Studies on chloroplast genomes of ferns and lycophytes are relatively few in comparison with those on seed plants. Although a basic phylogenetic framework of extant ferns is available, relationships among a few key nodes remain unresolved or poorly supported. The primary objective of this study is to explore the phylogenetic utility of large chloroplast gene data in resolving difficult deep nodes in ferns. We sequenced the chloroplast genomes from Cyrtomium devexiscapulae (Koidz.) Ching (eupolypod I) and Woodwardia unigemmata (Makino) Nakai (eupolypod II), and constructed the phylogeny of ferns based on both 48 genes and 64 genes. The trees based on 48 genes and 64 genes are identical in topology, differing only in support values for four nodes, three of which showed higher support values for the 48-gene dataset. Equisetum L. was resolved as the sister to the Psilotales-Ophioglossales clade, and Equisetales-Psilotales-Ophioglossales clade was sister to the clade of the leptosporangiate and marattioid ferns. The sister relationship between the tree fern clade and polypods was supported by 82% and 100% bootstrap values in the 64-gene and 48-gene trees, respectively. Within polypod ferns, Pteridaceae was sister to the clade of Dennstaedtiaceae and eupolypods with a high support value, and the relationship of Dennstaedtiaceae-eupolypods was strongly supported. With recent parallel advances in the phylogenetics of ferns using nuclear data, chloroplast phylogenomics shows great potential in providing a framework for testing the impact of reticulate evolution in the early evolution of ferns.

Comprising about 82% of the extant fern species diversity, Polypodiales are generally believed to have diversified in the Late Cretaceous. We estimated the divergence times of Polypodiales using both penalized likelihood and Bayesian methods, based on a dataset consisting of 208 plastomes representing all 28 families and 14 fossil constraints reflecting current interpretations of fossil record. Our plastome phylogeny recovered the same six major lineages as a recent nuclear phylogeny, but the position of Dennstaedtiineae was different. The present phylogeny showed high resolution of relationships among the families of Polypodiales, especially among those forming the Aspleniineae. The divergence time estimates supported the most recent common ancestor of Polypodiales and its closest relative dating back to the Triassic, establishment of the major lineages in the Jurassic, and a likely accelerated radiation during the late Jurassic and the Early Cretaceous. The estimated divergence patterns of Polypodiales and angiosperms converge to a scenario in which their main lineages were established simultaneously shortly before the onset of the Cretaceous Terrestrial Revolution, and further suggest a pre-Cretaceous hidden history for both lineages. The pattern of simultaneous diversifications shown here elucidate an important gap in our understanding of the Terrestrial Revolution that shaped today's ecosystems.

Adiantum consists of about 150-200 species mostly with a pantropical distribution, yet the classifications of Adiantum have been based primarily on regional studies. Confounding the clarity of reconstructing the evolutionary history of Adiantum is that previous molecular phylogenetic studies suggest that a separate and distinctive clade, the vittarioids, may be derived from within Adiantum. Five plastid markers (atpA, atpB, rbcL, trnL-F and rps4-trnS) are employed to assess the monophyly of Adiantum, and construct the molecular phylogeny of Chinese Adiantum. Our analyses support the monophyly of Adiantum. All temperate Adiantum species form a clade nested within the pantropical grade, suggesting a tropical origin of Adiantum. Six main clades are supported within Chinese Adiantum, which are only partially consistent with Lin's classification of the genus. Series Caudata is polyphyletic with series Gravesiana nested within one subgroup of series Caudata. The prolonged whip-like stolon at the apex of the fronds is the defining character for series Caudata, but it may have evolved multiple times. Adiantum reniforme with the simple fronds is sister to series Venusta, which has a decompound lamina with many flabellate to cuneate segments. Series Veneri-capilliformia is not monophyletic, with A. capillus-veneris sister to series Flabellulata except for A. diaphanum, and A. edentulum sister to series Pedata. Series Flabellulata is biphyletic with A. diaphanum nested within the pantropical grade. The phylogeny suggests that convergent evolution in frond architecture has occurred in Adiantum.

No abstract

Cyrtomium is an Asiatic genus characterized by anastomosing veins with included veinlets, and comprises about 40 species. We sequenced rbcL and trnL-F sequences of 19 species of Cyrtomium and eight species from related genera in order to elucidate a molecular phylogeny of the genus using maximum-parsimony methods. The phylogenetic trees did not agree with traditional classifications. Cyrtomium was resolved as paraphyletic, and a clade including subseries Balansana of Cyrtomium, Cyrtogonellum, Polystichum subacutidens and Cyrtomidictyum (the BCPC clade) and a second one containing Cyrtomium sensu stricto were monophyletic. The results also implied that: (1) C. uniseriale was synonymous with C. balansae; (2) C. falcatum was likely the female parent of C. devexiscapulae; and (3) based on the rbcL and trnL-F sequence data, C. nephrolepioides and C. grossum were the female parents of C. shingianum and C. chingianum, respectively, although other evidence is needed for the confirmation of this hypothesis.

