Coordinated ultrastructural and phylogenomic analyses shed light on the hidden phycobiont diversity of Trebouxia microalgae in Ramalina fraxinea

[EN] The precise boundary delineations between taxa in symbiotic associations are very important for evolutionary and ecophysiological studies. Growing evidence indicates that in many cases, the use of either morphological characters or molecular markers results in diversity underestimation. In lichen symbioses. Trebouxia is the most common genus of lichen phycobionts, however, the diversity within this genus has been poorly studied and as such there is no clear species concept. This study constitutes a multifaceted approach incorporating aspects of ultrastructural characterization by TEM and phylogenomics to evaluate the morphological and genetic diversity of phycobionts within the sexually reproducing lichen Ramalina fraxinea in the context of Mediterranean and temperate populations. Results reveal an association with at least seven different Trebouxia lineages belonging to at least two species. T. decolorans and T. jamesii, and diverse combinations of such lineages coexisting within the same thallus depending on the analysed sample. Some of these lineages are shared by several other non-related lichen taxa. Our findings indicate the existence of a highly diverse assemblage of Trebouxia algae associating with R. fraxinea and suggest a possible incipient speciation within T. decolorans rendering a number of lineages or even actual species. This study stresses the importance of coordinated ultrastructural and molecular analyses to improve estimates of diversity and reveal the coexistence of more than one Trebouxia species within the same thallus. lt is also necessary to have clearer species delimitation criteria within the genus Trebouxia and microalgae in general.This study was funded by the Ministerio de Economia y Competitividad (MINECO CGL2012-40058-0O2-01/02), FEDER, the Generalitat Valenciana (PROMETEOII2013/021, GVA) and the Direccion General de Universidades e Investigacion de la Consejeria de Educacion de la Comunidad de Madrid - Universidad de Alcala (CCG10-UAH/GEN-5904). Drs. Arantxa Matins and Patricia Moya (Universitat de Valencia) made helpful comments on the manuscript.Català, S.; Campo, ED.; Barreno, E.; García-Breijo, F.; Reig Armiñana, J.; Casano, L. (2016). Coordinated ultrastructural and phylogenomic analyses shed light on the hidden phycobiont diversity of Trebouxia microalgae in Ramalina fraxinea. Molecular Phylogenetics and Evolution. 94:765-777. https://doi.org/10.1016/j.ympev.2015.10.021S7657779

Lichen acclimation to changing environments:Photobiont switching vs. climate-specific uniqueness in Psora decipiens

Unraveling the complex relationship between lichen fungal and algal partners has been crucial in understanding lichen dispersal capacity, evolutionary processes, and responses in the face of environmental change. However, lichen symbiosis remains enigmatic, including the ability of a single fungal partner to associate with various algal partners. Psora decipiens is a characteristic lichen of biological soil crusts (BSCs), across semi-arid, temperate, and alpine biomes, which are particularly susceptible to habitat loss and climate change. The high levels of morphological variation found across the range of Psora decipiens may contribute to its ability to withstand environmental change. To investigate Psora decipiens acclimation potential, individuals were transplanted between four climatically distinct sites across a European latitudinal gradient for 2years. The effect of treatment was investigated through a morphological examination using light and SEM microscopy; 26S rDNA and rbcL gene analysis assessed site-specific relationships and lichen acclimation through photobiont switching. Initial analysis revealed that many samples had lost their algal layers. Although new growth was often determined, the algae were frequently found to have died without evidence of a new photobiont being incorporated into the thallus. Mycobiont analysis investigated diversity and determined that new growth was a part of the transplant, thus, revealing that four distinct fungal clades, closely linked to site, exist. Additionally, P.decipiens was found to associate with the green algal genus Myrmecia, with only two genetically distinct clades between the four sites. Our investigation has suggested that P.decipiens cannot acclimate to the substantial climatic variability across its environmental range. Additionally, the different geographical areas are home to genetically distinct and unique populations. The variation found within the genotypic and morpho-physiological traits of P.decipiens appears to have a climatic determinant, but this is not always reflected by the algal partner. Although photobiont switching occurs on an evolutionary scale, there is little evidence to suggest an active environmentally induced response. These results suggest that this species, and therefore, other lichen species, and BSC ecosystems themselves may be significantly vulnerable to climate change and habitat loss

Molecular studies on cyanobacterial diversity in lichen symbioses

Peer reviewe

Paleogene Radiation of a Plant Pathogenic Mushroom

Background: The global movement and speciation of fungal plant pathogens is important, especially because of the economic losses they cause and the ease with which they are able to spread across large areas. Understanding the biogeography and origin of these plant pathogens can provide insights regarding their dispersal and current day distribution. We tested the hypothesis of a Gondwanan origin of the plant pathogenic mushroom genus Armillaria and the currently accepted premise that vicariance accounts for the extant distribution of the species. Methods: The phylogeny of a selection of Armillaria species was reconstructed based on Maximum Parsimony (MP), Maximum Likelihood (ML) and Bayesian Inference (BI). A timeline was then placed on the divergence of lineages using a Bayesian relaxed molecular clock approach. Results: Phylogenetic analyses of sequenced data for three combined nuclear regions provided strong support for three major geographically defined clades: Holarctic, South American-Australasian and African. Molecular dating placed the initial radiation of the genus at 54 million years ago within the Early Paleogene, postdating the tectonic break-up of Gondwana. Conclusions: The distribution of extant Armillaria species is the result of ancient long-distance dispersal rather than vicariance due to continental drift. As these finding are contrary to most prior vicariance hypotheses for fungi, our result

