Lichen Photobionts Research Articles

A standardized approach for co-culturing dothidealean rock-inhabiting fungi and lichen photobionts in vitro

Black, rock inhabiting fungi (RIF) are polyextremotolerant, oligotrophic organisms which colonize bare rocks and are specialized to grow in niches precluded to other microorganisms in the harshest environments. In many cases RIF share this environment with green algae and cyanobacteria forming subaerial biofilms; some of them have also been found to be associated with lichen thalli. The RIF genus Lichenothelia is of particular interest because it includes lichen parasites and species which are loosely associated with algae or grow independently on rocks. Here, in vitro culture experiments studied the development of three Lichenothelia species when co-cultured with two different lichen photobionts on growth media differing in nutrient content. The growth rates of these fungi were statistically evaluated and the structure of the mixed cultures was analyzed by light and scanning electron microscopy. The results show that the presence of algae neither influence the growth rate of fungi nor the formation of any lichen-like structure; tight contacts between the hyphae and the algal cells were also not detected. Since multiple trials were carried out on well characterized species of microcolonial fungi and the methodological procedures were established, standardized and coupled with morphological analyses, this approach proves suitable for future investigations on fungal-algal interactions in other systems.

Fungal tissue per se is stronger as a UV-B screen than secondary fungal extrolites in Lobaria pulmonaria

Abstract To test the hypotheses that (1) protective mycobiont tissues and/or (2) medullary UV-B-absorbing carbon-based secondary compounds (CBSCs) protect lichen photobionts against UV-B radiation, we quantified cortical UV-transmittance and ran a three-way factorial lab experiment with (1) three UV radiation regimes, (2) photobiont layers with/without a screening cortex, and (3) with natural/reduced CBSC-concentration. We used melanin-deficient Lobaria pulmonaria from shaded forests. Maximum photochemical efficiency of photosystem II ( F v/ F m) in photobionts inside thalli with natural CBSC-concentrations was not affected by any UV-regime, consistent with close to 0% measured cortical transmittance of wavelengths P P P = 0.001), mainly affecting exposed photobiont layers given short-wavelength UV radiation. Based on these findings, we conclude that the primary role of extrolites in L. pulmonaria is not to screen excess solar radiation.

Heveochlorella (Trebouxiophyceae): a little-known genus of unicellular green algae outside the Trebouxiales emerges unexpectedly as a major clade of lichen photobionts in foliicolous communities.

Foliicolous lichens are formed by diverse, highly specialized fungi that establish themselves and complete their life cycle within the brief duration of their leaf substratum. Over half of these lichen-forming fungi are members of either the Gomphillaceae or Pilocarpaceae, and associate with Trebouxia-like green algae whose identities have never been positively determined. We investigated the phylogenetic affinities of these photobionts to better understand their role in lichen establishment on an ephemeral surface. Thallus samples of Gomphillaceae and Pilocarpaceae were collected from foliicolous communities in southwest Florida and processed for sequencing of photobiont marker genes, algal cultivation and/or TEM. Additional specimens from these families and also from Aspidothelium (Thelenellaceae) were collected from a variety of substrates globally. Sequences from rbcL and nuSSU regions were obtained and subjected to Maximum Likelihood and Bayesian analyses. Analysis of 37 rbcL and 7 nuSSU algal sequences placed all photobionts studied within the provisional trebouxiophycean assemblage known as the Watanabea clade. All but three of the sequences showed affinities within Heveochlorella, a genus recently described from tree trunks in East Asia. The photobiont chloroplast showed multiple thylakoid stacks penetrating the pyrenoid centripetally as tubules lined with pyrenoglobuli, similar to the two described species of Heveochlorella. We conclude that Heveochlorella includes algae of potentially major importance as lichen photobionts, particularly within (but not limited to) foliicolous communities in tropical and subtropical regions worldwide. The ease with which they may be cultivated on minimal media suggests their potential to thrive free-living as well as in lichen symbiosis.

Cyanobacterial gardens: the liverwort Frullania asagrayana acts as a reservoir of lichen photobionts.