DNA barcoding is a fast-developing technique to identify species by using short and standard DNA sequences. Universal selection of DNA barcodes in ferns remains unresolved. In this study, five plastid regions (rbcL, matK, trnH-psbA, trnL-F and rps4-trnS) and eight nuclear regions (ITS, pgiC, gapC, LEAFY, ITS2, IBR3_2, DET1, and SQD1_1) were screened and evaluated in the fern genus Adiantum from China and neighboring areas. Due to low primer universality (matK) and/or the existence of multiple copies (ITS), the commonly used barcodes matK and ITS were not appropriate for Adiantum. The PCR amplification rate was extremely low in all nuclear genes except for IBR3_2. rbcL had the highest PCR amplification rate (94.33%) and sequencing success rate (90.78%), while trnH-psbA had the highest species identification rate (75%). With the consideration of discriminatory power, cost-efficiency and effort, the two-barcode combination of rbcL+ trnH-psbA seems to be the best choice for barcoding Adiantum, and perhaps basal polypod ferns in general. The nuclear IBR3_2 showed 100% PCR amplification success rate in Adiantum, however, it seemed that only diploid species could acquire clean sequences without cloning. With cloning, IBR3_2 can successfully distinguish cryptic species and hybrid species from their related species. Because hybridization and allopolyploidy are common in ferns, we argue for including a selected group of nuclear loci as barcodes, especially via the next-generation sequencing, as it is much more efficient to obtain single-copy nuclear loci without the cloning procedure.

Structural variation of plastid genomes (plastomes), particularly large inversions and gene losses, can provide key evidence for the deep phylogeny of plants. In this study, we investigated the structural variation of fern plastomes in a phylogenetic context. A total of 127 plastomes representing all 50 recognized families and 11 orders of ferns were sampled, making it the most comprehensive plastomic analysis of fern lineages to date. The samples included 42 novel plastomes of 15 families with a focus on Hymenophyllales and Gleicheniales. We reconstructed a well-supported phylogeny of all extant fern families, detected significant structural synapomorphies, including 9 large inversions, 7 invert repeat region (IR) boundary shifts, 10 protein-coding gene losses, 7 tRNA gene losses or anticodon changes, and 19 codon indels (insertions or deletions) across the deep phylogeny of ferns, particularly on the backbone nodes. The newly identified inversion V5, together with the newly inferred expansion of the IR boundary R5, can be identified as a synapomorphy of a clade composed of Dipteridaceae, Matoniaceae, Schizaeales, and the core leptosporangiates, while a unique inversion V4, together with an expansion of the IR boundary R4, was verified as a synapomorphy of Gleicheniaceae. This structural evidence is in support of our phylogenetic inference, thus providing key insight into the paraphyly of Gleicheniales. The inversions of V5 and V7 together filled the crucial gap regarding how the “reversed” gene orientation in the IR region characterized by most extant ferns (Schizaeales and the core leptosporangiates) evolved from the inferred ancestral type as retained in Equisetales and Osmundales. The tRNA genes trnR-ACG and trnM-CAU were assumed to be relicts of the early-divergent fern lineages but intact in most Polypodiales, particularly in eupolypods; and the loss of the tRNA genes trnR-CCG, trnV-UAC, and trnR-UCU in fern plastomes was much more prevalent than previously thought. We also identified several codon indels in protein-coding genes within the core leptosporangiates, which may be identified as synapomorphies of specific families or higher ranks. This study provides an empirical case of integrating structural and sequence information of plastomes to resolve deep phylogeny of plants.

Cyrtomium (Dryopteridaceae) is an Asiatic genus comprising about 40 species characterized by having anastomosing veins that form areolae with included veinlets. Twelve chromosome counts representing 11 taxa were made in the present study. The chromosome numbers of six species endemic to China are reported for the first time: C. uniseriale , 2 n = 164; C. guizhouense , 2 n = 82; C. shingianum , n = 82, 2 n = c .164; C. chingianum , 2 n = 164; C. urophyllum , 2 n = 82 and C. aequibasis , 2 n = 123. The results were used to show that (1) C. uniseriale should be reduced to varietal status under C. balansae ; (2) subseries Balansana is not closely related to any other Cyrtomium species and should be separated from Cyrtomium ; and (3) C. guizhouense and C. lonchitoides are basal groups when subseries Balansana is not considered. These results were also supported by morphology. A high level of differentiation is a distinct characteristic in Cyrtomium . The genus has two reproductive types: sexual and apogamous. Sexual species are distributed mostly in China. Southwestern China is a modern diversity centre of Cyrtomium , with 80.6% of known species occurring there. Many species are endemic, most of which are sexual tetraploids or apogamous triploids. Both the apogamous reproductive type and polyploid species are adaptions to limestone habitats.