The size diversity of the Pteridaceae family chloroplast genome is caused by overlong intergenic spacers.

BACKGROUND: While the size of chloroplast genomes (cpDNAs) is often influenced by the expansion and contraction of inverted repeat regions and the enrichment of repeats, it is the intergenic spacers (IGSs) that appear to play a pivotal role in determining the size of Pteridaceae cpDNAs. This provides an opportunity to delve into the evolution of chloroplast genomic structures of the Pteridaceae family. This study added five Pteridaceae species, comparing them with 36 published counterparts. RESULTS: Poor alignment in the non-coding regions of the Pteridaceae family was observed, and this was attributed to the widespread presence of overlong IGSs in Pteridaceae cpDNAs. These overlong IGSs were identified as a major factor influencing variations in cpDNA size. In comparison to non-expanded IGSs, overlong IGSs exhibited significantly higher GC content and were rich in repetitive sequences. Species divergence time estimations suggest that these overlong IGSs may have already existed during the early radiation of the Pteridaceae family. CONCLUSIONS: This study reveals new insights into the genetic variation, evolutionary history, and dynamic changes in the cpDNA structure of the Pteridaceae family, providing a fundamental resource for further exploring its evolutionary research.

A De Novo Transcriptome Assembly of Ceratopteris richardii Provides Insights into the Evolutionary Dynamics of Complex Gene Families in Land Plants.

As the closest extant sister group to seed plants, ferns are an important reference point to study the origin and evolution of plant genes and traits. One bottleneck to the use of ferns in phylogenetic and genetic studies is the fact that genome-level sequence information of this group is limited, due to the extreme genome sizes of most ferns. Ceratopteris richardii (hereafter Ceratopteris) has been widely used as a model system for ferns. In this study, we generated a transcriptome of Ceratopteris, through the de novo assembly of the RNA-seq data from 17 sequencing libraries that are derived from two sexual types of gametophytes and five different sporophyte tissues. The Ceratopteris transcriptome, together with 38 genomes and transcriptomes from other species across the Viridiplantae, were used to uncover the evolutionary dynamics of orthogroups (predicted gene families using OrthoFinder) within the euphyllophytes and identify proteins associated with the major shifts in plant morphology and physiology that occurred in the last common ancestors of euphyllophytes, ferns, and seed plants. Furthermore, this resource was used to identify and classify the GRAS domain transcriptional regulators of many developmental processes in plants. Through the phylogenetic analysis within each of the 15 GRAS orthogroups, we uncovered which GRAS family members are conserved or have diversified in ferns and seed plants. Taken together, the transcriptome database and analyses reported here provide an important platform for exploring the evolution of gene families in land plants and for studying gene function in seed-free vascular plants.

Cryptic diversity in the model fern genus Ceratopteris (Pteridaceae).

Cryptic species are present throughout the tree of life. They are especially prevalent in ferns, because of processes such hybridization, polyploidy, and reticulate evolution. In addition, the simple morphology of ferns limits phenotypic variation and makes it difficult to detect cryptic species. The model fern genus Ceratopteris has long been suspected to harbor cryptic diversity, in particular within the highly polymorphic C. thalictroides. Yet no studies have included samples from throughout its pan-tropical range or utilized genomic sequencing, making it difficult to assess the full extent of cryptic variation within this genus. Here, we present the first multilocus phylogeny of the genus using reduced representation genomic sequencing (RADseq) and examine population structure, phylogenetic relationships, and ploidy level variation. We recover similar species relationships found in previous studies, find support for the cryptic species C. gaudichaudii as genetically distinct, and identify novel genomic variation within two of the mostly broadly distributed species in the genus, C. thalictroides and C. cornuta. Finally, we detail the utility of our approach for working on cryptic, reticulate groups of ferns. Specifically, it does not require a reference genome, of which there are very few available for ferns. RADseq is a cost-effective way to work with study groups lacking genomic resources, and to obtain the thousands of nuclear markers needed to untangle species complexes.

An ontogenetic framework for functional studies in the model fern Ceratopteris richardii.

