A light and scanning electron microscopic diagnosis of leaf epidermal morphology and its systematic implications in Dryopteridaceae: Investigating 12 Pakistani taxa.

Dryopteris and Polystichum are the 2 complex taxonomic genera of Dryopteridaceae. The comparative foliar epidermal anatomy of 12 species of both genera from Pakistan were studied using standard protocols of light microscopy (LM) and scanning electron microscopy (SEM). The objective of which was systematic comparison and investigation to elucidate the taxonomic importance of foliar micromorphology, which may be useful to taxonomists for identifying complex Dryopteridaceae taxa. Principal component analysis and UPGMA clustering analysis were performed to test the validity of leaf anatomical features as method of separating species and genera. The foliar epidermal anatomy described here is a good source of taxonomic characters in both groups that can help genera and species delimitation. This is the first report on leaf micromorphology in most of these species. Observation of foliar anatomy showed that stomata are only present on the abaxial surface; i.e., leaves of all species are hypostomatic. The shapes of epidermal cells in all studied species are irregular. The anticlinal walls are strongly lobed, irregular wavy and elongated wavy. However, substantial variation in epidermal cell size and other stomatal features were observed on both upper and lower surfaces in all investigated species. Two types of stomata were observed in all studied species. The presence of polocytic stomata in Dryopteris and staurocytic stomata in Polystichum are the important characters for the segregation of these genera. Elongate elliptic stomatal shape, narrow kidney shaped guard cells and broad elliptic shaped stomatal pores are diagnostic for all five species of Dryopteris selected. On the other hand, size and number of epidermal cells, lobes per cell, stomatal size, subsidiary cell size, stomatal pore size and stomatal index are the key features for species differentiation in Polystichum. An identification key was developed in order to apply the foliar anatomical characters in the discrimination of the species studied.

Into Africa: Molecular phylogenetics and historical biogeography of sub-Saharan African woodferns (Dryopteris).

PREMISE OF THE STUDY: Our goal was to infer the phylogenetic relationships and historical biogeography of the genus Dryopteris with a focus on taxa in sub-Saharan Africa and neighboring islands. In general, little is known about the relationships between African fern species and their congeners in other geographic regions, and our aim was to determine whether the sub-Saharan African species of Dryopteris are monophyletic and evolved within Africa or arrived there via repeated dispersals into Africa from other regions. METHODS: We obtained sequence data for five chloroplast markers from 214 species of Dryopteris and 18 outgroups. We performed phylogenetic and molecular dating analyses using a Bayesian relaxed clock method in BEAST with fossil and secondary calibration points and estimated ancestral ranges for the genus globally by comparing multiple models in BioGeoBEARS. KEY RESULTS: We found that 22 of 27 accessions of sub-Saharan African Dryopteris belong to a large clade of 31 accessions that also includes taxa from Indian and Atlantic Ocean islands. Additional accessions of taxa from our regions of interest have Asian, Hawaiian, European, or North American species as their closest relatives. CONCLUSIONS: The majority of sub-Saharan African Dryopteris species are descended from a shared common ancestor that dispersed to Africa from Asia approximately 10 Ma. There have been subsequent dispersal events from the African mainland to islands in the Atlantic and Indian Oceans, including Madagascar. Several additional species are estimated to have descended from ancestors that reached Africa via separate events over the last roughly 20 million years.

[Identification of Dryopteridis Crassirhizomatis Rhizoma based on psbA-trnH barcode].

