Michele D. Piercey-Normore

Genetically divergent algae shape an epiphytic lichen community on Jack Pine in Manitoba

Algal genotypes should freely associate with different lichen fungi that grow in the same confined habitat giving the appearance of low levels of selectivity and specificity. If genetic compat- ibility between algal and fungal partners limits the combination of partners, then some degree of taxonomic specificity should be evident. This study examined the photobiont composition in a community of epiphytic lichens on Jack Pine to investigate selectivity and specificity. The objectives of the study were to infer algal identity, to infer photobiont dispersal, and to investigate the distribution of algal genotypes relative to the fungal partner. Photobiont variability was determined by Restriction Fragment Length Polymorphism (RFLP) and nucleotide sequences of the Internal Transcribed Spacer (ITS) of ribosomal DNA (rDNA). Seven species of lichen-forming fungi are reported to associate withfive divergent algal genotypes, with only one species,Evernia mesomorpha, showing some degree of selectivity and specificity. The algae represent at least two species (Trebouxia jamesii and T. impressa) for the area confined to 200cm 2 on the north side of 20 Jack Pine trees. Gene flow was inferred in this tightly defined community of lichenized algae. The algal sharing and inferred gene flow may suggest that soredia provide a means of algal transport and distribution among lichen-forming fungi in the habitat.

Evolution of ITS Ribosomal RNA Secondary Structures in Fungal and Algal Symbionts of Selected Species of Cladonia sect. Cladonia (Cladoniaceae, Ascomycotina)

Evolutionary studies in lichen associations follow that of the fungal symbiont (mycobiont), which is the symbiont after which the lichen is named and forms the majority of the thallus. However, evolution of the algal partner (photobiont) is important to maintain compatibility between symbionts and to optimize productivity of the lichen association. The internal transcribed spacer (ITS) regions of the nuclear ribosomal DNA (rDNA) were examined for primary DNA sequence patterns and for patterns in the secondary structure of the rRNA transcripts in both symbionts of the genus Cladonia. Fungal and algal symbionts show opposite trends in rates of evolution and fragment lengths. Both symbionts showed stronger conservation of the ITS2 structure than the ITS1 structure. Homology was evident in the secondary structures between the two highly divergent chlorophyte and ascomycete taxonomic groups. Most fungal species and all species complexes were polyphyletic. The ITS rDNA of the natural lichen algae from Manitoba and four known algal species is highly similar. The natural lichen algae segregate into highly supported clades by environmental features, suggesting that algae that are already adapted to the environment may associate with germinating fungal propagules in the genus Cladonia. Fungal plasticity may allow the mycobiont to adapt to the environment of the photobiont producing variation in lichen morphology. This might explain the incongruence of phylogenetic patterns between the algal and fungal partners tested and the polyphyly of the fungal species.

Cladonia pyxidata and C. pocillum; genetic evidence to regard them as conspecific

Species delimitation in lichen-forming fungi has been based largely on morphological differences of fungal structures and the lichen thallus with an assumption that morphology reflects hereditary changes. One of the distinguishing features between Cladonia pyxidata and C. pocillum (Cladoniaceae, Ascomycotina) is the shape of the primary squamules. Because these species may inhabit different types of soil the phylogeny of these species was examined in light of potential environmental influences from soil pH and photobiont. Samples were collected across North America, and soil pH was measured for a subset of samples. The nuclear ribosomal internal transcribed spacer of 49 fungal and 14 algal partners and a portion of the polyketide synthase gene of 18 fungi was sequenced and analyzed by phylogenetic methods. Restriction fragment length polymorphism (RFLP) profiles for 129 algae were examined for geographic variation and fungal specificity. Cladonia pyxidata and C. pocillum are not monophyletic but results show seven highly supported lineages. The photobiont and geographic distribution do not play a role in species delimitation. Soil pH shows a statistically significant difference between the extreme morphologies of the two species suggesting a possible link between morphology and environmental conditions.

Gene flow in symbionts of Cladonia arbuscula

Genetic diversity in symbionts of the lichen Cladonia arbuscula was investigated. Forty-eight specimens of Cladonia arbuscula located within a 2 km range at Payuk Lake in Manitoba were collected. Population structure and method of symbiont transmission in C. arbuscula were inferred by examining the presence or absence of introns in the nuclear small subunit (SSU) ribosomal DNA (rDNA) of the mycobiont and restriction fragment length polymorphism (RFLP) of the internal transcribed spacer (ITS) rDNA in the photobiont. The mycobiont showed a high level of genetic variation and the photobiont a low level of variation within the small geographic range studied. An analysis of molecular variance of fungal genotypes showed significant population subdivision for the mycobiont when the data were analysed at P=0·05. No population subdivision was detected when P=0·001. The photobiont also showed no population subdivision. Photobiont genotypes did not correlate with mycobiont genotypes suggesting the symbionts were not transmitted together in the same propagule. Multiple fungal genotypes were also detected in C. arbuscula and possible explanations were discussed.