As the sister group to seed plants, ferns are a phylogenetically informative lineage. Functional studies in representatives of the fern lineage are helping bridge the knowledge gap in developmental mechanisms between angiosperms and non-vascular plants. The fern life cycle has the advantage of combining a sizable free-living haploid gametophyte, more amenable for developmental studies than the reduced seed plant gametophyte, with an indeterminate and complex diploid sporophyte. Ceratopteris richardii has long been proposed as a model fern and has recently become tractable due to stable transgenesis and increasing genomic resources, allowing researchers to test explicit questions about gene function in a fern for the first time. As with any model system, a detailed understanding of wild-type morphology and a staged ontogeny are indispensable for the characterization of mutant phenotypes resulting from genetic manipulations. Therefore, the goal of this study is to provide a unified reference ontogeny for this emerging model fern as a tool for comparative evolutionary and developmental studies. It complements earlier research by filling gaps in major stages of development of the haploid gametophyte and diploid sporophyte generations, and provides additional descriptions of the shoot apical meristem and early leaf development. This resource is meant to facilitate not only studies of candidate genes within C. richardii, but also broader ontogenetic comparisons to other model plants.

Low-copy nuclear sequence data confirm complex patterns of farina evolution in notholaenid ferns (Pteridaceae).

Notholaenids are an unusual group of ferns that have adapted to, and diversified within, the deserts of Mexico and the southwestern United States. With approximately 40 species, this group is noted for being desiccation-tolerant and having "farina"-powdery exudates of lipophilic flavonoid aglycones-that occur on both the gametophytic and sporophytic phases of their life cycle. The most recent circumscription of notholaenids based on plastid markers surprisingly suggests that several morphological characters, including the expression of farina, are homoplasious. In a striking case of convergence, Notholaena standleyi appears to be distantly related to core Notholaena, with several taxa not before associated with Notholaena nested between them. Such conflicts can be due to morphological homoplasy resulting from adaptive convergence or, alternatively, the plastid phylogeny itself might be misleading, diverging from the true species tree due to incomplete lineage sorting, hybridization, or other factors. In this study, we present a species phylogeny for notholaenid ferns, using four low-copy nuclear loci and concatenated data from three plastid loci. A total of 61 individuals (49 notholaenids and 12 outgroup taxa) were sampled, including 31 out of 37 recognized notholaenid species. The homeologous/allelic nuclear sequences were retrieved using PacBio sequencing and the PURC bioinformatics pipeline. Each dataset was first analyzed individually using maximum likelihood and Bayesian inference, and the species phylogeny was inferred using *BEAST. Although we observed several incongruences between the nuclear and plastid phylogenies, our principal results are broadly congruent with previous inferences based on plastid data. By mapping the presence of farina and their biochemical constitutions on our consensus phylogenetic tree, we confirmed that the characters are indeed homoplastic and have complex evolutionary histories. Hybridization among recognized species of the notholaenid clade appears to be relatively rare compared to that observed in other well-studied fern genera.

Leaf epidermal micromorphology and its implications in systematics of certain taxa of the fern family Pteridaceae from Northern Pakistan.

The present study is insights into foliar epidermal anatomy for characterizing clades, and their utility in taxonomic segregation of certain species of Pteridaceae from Northern Pakistan. The leaf epidermal anatomy of 10 species of Pteridaceae representing four genera were examined using light and scanning electron microscope. A micromorphological matrix was constructed for eight qualitative and 12 quantitative characters. unweighted pair group method with arithmetic means and principal components analysis statistical analysis were performed to test the validity of foliar epidermal anatomical features as method of separating species and genera, and phylogenetic clusters among species are constructed using qualitative and quantitative traits. The qualitative characters described here are shape of epidermal cells, stomata, guard cell and subsidiary cells, anticlinal wall pattern, and trichomes types which is helpful in defining groups within Pteridaceae. In addition, the size of stomata, guard cells, subsidiary cells, stomatal pore epidermal cells, and trichomes are quantitatively analyzed. All species have hypostomatic leaves. Two types of stomata were observed in studied species, anomocytic and polocytic. Anomocytic stomata were observed in three genera namely: Adiantum, Onychium, and Chielanthes whereas Pteris can be discriminated from other genera by its polocytic stomata. On the basis of multivariate analysis present study does provides sufficient information on the taxonomic importance of foliar anatomy which validate its efficacy in species and genera discrimination. From result obtained here it is further possible to use leaf micromorphologic data in ferns phylogeny and providing basis for future taxonomic delimitation in other taxa.

