Orchidaceae is the 3rd largest family of angiosperms, an evolved young branch of monocotyledons. This family contains a number of economically-important horticulture and flowering plants. However, the limited availability of genomic information largely hindered the study of molecular evolution and phylogeny of Orchidaceae. In this study, we determined the evolutionary characteristics of whole chloroplast (cp) genomes and the phylogenetic relationships of the family Orchidaceae. We firstly characterized the cp genomes of four orchid species: Cremastra appendiculata, Calanthe davidii, Epipactis mairei, and Platanthera japonica. The size of the chloroplast genome ranged from 153,629 bp (C. davidi) to 160,427 bp (E. mairei). The gene order, GC content, and gene compositions are similar to those of other previously-reported angiosperms. We identified that the genes of ndhC, ndhI, and ndhK were lost in C. appendiculata, in that the ndh I gene was lost in P. japonica and E. mairei. In addition, the four types of repeats (forward, palindromic, reverse, and complement repeats) were examined in orchid species. E. mairei had the highest number of repeats (81), while C. davidii had the lowest number (57). The total number of Simple Sequence Repeats is at least 50 in C. davidii, and, at most, 78 in P. japonica. Interestingly, we identified 16 genes with positive selection sites (the psbH, petD, petL, rpl22, rpl32, rpoC1, rpoC2, rps12, rps15, rps16, accD, ccsA, rbcL, ycf1, ycf2, and ycf4 genes), which might play an important role in the orchid species’ adaptation to diverse environments. Additionally, 11 mutational hotspot regions were determined, including five non-coding regions (ndhB intron, ccsA-ndhD, rpl33-rps18, ndhE-ndhG, and ndhF-rpl32) and six coding regions (rps16, ndhC, rpl32, ndhI, ndhK, and ndhF). The phylogenetic analysis based on whole cp genomes showed that C. appendiculata was closely related to C. striata var. vreelandii, while C. davidii and C. triplicate formed a small monophyletic evolutionary clade with a high bootstrap support. In addition, five subfamilies of Orchidaceae, Apostasioideae, Cypripedioideae, Epidendroideae, Orchidoideae, and Vanilloideae, formed a nested evolutionary relationship in the phylogenetic tree. These results provide important insights into the adaptive evolution and phylogeny of Orchidaceae.

In general, the chloroplast genomes of angiosperms are considered to be highly conserved and affected little by adaptive evolution. In this study, we tested this hypothesis based on sequence differentiation and adaptive variation in the plastid genomes in the order Dipsacales. We sequenced the plastid genomes of one Adoxaceae species and six Caprifoliaceae species, and together with seven previously released Dipsacales chloroplasts, we determined the sequence variations, evolutionary divergence of the plastid genomes, and phylogeny of Dipsacales species. The chloroplast genomes of Adoxaceae species ranged in size from 157,074 bp (Sinadoxa corydalifolia) to 158,305 bp (Sambucus williamsii), and the plastid genomes of Caprifoliaceae varied from 154,732 bp (Lonicera fragrantissima var. lancifolia) to 156,874 bp (Weigela florida). The differences in the number of genes in Caprifoliaceae and Adoxaceae species were largely due to the expansion and contraction of inverted repeat regions. In addition, we found that the number of dispersed repeats (Adoxaceae = 37; Caprifoliaceae = 384) was much higher than that of tandem repeats (Adoxaceae = 34; Caprifoliaceae = 291) in Dipsacales species. Interestingly, we determined 19 genes with positive selection sites, including three genes encoding ATP protein subunits (atpA, atpB, and atpI), four genes for ribosome protein small subunits (rps3, rps7, rps14, and rps15), four genes for photosystem protein subunits (psaA, psaJ, psbC, and pabK), two genes for ribosome protein large subunits (rpl22 and rpl32), and the clpP, infA, matK, rbcL, ycf1, and ycf2 genes. These gene regions may have played key roles in the adaptation of Dipsacales to diverse environments. In addition, phylogenetic analysis based on the plastid genomes strongly supported the division of 14 Dipsacales species into two previously recognized sections. The diversification of Adoxaceae and Caprifoliaceae was dated to the late Cretaceous and Tertiary periods. The availability of these chloroplast genomes provides useful genetic information for studying taxonomy, phylogeny, and species evolution in Dipsacales.

