Xylogenesis reveals the genesis and ecological signal of IADFs in Pinus pinea L. and Arbutus unedo L.

Background and Aims: Mediterranean trees have patterns of cambial activity with one or more pauses per year, leading to intra-annual density fluctuations (IADFs) in tree rings. We analysed xylogenesis (January 2015-January 2016) in Pinus pinea L. and Arbutus unedo L., co-occurring at a site on Mt. Vesuvius (southern Italy), to identify the cambial productivity and timing of IADF formation. Methods: Dendrochronological methods and quantitative wood anatomy were applied and enabled IADF identification and classification. Key Results: We showed that cambium in P. pinea was productive throughout the calendar year. From January to March 2015, post-cambial (enlarging) earlywood-like tracheids were observed, which were similar to transition tracheids. The beginning of the tree ring was therefore not marked by a sharp boundary between latewood of the previous year and the new xylem produced. True earlywood tracheids were formed in April. L-IADFs were formed in autumn, with earlywood-like cells in latewood. In A. unedo, a double pause in cell production was observed, in summer and winter, leading to L-IADFs in autumn as well. Moreover, the formation of more than one IADF was observed in A. unedo. Conclusions: Despite having completely different wood formation models and different life strategies, the production of earlywood, latewood and IADF cells was strongly controlled by climatic factors in the two species. Such cambial production patterns need to be taken into account in dendroecological studies to interpret climatic signals in wood from Mediterranean trees.

Leaf anatomy, BVOC emission and CO2 exchange of arctic plants following snow addition and summer warming.

BACKGROUND AND AIMS: Climate change in the Arctic is projected to increase temperature, precipitation and snowfall. This may alter leaf anatomy and gas exchange either directly or indirectly. Our aim was to assess whether increased snow depth and warming modify leaf anatomy and affect biogenic volatile organic compound (BVOC) emissions and CO2 exchange of the widespread arctic shrubs Betula nana and Empetrum nigrum ssp. hermaphroditum METHODS: Measurements were conducted in a full-factorial field experiment in Central West Greenland, with passive summer warming by open-top chambers and snow addition using snow fences. Leaf anatomy was assessed using light microscopy and scanning electron microscopy. BVOC emissions were measured using a dynamic enclosure system and collection of BVOCs into adsorbent cartridges analysed by gas chromatography-mass spectrometry. Carbon dioxide exchange was measured using an infrared gas analyser. KEY RESULTS: Despite a later snowmelt and reduced photosynthesis for B. nana especially, no apparent delays in the BVOC emissions were observed in response to snow addition. Only a few effects of the treatments were seen for the BVOC emissions, with sesquiterpenes being the most responsive compound group. Snow addition affected leaf anatomy by increasing the glandular trichome density in B. nana and modifying the mesophyll of E. hermaphroditum The open-top chambers thickened the epidermis of B. nana, while increasing the glandular trichome density and reducing the palisade:spongy mesophyll ratio in E. hermaphroditum CONCLUSIONS: Leaf anatomy was modified by both treatments already after the first winter and we suggest links between leaf anatomy, CO2 exchange and BVOC emissions. While warming is likely to reduce soil moisture, melt water from a deeper snow pack alleviates water stress in the early growing season. The study emphasizes the ecological importance of changes in winter precipitation in the Arctic, which can interact with climate-warming effects.

Selective microspore abortion correlated with aneuploidy: an indication of meiotic drive.

Selective megaspore abortion (monomegaspory) probably arose once in seed plants and occurs routinely in more than 70% of angiosperm species, representing one of the key characters of a heterosporous life history. In contrast, selective microspore abortion leading to pollen dispersal as pseudomonads (here termed monomicrospory) apparently arose at least twice independently within angiosperms, though it occurs in a limited number of taxa. Remarkably, similar examples of monomicrospory occur in members of two distantly related angiosperm families: the sedge family (Cyperaceae) and the epacrid subfamily (Styphelioideae) of the eudicot family Ericaceae. In sedges, monomicrospory is derived directly from normal tetrads, whereas epacrid pseudomonads apparently evolved via an intermediate stage, in which variable sterility occurs in a single tetrad. Our comparison of these two examples of selective microspore abortion highlights a correlation with aneuploidy, indicating that non-random chromosome segregation caused by monomicrospory could drive chromosomal mutations to rapid fixation through meiotic drive.

