Algal Lichens Research Articles

Response to copper and cadmium stress in wild-type and copper tolerant strains of the lichen alga Trebouxia erici: metal accumulation, toxicity and non-protein thiols

Cysteine, glutathione (GSH) and phytochelatins were determined in the cells of both wild and copper tolerant strains of the lichen alga Trebouxia erici following short-term (24 h) exposure to copper and cadmium and long-term (4 weeks) exposure to copper. Both metals caused concentration dependent synthesis of phytochelatins (PC2–PC5), but cadmium was a more potent activator of phytochelatin synthesis, even inducing synthesis of PC5. The copper-tolerant strain did not reveal a higher degree of phytochelatin synthesis than the wild strain, and at 5 μM Cu production of phytochelatins was in fact significantly lower. Lower levels of phytochelatin correlated with significantly decreased intracellular copper content in the copper-tolerant strain. Both strains maintained high GSH levels even at a high copper concentration of 5 μM, and only the highest copper concentration (10 μM) was toxic for both strains, causing a decrease of GSH and PC content in algal cells. Cadmium had less effect on GSH in the cells of both tested strains. In the long term experiments, only relatively small amounts of PC2 were detected in both strains, but the copper-tolerant strain retained significantly higher levels of reduced glutathione, probably due to the lesser degree of oxidative stress caused by Cu. The significant increase of cysteine synthesis in the copper-tolerant strain found in the present study may be related to copper tolerance in T. erici, while decreased intracellular Cu uptake, detoxification by PCs and increased free proline levels for protection of chloroplast membranes may also be implicated.

Lichens from the Hudson Bay Lowlands: diversity in the southeastern peatlands of Wapusk National Park, Manitoba

Peatlands form an important component of the Northern Hemisphere. They provide substrate for lichen growth and are used by large mammals such as polar bears, caribou, and humans. This study provides the first annotated species list of lichens on the peatlands of Wapusk National Park, Manitoba. It also provides insight into the chemical and genetic diversity of members of Cladonia that dominate the peatlands. Ninety-three species of lichen-forming fungi are reported for the peatlands, fewer than that reported for the coastal beach ridges in Wapusk National Park. This study reports six species of cyanobacterial lichens and two species of Basidiomycete lichens, including two new occurrences for Manitoba, Leptogium intermedium (Arnold) Arnold and Lichenomphalia hudsoniana (H.S. Jenn.) Redhead, Lutzoni, Moncalvo, & Vilgalys. Both chemical diversity in the genus Cladonia and genetic diversity within the species, Cladonia arbuscula (Wallr.) Flotow, were lower than those of a comparable study in a more southerly location. The abundance of apothecia produced by crustose species on the peatlands suggests that crustose species were predominantly sexually reproducing. Foliose species showed production of both sexual and vegetative features, and the fruticose species were mainly vegetatively reproducing. The study raises questions on life history strategies, the influence of habitat on lichen chemistry, and genetic variation of lichen algae in northern environments.

Ice nucleation activity identified in some phytopathogenic Fusarium species

In order to know which species of Fusarium are ice nucleating and to determine the factors affecting their pathogenicity, ice nucleation activity (INA) was examined in Fusarium oxysporum, F. sporotrichioides, and F. tricinctum. Positive controls (lna+) used were F. acuminatum and F. avenaceum. The test for fungal INA was done with a simple and rapid tube nucleation assay. Twelve out of the 42 F. oxysporum isolates, and 8 out of 14 F. tricinctum isolates were lna+. No INA was detected in F sporotrichioides. In this test the threshold freezing temperature tended to increase with culture age, reaching a peak of -1°C in a few samples, which is as high as the warmest INA reported for bacteria, and higher than the INA detected in pure cultures of free-living fungi, lichen fungi, lichen algae and cyanobacteria. This is the first report of INA for F oxysporum.

