Dryopterisjinpingensis, a critically endangered diploid new species of Dryopteridaceae from Yunnan, China.

Dryopterisjinpingensis, a new species of diploid, sexually reproductive ferns of Dryopteridaceae from Yunnan, southwestern China, is described and illustrated. Morphologically, D.jinpingensis is similar to D.gaoligongensis but unique in elongated lanceolate laminae, sessile or subsessile pinna stalks, and overlapping membranous scales adnate to stipe base. Phylogenetic analyses based on both plastome and the nuclear AK1 gene sequences showed that D.jinpingensis is sister to D.gaoligongensis. A detailed taxonomic description with line drawings is provided, and its conservation status is evaluated to be critically endangered.

Plastome phylogenomic analysis reveals evolutionary divergences of Polypodiales suborder Dennstaedtiineae.

BACKGROUND: Polypodiales suborder Dennstaedtiineae contain a single family Dennstaedtiaceae, eleven genera, and about 270 species, and include some groups that were previously placed in Dennstaedtiaceae, Hypolepidaceae, Monachosoraceae, and Pteridaceae. The classification and phylogenetic relationships among these eleven genera have been poorly understood. To explore the deep relationships within suborder Dennstaedtiineae and estimate the early diversification of this morphologically heterogeneous group, we analyzed complete plastomes of 57 samples representing all eleven genera of suborder Dennstaedtiineae using maximum likelihood and Bayesian inference. RESULTS: The phylogenetic relationships of all the lineages in the bracken fern family Dennstaedtiaceae were well resolved with strong support values. All six genera of Hypolepidoideae were recovered as forming a monophyletic group with full support, and Pteridium was fully supported as sister to all the other genera in Hypolepidoideae. Dennstaedtioideae (Dennstaedtia s.l.) fell into four clades with full support: the Microlepia clade, the northern Dennstaedtia clade, the Dennstaedtia globulifera clade, and the Dennstaedtia s.s. clade. Monachosorum was strongly resolved as sister to all the remaining genera of suborder Dennstaedtiineae. Based on the well resolved relationships among genera, the divergence between Monachosorum and other groups of suborder Dennstaedtiineae was estimated to have occurred in the Early Cretaceous, and all extant genera (and clades) in Dennstaedtiineae, were inferred to have diversified since the Late Oligocene. CONCLUSION: This study supports reinstating a previously published family Monachosoraceae as a segregate from Dennstaedtiaceae, based on unique morphological evidence, the shady habitat, and the deep evolutionary divergence from its closest relatives.

Structural Variation of Plastomes Provides Key Insight Into the Deep Phylogeny of Ferns.

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.

A revision of Dryopteris sect. Diclisodon (Dryopteridaceae) based on morphological and molecular evidence with description of a new species.

Dryopteris sect. Diclisodon is a small section of ferns with about 12 species mainly distributed in East Asia. Here, we carried out morphological and phylogenetic analyses of this section. A new species from southwest China, D. gaoligongensis, is described and illustrated. Dryopteris gaoligongensis resembles D. indonesiana and D. sparsa, but differs by having a creeping rhizome and large 4-pinnate fronds. We also show that D. glabrior Ching & Z.Y. Liu is a distinct species; however, because it is a later homonym of D. glabrior Copel., it should be renamed D. renchangiana. We conclude that a species previously known as D. nitidula , also an illegitimate homonym, should be recognized with a new name, D. sinonepalensis. We resolve the phylogenetic position of D. yoroii as sister to other sampled species of D. sect. Diclisodon. Our phylogenetic analyses confirm the distinctiveness of D. gaoligongensis, D. renchangiana, and D. sinonepalensis. A key to species of D. sect. Diclisodon in China is provided.

Simultaneous diversification of Polypodiales and angiosperms in the Mesozoic.

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.

Extreme plastid RNA editing may confound phylogenetic reconstruction: A case study of Selaginella (lycophytes).

Cytidine-to-uridine (C-to-U) RNA editing is common in coding regions of organellar genomes throughout land plants. In most cases RNA editing alters translated amino acids or creates new start codons, potentially confounds phylogenetic reconstructions. In this study, we used the spike moss genus Selaginella (lycophytes), which has the highest frequency of RNA editing, as a model to test the effects of extreme RNA editing on phylogenetic reconstruction. We predicted the C-to-U RNA editing sites in coding regions of 18 Selaginella plastomes, and reconstructed the phylogenetic relationships within Selaginella based on three data set pairs consisted of plastome or RNA-edited coding sequences, first and second codon positions, and translated amino acid sequences, respectively. We predicted between 400 and 3100 RNA editing sites of 18 Selaginella plastomes. The numbers of RNA editing sites in plastomes were highly correlated with the GC content of first and second codon positions, but not correlated with the GC content of plastomes as a whole. Contrast phylogenetic analyses showed that there were substantial differences (e.g., the placement of clade B in Selaginella) between the phylogenies generated by the plastome and RNA-edited data sets. This empirical study provides evidence that extreme C-to-U RNA editing in the coding regions of organellar genomes alters the sequences used for phylogenetic reconstruction, and might even confound phylogenetic reconstruction. Therefore, RNA editing sites should be corrected when plastid or mitochondrial genes are used for phylogenetic studies, particularly in those lineages with abundant organellar RNA editing sites, such as hornworts, quillworts, spike mosses, and some seed plants.