Structural characteristics of root-fungus associations in two mycoheterotrophic species, Allotropa virgata and Pleuricospora fimbriolata (Monotropoideae), from southwest Oregon, USA.

All members of the Monotropoideae (Ericaceae), including the species, Allotropa virgata and Pleuricospora fimbriolata, are mycoheterotrophs dependent on associated symbiotic fungi and autotrophic plants for their carbon needs. Although the fungal symbionts have been identified for A. virgata and P. fimbriolata, structural details of the fungal-root interactions are lacking. The objective of this study was, therefore, to determine the structural features of these plant root-fungus associations. Root systems of these two species did not develop dense clusters of mycorrhizal roots typical of some monotropoid species, but rather, the underground system was composed of elongated rhizomes with first- and second-order mycorrhizal adventitious roots. Both species developed mantle features typical of monotropoid mycorrhizas, although for A. virgata, mantle development was intermittent along the length of each root. Hartig net hyphae were restricted to the host epidermal cell layer, and fungal pegs formed either along the tangential walls (P. fimbriolata) or radial walls (A. virgata) of epidermal cells. Plant-derived wall ingrowths were associated with each fungal peg, and these resembled transfer cells found in other systems. Although the diffuse nature of the roots of these two plants differs from some members in the Monotropoideae, the structural features place them along with other members of the Monotropoideae in the "monotropoid" category of mycorrhizas.

Accumulation and in vivo tissue distribution of pollutant elements in Erica andevalensis.

Erica andevalensis is an endemic shrub from an area in the southwest of Spain (Andalucia) characterized by acidic and contaminated soils. Scanning electron microscopy (SEM) of samples after conventional or cryo-fixation preparation protocols was used for morphological and anatomical studies. SEM coupled with EDX-analysis was employed to localise and quantify different elements within plant parts (leaves, stems and roots) in samples collected in the field. Morphological studies revealed that the species has typical adaptive structures to drought-stress such as rolled needle-like leaves, sunken stomata and a thick waxy cuticle on the upper epidermis. Roots were associated with fungi which formed intra and extra-cellular mycelia. The SEM studies showed that Cu was not sequestrated into the root tissues and was uniformly distributed in leaf tissues. Meanwhile, Pb was only localised within epidermal root tissues which indicates that its sequestration in an external matrix might represent a tolerance mechanism in this species. Iron was uniformly distributed throughout the leaves, while in roots it was predominantly retained on the epidermal cell walls. The exclusion and tolerance mechanisms adopted by this species to survive in mining areas indicate that it can be used successfully in the re-vegetation of contaminated areas.

Fungal associates of Pyrola rotundifolia, a mixotrophic Ericaceae, from two Estonian boreal forests.

Pyrola rotundifolia (Ericaceae, Pyroleae tribe) is an understorey subshrub that was recently demonstrated to receive considerable amount of carbon from its fungal mycorrhizal associates. So far, little is known of the identity of these fungi and the mycorrhizal anatomy in the Pyroleae. Using 140 mycorrhizal root fragments collected from two Estonian boreal forests already studied in the context of mixotrophic Ericaceae in sequence analysis of the ribosomal DNA internal transcribed spacer region, we recovered 71 sequences that corresponded to 45 putative species in 19 fungal genera. The identified fungi were mainly ectomycorrhizal basidiomycetes, including Tomentella, Cortinarius, Russula, Hebeloma, as well as some ectomycorrhizal and/or endophytic ascomycetes. The P. rotundifolia fungal communities of the two forests did not differ significantly in terms of species richness, diversity and nutritional mode. The relatively high diversity retrieved suggests that P. rotundifolia does not have a strict preference for any fungal taxa. Anatomical analyses showed typical arbutoid mycorrhizae, with variable mantle structures, uniseriate Hartig nets and intracellular hyphal coils in the large epidermal cells. Whenever compared, fungal ultrastructure was congruent with the molecular identification. Similarly to other mixotrophic and autotrophic pyroloids in the same forests, P. rotundifolia shares its mycorrhizal fungal associates with surrounding trees that are likely a carbon source for pyroloids.