Cyanobacteria are important mediators of unrelated lichen species, which form epiphytic communities that share the same cyanobiont. No study to date, however, has considered the role of cyanobacteria as mediator between lichens and bryophytes. In the present study, DNA barcoding (16S rDNA, rbcLX) was used to identify filamentous cyanobacteria living in close association with members of an epiphytic liverwort-lichen community on balsam fir in Newfoundland. This study is the first to confirm the presence of Rhizonema strains in boreal forests where they are associated with the liverwort Frullania asagrayana and several lichen species. The majority of cyanobacterial haplotypes can associate with the liverwort, however, some lichen species appear to be more selective for single or closely related haplotypes. Some Rhizonema strains were found exclusively in association with boreal lichens, while others seem to be globally distributed and involved in different lichen symbioses of unrelated fungal lineages and of varying ecological traits. Complex biological interactions in a cyanobacteria-mediated guild are proposed here, which explains composition and dynamics in bryophyte and lichen-dominated epiphytic communities.

New features of desiccation tolerance in the lichen photobiont Trebouxia gelatinosa are revealed by a transcriptomic approach.

Trebouxia is the most common lichen-forming genus of aero-terrestrial green algae and all its species are desiccation tolerant (DT). The molecular bases of this remarkable adaptation are, however, still largely unknown. We applied a transcriptomic approach to a common member of the genus, T. gelatinosa, to investigate the alteration of gene expression occurring after dehydration and subsequent rehydration in comparison to cells kept constantly hydrated. We sequenced, de novo assembled and annotated the transcriptome of axenically cultured T. gelatinosa by using Illumina sequencing technology. We tracked the expression profiles of over 13,000 protein-coding transcripts. During the dehydration/rehydration cycle c. 92% of the total protein-coding transcripts displayed a stable expression, suggesting that the desiccation tolerance of T. gelatinosa mostly relies on constitutive mechanisms. Dehydration and rehydration affected mainly the gene expression for components of the photosynthetic apparatus, the ROS-scavenging system, Heat Shock Proteins, aquaporins, expansins, and desiccation related proteins (DRPs), which are highly diversified in T. gelatinosa, whereas Late Embryogenesis Abundant Proteins were not affected. Only some of these phenomena were previously observed in other DT green algae, bryophytes and resurrection plants, other traits being distinctive of T. gelatinosa, and perhaps related to its symbiotic lifestyle. Finally, the phylogenetic inference extended to DRPs of other chlorophytes, embryophytes and bacteria clearly pointed out that DRPs of chlorophytes are not orthologous to those of embryophytes: some of them were likely acquired through horizontal gene transfer from extremophile bacteria which live in symbiosis within the lichen thallus.

Lichen photobiont diversity and selectivity at the southern limit of the maritime Antarctic region (Coal Nunatak, Alexander Island)

Antarctic ice-free inland sites provide a unique perspective on the strategies coevolving organisms have developed for survival at the limits of life. Here, we provide the first combined description of the ecological and genetic diversity of lichen photobionts colonising an isolated Antarctic inland site, Coal Nunatak, on south-east Alexander Island (Antarctic Peninsula). Photobionts of 14 lichen species (42 samples), all belonging to the group of coccal green algae, representing the entire lichen community of Coal Nunatak were investigated using the internal transcribed spacer region (ITS) of the nuclear ribosomal DNA. The study attempted to address the hypothesis that mycobiont selectivity for the photobiont partner is lower in more extreme environments. This hypothesis did not appear to hold true for the entire lichen community except one species. Another aspect focuses on the relevance of the reproduction modus concerning the distribution of photobiont haplotypes in the lichen community.

Evaluation of Аir Рollution by Вiological Тesting Мethods

The possibility of using 2 test kits – the display on the basis of the characteristics of lichens and bioassays based on luminescent bacteria for rapid assessment of the state of air pollution in the example area in Shchelkovo district, Moscow region. The number of detected lichen species, the classes of their resistance to pollution and changes in the morphology of lichens thalli and cell diameters of lichen photobionts and toxicity of tree bark in the allocation of lichens was estimated.