A new method to isolate lichen algae by using Percoll®gradient centrifugation

Abstract:A rapid method to isolate intact functional algae from the lichens Evernia prunastri and Ramalina farinacea has been developed. This method is based on the use of Percoll®gradients after mechanical disruption of lichen thalli. Results obtained show that the algal preparations were virtually free of contamination by fungal hyphae. The purified algal cells were photosynthetically active and without symptoms of photoinhibition, which indicates their functional integrity. This method may be used for the isolation of intact algae from a broad range of lichen species.

Phylogenetic Species, Reproductive Mode, and Specificity of the Green AlgaTrebouxiaForming Lichens with the Fungal GenusLetharia

The green algal symbionts of the lichenized fungus Letharia are identified as members of the morphospecies Trebouxia jamesii, based on ITS sequences that place them in a molecularly diverse clade with other lichen algae also identified as T. jamesii. However, based on additional sequence from a second locus, actin, T. jamesii is seen to consist of many phylogenetic species. A clade of six to seven closely related phylogenetic species of T. jamesii are symbiotic with five of the six phylogenetic species of Letharia. Within this broad level of specificity, there are also narrower patterns of specificity. For example, three cryptic species of T. jamesii were found only in two species of Letharia in California, a pattern that may be due either to biogeography, or to Letharia phylogenetically tracking Trebouxia. Allopatric Letharia vulpina has switched to a genetically distant phylogenetic species within the T. jamesii morphospecies, suggesting that habitat plays a role in specificity. One phylogenetic species of T. jamesii was found to have a recombining population structure, as two alleles of two loci were found in all four pairwise combinations among the haploid genotypes of that species. This result suggests that Trebouxia, a genus in which sexual structures have never been observed, is nevertheless recombining.

USING RDNA GENES TO UNDERSTAND THE ORIGIN AND EVOLUTION OF SPLICEOSOMAL AND GROUP I INTRONS

Ribosomal DNA genes in lichen algae and lichen fungi are astonishingly rich in spliceosomal and group I introns. We use phylogenetic, secondary structure, and biochemical analyses to understand the evolution of these introns. Despite the widespread distribution of spliceosomal introns in nuclear pre‐mRNA genes, their general mechanism of origin remains an open question because few proven cases of recent and pervasive intron origin have been documented. The lichen introns are valuable in this respect because they are undoubtedly of a “recent” origin and limited to the Euascomycetes. Our analyses suggest that rDNA spliceosomal introns have arisen through aberrant reverse‐splicing (in trans) of free pre‐mRNA introns into rRNAs. We propose that the spliceosome itself (and not an external agent; e.g. transposable elements, group II introns) has given rise to the introns. The rDNA introns are found most often between the flanking sequence G (78%) ‐ intron‐G (72%), and their clustered positions on secondary structures suggest that particular rRNA regions are preferred sites (i.e., proto‐splice sites) for insertion. Mapping of intron positions on the newly available tertiary structures show that they are found most often in exposed regions of the ribosomes. This again is consistent with an intron origin through reverse‐splicing. Remarkably, the distribution and phylogenetic relationships of most group I introns in nuclear rDNA genes are also consistent with a reverse‐splicing origin. These data underline the value of lichens as a model system for understanding intron origin and stress the importance of RNA‐level processes in the spread of these sequences in nuclear coding regions.

The Distribution of Group I Introns in Lichen Algae Suggests That Lichenization Facilitates Intron Lateral Transfer

The nuclear-encoded small subunit ribosomal DNA gene of many lichen-forming green algae in the genus Trebouxia contains a group I intron at Escherichia coli genic position 1512. We studied the evolutionary history of the 1512 intron in Trebouxia spp. (Trebouxiophyceae) by analyzing intron and “host” cell phylogenies. The host trees were constructed by comparing internal transcribed spacer regions of rDNA. Maximum-likelihood, maximum-parsimony, and distance analyses suggest that the 1512 intron was present in the common ancestor of the green algal classes Trebouxiophyceae, Chlorophyceae, and Ulvophyceae. The 1512 intron, however, was laterally transferred at least three times among later-diverging Trebouxia spp. that form lichen partnerships. Intron secondary structure analyses are consistent with this result. Our results support the hypothesis that lichenization may facilitate 1512 group I intron lateral transfer through the close cell-to-cell contact that occurs between the lichen algal and fungal symbionts in the developing lichen thallus.