Pityopus californicus: structural characteristics of seed and seedling development in a myco-heterotrophic species.

Pityopus californicus (Eastw.) H. F. Copel., a monotypic member of the Monotropoideae in the family Ericaceae, is a myco-heterotrophic species with distribution limited to the Pacific Northwest of the USA. Young embryos of P. californicus developed mycorrhizal associations in seed packets that had been buried for up to 681 days, suggesting that seeds of P. californicus may require the presence of a fungus to achieve germination. Samples of nongerminated seeds and early stages in embryo and root development were subsequently processed for light microscopy, histochemistry, and transmission electron microscopy (TEM). Nongerminated seeds possessed a thick testa, lacked a shoot and root meristem, and consisted of an embryo with large parenchymatous cells containing protein bodies and starch grains as storage reserves. In the earliest developmental stage (seed coat still attached), fungal hyphae were present on the testa surface and between the testa and embryo. This stage was followed by embryo elongation, the organization of a root apical meristem, and the development of a well-developed fungal mantle surrounding the elongated embryo. At least two morphotypes were identified based on structural characteristics of the mantle. One of these, with ascomycetous septa, had Cenococcum-like features. Late-stage embryo/early root development revealed a typical mantle and Hartig net, with fungal pegs penetrating the outer tangential walls of epidermal cells. Transfer cell-like deposits of wall material, similar to those described in Monotropa spp., enclosed fungal pegs. The development of a Hartig net and fungal pegs suggests that nutrient exchange interfaces are required for seedling development.

Sebacinales are common mycorrhizal associates of Ericaceae.

Previous reports of sequences of Sebacinales (basal Hymenomycetes) from ericoid mycorrhizas raised the question as to whether Sebacinales are common mycorrhizal associates of Ericaceae, which are usually considered to associate with ascomycetes. Here, we sampled 239 mycorrhizas from 36 ericoid mycorrhizal species across the world (Vaccinioideae and Ericoideae) and 361 mycorrhizas from four species of basal Ericaceae lineages (Arbutoideae and Monotropoideae) that do not form ericoid mycorrhizas, but ectendomycorrhizas. Sebacinales were detected using sebacinoid-specific primers for nuclear 28S ribosomal DNA, and some samples were investigated by transmission electron microscopy (TEM). Diverging Sebacinales sequences were recovered from 76 ericoid mycorrhizas, all belonging to Sebacinales clade B. Indeed, some intracellular hyphal coils had ultrastructural TEM features expected for Sebacinales, and occurred in living cells. Sebacinales belonging to clade A were found on 13 investigated roots of the basal Ericaceae, and TEM revealed typical ectendomycorrhizal structures. Basal Ericaceae lineages thus form ectendomycorrhizas with clade A Sebacinales, a clade that also harbours ectomycorrhizal fungi. This further supports the proposition that Ericaceae ectendomycorrhizas involve ectomycorrhizal fungal taxa. When ericoid mycorrhizas evolved secondarily in Ericaceae, a shift of mycobionts occurred to ascomycetes and clade B Sebacinales, hitherto not described as ericoid mycorrhizal fungi.

Optimal conditions for visualizing water-conducting pathways in a living tree by the dye injection method.