Lichen photobionts of the rocky outcrops of Karadag massif (Crimean Peninsula)

Despite the fact that lichen algae have been explored for over100 years, their diversity, taxonomy and ecology still remains understudied. In present study we analyzed 114 specimens representing 72 lichen taxa collected on the Crimean Peninsula, culturing and sequencing their algal partners. In total we identified 26 taxa of photobionts representing 10 algal genera (Asterochloris, Chloroidium, Dilabifilum, Diplosphaera, Gloeocapsa, Myrmecia, Nostoc, Printzina, Stichococcus, Trebouxia). As most of the studied lichens (more than 80 % of studied lichen species) contained Trebouxia, for which morphology-based identification is often difficult and insufficient, we additionally used molecular methods. According to the molecular phylogeny of internal transcribed spacer (ITS) rDNA, Trebouxia species were related to three main clades: “Arboricola” (88.9 % of the investigated Trebouxia-containing lichens), “Impressa” (4.4 %) and “Simplex” (6.7 %). Based on results of molecular phylogeny and morphology three new species of Trebouxia, namely T. solaris, T. vagua and T. cretacea were described. In several lichens, two coexisting Trebouxia species were revealed in the same thallus.

Vulcanochloris (Trebouxiales, Trebouxiophyceae), a new genus of lichen photobiont from La Palma, Canary Islands, Spain

This paper describes a new genus of lichen photobionts, Vulcanochloris, with three newly proposed species, V. canariensis, V. guanchorum and V. symbiotica. These algae have been discovered as photobionts of lichen Stereocaulon vesuvianum growing on slopes of volcanos and lava fields on La Palma, Canary Islands, Spain. Particular species, as well as the newly proposed genus, are delimited based on ITS rDNA, 18S rDNA and rbcL sequences, chloroplast morphology, and ultrastructural features. Phylogenetic analyses infer the genus Vulcanochloris as a member of Trebouxiophycean order Trebouxiales, in a sister relationship with the genus Asterochloris. Our data point to the similar lifestyle and morphology of these two genera; however, Vulcanochloris can be well distinguished by a unique formation of spherical incisions within the pyrenoid. Mycobiont specificity and geographical distribution of the newly proposed genus is further discussed.

Assembling the challenging puzzle of algal biodiversity: species delimitation within the genus Asterochloris (Trebouxiophyceae, Chlorophyta).

The genus Asterochloris represents one of the most common, widespread, and diverse taxa of lichen photobionts. In this report, we describe and characterize six new species (A.echinata, A.friedlii, A.gaertneri, A.leprarii, A.lobophora, and A.woessiae) that were identified during our recent investigation of photobiont diversity. We found that the species differed genetically, morphologically, ecologically, and with respect to their mycobiont partners. Statistical analyses revealed significant morphological differentiation of all six newly described species, as well as their separation from previously described Asterochloris species. Chloroplast morphology represented the best morphological marker for species delineation. In fact, each species can be recognized by the dominance and unique assemblage of particular chloroplast types. Although genetically well recognized by rapidly evolving internal transcribed spacer rDNA and actin intron markers, all 13 investigated Asterochloris species shared identical small subunit rDNA sequences. We therefore demonstrated that morphologically and ecologically diverse species can frequently be grouped into a single taxonomic unit in whole-transcriptome sequencing studies, considerably affecting the resulting estimates of species diversity. Finally, we demonstrated the presence of isogamous sexual reproduction in Asterochloris, disputing the current symbiotic dogma of the loss of sexual reproduction in algal symbionts.

Production of UV-B screens and changes in photosynthetic efficiency in Antarctic Nostoc commune colonies and a lichen Xanthoria elegans depend on a dose and duration of UV-B stress

The survival of non-vascular autotrophs in the extreme polar conditions and the principles of their high tolerance to extreme physical factors have been intriguing scientists in last decades. Therefore, this study focuses on the capacity of production of UV-B screening pigments in two model Antarctic species, one algal lichen, and colony of a cyanobacterium. Dose-dependent activation of protective mechanisms of Antarctic cyanobacterium (Nostoc commune) and algal lichen (Xanthoria elegans), synthesis of UV-B screening compounds in particular, were studied together with the changes in photosynthetic efficiency induced by a background photosynthetically active radiation (PAR) supplemented with UV-B radiation. The samples were exposed to different doses of UV-B (280–320 nm), low (0.7 W m-2), medium (1.5 W m-2) and high (3.0 W m-2) for 5 days. Untreated samples (control) were shielded from UV-B radiation during experiment. Chlorophyll fluorescence parameters and secondary UV-B protective metabolites were analysed in the intervals of 24 h, 48 h and 120 h. Amount of UV-B screening pigments was measured spectrophotometrically using several specific wavelengths in UV-B absorption range. Results showed that if exposed to a low dose of UV-B radiation or a short-term treatment, both species exhibited an increase in UV-B screening pigments to protect the lichen photobiont against UV-B damage. However, if exposed to a high dose of UV-B radiation or a long-term treatment, a decrease of UV-B screening compounds occured. This implies that Antarctic lichen and cyanobacterium can protect themselves against an increase of stress factors ranging within physiological limits, like e.g. increased synthesis of UV-B screening compounds thanks to a thinning of the ozone layer and consequent increase in UV radiation doses incident on Antarctic terrestrial ecosystems. Nevertheless, the likely increased UV-B radiation due to more intense depletion of stratospheric ozone layer may lead to alterations in UV-B tolerance in Antarctic lichens in future.