To identify origin of the medicinal materials Dryopteridis Crassirhizomatis Rhizoma by using the psbA-trnH sequence, the polymerase chain reaction (PCR) amplification and product sequencing of the experimental samples were performed. In order to expand the scope of the study, the psbA-trnH sequences of 8 genera and 3 species were downloaded from GenBank for analysis. DNAMAN 8.0 software was used to show splicing and comparison results of the peak diagrams with analysis of them, and MEGA 6.0 software was to calculate K2P genetic distances and establish clustering tree adjacent genus. The results showed that by using the psbA-trnH sequence, Dryopteridis Crassirhizomatis Rhizoma, its original plant and other easy-confused medicinal materials and plants can be distinguished with each other obviously, with the psbA-trnH sequence of Dryopteridis Crassirhizomatis Rhizoma completely consistent with that of its original plant. Consequently, it is revealed that it's feasible to identify Dryopteridis Crassirhizomatis Rhizoma and its original plant, and separate from its adulterants by means of the psbA-trnH sequence, which can provide more scientific bases for the further study of the identification of the ferny medicinal herbs.

Molecular circumscription and major evolutionary lineages of the fern genus Dryopteris (Dryopteridaceae).

BACKGROUND: The fern genus Dryopteris (Dryopteridaceae) is among the most common and species rich fern genera in temperate forests in the northern hemisphere containing 225-300 species worldwide. The circumscription of Dryopteris has been controversial and various related genera have, over the time, been included in and excluded from Dryopteris. The infrageneric phylogeny has largely remained unclear, and the placement of the majority of the supraspecific taxa of Dryopteris has never been tested using molecular data. RESULTS: In this study, DNA sequences of four plastid loci (rbcL gene, rps4-trnS spacer, trnL intron, trnL-F spacer) were used to reconstruct the phylogeny of Dryopteris. A total of 122 accessions are sampled in our analysis and they represent 100 species of the expanded Dryopteris including Acrophorus, Acrorumohra, Diacalpe, Dryopsis, Nothoperanema, and Peranema. All four subgenera and 19 sections currently recognized in Dryopteris s.s. are included. One species each of Arachniodes, Leptorumohra, and Lithostegia of Dryopteridaceae are used as outgroups. Our study confirms the paraphyly of Dryopteris and provides the first strong molecular evidence on the monophyly of Acrophorus, Diacalpe, Dryopsis, Nothoperanema, and Peranema. However, all these monophyletic groups together with the paraphyletic Acrorumohra are suggested to be merged into Dryopteris based on both molecular and morphological evidence. Our analysis identified 13 well-supported monophyletic groups. Each of the 13 clades is additionally supported by morphological synapomophies and is inferred to represent a major evolutionary lineage in Dryopteris. In contrast, monophyly of the four subgenera and 15 out of 19 sections currently recognized in Dryopteris s.s is not supported by plastid data. CONCLUSIONS: The genera, Acrophorus, Acrorumohra, Diacalpe, Dryopsis, Nothoperanema, and Peranema, should all be merged into Dryopteris. Most species of these genera share a short rhizome and catadromic arrangement of frond segments, unlike the sister genus of Dryopteris s.l., Arachniodes, which has anadromic arrangement of frond segments. The non-monophyly of the 19 out of the 21 supraspecific taxa (sections, subgenera) in Dryopteris strongly suggests that the current taxonomy of this genus is in need of revision. The disagreement between the previous taxonomy and molecular results in Dryopteris may be due partly to interspecific hybridization and polyplodization. More morphological studies and molecular data, especially from the nuclear genome, are needed to thoroughly elucidate the evolutionary history of Dryopteris. The 13 well-supported clades identified based on our data represent 13 major evolutionary lineages in Dryopteris that are also supported by morphological synapomophies.

Unraveling reticulate evolution in North American Dryopteris (Dryopteridaceae).