To elucidate the water-conducting pathways in living trees by the dye injection method, suitable sample preparation procedures are needed. We evaluated quantitatively the properties and concentrations of three dyes (acid fuchsin, basic fuchsin and safranin) widely used for this purpose, and determined the optimal conditions required to avoid artifacts after dye injection into the sap stream of Pieris japonica D. Don. Among the dyes tested, an aqueous solution of acid fuchsin at a concentration of 0.1% or more was the most useful for delineating water movement. In non-transpiring stem segments, the vertical movement of acid fuchsin by capillarity and diffusion from the dye injection site was limited. However, acid fuchsin moved rapidly in the horizontal direction by capillarity and diffusion, and most xylem cells were stained within 2 h. A delay of more than 2 h between dye injection and examination of the tissues greatly reduces the precision of the method. Use of the dye injection method without appropriate, well-defined experimental procedures may give rise to misleading information about the functional water-conducting pathway in living trees.

The micromorphology of pit membranes in tracheary elements of ericales: new records of tori or pseudo-tori?

BACKGROUND AND AIMS: Intervascular pit membranes were examined within Ericales to determine the distribution and structure of torus-like thickenings. METHODS: Forty-nine species representing 12 families of the order Ericales were investigated using light, scanning and transmission electron microscopy. They were compared with four species of Oleaceae to determine the true nature of the thickenings. KEY RESULTS: Pit membranes with torus-like thickenings were observed in seven species of Ericaceae and were found to be amorphous, plasmodesmata-associated structures with an irregular distribution. These pseudo-tori show major differences compared with true tori with respect to their distribution and ultrastructure. Genuine tori, which are strongly correlated with round pit apertures in narrow tracheary elements, were found in two species of Osmanthus (Oleaceae). CONCLUSIONS: The pseudo-tori found in some Ericaceae are considered to be similar to pit membrane thickenings previously recorded in Rosaceae. While true tori appear to be functionally significant in terms of efficiency and safety of water transport, the possible function of pseudo-tori could be associated with the role of plasmodesmata during differentiation of tracheids, fibre-tracheids or narrow vessels.

Structural features of mycorrhizal associations in two members of the Monotropoideae, Monotropa uniflora and Pterospora andromedea.

Species in the subfamily Monotropoideae (family Ericaceae) are achlorophyllous and myco-heterotrophic. They have become highly specialized in that each plant species is associated with a limited number of fungal species which in turn are linked to autotrophic plants. This study provides an updated and comprehensive examination of the anatomical features of two species that have recently received attention with respect to their host-fungal specificity. Root systems of Monotropa uniflora and Pterospora andromedea collected from the field were characterized by light microscopy and scanning electron microscopy. All roots of both species were associated with fungi, each root having a well-developed mantle, paraepidermal Hartig net, and intracellular "fungal pegs" within epidermal cells. The mantle of M. uniflora was multi-layered and numerous outer mantle hyphae developed into cystidia of two distinct morphologies. Large calcium oxalate crystals were present, primarily on the mantle surface. The outer mantle of P. andromedea was more loosely organized, lacked cystidia, and had smaller plate-like as well as cylindrical crystals on the surface and between outer mantle hyphae. Fungal pegs in M. uniflora originated from inner mantle hyphae that penetrated the outer tangential wall of epidermal cells; in P. andromedea, these structures were initiated either from inner mantle hyphae or Hartig net hyphae and penetrated radial walls of epidermal cells. With respect to function, fungal pegs occurred frequently in both host species and, although presumed to be the sites of active nutrient exchange, no direct evidence exists to support this. Differences between these two monotropoid hosts, resulting from the mycorrhizal fungi with which each associates, are discussed.

Re-publication of a translation of 'The vegetative organs of Monotropa hypopitys L.' published by F. Kamienski in 1882, with an update on Monotropa mycorrhizas.