Desiccation and low temperature attenuate the effect of UVC254 nm in the photobiont of the astrobiologically relevant lichens Circinaria gyrosa and Buellia frigida

AbstractSeveral investigations on lichen photobionts (PBs) after exposure to simulated or real-space parameters consistently reported high viability and recovery of photosynthetic activity. These studies focused on PBs within lichen thalli, mostly exposed in a metabolically inactive state. In contrast, a recent study exposed isolated and metabolically active PBs to the non-terrestrial stressor UVC254 nm and found strong impairment of photosynthetic activity and photo-protective mechanisms (Meeßen et al. in 2014b). Under space and Mars conditions, UVC is accompanied by other stressors as extreme desiccation and low temperatures. The present study exposed the PBs of Buellia frigida and Circinaria gyrosa, to UVC in combination with desiccation and subzero temperatures to gain better insight into the combined stressors' effect and the PBs' inherent potential of resistance. These effects were examined by chlorophyll a fluorescence which is a good indicator of photosynthetic activity (Lüttge & Büdel in 2010) and widely used to test the viability of PBs after (simulated) space exposure. The present results reveal fast recovery of photosynthetic activity after desiccation and subzero temperatures. Moreover, they demonstrate that desiccation and cold confer an additional protective effect on the investigated PBs and attenuate the PBs' reaction to another stressor – even if it is a non-terrestrial one such as UVC. Besides other protective mechanisms (anhydrobiosis, morphological–anatomical traits and secondary lichen compounds), these findings may help to explain the high resistance of lichens observed in astrobiological studies.

Viability of the lichenXanthoria elegansand its symbionts after 18 months of space exposure and simulated Mars conditions on the ISS

AbstractThe lichenXanthoria eleganshas been exposed to space conditions and simulated Mars-analogue conditions in the lichen and fungi experiment (LIFE) on the International Space Station (ISS). After several simulations and short space exposure experiments such as BIOPAN, this was the first long-term exposure of eukaryotic organisms to the hostile space conditions of the low Earth orbit (LEO). The biological samples were integrated in the EXPOSE-E facility and exposed for 1.5 years outside the ISS to the combined impact of insolation, ultraviolet (UV)-irradiation, cosmic radiation, temperatures and vacuum conditions of LEO space. Additionally, a subset ofX. eleganssamples was exposed to simulated Martian environmental conditions by applying Mars-analogue atmosphere and suitable solar radiation filters. After their return to Earth the viability of the lichen samples was ascertained by viability analysis of LIVE/DEAD staining and confocal laser-scanning microscopy, but also by analyses of chlorophyllafluorescence. According to the LIVE/DEAD staining results, the lichen photobiont showed an average viability rate of 71%, whereas the even more resistant lichen mycobiont showed a rate of 84%. Post-exposure viability rates did not significantly vary among the applied exposure conditions. This remarkable viability is discussed in the context of particular protective mechanisms of lichens such as anhydrobiosis and UV-screening pigments.

Ecological specialization in Trebouxia (Trebouxiophyceae) photobionts of Ramalina menziesii (Ramalinaceae) across six range-covering ecoregions of western North America.

• Premise of the study: Many lichens exhibit extensive ranges spanning several ecoregions. It has been hypothesized that this wide ecological amplitude is facilitated by fungal association with locally adapted photobiont strains.• Methods: We studied the identity and geographic distribution of photobionts of the widely distributed North American lichen Ramalina menziesii based on rbcL (chloroplast DNA) and nuclear ribosomal ITS DNA sequences. To test for ecological specialization, we associate photobiont genotypes with local climate and phorophyte.• Key results: Of the photobiont lineages of R. menziesii, 94% belong to a clade including Trebouxia decolorans. The remaining are related to T. jamesii. The photobionts showed (1) significant structure according to ecoregion and phorophyte species and (2) genetic associations with phorophyte species and climate.• Conclusions: Geography, climate, and ecological specialization shape genetic differentiation of lichen photobionts. One great advantage of independent dispersal of the fungus is symbiotic association with locally adapted photobiont strains.