Vegetatively reproducing fungi in three genera of the Parmeliaceae share divergent algal partners

Abstract The taxonomic pattern of algal association with lichen fungi may be explained by the fungal selection of algae that are available in the habitat, genetic compatibility between symbionts or mode of reproduction of the lichen. This study examined algal selection among species in the genera Evernia, Pseudevernia and Imshaugia of the Parmeliaceae using nucleotide sequences of the algal Internal Transcribed Spacer (ITS) of the nuclear ribosomal DNA (rDNA), the algal actin gene, the 1512 group I intron and secondary RNA structures. The hypothesis of high selectivity within one species of Evernia examined, E. mesomorpha, was supported suggesting that E. mesomorpha exhibits high selectivity toward its photobiont, which is not shared with other species of lichenized fungi in the same habitat. The eight strongly supported ITS clades correspond with two newly described algal species broadly known as Trebouxia jamesii s.l. and one existing species, T. simplex. The possibility of lateral algal transfer is discussed with reference to algal availability and mode of reproduction of the lichen.

Convergent evolution in Cladonia gracilis and allies

Members of the Cladonia gracilis group of lichen fungi are common terrestrial lichens where morphological features are more similar between members of the C. gracilis species complex and allied species outside the complex than they are between subspecies within the complex. The objectives of this study were to examine whether the Cladonia gracilis species complex is monophyletic, to determine whether morphological similarity is supported by genetic variation, and to examine the utility of the polyketide synthase (PKS) gene for phylogenetic studies among closely related species. Two loci, the ketosynthase region of the PKS gene and the internal transcribed spacer (ITS) region of nuclear ribosomal DNA, were sequenced and analysed by Maximum Parsimony, Bayesian and haplotype network analyses. Functional differences were also inferred through ITS2 RNA secondary structures and non-synonymous changes in translated PKS amino acid sequences. The monophyly of the C. gracilis complex is supported by 71% bootstrap in the ITS phylogeny, and 92% bootstrap with greater than 95% posterior probability in the PKS phylogeny. Morphological similarity is not always supported by genetic similarity. The PKS gene is less variable than the ITS but the PKS supports species hypotheses that are reflected in the ITS2 RNA model. We conclude that monophyly of the C. gracilis complex can be supported if C. cornuta, C. coniocraea and C. ochrochlora are included in the complex. In addition, C. maxima, C. phyllophora and C. subchordalis are supported as monophyletic species outside the C. gracilis complex. Cladonia maxima may form a separate species and variation among podetial morphology may be explained by convergent evolution.

Effect of Environmental Change on Secondary Metabolite Production in Lichen-Forming Fungi

The production and regulation of secondary metabolites in non-lichenized fungi, mainly ascomycetes, has been reviewed by a number of authors with an emerging understanding of the biosynthesis and the pathways involved in regulation (Keller et al., 2005; Yu & Keller 2005; and others). However, lichenized fungi make up almost half of all known ascomycetes (Kirk et al., 2001) and are known to produce over 800 secondary metabolites, most of which are unique to lichenized fungi. Many of these compounds have bioactive properties (Huneck, 1999) and some studies have shown or suggested that secondary metabolite production is influenced by changes in culture conditions, which might be regarded as environmental changes. Intense investigation of the changes in production of these unique bioactive secondary metabolites from lichen fungi have been hampered by problems associated with isolating and growing cultures of lichen fungi. Lichens have been studied for more than two centuries as morphological entities but experimental lichenology has remained a nearly unexplored scientific field for many decades because of the slow growing nature of lichens. Thomas (1939 in Stocker-Worgotter, 2001) reported the first successful resynthesis of Cladonia pyxidata. Since the 1970’s, one major goal of experimental lichenology has been the improvement and optimization of culture conditions of lichen fungi. Culture techniques for lichen fungi have improved in recent years allowing for further research on these challenging organisms. Therefore, with greater access to cultures of lichen symbionts and progression of knowledge of non-lichenized fungi, studies are just beginning to accumulate on genes involved in production of secondary metabolites from lichen fungi; and the effects of the environment on the expression of these genes by observations in ecological studies, and through experimentation by manipulating culture conditions. Fungal secondary metabolism is covered by extensive body of literature (see Bennett & Ciegler, 1983). Secondary metabolism is not required for survival and its products are dispensible whereas primary metabolism is essential for survival with anabolic and catabolic activities to maintain life. Secondary metabolites are chemically diverse but are produced from a few key intermediates of primary metabolism, and are generally categorized by the intermediates from which they are produced. Bennett and Ciegler (1983) summarize six categories of secondary metabolites derived from different primary intermediates. Although fungal secondary metabolites are extensive, they are generally