BACKGROUND: The thirteen species of Dryopteris in North America have long been suspected of having undergone a complicated history of reticulate evolution via allopolyploid hybridization. Various explanations for the origins of the allopolyploid taxa have been suggested, and though most lines of evidence have supported the so-called "semicristata" hypothesis, contention over the group's history has continued in several recent, conflicting studies. RESULTS: Sequence data from nine plastid and two nuclear markers were collected from 73 accessions representing 35 species of Dryopteris. Sequences from each of the allopolyploids are most closely related to their progenitor species as predicted by the "semicristata" hypothesis. Allotetraploid D. campyloptera appears to be derived from a hybrid between diploid D. expansa and D. intermedia; D. celsa, from diploid D. ludoviciana x D. goldiana; and D. carthusiana and D. cristata, from diploid "D. semicristata" x D. intermedia and D. ludoviciana, respectively. Allohexaploid D. clintoniana appears to be derived from D. cristata x D.goldiana. The earliest estimated dates of formation of the allopolyploids, based on divergence time analyses, were within the last 6 Ma. We found no evidence for recurrent formation of any of the allopolyploids. The sexual allopolyploid taxa are derived from crosses between parents that show intermediate levels of genetic divergence relative to all pairs of potential progenitors. In addition, the four allotetraploids are transgressive with respect to geographic range relative to one or both of their parents (their ranges extend beyond those of the parents), suggesting that ecological advantages in novel habitats or regions may promote long-term regional coexistence of the hybrid taxa with their progenitors. CONCLUSIONS: This study provides the first thorough evaluation of the North American complex of woodferns using extensive sampling of taxa and genetic markers. Phylogenies produced from each of three datasets (one plastid and two nuclear) support the "semicristata" hypothesis, including the existence of a missing diploid progenitor, and allow us to reject all competing hypotheses. This study demonstrates the value of using multiple, biparentally inherited markers to evaluate reticulate complexes, assess the frequency of recurrent polyploidization, and determine the relative importance of introgression vs. hybridization in shaping the histories of such groups.

Reticulate evolution on a global scale: a nuclear phylogeny for New World Dryopteris (Dryopteridaceae).

Reticulate, or non-bifurcating, evolution is now recognized as an important phenomenon shaping the histories of many organisms. It appears to be particularly common in plants, especially in ferns, which have relatively few barriers to intra- and interspecific hybridization. Reticulate evolutionary patterns have been recognized in many fern groups, though very few have been studied rigorously using modern molecular phylogenetic techniques in order to determine the causes of the reticulate patterns. In the current study, we examine patterns of branching and reticulate evolution in the genus Dryopteris, the woodferns. The North American members of this group have long been recognized as a classic example of reticulate evolution in plants, and we extend analysis of the genus to all 30 species in the New World, as well as numerous taxa from other regions. We employ sequence data from the plastid and nuclear genomes and use maximum parsimony (MP), maximum likelihood (ML), Bayesian inference (BI), and divergence time analyses to explore the relationships of New World Dryopteris to other regions and to reconstruct the timing and events which may have led to taxa displaying reticulate rather than strictly branching histories. We find evidence for reticulation among both the North and Central/South American groups of species, and our data support a classic hypothesis for reticulate evolution via allopolyploid speciation in the North America taxa, including an extinct diploid progenitor in this group. In the Central and South American species, we find evidence of extensive reticulation involving unknown ancestors from Asia, and we reject deep coalescent processes such as incomplete lineage sorting in favor of more recent intercontinental hybridization and chloroplast capture as an explanation for the origin of the Latin American reticulate taxa.

Origin of Dryopteris shibipedis (Dryopteridaceae), a fern species extinct in the wild.

Dryopteris shibipedis was once treated as an extinct species in the Red List (2007) by the Japanese Ministry of the Environment, but 'rediscovered' in the Tsukuba Botanical Garden. To clarify its origin, using 'overlooked' cultivated stocks we analyzed nuclear PgiC intron sequences. As the PgiC genotype of D. shibipedis can be explained by a combination of alleles of D. kinkiensis and those of D. pacifica, the hypothesis of hybrid origin of the species is supported.

