycf1, ycf2) were examined with positive selection, and nine divergent hotpot regions (psbL, accD, rpl23, ycf2, ycf3, rbcL-accD, trnI-CAU-ycf2, ndhH-rps15, and rps18 intron) were potentially valuable for DNA barcodes. Contrasted to the variability in plastome sequences, mitogenomes contained 12 protein-coding genes with limited indels and nucleotide substitutions, and no gene was found under positive selection. Genes in organellar genomes tended to have a similar pattern of codon usage bias, with a preference of A/U ending codons. Phylogenetic trees constructed with plastomes, mitogenomes, and ITS sequences consistently supported that Dipelta was monophyletic, and Dipelta elegans was sister to the other two taxa. Interspecific divergences were estimated at about 33-37 Ma in the Eocene/Oligocene boundary, suggesting the paleo-endemism of the extant species as "living fossils" of the East Asian Flora. Our study well-exhibited that genome skimming could provide valuable genomic information to elucidate the evolutionary history of the complex group in a cost-efficient way.

Framework Phylogeny, Evolution and Complex Diversification of Chinese Oaks.

Oaks (Quercus L.) are ideal models to assess patterns of plant diversity. We integrated the sequence data of five chloroplast and two nuclear loci from 50 Chinese oaks to explore the phylogenetic framework, evolution and diversification patterns of the Chinese oak's lineage. The framework phylogeny strongly supports two subgenera Quercus and Cerris comprising four infrageneric sections Quercus, Cerris, Ilex and Cyclobalanopsis for the Chinese oaks. An evolutionary analysis suggests that the two subgenera probably split during the mid-Eocene, followed by intergroup divergence within the subgenus Cerris around the late Eocene. The initial diversification of sections in the subgenus Cerris was dated between the mid-Oligocene and the Oligocene-Miocene boundary, while a rapid species radiation in section Quercus started in the late Miocene. Diversification simulations indicate a potential evolutionary shift on section Quercus, while several phenotypic shifts likely occur among all sections. We found significant negative correlations between rates of the lineage diversification and phenotypic turnover, suggesting a complex interaction between the species evolution and morphological divergence in Chinese oaks. Our infrageneric phylogeny of Chinese oaks accords with the recently proposed classification of the genus Quercus. The results point to tectonic activity and climatic change during the Tertiary as possible drivers of evolution and diversification in the Chinese oak's lineage.

The complete chloroplast genome sequence of Liparis japonica (Orchidaceae).

The Orchidaceae contains numerous species with great ecological and economic values. In this study, the complete chloroplast genome of Liparis japonica was presented by next-generation sequencing technologies. The cpDNA is 152,084 bp in length with a large single copy region (LSC) of 85,398 bp, a small single copy region (SSC) of 14,774 bp, and a pair of inverted repeat regions (IRs) of 25,956 bp. It contains 132 genes, including 79 protein-coding genes, 38 tRNA genes, 8 rRNA genes, and 7 pseudogenes. The overall GC content is 37.0%, while the corresponding values in the LSC, SSC, and IR region are 34.3, 30.0, 43.5%, respectively. The phylogenetic analysis shows that Liparis japonica is sister to Liparis loeselii and the genus Liparis is closely related to Dendrobium.

The complete chloroplast genome sequence of Clintonia udensis (Liliaceae).

The whole chloroplast genome of Clintonia udensis, an import Chinese medicinal herb, was determined by Illumina sequencing data in this study. The cp genome is 153,160 bp in length, with a large single-copy region (LSC) of 83,901 bp, a small single-copy region (SSC) of 17,241 bp and a pair of inverted repeat regions (IRs) of 26,009 bp. It contains 132 genes, including 83 protein-coding genes, 36 tRNA genes, 8 rRNA genes, and 5 pseudogenes. The overall GC content was 37.3%, while the corresponding values in the LSC, SSC and IR region are 35.1, 31.0, and 42.8%, respectively. Phylogenetic analysis indicated that Clintonia udensis was related to Lilioideae species in Liliaceae.

The first complete chloroplast genome sequence from Polemonium chinense (Polemoniaceae).

In this study, the complete chloroplast genome of Polemonium chinense was determined by next-generation sequencing technology. The plastome is 155,578 bp in length, with a large single-copy region (LSC) of 85,864 bp and a small single-copy region (SSC) of 18,246 bp, separated by a pair of inverted repeat regions (IRs) of 25,734 bp. It contains 135 genes, including 89 protein-coding genes, 37 tRNA genes, 8 rRNA genes, and 1 pseudogene. The overall GC content is 37.1%, while the corresponding values in the LSC, SSC and IR region are 35.1, 30.3, and 42.9%, respectively. The phylogenetic tree shows that Polemoniaceae is sister to Fouquieriaceae.

The complete chloroplast genome of Parnassia wightiana (Celastraceae).