Species diversity and genetic variation of terrestrial lichens and bryophytes in a boreal jack pine forest of central Canada

Abstract Little is known about species diversity and genetic structure of populations of lichens and bryophytes in the Sandilands Provincial Forest in Manitoba. This study investigated the current species diversity of lichens and bryophytes in the area. It also examined the population structure and gene flow in the lichen (Cladonia arbuscula) with the highest cover and second most frequent occurrence, and bryophyte (Pleurozium schreberi) with the highest frequency and percent ground cover. Analysis of Molecular Variance (AMOVA) of the presence and absence of group I introns in the fungal small subunit (SSU) nuclear ribosomal DNA (rDNA) and Restriction Fragment Length Polymorphisms (RFLP) of the algal Internal Transcribed Spacer (ITS) rDNA suggests that low levels of gene flow were occurring in the fungus while a single algal genotype was maintaining the lichen association. Similarly, Interspersed Simple Sequence Repeat (ISSR) markers of the bryophyte suggested low levels of gene flow. The examination of variation in both lichen symbionts and a bryophyte, dominant within the same habitat, is a novel aspect of this study. Both these widespread species, dominating the understory in mature jack pine forests, exhibited low levels of sexual reproduction.

Role of pH on antioxidants production by Spirulina (Arthrospira) platensis

Algae can tolerate a broad range of growing conditions but extreme conditions may lead to the generation of highly dangerous reactive oxygen species (ROS), which may cause the deterioration of cell metabolism and damage cellular components. The antioxidants produced by algae alleviate the harmful effects of ROS. While the enhancement of antioxidant production in blue green algae under stress has been reported, the antioxidant response to changes in pH levels requires further investigation. This study presents the effect of pH changes on the antioxidant activity and productivity of the blue green alga Spirulina (Arthrospira) platensis. The algal dry weight (DW) was greatly enhanced at pH 9.0. The highest content of chlorophyll a and carotenoids (10.6 and 2.4 mg/g DW, respectively) was recorded at pH 8.5. The highest phenolic content (12.1 mg gallic acid equivalent (GAE)/g DW) was recorded at pH 9.5. The maximum production of total phycobiliprotein (159 mg/g DW) was obtained at pH 9.0. The antioxidant activities of radical scavenging activity, reducing power and chelating activity were highest at pH 9.0 with an increase of 567, 250 and 206% compared to the positive control, respectively. Variation in the activity of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) was also reported. While the high alkaline pH may favor the overproduction of antioxidants, normal cell metabolism and membrane function is unaffected, as shown by growth and chlorophyll content, which suggests that these conditions are suitable for further studies on the harvest of antioxidants from S. platensis.

Photobiont Relationships and Phylogenetic History of Dermatocarpon luridum var. luridum and Related Dermatocarpon Species

Members of the genus Dermatocarpon are widespread throughout the Northern Hemisphere along the edge of lakes, rivers and streams, and are subject to abiotic conditions reflecting both aquatic and terrestrial environments. Little is known about the evolutionary relationships within the genus and between continents. Investigation of the photobiont(s) associated with sub-aquatic and terrestrial Dermatocarpon species may reveal habitat requirements of the photobiont and the ability for fungal species to share the same photobiont species under different habitat conditions. The focus of our study was todetermine the relationship between Canadian and Austrian Dermatocarpon luridum var. luridum along with three additional sub-aquatic Dermatocarpon species, and to determine the species of photobionts that associate with D. luridum var. luridum. Culture experiments were performed to identify the photobionts. In addition, the question of the algal sharing potential regarding different species of Dermatocarpon was addressed. Specimens were collected from four lakes in northwestern Manitoba, Canada and three streams in Austria. Three Canadian and four Austrian thalli of D. luridum var. luridum were selected for algal culturing. The nuclear Internal Transcribed Spacer (ITS) rDNA gene of the fungal partner along with the algal ITS rDNA gene was sequenced to confirm the identity of the lichen/photobiont and afterwards the same data sets were used in phylogenetic analyses to assess algal sharing. The green algal photobiont was identified as Diplosphaera chodatii (Trebouxiophyceae). The phylogenetic analyses of Canadian and Austrian D. luridum var. luridum revealed that ITS sequences are identical despite the vast geographic distance. Phylogenetic placement of D. luridum var. decipiens and D. arnoldianum suggested that a re-examination of the species status might be necessary. This study concluded that additional photobiont culture experiments should be conducted to answer the question of whether multiple photobionts are present within the genus Dermatocarpon.