Allelopathic effects of lichen secondary metabolites and their naturally occurring mixtures on cultures of aposymbiotically grown lichen photobiont Trebouxia erici (Chlorophyta)

In the present work, the phytotoxic effects of secondary metabolites extracted from five lichen species (Cladonia arbuscula var. mitis, Cladonia furcata, Hypogymnia physodes, Evernia prunastri and Ramalina farinacea) on cultures of the aposymbiotically grown lichen photobiont Trebouxia erici were evaluated. Toxicity of single secondary metabolites, as well as their naturally occurred mixtures in the lichens was tested at the two selected doses, 0.1mg/disk and 0.01mg/disk. Application of all tested metabolites decreased the growth of photobiont cells in a dose dependent manner. However, based on selected physiological parameters, e.g. composition of assimilation pigments, fluorescence of chlorophyll a, content of soluble proteins, production of TBARS and ROS, differential phytotoxicity of tested compounds and their mixtures was confirmed. It appears that most of the secondary metabolites tested in the present study may be functioning as allelochemicals which are controlling the cell division of the algal partner inside the thallus. The allelochemicals may play a key role in maintaining the balance between the symbionts forming the lichen thallus.

New insights into diversity and selectivity of trentepohlialean lichen photobionts from the extratropics.

Aerial green algae of Trentepohliaceae can form conspicuous free-living colonies, be parasites of plants or photobionts of lichen-forming ascomycetes. So far, their diversity in temperate regions is still poorly known as it has been mostly studied by phenotypic approaches only. We present new insights in the phylogenetic relationships of lichenized representatives from temperate and Mediterranean parts of Europe by analysis of 18S rRNA and rbcL gene fragments, and nuclear ITS sequence data. For this purpose we isolated the trentepohlialean photobionts from lichens representing different genera. Algal cultures from lichenized and free-living Trentepohliaceae were used to design new primers for amplification of the marker loci. We constructed a phylogenetic hypothesis to reveal the phylogenetic placements of lichenized lineages with 18S rRNA and rbcL sequences. ITS variation among the clades was substantial and did not allow including them in the general phylogenetic assessment, yet ITS appears to be a promising marker for DNA-barcoding approaches. Specific algae were found in particular lichen but the overall diversity of photobionts was limited. The multilocus tree does not support the current morphological classification of genera in Trentepohliaceae, suggesting that morphology is more variable than previously thought in this group of algae.Electronic supplementary materialThe online version of this article (doi:10.1007/s13199-014-0285-z) contains supplementary material, which is available to authorized users.

Integrating coalescent and phylogenetic approaches to delimit species in the lichen photobiont Trebouxia

The accurate assessment of species boundaries in symbiotic systems is a prerequisite for the study of speciation, co-evolution and selectivity. Many studies have shown the high genetic diversity of green algae from the genus Trebouxia, the most common photobiont of lichen-forming fungi. However, the phylogenetic relationships, and the amount of cryptic diversity of these algae are still poorly understood, and an adequate species concept for trebouxiophycean algae is still missing. In this study we used a multifaceted approach based on coalescence (GMYC, STEM) and phylogenetic relationships to assess species boundaries in the trebouxioid photobionts of the lichen-forming fungus Lasallia pustulata. We further investigated whether putative species of Trebouxia found in L. pustulata are shared with other lichen-forming fungi. We found that L. pustulata is associated with at least five species of Trebouxia and most of them are shared with other lichen-forming fungi, showing different patterns of species-to-species and species-to-community interactions. We also show that one of the putative Trebouxia species is found exclusively in association with L. pustulata and is restricted to thalli from localities with Mediterranean microclimate. We suggest that the species delimitation method presented in this study is a promising tool to address species boundaries within the heterogeneous genus Trebouxia.