Photobionts in Lichens: Possible Indicators of Phylogenetic Relationships?

A compilation of records of photobionts that are associated in lichens of the order Lecanorales shows that mycobionts, when seen at the levels of suborders, families, and genera, are strongly selective towards their photobionts. The recently proposed split of the genus Trebouxia into two genera, Asterochloris and Trebouxia, is reflected by the choice of mycobionts. While Asterochloris is mostly found in the suborder Cladoniineae, Trebouxia is mainly confined to the Lecanoriineae. The symbiosis with coccoid green algae other than Trebouxia or Asterochloris might indicate the basal position of a lichen family within Lecanorales. For instance, Dictyochloropsis and Coccomyxa are confined to the Lecanoralean families Biatoraceae (Lecanorineae), Megalosporaceae (cf Teloschistineae), Peltigeraceae and Nephromataceae (Peltigerineae), and Catillariaceae (fam. inc. sed.). The most important cyanobacterial photobionts in Lecanorales are Nostoc and Scytonema as primary and accessory photobionts, while Gloeocapsa and Stigonema are more frequent as accessory photobionts in a few suborders and families of Lecanorales. The co-occurrence of cyanobacteria with Trentepohlia as a primary photobiont in lichen symbioses is not known from families of lichenized ascomycetes. This may reflect differences of ecological preferences of both photobiont groups and of the mycobiont groups that belong to natural relationships. These examples show that the knowledge of photobionts is valuable information for systematics of lichens and therefore, exploration of photobiont diversity in lichens needs to be expanded. Within the last years, lichen photobionts have attracted increasing interest that has led to the unraveling of their diversity and evolutionary relationships using microscopy, ultrastructure, and molecular techniques (Btidel 1992; Ettl & Giirtner 1995; Friedl 1995; Friedl & Biudel 1996; Melkonian & Peveling 1988; Tschermak-Woess 1988). In the green algal genus Trebouxia, the most common photobiont, the recently demonstrated congruence of morphological and ultrastructural characters with rDNA sequence data resulted in a revised systematic concept of the genus (Friedl & Rokitta 1997). However, the question whether photobionts are important markers for the evolution and systematics of lichens, has been controversially discussed (Ahmadjian 1993). The capability of lichen forming fungi to specifically select their appropriate partners among the algal taxa common in subaerial habitats may be an important phylogenetic trait. From a closer examination of the distribution of algal taxa symbiotic in lichens may become obvious that only a single algal species or a very narrow spectrum of algal taxa may cause transformation of a fungus into a lichen thallus. Chodat (1913), an early pioneer in the study of lichen algae, felt that his investigations indicated specificity of lichen-forming fungi with respect to their algal symbionts. Later, observations of Jaag (1933) showed that lichen species belonging to the same genus (e.g., Cladonia, Parmelia, Solorina and Umbilicaria) always contain the same certain type of 'gonidia.' He thought that this phenomenon may also hold true at the level of lichen families. In the following, however, the investigators of lichen photobionts stated that specificity of lichen-forming fungi with respect to their algal symbionts is rather poor. Wang-Yang and Ahmadjian (1972) found different species or strains of algae in morpholog1 This paper was presented as part of a symposium entitled Bridging the Gap Between Phylogeny and the Classification of Lichen-forming Ascomycetes organized by Frangois Lutzoni in Montreal, Canada at the August, 1997, ABLS annual meeting. 0007-2745/98/392-397$0.75/0 This content downloaded from 157.55.39.249 on Wed, 03 Aug 2016 05:52:48 UTC All use subject to http://about.jstor.org/terms 1998] RAMBOLD ET AL.: PHOTOBIONTS IN LICHENS 393 ically identical lichen thalli and conversely subspecies of a particular lichen may contain the same type of algal symbiont. The authors felt that the contribution of knowledge of photobionts to lichen systematics might be poor. Recently, Ahmadjian (1993) stated that it is unlikely that taxonomists can find much value in the types of photobionts that occur in lichens, at least at the species