A Survey of Lichens and Bryophytes in White Spruce, Picea glauca, Tree Islands on a Calcareous Beach Ridge in Northeastern Manitoba

Studies on the diversity of lichens and bryophytes in northern ecosystems have focused on open tundra and boreal forests. Krummholz tree islands have received little attention. This study examined species’ diversity of lichens and bryophytes among islands of White Spruce trees in northern Manitoba. Thirty-three species were found in the tree islands with 11 additional species forming the understorey layer. The trees were dominated by epiphytic lichens and the understorey was dominated by pleurocarpous mosses. The most common lichens were widespread species. Rare lichens in the study area included Protopannaria pezizoides and Alectoria nigricans growing in the shaded interior of the tree islands. The age and density of the tree islands may produce habitat conditions different from those in mature White Spruce boreal forests. Comparative studies between White Spruce krummholz and other White Spruce stands are needed

Molecular evolution, identification and epidemiology of North American species of a root-infecting fungus, Armillaria

Armillaria (Fr.:Fr.) Staude is a genus of root-infecting fungal pathogens which cause disease in forests and orchard plantations. Biological species of Armillaria are identified by sexual incompatibility mating interactions. Epidemiological studies of North American species of Armillaria indicated that some are saprotrophic, some mycotrophic, and others are necrotrophic pathogens often producing death of the host. Stress of the host tree is often considered a critical factor in disease development. -- Three objectives to this study examined different aspects of the pathogenicity of Armillaria. The first was to estimate phylogenetic relationships among North American biological species (NABS) of Armillaria. This allowed inference of inheritance of pathogenic traits among species. The second was to develop species-specific molecular markers for NABS Armillaria. This would provide a method of identification for pathogenic species. The third was to examine disease development of Armillaria inoculum using different types and degrees of stress inflicted on the host tree. Molecular techniques were employed to examine the first two objectives. The third objective involved a field inoculation trial with two Newfoundland isolates of A. ostoyae. -- This study is consistent with previous phylogenetic hypotheses concerning relationships among species of Armillaria. Phylogenetic analysis of randomly amplified DNA regions of unknown function provided strong support for intraspecies clustering. Most NABS Armillaria were resolved using four anonymous nucleotide sequences combined within a single data set. There was stong support for the clustering of NABS I and II, as well as NABS III and VII. Isolates of NABS V showed sequence polymorphism. -- Species-specific molecular markers were developed for most NABS Armillaria. PCR amplification using a combination of different random primer sets in each of the reaction mixtures, yielded species size-specific bands on an agarose gel for each of NABS I, II and VI. A common band was found specific for NABS III and VII. Although NABS V, IX and X contained less variation, 10 nucleotide primers could be used to confirm their identity. Results from inverse PCR suggested that secondary DNA structure and primer/template competition played a significant role in determining species-specificity with SWAPP 10 nucleotide primers. -- Results from the field inoculation trials suggested that two years was sufficient time to allow forest managers to make informed decisions regarding stand management. Host stress appeared to influence Armillaria root disease development. The black spruce plantation had more infection than the naturally regenerated stand. There was more disease in the balsam fir thinned stand than the un-thinned stand. Significant correlation between infection and above ground tree symptoms occurred in a very severely defoliated balsam fir sawfly plot only. Well-drained sandy soil seemed to increase the aggressiveness of the Armillaria isolate used as inoculum. -- The utilization of molecular techniques, combined with knowledge of ecological processes, would greatly enhance the efficiency of forest management

Effects of damage-induced and developmental changes of white birch (Betula papyrifera Marsh.) on populations of birch casebearer (Coleophora serratella L.) (Lepidoptera: Coleophoridae)

Insects feeding on a plant may induce changes within the leaves which affect success of insects subsequently feeding on that plant. The objective of this study was to demonstrate if birch casebearer feeding on birch leaves induced changes in the tree that would deter subsequent feeding by the same insect species. -- White birch trees were divided into four treatment groups of five trees each. Treatments consisted of birch leaves being severely damaged by insect feeding, lightly damaged by insect feeding, mechanically damaged with no insects present, and control with neither type of damage. Comparisons were made among treatment groups for 1) changes in the leaves, 2) population size of the birch casebearer at various times of the season, and 3) effects of damage-induced leaf changes on larval size. Damage-induced changes in the leaves were evident in nitrogen and phosphorus levels between 1991 and 1992, long-shoot length and number of buds, and bud development and leaf senescence. These changes were likely a result of nutrient depletion by insect feeding the previous year, reducing the tree's ability to compensate for losses. Population size of the birch casebearer between treatment groups changed very little throughout 1991 and 1992 suggesting that moths did not move far from the site of pupation. Positive correlation between nitrogen levels and egg density suggested that female moths might be able to distinguish between high and low leaf nitrogen levels, though this could occur within the same tree. Since headcapsule widths of Instar II larvae did not differ significantly between treatment groups, it appeared that damage-induced changes of white birch had no measurable effect on larval size. -- Leaf developmental changes, measured as water potential, water content, and fresh and dry leaf weights, were compared with seasonal changes in plant and insect life history stages. Egg hatch coincided with second leaf flush. Since eggs were laid on old leaves, it was suggested that Instar I larvae took advantage of nutrients being mobilized out of the older leaves and moving into younger leaves. -- The study showed no evidence for damage-induced leaf changes affecting success of the birch casebearer. However, there was speculation that the unusual life history pattern of the birch casebearer was adapted so that Instar I and II larvae could take advantage of mobilized leaf nutrients, lower water contents, and water potentials late in the season. Instar III and IV larvae, with faster growth, could take advantage of high leaf nutrients early in the season