The infrageneric relationships of the genus Parnassia were not clearly identified due to limited available genetic data. In this study, we presented the complete chloroplast genome of Parnassia wightiana for future phylogenetic study. The plastome of P. wightiana was 152,043 bp in length, with a large single-copy region (LSC) of 82,737 bp and a small single-copy region (SSC) of 19,030 bp, separated by a pair of inverted repeat regions (IRs) of 25,138 bp. It contained 134 genes, including 87 protein-coding genes, 37 tRNA genes, 8 rRNA genes, and 2 pseudogenes. The overall GC content was 37.1%, while the corresponding values in the LSC, SSC, and IR region were 35.0%, 30.8%, and 42.9%, respectively. The phylogenetic analysis supported the monophyly of Parnassia.

The complete chloroplast genome sequence of Cypripedium tibeticum King Ex Rolfe (Orchidaceae).

In this study, we determined the complete chloroplast genome of Cypripedium tibeticum, an endangered species in China. The plastome is 159,223 bp in length, with a large single-copy region (LSC) of 86,537 bp, a small single-copy region (SSC) of 17,552 bp, and a pair of inverted repeat regions (IRs) of 27,567 bp each. It contains 133 genes, including 87 protein-coding genes, 38 tRNA genes, and 8 rRNA genes. The overall GC content was 36.9%, while the corresponding values in the LSC, SSC, and IR regions are 34.6, 30.4, and 42.6%, respectively. The phylogenetic analysis showed that C. tibeticum was closely related to its congeners and the classification of five subfamilies of Orchidaceae was also highly supported.

The complete chloroplast genomes of Trollius farreri and Anemone taipaiensis (Ranunculaceae).

Ranunculaceae, with high morphological biodiversity and widely ecological amplitude tolerance, has been considered as a new good model for adaptive evolution study. In this study, two high-quality chloroplast genomes of Ranunculaceae were determined by next-generation sequencing technologies. The plastomes of Trollius farreri and Anemone taipaiensis exhibit a conserved quadripartite structure, with 160,612 bp and 160,214 bp in length, presenting similar GC contents (38% and 37.6%). The chloroplast genome of Trollius farreri contains 131 genes, including 87 protein-coding genes, 8 rRNA, and 36 tRNA genes, while the plastome of Anemone taipaiensis harbours 137 genes, including 93 protein-coding genes, 8 rRNA, and 36 tRNA genes. Phylogenetic tree constructed with 21 plastomes of Ranunculaceae species indicates that Trollius farreri and Anemone taipaiensis are closely related to their congeneric species, respectively. The phylogenetic relationships are inconsistent with the current classification of Ranunculaceae.

Complete chloroplast genome of the Meconopsis quintuplinervia (Papaveraceae), a traditional medicine of Tibetan.

As a medicinal herb of Tibetan, Meconopsis quintuplinervia is often utilized for treating pneumonia. In this study, the complete chloroplast genome of M. quintuplinervia was determined by next-generation sequencing technology. The overall genome was 154,997 bp in size, including a large single copy (LSC), a small single copy (SSC) and two inverted repeat (IR) regions, which were 85,153 bp, 17,876 bp, and 25,984 bp in length, respectively. The circular chloroplast genome owned 129 genes, comprising 84 protein-coding genes, 8 ribosomal RNA genes (four rRNA species), and 37 transfer RNA genes. The GC contents of the entire sequence, LSC, SSC, and IR region were 38.5%, 37.1%, 32.8%, and 43%, separately. The maximum likelihood tree revealed that M. quintuplinervia was closely related to M. racemosa with strong support value.

The Complete Chloroplast Genome Sequences of Six Rehmannia Species.

Rehmannia is a non-parasitic genus in Orobanchaceae including six species mainly distributed in central and north China. Its phylogenetic position and infrageneric relationships remain uncertain due to potential hybridization and polyploidization. In this study, we sequenced and compared the complete chloroplast genomes of six Rehmannia species using Illumina sequencing technology to elucidate the interspecific variations. Rehmannia plastomes exhibited typical quadripartite and circular structures with good synteny of gene order. The complete genomes ranged from 153,622 bp to 154,055 bp in length, including 133 genes encoding 88 proteins, 37 tRNAs, and 8 rRNAs. Three genes (rpoA, rpoC2, accD) have potentially experienced positive selection. Plastome size variation of Rehmannia was mainly ascribed to the expansion and contraction of the border regions between the inverted repeat (IR) region and the single-copy (SC) regions. Despite of the conserved structure in Rehmannia plastomes, sequence variations provide useful phylogenetic information. Phylogenetic trees of 23 Lamiales species reconstructed with the complete plastomes suggested that Rehmannia was monophyletic and sister to the clade of Lindenbergia and the parasitic taxa in Orobanchaceae. The interspecific relationships within Rehmannia were completely different with the previous studies. In future, population phylogenomic works based on plastomes are urgently needed to clarify the evolutionary history of Rehmannia.