Phylogeny and systematics of the brake fern genus Pteris (Pteridaceae) based on molecular (plastid and nuclear) and morphological evidence.

The brake fern genus Pteris belongs to Pteridaceae subfamily Pteridoideae. It is one of the largest fern genera and has been estimated to contain 200-250 species distributed on all continents except Antarctica. Previous studies were either based on plastid data only or based on both plastid and nuclear data but the sampling was small. In addition, an infrageneric classification of Pteris based on morphological and molecular evidence has not been available yet. In the present study, based on molecular data of eight plastid markers and one nuclear marker (gapCp) of 256 accessions representing ca. 178 species of Pteris, we reconstruct a global phylogeny of Pteris. The 15 major clades identified earlier are recovered here and we further identified a new major clade. Our nuclear phylogeny recovered 11 of these 16 major clades, seven of which are strongly supported. The inclusion of Schizostege in Pteris is confirmed for the first time. Based on the newly reconstructed phylogeny and evidence from morphology, distribution and/or ecology, we classify Pteris into three subgenera: P. subg. Pteris, P. subg. Campteria, and P. subg. Platyzoma. The former two are further divided into three and 12 sections, respectively.

Phylogeny of the fern subfamily Pteridoideae (Pteridaceae; Pteridophyta), with the description of a new genus: Gastoniella.

As the second most genera-rich fern family, Pteridaceae contain more than 1000 species contributing to ca. 10% of extant leptosporangiate fern diversity. The subfamily Pteridoideae is one of the five subfamilies often recognized. The circumscription of Pteridoideae has not been clear. A large number of species have not yet been included in any molecular analyses before. In this study, DNA sequences of six plastid loci of 154 accessions representing ca. 87 species in 14 genera of Pteridaceae subfam. Pteridoideae and four accessions representing two species in subfam. Parkerioideae and one species of subfam. Adiantoideae as outgroups were used to infer a phylogeny using maximum likelihood and maximum parsimony. Our analyses show that (1) Pteridoideae is monophyletic and the newly defined subfamily is composed of 14 genera including a newly described genus; (2) Pteridoideae is resolved into four strongly supported monophyletic clades: the Pteris clade, the Actiniopteris+Onychium clade, the JAPSTT clade, and the GAPCC clade, these being supported by not only molecular data but also morphological features and distribution information; (3) Onychium is confirmed as monophyletic and accessions of Onychium are resolved into two strongly supported clades, the O. cryptogrammoides clade and the O. siliculosum clade; and (4) Accessions of the traditionally defined Anogramma are resolved as paraphyletic in relation to Cerosora, Cosentinica, and Pityrogramma. Three species traditionally treated in Anogramma are in fact more closely related to Cerosora and Pityrogramma than they are to Anogramma. Gastoniella Li Bing Zhang & Liang Zhang, gen. nov. is described to accommodate these species and three new combinations are provided. Three currently known species of Gastoniella are distributed in the Ascension Island in South Atlantic Ocean, central Mexico, and tropical America, respectively. The new genus is distinct from Anogramma s.s. in having ultimate segments linear not obviously broadening toward the upper portion.

Convergent Evolution of Fern-Specific Mitochondrial Group II Intron atp1i361g2 and Its Ancient Source Paralogue rps3i249g2 and Independent Losses of Intron and RNA Editing among Pteridaceae.