Characterization of Chasmoendolithic Community in Miers Valley, McMurdo Dry Valleys, Antarctica

The Antarctic Dry Valleys are unable to support higher plant and animal life and so microbial communities dominate biotic ecosystem processes. Soil communities are well characterized, but rocky surfaces have also emerged as a significant microbial habitat. Here, we identify extensive colonization of weathered granite on a landscape scale by chasmoendolithic microbial communities. A transect across north-facing and south-facing slopes plus valley floor moraines revealed 30-100 % of available substrate was colonized up to an altitude of 800 m. Communities were assessed at a multidomain level and were clearly distinct from those in surrounding soils and other rock-inhabiting cryptoendolithic and hypolithic communities. All colonized rocks were dominated by the cyanobacterial genus Leptolyngbya (Oscillatoriales), with heterotrophic bacteria, archaea, algae, and fungi also identified. Striking patterns in community distribution were evident with regard to microclimate as determined by aspect. Notably, a shift in cyanobacterial assemblages from Chroococcidiopsis-like phylotypes (Pleurocapsales) on colder-drier slopes, to Synechococcus-like phylotypes (Chroococcales) on warmer-wetter slopes. Greater relative abundance of known desiccation-tolerant bacterial taxa occurred on colder-drier slopes. Archaeal phylotypes indicated halotolerant taxa and also taxa possibly derived from nearby volcanic sources. Among the eukaryotes, the lichen photobiont Trebouxia (Chlorophyta) was ubiquitous, but known lichen-forming fungi were not recovered. Instead, fungal assemblages were dominated by ascomycetous yeasts. We conclude that chasmoendoliths likely constitute a significant geobiological phenomenon at lower elevations in granite-dominated Antarctic Dry Valley systems.

Molecular phylogeny and symbiotic selectivity of the green algal genus Dictyochloropsis s.l. (Trebouxiophyceae): a polyphyletic and widespread group forming photobiont‐mediated guilds in the lichen family Lobariaceae

Dictyochloropsis s.l. is an ecologically important, common but little-studied genus of green algae. Here, we examined the diversity and host selectivity of algae attributed to this genus at both species-to-species and species-to-community levels. We conducted a molecular investigation of 15 cultured strains and several lichen photobionts, using 18S rRNA, rbcL and ITS sequence data. We further used seven alga-specific microsatellite markers to study algal sharing among fungi of the family Lobariaceae in two populations in Madeira and Taiwan (454 lichens). We found that the genus Dictyochloropsis s.l. is polyphyletic. Dictyochloropsis clade 1 comprises only free-living algae whereas Dictyochloropsis clade 2 includes lichenized algae as well as free-living algae. Fungal selectivity towards algae belonging to Dictyochloropsis clade 2 is high. Selectivity varies geographically, with photobionts being restricted to a single region. Finally, we showed that Dictyochloropsis clade 2 individuals are shared among different fungal hosts in communities of lichens of the Lobariaceae. As for other green algal lineages, there is a high amount of cryptic diversity in Dictyochloropsis. Furthermore, co-evolution between Dictyochloropsis clade 2 algae and representatives of the Lobariaceae is manifested at the community level, with several unrelated fungal species being horizontally connected by shared photobiont clones.

Sensitivity of Lichens to Diesel Exhaust under Laboratory Conditions

Lichen vegetation reacts very sensitively to a variety of air pollutants including increased nitrogen concentrations as well as to traffic exhaust in general, which makes lichens reliable monitoring organisms for atmospheric pollution. Recent environmental studies have shown that decreasing abundance of acidophytic lichen species and the increase of nitrophytic lichens can be explained by elevated levels of atmospheric nitric-compounds adsorbed onto nanoparticles. One major source of these atmospheric compounds amongst a wider pollution inventory is diesel exhaust—a mixture of gases and particle matter. This study aimed to shed light on the impact of diesel exhaust on the viability of six differently sensitive lichen species. Diesel exhaust particle concentrations in the laboratory experiments resembled those at a local highway during rush hour. By incubation in a closed stainless steel chamber we could exclude influences from other pollutants than diesel exhaust providing explicit data about the effects of diesel exhaust on lichens. The investigations revealed effects on the photosynthesis of the lichen photobionts and hence the lichen vitality. The conclusions of this study are that 1) the photobiont is affected stronger as the mycobiont and 2) older parts of the lichen are damaged first. Another remarkable result of this study is that 3) these lichens are regenerating to some extent during incubation-free periods—unless the organism is not damaged too much to restore photosynthetic activity. To our knowledge this is the first study evaluating the impact of diesel exhaust on lichens under laboratory conditions separate from other interfering pollutants.