Complete plastome of an endemic fern species from China: Neocheiropteris palmatopedata (Polypodiaceae).

Neocheiropteris palmatopedata (Baker) Christ is an endangered fern species endemic to southwest China. In this study, we sequenced the complete plastid genome of N. palmatopedata. The gene order and structure of the N. palmatopedata plastome are similar to those published plastomes in Polypodiales. The complete plastome is 153,344 bp in length, and the GC content is 42.1%. The plastome comprises 113 unique genes (83 protein-coding genes, 29 tRNA genes and four rRNA genes).

Applying DNA Barcodes to Identify Closely Related Species of Ferns: A Case Study of the Chinese Adiantum (Pteridaceae).

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.

Phylogenetic relationships of Chinese Adiantum based on five plastid markers.

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.

Biogeographic disjunction between eastern Asia and North America in the Adiantum pedatum complex (Pteridaceae).

PREMISE OF THE STUDY: Biogeographic analyses of ferns with an eastern Asian-North American disjunction are few. The Adiantum pedatum complex has such a disjunct distribution. The monophyly of the complex needs to be tested and diversification history of the four species needs to be reconstructed. METHODS: Plastid (atpA, atpB, rbcL, trnL-F, and rps4-trnS) sequences of 100 accessions representing the biogeographic diversity of Adiantum were analyzed with parsimony and Bayesian inference. Biogeography of the Adiantum pedatum complex was inferred using programs DIVA and LAGRANGE. Divergence times of clades were estimated with the program BEAST. KEY RESULTS: The A. pedatum complex is monophyletic and sister to the eastern Asian A. edentulum. Accessions of A. pedatum do not form a clade; instead three subgroups are recognizable. The clade of A. aleuticum and A. viridimontanum is nested within A. pedatum. The Asian A. myriosorum is sister to the A. pedatum-A. aleuticum clade. Both DIVA and LAGRANGE analyses suggest an eastern Asian origin of the A. pedatum complex. The age of the crown A. pedatum complex is dated to be at 4.27 (2.24-6.57) million years ago. CONCLUSIONS: The currently recognized eastern Asian-North American disjunct species A. pedatum needs to be segregated into three species, corresponding to populations in eastern North America, China, and Japan. The eastern Asian-North American disjunction in the complex is inferred to be the result of two intercontinental migrations, one from eastern Asia into North America in the late Tertiary and the other from North America back to eastern Asia in the Pleistocene.

[Relationship between longevity and soil environment in Xiayi County, Henan Province, China].

Based on field investigation and laboratory analysis, exploration was done of distribution of macro and micro elements in the soils and distribution of longevity population in Xiayi County, Henan Province, China, and relationship between the spatial variation of the distribution of soil elements and the distribution of the longevity population. It was found that longevity population was distributed in a belt running across Xiayi county from northeast to southwest. In the northeast part, the longevity rate (over 95 years old) reached 187 per million and while in the northwest part, it was only 83. The concentrations of heavy metals such as Cd, Cr, Cu, Ni, Pb, Zn in Xiayi soils were (0.177 +/- 0.057), (63.9 +/- 7.48), (23.6 +/- 5.63), (29.5 +/- 3.80), (21.8 +/- 3.37) and (64.80 +/- 8.81) mg x kg(-1), respectively, all up to the criteria for grade II soils in the National Standard for Soil Environment Quality of China; Moreover, the soils in longevous areas were rich in Cr, Zn, K and Mg. K and Mg in Xiayi soils were 1.30 and 1.79 as times as high as the background value of Henan Province and 1.21 and 2.62 times that of the country, respectively. In Liji, Huodian and Zhongfeng where the centenarian rate was relatively low, the soils were low in Cu, Se and Zn, but high in Na. The findings clearly indicate that the soil rich in Cr, Zn, K and Mg and free of any heavy metal pollution is a major contributor to the longevity in the area, while uneven distribution of Se, Zn, Cu and Na in the soil is the key factor affecting the distribution of longevity population in Xiayi County.

Molecular authentication of the traditional Tibetan medicinal plant Swertia mussotii.

Swertia mussotii is an important species in Tibetan folk medicine. However, it is quite expensive and frequently adulterated, so reliable methods for authentication of putative specimens and preparations of the species are needed to protect consumers and to support conservation measures. We show here that the chloroplast (cp) DNA RPL16 intron has limited utility for differentiating S. mussotii from closely related species, since the cpDNA RPL16 sequences are identical in S. mussotii and two other species of Swertia. However, the rDNA internal transcribed spacer (ITS) sequences differ significantly between S. mussotii and all of 13 tested potential adulterants. Thus, the ITS region provides a robust molecular marker for differentiating the medicinal S. mussotii from related adulterants. Therefore, a pair of allele-specific diagnostic primers based on the divergent ITS region was designed to distinguish S. mussotii from the other species. Authentication by allele-specific diagnostic PCR using these primers is convenient, effective and both simpler and less time-consuming than sequencing the ITS region.

Paraphyly of Cyrtomium (Dryopteridaceae): evidence from rbcL and trnL-F sequence data.

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.