Temperate rainforest lichens in New Zealand: Light response of photosynthesis

The photosynthetic response to photosynthetically active photon flux density (PPFD) was measured in the field for nine New Zealand rain forest lichen species spanning environments from the deep shade within the forest through the forest margin to outside, open ground sites. Light compensation (PPFDcomp) and light saturation (PPFDsat) were much higher for the species of the open sites (over 50 and 500 μmol/m2.s, respectively). Lichens at forest margin or within the forest had PPFDsat of 64.3-29, and 61–162, μmol/m2.s, respectively, but showed no apparent adaptive trends for these parameters. PPFD response of photosynthesis was measured at several thallus water contents (WC) for one species, Pseudocyphellaria coronata, which showed severe depression of photosynthesis at high WC because of increased diffusion resistances, at all PPFD above compensation. PPFDsat was also depressed but not dark respiration, PPFDcomp, or quantum efficiency. Green algal and cyanobacterial lichens showed no relationship between nitrogen and chlorophyll contents but green algal species were always ≤0.7% nitrogen. A third group, cephalodiate (nitrogen fixing) lichens, had nitrogen contents of 1.5–4.4% dry weight with a clear, positive relationship with chlorophyll content. Chlorophyll and nitrogen contents both increased at more shaded sites within the forest. It is suggested that the possession of cephalodia providing a nitrogen source is advantageous within these evergreen forests by allowing a greater contribution to be made to photosynthetic machinery. The PPFDsat of forest lichens seems to be too high for the prevailing PPFD environment and the lichens would rarely be able to photosynthesise at optimal PPFD. It is suggested that this is a protection against transient high light in sunflecks.

ASSESSING THE RELATIONSHIPS OF SOME COCCOID GREEN LICHEN ALGAE AND THE MICROTHAMNIALES (CHLOROPHYTA) WITH 18S RIBOSOMAL RNA GENE SEQUENCE COMPARISONS1

ABSTRACTEight complete nuclear‐encoded small‐subunit ribosomal RNA (18S rRNA) gene sequences were determined for four genera of the Microthamniales (Pleurastrophyceae) and forGloeotilopsis planctonicaIyengar & Philipose (Ulvophyceae, Ulotrichales) to investigate evolutionary relationships within the Microthamniales and the taxonomic position of this order within the green algae. Phylogenies inferred from these data revealed specific relationships at the level of genera and species that disagree with those inferred from vegetative cell morphology but agree with those inferred from motile cell characters. The rRNA phylogenies provide even better resolution than that gained from morphology alone. The coccoid lichen algaTrebouxiaspp. is specifically related to other coccoid lichen and soil algae (i.e.Myrmecia biatorellaeBoye‐Petersen andFriedmannia israelensisChantanachat & Bold), forming the “Lichen Algae Group,” an evolutionarily distinct lineage within the Microthamniales.Trebouxiais a paraphyletic andPleurastruma polyphyletic genus in rRNA phylogenies. In contrast to previous hypotheses based on morphology,Pleurastrum terrestreFritsch & John is not closely related toTrebouxiabut occupies an isolated position within the Microthamniales. The filamentous algaMicrothamnion kuetzingianumis not ancestral to coccoid members of the Microthamniales but is closely related with the coccoidFusochloris perforata(Lee & Bold) Floyd, Watanabe & Deason. The Microthamniales are inferred to be an array of independent lineages that radiate nearly simultaneously and may also include some autosporic coccoid taxa previously classified in the Chlorophyceae. Monophyly of the Microthamniales could not be demonstrated unequivocally. In contrast to a hypothesis based on ultrastructure, the Microthamniales are evolutionarily distinct from the Ulotrichales. The latter are ancestral to the radiation of the Microthamniales and the Chlorophyceae in the rRNA phylogenies.

