A global plastid phylogeny of the fern genus Asplenium (Aspleniaceae).

The infrageneric relationships and taxonomy of the largest fern genus, Asplenium (Aspleniaceae), have remained poorly understood. Previous studies have focused mainly on specific species complexes involving a few or dozens of species only, or have achieved a large taxon sampling but only one plastid marker was used. In the present study, DNA sequences from six plastid markers (atpB, rbcL, rps4, rps4-trnS, trnL and trnL-F) of 1030 accessions (616 of them newly sequenced here) representing c. 420 species of Asplenium (60% of estimated species diversity), 16 species of Hymenasplenium, three Diplaziopsidaceae, and four Rhachidosoraceae were used to produce the largest genus-level phylogeny yet for ferns. Our major results include: (i) Asplenium as broadly circumscribed is monophyletic based on our inclusion of representatives of 32 of 38 named segregate genera; (ii) 11 major clades in Asplenium are identified, and their relationships are mostly well-resolved and strongly supported; (iii) numerous species, unsampled in previous studies, suggest new relationships and numerous cryptic species and species complexes in Asplenium; and (iv) the accrued molecular evidence provides an essential foundation for further investigations of complex patterns of geographical diversification, speciation and reticulate evolution in this family.

Comparative in situ analysis reveals the dynamic nature of sclerenchyma cell walls of the fern Asplenium rutifolium.

Background and Aims: A key structural adaptation of vascular plants was the evolution of specialized vascular and mechanical tissues, innovations likely to have generated novel cell wall architectures. While collenchyma is a strengthening tissue typically found in growing organs of angiosperms, a similar tissue occurs in the petiole of the fern Asplenium rutifolium. Methods: The in situ cell wall (ultra)structure and composition of this tissue was investigated and characterized mechanically as well as structurally through nano-indentation and wide-angle X-ray diffraction, respectively. Key Results: Structurally the mechanical tissue resembles sclerenchyma, while its biomechanical properties and molecular composition both share more characteristics with angiosperm collenchyma. Cell wall thickening only occurs late during cell expansion or after cell expansion has ceased. Conclusions: If the term collenchyma is reserved for walls that thicken during expansive growth, the mechanical tissue in A. rutifolium represents sclerenchyma that mimics the properties of collenchyma and has the ability to modify its mechanical properties through sclerification. These results support the view that collenchyma does not occur in ferns and most probably evolved in angiosperms.

The complete chloroplast genome sequence of Huperzia javanica (sw.) C. Y. Yang in Lycopodiaceae.

Huperzia javanica (Sw.) C. Y. Yang is a valuable medical herb used for treating Alzheimer's disease. Here, we described the complete chloroplast genome of H. javanica using Illumina paired-end sequencing. The total genome length is 154,415 bp, containing 119 unique genes, with 86 protein-coding genes, 29 tRNA genes, and 4 rRNA genes. The gene content and their order are consistent with two previously reported Huperzia genomes. The overall GC content of the chloroplast genome of H. javanica is 36.4%. The topology of our maximum-likelihood tree is consistent with topologies found in previous studies, with H. javanica sister to a clade of H. serrata and H. lucidula. We support the recognition of H. javanica as an independent species. Huperzia serrata is more closely related to H. lucidula than to H. javanica.

Comparative glycan profiling of Ceratopteris richardii 'C-Fern' gametophytes and sporophytes links cell-wall composition to functional specialization.

BACKGROUND AND AIMS: Innovations in vegetative and reproductive characters were key factors in the evolutionary history of land plants and most of these transformations, including dramatic changes in life cycle structure and strategy, necessarily involved cell-wall modifications. To provide more insight into the role of cell walls in effecting changes in plant structure and function, and in particular their role in the generation of vascularization, an antibody-based approach was implemented to compare the presence and distribution of cell-wall glycan epitopes between (free-living) gametophytes and sporophytes of Ceratopteris richardii 'C-Fern', a widely used model system for ferns. METHODS: Microarrays of sequential diamino-cyclohexane-tetraacetic acid (CDTA) and NaOH extractions of gametophytes, spores and different organs of 'C-Fern' sporophytes were probed with glycan-directed monoclonal antibodies. The same probes were employed to investigate the tissue- and cell-specific distribution of glycan epitopes. KEY RESULTS: While monoclonal antibodies against pectic homogalacturonan, mannan and xyloglucan widely labelled gametophytic and sporophytic tissues, xylans were only detected in secondary cell walls of the sporophyte. The LM5 pectic galactan epitope was restricted to sporophytic phloem tissue. Rhizoids and root hairs showed similarities in arabinogalactan protein (AGP) and xyloglucan epitope distribution patterns. CONCLUSIONS: The differences and similarities in glycan cell-wall composition between 'C-Fern' gametophytes and sporophytes indicate that the molecular design of cell walls reflects functional specialization rather than genetic origin. Glycan epitopes that were not detected in gametophytes were associated with cell walls of specialized tissues in the sporophyte.

Ceratopteris richardii (C-fern): a model for investigating adaptive modification of vascular plant cell walls.

Plant cell walls are essential for most aspects of plant growth, development, and survival, including cell division, expansive cell growth, cell-cell communication, biomechanical properties, and stress responses. Therefore, characterizing cell wall diversity contributes to our overall understanding of plant evolution and development. Recent biochemical analyses, concomitantly with whole genome sequencing of plants located at pivotal points in plant phylogeny, have helped distinguish between homologous characters and those which might be more derived. Most plant lineages now have at least one fully sequenced representative and although genome sequences for fern species are in progress they are not yet available for this group. Ferns offer key advantages for the study of developmental processes leading to vascularisation and complex organs as well as the specific differences between diploid sporophyte tissues and haploid gametophyte tissues and the interplay between them. Ceratopteris richardii has been well investigated building a body of knowledge which combined with the genomic and biochemical information available for other plants will progress our understanding of wall diversity and its impact on evolution and development.

