Ronald L. L. Viane

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.

An extensin-rich matrix lines the carinal canals in Equisetum ramosissimum, which may function as water-conducting channels.

BACKGROUND AND AIMS The anatomy of Equisetum stems is characterized by the occurrence of vallecular and carinal canals. Previous studies on the carinal canals in several Equisetum species suggest that they convey water from one node to another. METHODS Cell wall composition and ultrastructure have been studied using immunocytochemistry and electron microscopy, respectively. Serial sectioning and X-ray computed tomography were employed to examine the internode-node-internode transition of Equisetum ramosissimum. KEY RESULTS The distribution of the LM1 and JIM20 extensin epitopes is restricted to the lining of carinal canals. The monoclonal antibodies JIM5 and LM19 directed against homogalacturonan with a low degree of methyl esterification and the CBM3a probe recognizing crystalline cellulose also bound to this lining. The xyloglucan epitopes recognized by LM15 and CCRC-M1 were only detected in this lining after pectate lyase treatment. The carinal canals, connecting consecutive rings of nodal xylem, are formed by the disruption and dissolution of protoxylem elements during elongation of the internodes. Their inner surface appears smooth compared with that of vallecular canals. CONCLUSIONS The carinal canals in E. ramosissimum have a distinctive lining containing pectic homogalacturonan, cellulose, xyloglucan and extensin. These canals might function as water-conducting channels which would be especially important during the elongation of the internodes when protoxylem is disrupted and the metaxylem is not yet differentiated. How the molecularly distinct lining relates to the proposed water-conducting function of the carinal canals requires further study. Efforts to elucidate the spatial and temporal distribution of cell wall polymers in a taxonomically broad range of plants will probably provide more insight into the structural-functional relationships of individual cell wall components or of specific configurations of cell wall polymers.

A new preparation method to study fresh plant structures with X-ray computed tomography

Since the development of X‐ray computed tomography as a medical diagnostic tool, it was adapted and extended for many scientific applications, including plant structure research. As for many biological studies, sample preparation is of major importance to obtain good‐quality images. Therefore, we present a new preparation method for fresh material which includes critical point drying and heavy metal staining. This technique enhances the contrast of fresh tissues, prevents artefacts such as tissue compression, and requires no embedding.

Critical phases in the seed development of common juniper (Juniperus communis)

Common juniper (Juniperus communis L.) populations in northwest European lowlands are currently declining in size and number. An important cause of this decline is a lack of natural regeneration. Low seed viability seems to be one of the main bottlenecks in this process. Previous research revealed a negative relation between seed viability and both temperature and nitrogen deposition. Additionally, the seeds of common juniper have a variable ripening time, which possibly influences seed viability. However, the underlying mechanisms remain unresolved. In order to elucidate this puzzle, it is important to understand in which phases of seed production the main defects are situated, together with the influence of ripening time. In this study, we compared seed viability of populations with and without successful recruitment. We examined three seed phases: (i) gamete development; (ii) fertilisation and early-embryo development; and (iii) late-embryo development. After the first two phases, we found no difference in the percentage viable seeds between populations with or without recruitment. After late-embryo development, populations without recruitment showed a significantly lower percentage of viable seeds. These results suggest that late-embryo development is a bottleneck in seed development. However, the complex interaction between seed viability and ripening time suggest that the causes should be in the second seed phase, as the accelerated development of male and female gametophytes may disturb the male-female synchrony for successful mating.