Sulphite-induced changes of sulphur metabolism in the lichen alga Trebouxia sp.

Solutions of sodium sulphite have been used to simulate the effect of gaseous sulphur dioxide on the lichen alga Trebouxia sp. The uptake and the metabolism of inorganic and organic [35 S]compounds such as sulphate, sulphite, and cysteine were investigated in cells of Trebouxia under conditions of sulphite preincubation (sulphite-stressed cells: 3 d pretreatment with 50 mmol m-3 and 5 mol m-3 solutions of sodium sulphite). Metabolism was similar, regardless of whether the Trebouxia was provided with [35 S]sulphate or -sulphite. In sulphite-stressed cells, uptake and incorporation into ethanol-soluble compounds, especially cysteine and methionine, was significantly decreased. In Trebouxia exogenous [35 S]cysteine is metabolized only at a very low rate.

Effects of Light and Dark on the Ultrastructure of Lichen Algae

Observation chez deux especes de lichens des modifications qualitatives et quantitatives induites par l'alternance de lumiere et d'obscurite, au niveau des structures ayant des fonctions de mise en reserve

Isoenzyme analysis of lichen algae in immobilized pH gradients

A base for a modern species' concept of chlorococcal algae can be obtained not by morphological analysis, but by biochemical characters, e.g. isoenzyme banding patterns. From isolated lichen algae of the genus Trebouxia de Puymaly a set of five such enzymes has been studied by isoelectric focusing in immobilized pH gradients (IPG): phosphoglucomutase, phosphoglucose isomerase, malate dehydrogenase, mannitol dehydrogenase and leucine aminopeptidase. The first four are resolved into isoforms in a pH 4-7 IPG interval, while the last one is analyzed in an IPG pH 3.5-5 span. The patterns are specific for distinct populations, inter- and intraspecifically varying in dependence from their geographical distribution or the lichen species from which they have been isolated. Their limited heterogeneity (one to four isoforms) suggests that they are the products of specific genes rather than artefacts of the extraction procedure or the IPG analysis. Sharp isozyme patterns can only be obtained in a mixed-bed, carrier-ampholyte (CA)-IPG gel and by anodic application, suggesting that the recently proposed mechanism of hydrophobic protein-IPG matrix interaction (Electrophoresis, 1987, 8, 62-70) is fully operative here. As an additional mechanism, it is proposed that, in some cases, CA might simply act, when added to an IPG gel, by buffering, in the transient state, the sample zone before the protein migrates from the liquid phase into the IPG matrix.

Isoenzyme patterns as a tool in taxonomy of chlorococcal algae.

From isolated lichen algae of the genus Trebouxia, De Puymaly isoenzymes of phosphoglucomutase, malate dehydrogenase and superoxide dismutase were studied by isoelectric focusing in hybrid immobilized pH gradients. Phosphoglucomutase has proven to be a suitable marker since it is highly variable whereas malate dehydrogenase and superoxide dismutase are less variable. The focusing patterns are species specific and provide a basis for a reevaluation of species classification of Trebouxia.

An improved method to isolate lichen algae by gel filtration

Photobiont cells of the lichen Evernia prunastri have completely been separated from their fungal partner by filtration through a bed of Sepharose 2B. Both mannitol and ribitol have been quantified by gas-liquid chromatography in the different steps of the isolation procedure. Absence of mannitol, which is exclusively produced by the mycobiont, has been used as the best probe to monitor isolation.

Carbon dioxide exchange in lichens: Estimation of internal thallus CO2 transport resistances

The gaseous exchange pathways of Sticla latifrons Rich. and Pseudocyphellaria amphisticta Kremp. were examined using both light and scanning electron microscopes. The size and frequency of the pores in the gas exchange structures (cyphellae and pseudocyphellae) and in the medulla were measured and from these CO2 diffusion resistances were calculated. Pseudocyphellae were found to be smaller and more widely spaced than cyphellae, consequently the resistance of the pseudocyphellae, was much greater than that of the cyphellae. Medulla resistances were low in both lichens and are probably unimportant, even at high water contents. No evidence of hyphal swelling was found. Gas exchange structure resistances were more than five fold greater than medulla resistances. It is suggested that this arrangement of resistances may simultaneously encourage refixation of respired CO2 and maintain a non desiccating environment for the lichen algae. The internal transport resistances calculated in this work approximate experimentally obtained values.