Mitochondrial intron patterns are highly divergent between the major land plant clades. An intron in the atp1 gene, atp1i361g2, is an example for a group II intron specific to monilophytes (ferns). Here, we report that atp1i361g2 is lost independently at least 4 times in the fern family Pteridaceae. Such plant organelle intron losses have previously been found to be accompanied by loss of RNA editing sites in the flanking exon regions as a consequence of genomic recombination of mature cDNA. Instead, we now observe that RNA editing events in both directions of pyrimidine exchange (C-to-U and U-to-C) are retained in atp1 exons after loss of the intron in Pteris argyraea/biaurita and in Actiniopteris and Onychium We find that atp1i361g2 has significant similarity with intron rps3i249g2 present in lycophytes and gymnosperms, which we now also find highly conserved in ferns. We conclude that atp1i361g2 may have originated from the more ancestral rps3i249g2 paralogue by a reverse splicing copy event early in the evolution of monilophytes. Secondary structure elements of the two introns, most characteristically their domains III, show strikingly convergent evolution in the monilophytes. Moreover, the intron paralogue rps3i249g2 reveals relaxed evolution in taxa where the atp1i361g2 paralogue is lost. Our findings may reflect convergent evolution of the two related mitochondrial introns exerted by co-evolution with an intron-binding protein simultaneously acting on the two paralogues.

Identifying multiple origins of polyploid taxa: a multilocus study of the hybrid cloak fern (Astrolepis integerrima; Pteridaceae).

PREMISE OF THE STUDY: Molecular studies have shown that multiple origins of polyploid taxa are the rule rather than the exception. To understand the distribution and ecology of polyploid species and the evolutionary significance of polyploidy in general, it is important to delineate these independently derived lineages as accurately as possible. Although gene flow among polyploid lineages and backcrossing to their diploid parents often confound this process, such post origin gene flow is very infrequent in asexual polyploids. In this study, we estimate the number of independent origins of the apomictic allopolyploid fern Astrolepis integerrima, a morphologically heterogeneous species most common in the southwestern United States and Mexico, with outlying populations in the southeastern United States and the Caribbean. METHODS: Plastid DNA sequence and AFLP data were obtained from 33 A. integerrima individuals. Phylogenetic analysis of the sequence data and multidimensional clustering of the AFLP data were used to identify independently derived lineages. KEY RESULTS: Analysis of the two datasets identified 10 genetic groups within the 33 analyzed samples. These groups suggest a minimum of 10 origins of A. integerrima in the northern portion of its range, with both putative parents functioning as maternal donors, both supplying unreduced gametes, and both contributing a significant portion of their genetic diversity to the hybrids. CONCLUSIONS: Our results highlight the extreme cryptic genetic diversity and systematic complexity that can underlie a single polyploid taxon.

rbcL and matK earn two thumbs up as the core DNA barcode for ferns.

BACKGROUND: DNA barcoding will revolutionize our understanding of fern ecology, most especially because the accurate identification of the independent but cryptic gametophyte phase of the fern's life history--an endeavor previously impossible--will finally be feasible. In this study, we assess the discriminatory power of the core plant DNA barcode (rbcL and matK), as well as alternatively proposed fern barcodes (trnH-psbA and trnL-F), across all major fern lineages. We also present plastid barcode data for two genera in the hyperdiverse polypod clade--Deparia (Woodsiaceae) and the Cheilanthes marginata group (currently being segregated as a new genus of Pteridaceae)--to further evaluate the resolving power of these loci. PRINCIPAL FINDINGS: Our results clearly demonstrate the value of matK data, previously unavailable in ferns because of difficulties in amplification due to a major rearrangement of the plastid genome. With its high sequence variation, matK complements rbcL to provide a two-locus barcode with strong resolving power. With sequence variation comparable to matK, trnL-F appears to be a suitable alternative barcode region in ferns, and perhaps should be added to the core barcode region if universal primer development for matK fails. In contrast, trnH-psbA shows dramatically reduced sequence variation for the majority of ferns. This is likely due to the translocation of this segment of the plastid genome into the inverted repeat regions, which are known to have a highly constrained substitution rate. CONCLUSIONS: Our study provides the first endorsement of the two-locus barcode (rbcL+matK) in ferns, and favors trnL-F over trnH-psbA as a potential back-up locus. Future work should focus on gathering more fern matK sequence data to facilitate universal primer development.