Ultrastructure and composition of cell wall appositions in the roots of Asplenium (Polypodiales)

Cell wall appositions (CWAs), formed by the deposition of extra wall material at the contact site with microbial organisms, are an integral part of the response of plants to microbial challenge. Detailed histological studies of CWAs in fern roots do not exist. Using light and electron microscopy we examined the (ultra)structure of CWAs in the outer layers of roots of Asplenium species. All cell walls studded with CWAs were impregnated with yellow-brown pigments. CWAs had different shapes, ranging from warts to elongated branched structures, as observed with scanning and transmission electron microscopy. Ultrastructural study further showed that infecting fungi grow intramurally and that they are immobilized by CWAs when attempting to penetrate intracellularly. Immunolabelling experiments using monoclonal antibodies indicated pectic homogalacturonan, xyloglucan, mannan and cellulose in the CWAs, but tests for lignins and callose were negative. We conclude that these appositions are defense-related structures made of a non-lignified polysaccharide matrix on which phenolic compounds are deposited in order to create a barrier protecting the root against infections.

Non-lignified helical cell wall thickenings in root cortical cells of Aspleniaceae (Polypodiales): histology and taxonomical significance

BACKGROUND AND AIMS Extraxylary helical cell wall thickenings in vascular plants are not well documented, except for those in orchid velamen tissues which have been studied extensively. Reports on their occurrence in ferns exist, but detailed information is missing. The aim of this study is to focus on the broad patterns of structure and composition and to study the taxonomic occurrence of helical cell wall thickenings in the fern family Aspleniaceae. METHODS Structural and compositional aspects of roots have been examined by means of light, electron, epifluorescence and laser scanning confocal microscopy. To assess the taxonomical distribution of helical cell wall thickenings a molecular phylogenetic analysis based on rbcL sequences of 64 taxa was performed. KEY RESULTS The helical cell wall thickenings of all examined species showed considerable uniformity of design. The pattern consists of helical, regularly bifurcating and anastomosing strands. Compositionally, the cell wall thickenings were found to be rich in homogalacturonan, cellulose, mannan and xyloglucan. Thioacidolysis confirmed our negative phloroglucinol staining tests, demonstrating the absence of lignins in the root cortex. All taxa with helical cell wall thickenings formed a monophyletic group supported by a 100 % bootstrap value and composed of mainly epiphytic species. CONCLUSIONS This is the first report of non-lignified pectin-rich secondary cell walls in ferns. Based on our molecular analysis, we reject the hypothesis of parallel evolution of helical cell wall thickenings in Aspleniaceae. Helical cell wall thickenings can mechanically stabilize the cortex tissue, allowing maximal uptake of water and nutrients during rainfall events. In addition, it can also act as a boundary layer increasing the diffusive pathway towards the atmosphere, preventing desiccation of the stele of epiphytic growing species.

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.

A sandwich-embedding method for oriented sectioning.

High‐quality sections are indispensable for many scientific studies. Most published methods are often time‐consuming or require special devices. We present an easy, quick and low‐cost method for oriented embedding of thin structures using glycol methacrylate resin and self‐constructed, reusable embedding tools made of overhead transparencies. This technique allows for more flexibility in orientation than other methods, enabling precise transverse, longitudinal and even oblique sectioning.

Asplenium ceterach and A-octoploideum on the Canary Islands (Aspleniaceae, Pteridophyta)

Abstract Isozyme and plastid DNA analysis prove that true A. ceterach occurs on the Canary Islands, in addition to A. aureum and an octoploid taxon. Combining morphological and cytological observations leads to correct determination, but the exospore length alone also allows reliable identification of these Canarian species. Our allozyme data suggest that the Canarian A. ceterach population is not completely genetically isolated from the European ones. The holotype of Ceterach aureum var. parvifolium, formerly regarded as an octoploid taxon, proved to be A. ceterach, leaving the octoploid without a correct name. The recently described A. octoploideum shows monomorphic, presumably fixed heterozygosity for a combination of the patterns seen in A. ceterach and A. aureum at four loci (Aat, Skdh, Me, and Pgi-2) confirming its allo-octoploid nature. It most probably originated by chromosome doubling in a tetraploid hybrid between A. aureum and A. ceterach or via the union of their unreduced gametes. Pgi-2 indicates multiple origins of the allo-octoploid, implicating recurrent gene flow from tetraploids to octoploids.

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.