THE CONTRIBUTION OF ORGANIC SOLUTES TO OSMOTIC BALANCE IN SOME GREEN AND EUSTIGMATOPHYTE ALGAE1

ABSTRACTSorbitol and proline accumulate to osmotically significant concentrations in Stichococcus bacillaris Nägeli, Stichococcus chloranthus Krüger and Klebsormidium marinum (Deason) Silva, Mattox & Blackwell. The extent of osmotic balance by these and other organic solutes depends on the isolate for these three species of essentially non‐vacuolate algae. Sucrose, proline and sorbitol accumulate in the lichen alga Hyalococcus dermatocarponis Warén, but concentrations on an osmotic basis are low. Sucrose and glutamic acid are significant solutes in the likely non‐vacuolate K. flaccidum (Braun) Silva, Mattox & Blackwell. A similar set of solutes accumulates to an osmotically significant level in Ulothrix fimbriata Bold, a highly vacuolate alga. Two small eustigmatophycean algae (strains WHOI GSB‐Sticho and WHOI Car‐A) and one small chlorophyte (strain UWASH 20‐2‐2) accumulate organic solutes to a moderate degree during salinity stress; mannitol and proline in the eustigmatophytes and proline in the chlorophyte. The highly vacuolate seaweed Enteromorpha intestinalis (L.) Link accumulates sucrose, but at low levels. Osmotic balance in these algae may also include other solutes, compartmentation (especially in vacuolated forms), or other factors. The role of organic solutes in osmotic balance is variable, and depends on details of physiology and ultrastructure.

Notes on the Instability of Extracted Chlorophyll and A Reported Effect of Ozone on Lichen Algae

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LICHEN PHYSIOLOGY

SummaryThe pattern of 14C fixation and release by algae after isolation from lichens was studied using Nostoc from Peltigera canina, Hyalococcus from Dermatocarpon miniatum. Coccomyxa from Peltigera aphthosa and Trebouxia from Xanthoria aureola; cultured strains of these algae were also investigated. In all cases, the following changes occur as soon as the algae are isolated: (a) release of fixed 14C to the medium declines sharply; (b) the proportion of released 14C incorporated into simple carbohydrates also falls rapidly; and (c) incorporation into ethanol‐insoluble material in the cell rises substantially. The direction of these changes is towards the behaviour of cultured algae. It is concluded the lichen algae begin to show substantial changes from their behaviour in symbiosis as soon as they are separated from the fungus.

Effects of gamma radiation on lichens—i. acute gamma radiation on lichen algae and fungi

Gannutz T.P. Effects of gamma radiation on lichens—I. Acute gamma radiation on lichen algae and fungi. Radiation Botany 12, 331–338. 1972.-The effects of acute gamma irradiation on isolated cultures of lichen algae and fungi have been determined quantatively. Lichen algae were more radiosensitive than the lichen fungi, even among components from the same species of lichen. This is in direct correlation to the effects of natural environmental stress. Although the lichen fungi have similar radiosensitivities to similar free-living fungi, the irradiated lichen fungi were entirely in the vegetative state (i.e. not in stages of reproduction or sporulation). Consequently, it is apparent that the lichen fungi are less radiosensitive than similar free-living fungi. Radiosensitivities of lichen algae and free-living algae were similar. When comparisons are made with intact thalli growing in a chronically irradiated situation, it appears that the intact thallus is more radiosensitive than either the algal or fungal component in culture. It is postulated that this is due to the addition of another stress (i.e. radiation) on an already strained symbiotic relationship. Correlations were attempted between the various species of algae and fungi, their radiosensitivities, and water, lipid, and DNA contents.