This paper begins with a brief comparison of Franz Kamienski's 1882 view of the fungus-root associations and nutrition of Monotropa hypopitys with our current understanding. The rest of this paper is a re-publication of Shannon Berch's 1985 translation of Kamienski's breakthrough paper in which it was asserted that Monotropa forms a mutualistic symbiosis and is nourished by fungi associated with the roots of neighbouring trees.

Ozone effects on the ultrastructure of peatland plants: Sphagnum mosses, Vaccinium oxycoccus, Andromeda polifolia and Eriophorum vaginatum.

BACKGROUND AND AIMS: Ozone effects on peatland vegetation are poorly understood. Since stress responses are often first visible in cell ultrastructure, electron microscopy was used to assess the sensitivity of common peatland plants to elevated ozone concentrations. METHODS: Three moss species (Sphagnum angustifolium, S. magellanicum and S. papillosum), a graminoid (Eriophorum vaginatum) and two dwarf shrubs (Vaccinium oxycoccus and Andromeda polifolia), all growing within an intact canopy on peat monoliths, were exposed to a concentration of 0, 50, 100 or 150 ppb ozone in two separate growth chamber experiments simulating either summer or autumn conditions in central Finland. After a 4- or 5-week-long exposure, samples were photographed in a transmission electron microscope and analysed quantitatively using image processing software. KEY RESULTS: In the chlorophyllose cells of the Sphagnum moss leaves from the capitulum, ozone exposure led to a decrease in chloroplast area and in granum stack thickness and various changes in plastoglobuli and cell wall thickness, depending on the species and the experiment. In E. vaginatum, ozone exposure significantly reduced chloroplast cross-sectional areas and the amount of starch, whereas there were no clear changes in the plastoglobuli. In the dwarf shrubs, ozone induced thickening of the cell wall and an increase in the size of plastoglobuli under summer conditions. In contrast, under autumn conditions the cell wall thickness remained unchanged but ozone exposure led to a transient increase in the chloroplast and starch areas, and in the number and size of plastoglobuli. CONCLUSIONS: Ozone responses in the Sphagnum mosses were comparable to typical ozone stress symptoms of higher plants, and indicated sensitivity especially in S. angustifolium. The responses in the dwarf shrubs suggest stimulation of photosynthesis by low ozone concentrations and ozone sensitivity only under cool autumn conditions.

Comparative wood anatomy of epacrids (Styphelioideae, Ericaceae s.L.).

The wood anatomy of 16 of the 37 genera within the epacrids (Styphelioideae, Ericaceae s.l.) is investigated by light and scanning electron microscopy. Several features in the secondary xylem occur consistently at the tribal level: arrangement of vessel-ray pits, distribution of axial parenchyma, ray width, and the presence and location of crystals. The primitive nature of Prionoteae and Archerieae is supported by the presence of scalariform perforation plates with many bars and scalariform to opposite vessel pitting. The wood structure of Oligarrheneae is similar to that of Styphelieae, but the very narrow vessel elements, exclusively uniseriate rays and the lack of prismatic crystals in Oligarrheneae distinguish these two tribes. The secondary xylem of Monotoca tamariscina indicates that it does not fit in Styphelieae; a position within Oligarrheneae is possible. Like most Cosmelieae, all Richeeae are characterized by exclusively scalariform perforation plates with many bars, a very high vessel density and paratracheal parenchyma, although they clearly differ in ray width (exclusively uniseriate rays in Cosmelieae vs. uniseriate and wide multiseriate rays in Richeeae). Several wood anatomical features confirm the inclusion of epacrids in Ericaceae s.l. Furthermore, there are significant ecological implications. The small vessel diameter and high vessel frequency in many epacrids are indicative of a high conductive safety to avoid embolism caused by freeze-thaw cycles, while the replacement of scalariform by simple vessel perforation plates and an increase in vessel diameter would suggest an increased conductive efficiency, which is especially found in mesic temperate or tropical Styphelieae.