The base number of 'loxoscaphoid' Asplenium species and its implication for cytoevolution in Aspleniaceae.

BACKGROUND AND AIMS: 'Loxoscaphoid' Asplenium species are morphologically a remarkably distinct group of Aspleniaceae. Except for two preliminary chromosome counts of Asplenium theciferum, the cytology of this group of species has, however, been largely unstudied. METHODS: Chromosome counts were obtained by acetocarmine squash preparations of one mitotic cell and several meiotic cells. Relative DNA content of gametophytic and sporophytic cells was determined by flow cytometry. The phylogenetic placement of A. loxoscaphoides, A. rutifolium s.l. and A. theciferum s.l. was investigated through an analysis of rbcL sequences. KEY RESULTS: The dysploid base number is reported to be x = 35 in Asplenium centrafricanum, A. loxoscaphoides, A. sertularioides and A. theciferum. Analysis of rbcL sequences confirms that 'loxoscaphoids' nest robustly within Asplenium. Several high ploidy levels exceeding the tetraploid level were found in A. theciferum s.l. and A. rutifolium s.l. All taxa proved to be sexual. CONCLUSIONS: Four base numbers are known at present for Aspleniaceae: x = 39, 38, 36 and 35. The dysploid base number x = 35 found in the 'loxoscaphoid' Asplenium spp. sheds a novel light on the cytoevolution of the whole family. We postulate a recurrent descending dysploid evolution within Aspleniaceae, leading to speciation at the (sub)generic and species/group level.

Intercellular pectic protuberances in Asplenium: new data on their composition and origin.

BACKGROUND AND AIMS: Projections of cell wall material into the intercellular spaces between parenchymatic cells have been observed since the mid-19th century. Histochemical staining suggested that these intercellular protuberances are probably pectic in nature, but uncertainties about their origin, composition and biological function(s) have remained. METHODS: Using electron and light microscopy, including immunohistochemical methods, the structure and the presence of some major cell wall macromolecules in the intercellular pectic protuberances (IPPs) of the cortical parenchyma have been studied in a specimen of the Asplenium aethiopicum complex. KEY RESULTS: IPPs contained pectic homogalacturonan, but no evidence for pectic rhamnogalacturonan-I or xylogalacturonan epitopes was obtained. Arabinogalactan-proteins and xylan were not detected in cell walls, middle lamellae or IPPs of the cortical parenchyma, whereas xyloglucan was only found in its cell walls. Extensin (hydroxyproline-rich glycoproteins) LM1 and JIM11 and JIM20 epitopes were detected specifically in IPPs but not in their adjacent cell walls or middle lamellae. CONCLUSIONS: It is postulated that IPPs do not originate exclusively from the middle lamellae because extensins were only found in IPPs and not in surrounding cell walls, intercellular space linings or middle lamellae, and because IPPs and their adjacent cell walls are discontinuous.

Phylogenetic analysis of Asplenium subgenus Ceterach (Pteridophyta: Aspleniaceae) based on plastid and nuclear ribosomal ITS DNA sequences.

Phylogenetic relationships among 20 taxa of the fern genus Asplenium subgenus Ceterach (Filicopsida, represented by 73 accessions) were investigated using DNA sequence data from the nuclear ribosomal internal transcribed spacers (ITS nDNA) and plastid trnL-F intergenic spacer. In addition, a single sample per taxon was used in an analysis of the plastid rbcL gene. Chromosome counts were determined for all the samples, and these demonstrated a range from diploid to octoploid. Analyses of the DNA sequence data indicated that Asplenium subgenus Ceterach is polyphyletic, implicating homoplasy in the characters previously used to circumscribe this taxon. Plastid trnL-F and rbcL analyses resulted in identical tree topologies. The trees produced from the separate plastid and nuclear matrices agree in (1) the recognition of identical groups of accessions corresponding to A. dalhousiae, A. ceterach, A. aureum, A. cordatum, A. phillipsianum, and A. haughtonii; (2) the division of A. subg. Ceterach into two subclades, a Eurasian-Macaronesian and a strictly African alliance; (3) the position of A. dalhousiae as a member of the former subclade; (4) the lack of genetic variation in A. cordatum despite its morphological variability; and (5) the clustering of each autopolyploid with their diploid ancestor. However, the plastid and nuclear trees differ in their placement of A. haughtonii and A. dalhousiae, which might be due to different evolutionary histories of nuclear and plastid genomes, and is possibly an indication of ancient hybridization. The analyses confirm the existence of several strictly African taxa. Asplenium phillipsianum and A. cordatum each form species complexes of diploid and autopolyploid taxa, from which a third, morphologically intermediate, allotetraploid species has originated. Asplenium haughtonii is a distinct endemic species from Saint Helena. The maternally inherited plastid sequences support the hypothesis that A. aureum is an ancestor of A. lolegnamense and of A. octoploideum. Because gene conversion did not eliminate divergent ITS alleles in the allopolyploids, their reticulate ancestry could be demonstrated. Biparentally inherited nrITS sequences support the allopolyploid status of A. aureum, A. lolegnamense, and A. punjabense, indicating they share the ancestral A. javorkeanum genome.