We have determined the complete chloroplast (cp) genome sequence of Cephalotaxus oliveri. The genome is 134,337 bp in length, encodes 113 genes, and lacks inverted repeat (IR) regions. Genome-wide mutational dynamics have been investigated through comparative analysis of the cp genomes of C. oliveri and C. wilsoniana. Gene order transformation analyses indicate that when distinct isomers are considered as alternative structures for the ancestral cp genome of cupressophyte and Pinaceae lineages, it is not possible to distinguish between hypotheses favoring retention of the same IR region in cupressophyte and Pinaceae cp genomes from a hypothesis proposing independent loss of IR_A and IR_B. Furthermore, in cupressophyte cp genomes, the highly reduced IRs are replaced by short repeats that have the potential to mediate homologous recombination, analogous to the situation in Pinaceae. The importance of repeats in the mutational dynamics of cupressophyte cp genomes is also illustrated by the accD reading frame, which has undergone extreme length expansion in cupressophytes. This has been caused by a large insertion comprising multiple repeat sequences. Overall, we find that the distribution of repeats, indels, and substitutions is significantly correlated in Cephalotaxus cp genomes, consistent with a hypothesis that repeats play a role in inducing substitutions and indels in conifer cp genomes.

Background: Ferns have generally been neglected in studies of chloroplast genomics. Before this study, only one polypod and two basal ferns had their complete chloroplast (cp) genome reported. Tree ferns represent an ancient fern lineage that first occurred in the Late Triassic. In recent phylogenetic analyses, tree ferns were shown to be the sister group of polypods, the most diverse group of living ferns. Availability of cp genome sequence from a tree fern will facilitate interpretation of the evolutionary changes of fern cp genomes. Here we have sequenced the complete cp genome of a scaly tree fern Alsophila spinulosa (Cyatheaceae). Results: The Alsophila cp genome is 156,661 base pairs (bp) in size, and has a typical quadripartite structure with the large (LSC, 86,308 bp) and small single copy (SSC, 21,623 bp) regions separated by two copies of an inverted repeat (IRs, 24,365 bp each). This genome contains 117 different genes encoding 85 proteins, 4 rRNAs and 28 tRNAs. Pseudogenes of ycf66 and trnT-UGU are also detected in this genome. A unique trnR-UCG gene (derived from trnR-CCG) is found between rbcL and accD. The Alsophila cp genome shares some unusual characteristics with the previously sequenced cp genome of the polypod fern Adiantum capillus-veneris, including the absence of 5 tRNA genes that exist in most other cp genomes. The genome shows a high degree of synteny with that of Adiantum, but differs considerably from two basal ferns (Angiopteris evecta and Psilotum nudum). At one endpoint of an ancient inversion we detected a highly repeated 565-bp-region that is absent from the Adiantum cp genome. An additional minor inversion of the trnD-GUC, which is possibly shared by all ferns, was identified by comparison between the fern and other land plant cp genomes. Conclusion: By comparing four fern cp genome sequences it was confirmed that two major rearrangements distinguish higher leptosporangiate ferns from basal fern lineages. The Alsophila cp genome is very similar to that of the polypod fern Adiantum in terms of gene content, gene order and GC content. However, there exist some striking differences between them: the trnR-UCG gene represents a putative molecular apomorphy of tree ferns; and the repeats observed at one inversion endpoint may be a vestige of some unknown rearrangement(s). This work provided fresh insights into the fern cp genome evolution as well as useful data for future phylogenetic studies.