[Phylogenetic analysis and detection of selective pressure on the CVNH domains].

Cyanovirin-N (CV-N) is a novel protein with broad-spectrum antiviral activity. Its homologs constitute a protein family known as CVNH (Cyanovirin-N homology), which possess the evolutionarily conserved anti-HIV (Human immunodeficiency virus) domain. In this study, more details about the patchy organism distribution of CVNH domain were explored by reconstructing gene trees. Duplicated CVNH sequences were also identified in a wide range of species including Aspergillus niger, Neosartorya fischeri NRRL 181, Penicillium chrysogenum Wisconsin 54-1255, Neurospora crassa, Cyanothece sp. PCC, and Ceratopteris richardii. Besides these findings, both the mechanistic and mechanistic-empirical combination (MEC) models were used to analyze the adaptive evolution of amino acid sites in the CVNH domain. Our results showed that: (1) neither model reveals significant sites undergoing positive selection; (2) purifying selection has played a dominant role during CVNH evolution; and (3) the MEC model better fits the CVNH data set. Furthermore, the ancestral branch leading to Cyanothece sp. PCC 7822 and 7424 were examined using "branch-specific" and "branch-site" models. Six positively selected sites (34L, 63L, 13H, 76C, 78K, and 80I) were identified on the branch.

Cryptic species in the fern Ceratopteris thalictroides (Parkeriaceae). III. Referential diagnostic characters of three cryptic species.

Three cryptic species of Ceratopteris thalictroides, named the south type, the north type and the third type, were examined for their morphological characteristics, using sporophytes cultivated under common conditions. The discriminant analysis for leaf characters followed by one-way layout ANOVA or Kruskal-Wallis test for selected combinations of characters revealed that the following characters may be effective for identifying the three types: the relative lengths of stipe to blade and to pinna, the degree of dissection, the segment densities on rachis and pinna rachis, and the elongation degree of ultimate segments. The number of annulus cells on sporangia also proved to be a possible distinguishing character. As morphological data were obtained from a limited number of cultivated sporophytes, they are regarded as not definitive, but only referential diagnostic characters of the types and should be utilized not solely, but collectively, to avoid identification errors of the types. An identification trial using herbarium specimens proved these diagnostic characters to be useful to a considerable degree.

A molecular phylogeny of the fern family Pteridaceae: assessing overall relationships and the affinities of previously unsampled genera.

The monophyletic Pteridaceae accounts for roughly 10% of extant fern diversity and occupies an unusually broad range of ecological niches, including terrestrial, epiphytic, xeric-adapted rupestral, and even aquatic species. In this study, we present the results of the first broad-scale and multi-gene phylogenetic analyses of these ferns, and determine the affinities of several previously unsampled genera. Our analyses of two newly assembled data sets (including 169 newly obtained sequences) resolve five major clades within the Pteridaceae: cryptogrammoids, ceratopteridoids, pteridoids, adiantoids, and cheilanthoids. Although the composition of these clades is in general agreement with earlier phylogenetic studies, it is very much at odds with the most recent subfamilial classification. Of the previously unsampled genera, two (Neurocallis and Ochropteris) are nested within the genus Pteris; two others (Monogramma and Rheopteris) are early diverging vittarioid ferns, with Monogramma resolved as polyphyletic; the last previously unsampled genus (Adiantopsis) occupies a rather derived position among cheilanthoids. Interestingly, some clades resolved within the Pteridaceae can be characterized by their ecological preferences, suggesting that the initial diversification in this family was tied to ecological innovation and specialization. These processes may well be the basis for the diversity and success of the Pteridaceae today.