Anatomía de seis especies de helechos del género Dryopteris (Dryopteridaceae) de México / Anatomy of six species of Dryopteris ferns (Dryopteridaceae) from Mexico

Rhizome and foliar anatomy of the Mexican Dryopteris Adans. species were studied and compared with other Dryopteridaceae and other fern families to identify anatomical features with diagnostic value. The anatomy of rhizome, stipe, and blade is similar in species of the Dryopteris patula complex. The cells with un-lignified, thickened wall, with cap or U-shape around the meristeles belong to the collenchyma, in contrast with other fern families. Dryopteris wallichiana (Spreng.) Hyl. is anatomically distinguished from the other studied species by having more layers of sclerenchyma and meristeles on the stipe, and by the lack of sclereid nests on the rhizome. Dryopteris rossii C. Chr. and D. maxonii Underw. & C. Chr. are characterized by the presence of crystals on the periphery of rhizome nests. D. maxonni and D. wallichiana lack blade glands.

Se estudió la anatomía del rizoma y hoja de especies mexicanas de Dryopteris Adans. y se comparó con la información disponible para Dryopteridaceae y otras familias de helechos en busca de caracteres anatómicos con valor diagnóstico. La anatomía de rizoma, pecíolo y lámina es similar en las especies del complejo Dryopteris patula. Las células con pared engrosada, no lignificada y en forma de casquete o de “U” presentes alrededor de las meristelas corresponden a colénquima, a diferencia de lo informado para otras familias de helechos. Dryopteriswallichiana (Spreng.) Hyl. se distingue anatómicamente de las otras especies estudiadas por presentar un mayor número de capas de esclerénquima y de meristelas en el pecíolo, además de carecer de nidos de esclereidas en el rizoma. Dryopteris rossii y D. maxonii se caracterizan por la presencia de cristales en la periferia de los nidos en el rizoma. Las glándulas en la lámina están ausentes en D. maxonii y D. wallichiana.

Phylogenetics of Chinese Dryopteris (Dryopteridaceae) based on the chloroplast rps4-trnS sequence data.

Dryopteris is one of the largest and most taxonomically complex fern genera in the Dryopteridaceae, with 127 species occurring throughout temperate, sub-temperate, subtropical, and tropical China. Investigations of the evolutionary relationships of a subset of these Chinese Dryopteris species, using DNA sequence-based methods, specifically tested the monophyly of the genus and the validity of the previous subgeneric classifications. Sixty species of Dryopteris, four closely related non-Dryopteris and three species of Arachniodes, were used as outgroup taxa. The rps4-trnS region of the chloroplast genome was sequenced in these species for the first time. Both maximum parsimony (MP) and neighbor-joining (NJ) analyses identified six polyphyletic clades that contained Dryopteris species. These results were supported by a Bayesian analysis of the same data set. The phylogenetic patterns strongly suggest the polyphyletic status of Dryopteris; the monophyletic groupings of the species do not correspond with either Fraser-Jenkins [In: Bull Brit Mus (Nat Hist) Bot 14(3):183-218, 1986} or Wu (In: Flora Reipublica Popularis Sinicae Tomus 5 (1) pp 1-241, 2000] subgeneric classification of Dryopteris, except in a few specific cases. This work represents the first molecular systematic analyses of Chinese Dryopteris, and we propose the next steps necessary to recognize new subgenera of the genus.

[Anatomy of six species of Dryopteris ferns (Dryopteridaceae) from Mexico]. / Anatomía de seis especies de helechos del género Dryopteris (Dryopteridaceae) de México.

Rhizome and foliar anatomy of the Mexican Dryopteris Adans. species were studied and compared with other Dryopteridaceae and other fern families to identify anatomical features with diagnostic value. The anatomy of rhizome, stipe, and blade is similar in species of the Dryopteris patula complex. The cells with un-lignified, thickened wall, with cap or U-shape around the meristeles belong to the collenchyma, in contrast with other fern families. Dryopteris wallichiana (Spreng.) Hyl. is anatomically distinguished from the other studied species by having more layers of sclerenchyma and meristeles on the stipe, and by the lack of sclereid nests on the rhizome. Dryopteris rossii C. Chr. and D. maxonii Underw. & C. Chr. are characterized by the presence of crystals on the periphery of rhizome nests. D. maxonni and D. wallichiana lack blade glands.