Chloroplast genome sequences have been used to understand evolutionary events and to infer efficiently phylogenetic relationships.Callitropsis funebris(Cupressaceae) is an endemic species in China. Its phylogenetic position is controversial due to morphological characters similar to those ofCupressus,Callitropsis,andChamaecyparis. This study used next‐generation sequencing technology to sequence the complete chloroplast genome ofCa. funebrisand then constructed the phylogenetic relationship betweenCa. funebrisand its related species based on a variety of data sets and methods. Simple sequence repeats (SSRs) and adaptive evolution analysis were also conducted. Our results showed that the monophyletic branch consisting ofCa. funebrisandCupressus tonkinensisis a sister toCupressus,whileCallitropsisis not monophyletic;Ca. nootkatensisandCa. vietnamensisare nested in turn at the base of the monophyletic groupHesperocyparis. The statistical results of SSRs supported the closest relationship betweenCa. funebrisandCupressus. By performing adaptive evolution analysis under the phylogenetic background of Cupressales, the Branch model detected three genes and the Site model detected 10 genes under positive selection; and the Branch‐Site model uncovered thatrpoA has experienced positive selection in theCa. funebriesbranch. Molecular analysis from the chloroplast genome highly supported thatCa. funebrisis at the base ofCupressus. Of note, SSR features were found to be able to shed some light on phylogenetic relationships. In short, this chloroplast genomic study has provided new insights into the phylogeny ofCa. funebrisand revealed multiple chloroplast genes possibly undergoing adaptive evolution.

Why some species become successful invaders is an important issue in invasive biology. However, limited genomic resources make it very difficult for identifying candidate genes involved in invasiveness. Mikania micrantha H.B.K. (Asteraceae), one of the world's most invasive weeds, has adapted rapidly in response to novel environments since its introduction to southern China. In its genome, we expect to find outlier loci under selection for local adaptation, critical to dissecting the molecular mechanisms of invasiveness. An explorative amplified fragment length polymorphism (AFLP) genome scan was used to detect candidate loci under selection in 28 M. micrantha populations across its entire introduced range in southern China. We also estimated population genetic parameters, bottleneck signatures, and linkage disequilibrium. In binary characters, such as presence or absence of AFLP bands, if all four character combinations are present, it is referred to as a character incompatibility. Since character incompatibility is deemed to be rare in populations with extensive asexual reproduction, a character incompatibility analysis was also performed in order to infer the predominant mating system in the introduced M. micrantha populations. Out of 483 AFLP loci examined using stringent significance criteria, 14 highly credible outlier loci were identified by Dfdist and Bayescan. Moreover, remarkable genetic variation, multiple introductions, substantial bottlenecks and character compatibility were found to occur in M. micrantha. Thus local adaptation at the genome level indeed exists in M. micrantha, and may represent a major evolutionary mechanism of successful invasion. Interactions between genetic diversity, multiple introductions, and reproductive modes contribute to increase the capacity of adaptive evolution.

The trans-splicing rps12 gene of fern plastomes (plastid genomes) exhibits a unique structure owing to its variations in intragenic exon location and intron content, and thus, it provides an excellent model system for examining the effect of plastid gene structure on rates and patterns of molecular evolution. In this study, 16 complete fern plastome sequences were newly generated via the Illumina HiSeq sequencing platform. We reconstructed the phylogeny of ferns and inferred the patterns and rates of plastid rps12 gene evolution in a phylogenetic context by combining these plastome data with those of previously published fern species. We uncovered the diversity of fern plastome evolution by characterizing the structures of these genomes and obtained a highly supported phylogenetic framework for ferns. Furthermore, our results revealed molecular evolutionary patterns that were completely different from the patterns revealed in previous studies. There were significant differences in the patterns and rates of nucleotide substitutions in both intron-containing and intron-less rps12 alleles. Rate heterogeneity between single-copy (SC) and inverted repeat (IR) exons was evident. Unexpectedly, however, IR exons exhibited significantly higher synonymous substitution rates (dS) than SC exons, a pattern that contrasts the regional effect responsible for decreased rates of nucleotide substitutions in IRs. Our results reveal that structural changes in plastid genes have important effects on evolutionary rates, and we propose possible mechanisms to explain the variations in the nucleotide substitution rates of this unusual gene.