Efecto hipoglicemiante y sobre la lipidemia de Notholaena Nivea, Cuti Cuti / Hipoglicemiante effect and on lipidemia of Notholaena Nivea, Cuti Cuti

Se evaluó la toxicidad aguda, oral, en ratones y el efecto metabólico sobre glicemia y lipidemia del extracto metanólico de Notholaena Nivea, "Cuti-Cuti", al 10 por ciento en modelos del CYTED. El efecto sobre la glicemia fue evaluado en ratas normo e hiperglicémicas, frente a grupos controles a los cuales se les administró suero fisiológico. La hiperglicemia fue inducida con aloxano, administrados por vía intraperitoneal a la dosis de 150 mg/kg de peso. Utilizamos 60 ratas distribuidas en tres grupos (I, II y III), de 20 ratas cada uno. Todos los animales fueron sometidos a ayuno de 24 horas, previo al inicio de los experimentos. El grupo I fue subdividido en dos subgrupos de 10 animales cada uno y recibieron suero fisiológico o extracto de Cuti-Cuti, para observar su efecto sobre la glicemia normal. El segundo grupo recibió aloxano a la dosis de 150mg/kg de peso, vía intraperitoneal, para inducir hiperglicemia experimental; los valores de glicemia y lipidemia fueron determinados a la 1,2,4 y 24 horas. Al tercer grupo de ratas se les administró extracto aloxano por vía intraperitoneal a la dosis de 150 mg/kg de peso; cuando los niveles de glicemia eran superiores a 300 mg por ciento, administramos a 10 ratas suero fisiológico y a otras 10 Cuti-Cuti a la dosis de 250 mg/kg de peso, vía oral. La determinación de la glicemia, colesterol total, HDL y triglicéridos fue realizada en el autoanalizador bioquímico Vitalab Selectra 2. La DL50 fue de 11,099 con un límite superior e inferior al 95 por ciento de 13,009.72 y 9,470.59 respectivamente...

We evaluated the acute oral toxicity and the metabolic effect on glycemia and lipidemia in mice, of the metanolic extract of Notholaena Nivea, "Cuti-Cuti" to 10%, in CYTED models. The effect on plasma glucose was evaluated in normoglycemic and hyperglycemic rats, compared with control groups which received physiological serum. Hyperglycemia was induced with Alloxan, administered by intraperitoneal route to the dose of 150 mg/kg of weight. We used 60 rats distributed in three groups (I, II and Ill), of 20 rats each one. All the animals were put under 24 hours fasting, previous to the beginning of the experiments. Group I was subdivided in two sub-groups of 10 animals each one and received physiological serum or extract of Cuti-Cuti, to observe its effects on normal blood glucose. The second group received alloxan to the dose of 150mg/Kg of weight, by intraperitoneal route, to induce experimental hyperglycemia; the values of blood glucose and lipids were determined at 1, 2, 4 and 24 hours. The third group of rats received 150 mg/kg of alloxan by intraperitoneal route. When the blood glucose level was over 300 mg%, 10 of them received 250 mg/kg of Cuti-Cuti and the others 10 received physiological serum by oral route. Determination of glucose, total cholesterol, HDL and triglycerids in plasma was made in the biochemical Autoanalizador Vitalab Selectra 2. The DL50 with superior and inferior limit of 95% was 13,009 and 9,470 mg/kg of weight with an average of 11,099 mg/kg of weight...

[Reports in Pteridaceae (Filacales) family in the Neotropics]. / Informes en la familia Pteridaceae (Filicales) para el Neotrópico.

Several contributions to the Neotropical pteridophyte flora are made in this paper with the range extensions for four species of Adiantum, two species of Doryopteris and one species of Eriosorus. The species concerned are: A. polyphyllum Willd. for Nicaragua and Costa Rica; A. trichochlaenum Mickel et Beitel for Guatemala and Costa Rica; A. villosissimum Mett. ex Kuhn for Costa Rica; A. wilesianum Hook. for Nicaragua; D. nobilis (T. Moore) C. Chr. for Costa Rica, Colombia and Venezuela; D. redivida Fée for Panama; and E. hirtus (Kunth) Copel. for Costa Rica. The concept of D. pedata (L.) Fée is redefined.