Cotton is one of the most economically important fiber crop plants worldwide. The genus Gossypium contains a single allotetraploid group (AD) and eight diploid genome groups (A–G and K). However, the evolution of repeat sequences in the chloroplast genomes and the phylogenetic relationships of Gossypium species are unclear. Thus, we determined the variations in the repeat sequences and the evolutionary relationships of 40 cotton chloroplast genomes, which represented the most diverse in the genus, including five newly sequenced diploid species, i.e., G. nandewarense (C_1-n), G. armourianum (D_2-1), G. lobatum (D₇), G. trilobum (D₈), and G. schwendimanii (D₁₁), and an important semi-wild race of upland cotton, G. hirsutum race latifolium (AD₁). The genome structure, gene order, and GC content of cotton species were similar to those of other higher plant plastid genomes. In total, 2860 long sequence repeats (>10 bp in length) were identified, where the F-genome species had the largest number of repeats (G. longicalyx F₁: 108) and E-genome species had the lowest (G. stocksii E₁: 53). Large-scale repeat sequences possibly enrich the genetic information and maintain genome stability in cotton species. We also identified 10 divergence hotspot regions, i.e., rpl33-rps18, psbZ-trnG (GCC), rps4-trnT (UGU), trnL (UAG)-rpl32, trnE (UUC)-trnT (GGU), atpE, ndhI, rps2, ycf1, and ndhF, which could be useful molecular genetic markers for future population genetics and phylogenetic studies. Site-specific selection analysis showed that some of the coding sites of 10 chloroplast genes (atpB, atpE, rps2, rps3, petB, petD, ccsA, cemA, ycf1, and rbcL) were under protein sequence evolution. Phylogenetic analysis based on the whole plastomes suggested that the Gossypium species grouped into six previously identified genetic clades. Interestingly, all 13 D-genome species clustered into a strong monophyletic clade. Unexpectedly, the cotton species with C, G, and K-genomes were admixed and nested in a large clade, which could have been due to their recent radiation, incomplete lineage sorting, and introgression hybridization among different cotton lineages. In conclusion, the results of this study provide new insights into the evolution of repeat sequences in chloroplast genomes and interspecific relationships in the genus Gossypium.

Notopterygium H. de Boissieu (Apiaceae) is an endangered perennial herb endemic to China. A good knowledge of phylogenetic evolution and population genomics is conducive to the establishment of effective management and conservation strategies of the genus Notopterygium. In this study, the complete chloroplast (cp) genomes of four Notopterygium species (N. incisum C. C. Ting ex H. T. Chang, N. oviforme R. H. Shan, N. franchetii H. de Boissieu and N. forrestii H. Wolff) were assembled and characterized using next-generation sequencing. We investigated the gene organization, order, size and repeat sequences of the cp genome and constructed the phylogenetic relationships of Notopterygium species based on the chloroplast DNA and nuclear internal transcribed spacer (ITS) sequences. Comparative analysis of plastid genome showed that the cp DNA are the standard double-stranded molecule, ranging from 157,462 bp (N. oviforme) to 159,607 bp (N. forrestii) in length. The circular DNA each contained a large single-copy (LSC) region, a small single-copy (SSC) region, and a pair of inverted repeats (IRs). The cp DNA of four species contained 85 protein-coding genes, 37 transfer RNA (tRNA) genes and 8 ribosomal RNA (rRNA) genes, respectively. We determined the marked conservation of gene content and sequence evolutionary rate in the cp genome of four Notopterygium species. Three genes (psaI, psbI and rpoA) were possibly under positive selection among the four sampled species. Phylogenetic analysis showed that four Notopterygium species formed a monophyletic clade with high bootstrap support. However, the inconsistent interspecific relationships with the genus Notopterygium were identified between the cp DNA and ITS markers. The incomplete lineage sorting, convergence evolution or hybridization, gene infiltration and different sampling strategies among species may have caused the incongruence between the nuclear and cp DNA relationships. The present results suggested that Notopterygium species may have experienced a complex evolutionary history and speciation process.

Plant plastomes play crucial roles in species evolution and phylogenetic reconstruction studies due to being maternally inherited and due to the moderate evolutionary rate of genomes. However, patterns of sequence divergence and molecular evolution of the plastid genomes in the horticulturally- and economically-important Lonicera L. species are poorly understood. In this study, we collected the complete plastomes of seven Lonicera species and determined the various repeat sequence variations and protein sequence evolution by comparative genomic analysis. A total of 498 repeats were identified in plastid genomes, which included tandem (130), dispersed (277), and palindromic (91) types of repeat variations. Simple sequence repeat (SSR) elements analysis indicated the enriched SSRs in seven genomes to be mononucleotides, followed by tetra-nucleotides, dinucleotides, tri-nucleotides, hex-nucleotides, and penta-nucleotides. We identified 18 divergence hotspot regions (rps15, rps16, rps18, rpl23, psaJ, infA, ycf1, trnN-GUU-ndhF, rpoC2-rpoC1, rbcL-psaI, trnI-CAU-ycf2, psbZ-trnG-UCC, trnK-UUU-rps16, infA-rps8, rpl14-rpl16, trnV-GAC-rrn16, trnL-UAA intron, and rps12-clpP) that could be used as the potential molecular genetic markers for the further study of population genetics and phylogenetic evolution of Lonicera species. We found that a large number of repeat sequences were distributed in the divergence hotspots of plastid genomes. Interestingly, 16 genes were determined under positive selection, which included four genes for the subunits of ribosome proteins (rps7, rpl2, rpl16, and rpl22), three genes for the subunits of photosystem proteins (psaJ, psbC, and ycf4), three NADH oxidoreductase genes (ndhB, ndhH, and ndhK), two subunits of ATP genes (atpA and atpB), and four other genes (infA, rbcL, ycf1, and ycf2). Phylogenetic analysis based on the whole plastome demonstrated that the seven Lonicera species form a highly-supported monophyletic clade. The availability of these plastid genomes provides important genetic information for further species identification and biological research on Lonicera.