Isolation and Identification of Lichen Photobionts Collected from Different Environments in North of Portugal and Evaluation of Bioactivities of Their Extracts.

Lichens are organisms constituted by a symbiotic relationship between a fungus (mycobiont) and a photoautotrophic partner (photobiont). Lichens produce several bioactive compounds; however, the biotechnological exploitation of this organism is hampered by its slow growth. To start studying the possibility of exploiting lichens as alternative sources of bioactive compounds, eighteen lichens were collected in the north of Portugal in order to isolate and study the bioactivity of their photobionts. It was possible to isolate and cultivate only eight photobionts. Three of them, LFR1, LFA2 and LCF3, belong to the Coelastrella genus, the other two (LFA1 and LCF1) belong to the Chlorella genus and for the remaining three photobionts, LFS1, LCA1 and LCR1, it was impossible to isolate their microalgae. These only grow in consortium with bacteria and/or cyanobacteria. All extracts showed antioxidant activity, mainly at a concentration of 10 mg.mL-1. LFS1, a consortium extract, showed the highest antioxidant power, as well as the highest concentration of phenolic compounds (5.16 ± 0.53 mg of gallic acid equivalents (GAE).g-1). The extracts under study did not show significant antibacterial activity against Escherichia coli, Listeria or Salmonella. The Coelastrella sp. and LFA1 extracts showed the highest hyaluronidase inhibition. The LFR1 extract at a concentration of 5 mg.mL-1 showed the highest anti-inflammatory activity (79.77 ± 7.66%). The extracts of Coelastrella sp. and LFA1 also showed greater antidiabetic activity, demonstrating the high inhibitory power of α-amylase and α-glucosidase. LFR1 at a concentration of 5 mg.mL-1, due to its selective cytotoxicity inhibiting the growth of cancer cells (Caco-2 cells), is a promising anticancer agent.

Additional new species and new records of the genus Sticta (lichenised Ascomycota, lobarioid Peltigeraceae) from Bolivia.

Four species of the genus Sticta are described as new from Bolivia, based on morphological examination and phylogenetic analysis of the fungal ITS barcoding marker. Additionally, two species are reported as new to Bolivia (their identification confirmed by molecular data) and one previously reported species is confirmed by molecular data for the first time. Detailed morphological and anatomical descriptions are provided for all new species. Two of the new species, S.isidiolobulata Ossowska, B. Moncada, Lücking & Kukwa and S.madidiensis Ossowska, B. Moncada, Lücking & Kukwa belong to clade I, as defined in previous studies. In contrast, S.montepunkuensis Ossowska, B. Moncada, Lücking & Kukwa and S.macrolobata Ossowska, B. Moncada, Lücking & Kukwa, also described here as new to science, belong to clade III. Stictaisidiolobulata has an irregular to suborbicular thallus of medium size, with isidia developing into spathulate lobules, cyanobacterial photobiont and apothecia with entire to weakly-crenate margins. The large irregular thallus of the cyanobacteria-associated S.macrolobata has broad lobes, apothecia with verrucous to tomentose margins and cyphellae with raised margins, whereas S.madidiensis has a medium-sized, palmate to irregular thallus with a stipe, but without vegetative propagules and apothecia. Stictamontepunkuensis has large and irregular thalli with green algae as photobiont, apothecia with crenate to verrucous margins and urceolate cyphellae with a wide pore and a scabrid basal membrane. Two species, S.beauvoisii Delise and S.riparia Merc.-Díaz are reported as new to Bolivia (the latter also as new to South America) and belong to clade III. Stictatomentosa (Sw.) Ach., species confirmed from Bolivia by molecular data, belongs to clade II. Stictabeauvoisii is characterised by a smooth yellowish-brown upper surface with darker apices and abundant, marginal isidia and a brown lower surface with golden-chocolate brown primary tomentum and sparse, golden-brown rhizines. Stictariparia has a strongly branched thallus, with undulate lobes and abundant, marginal, palmate, grey to dark brown phyllidia and greyish-brown lower surface with the primary tomentum absent towards the margins. Stictatomentosa has palmate, bluish thalli with white cilia and abundant, submarginal apothecia and creamy-white lower surface with a sparse, white primary tomentum.

The complete mitochondrial genome of Clonostachys farinosa (Bionectriaceae, Hypocreales).

The complete mitochondrial genome of Clonostachys farinosa was assembled using the strain CSC22A0184 that was isolated from the lichen Parmotrema clavuliferum. The total length of the mitogenome was 51,551 bp and contained 49 genes: 15 protein-coding genes, two ribosomal RNA genes, 26 transfer RNA genes, and six open reading frames. The GC content of the mitogenome was 28.5% and had a base composition of 36.4% A, 12.6% C, 15.9% G, and 35.1% T. Phylogenetic analysis based on 14 protein-coding genes indicates that C. farinosa is clustered in the family Bionectriaceae. This is the first study of the mitogenome of C. farinosa, which is helpful for understanding the evolution of species within the genus Clonostachys.

Assessing Tunisia's urban air quality using combined lichens and Sentinel-5 satellite integration.

In Tunisia, urban air pollution is becoming a bigger problem. This study used a combined strategy of biomonitoring with lichens and satellite mapping with Sentinel-5 satellite data processed in Google Earth Engine (GEE) to assess the air quality over metropolitan Tunis. Lichen diversity was surveyed across the green spaces of the Faculty of Science of Tunisia sites, revealing 15 species with a predominance of pollution-tolerant genera. The Index of Atmospheric Purity (IAP) calculated from the lichen data indicated poor air quality. Spatial patterns of pollutants sulfur dioxide (SO2), ozone (O3), nitrogen dioxide (NO2), carbon monoxide (CO), and aerosol index across Greater Tunis were analyzed from Sentinel-5 datasets on the GEE platform. The higher values of these indices in the research area indicate that it may be impacted by industrial activity and highlight the considerable role that vehicle traffic plays in air pollution. The results of the IAP, IBL, and the combined ground-based biomonitoring and satellite mapping techniques confirm poor air quality and an environment affected by atmospheric pollutants which will enable proactive air quality management strategies to be put in place in Tunisia's rapidly expanding cities.

Chemical composition and antioxidant, enzyme inhibition and cytoprotective activity of two Antarctic lichens of the genus Psoroma (Pannariaceae).

Lichens contain different types of chemical compounds with multiple biological activities that demonstrate their potential pharmacological use. This research aims to report the metabolomic identification of the ethanolic extracts of P. antarcticum and P. hypnorum, their antioxidant, enzyme inhibitory, and their cytoprotection activity. Sixteen metabolites were identified in P. antarcticum and twelve in P. hypnorum; the extracts reported variable antioxidant activity with IC50 >350 µg/mL in DPPH·, values >18 µmol Trolox/g in ORAC and >40 µmol Trolox/g in FRAP and a phenolic compound content >10 mg GAE/g, as well as significant results in cholinesterases, α-glucosidase, pancreatic lipase, α-amylase, and tyrosinase enzyme inhibition activities with IC50 ranging from 18 to 510 µg/mL, and which were complemented by molecular docking experiments. Both extracts showed improved cytoprotection at the concentrations of 0.5 to 1.0 µg/mL. This study contributes to the knowledge of the chemical diversity of Antarctic lichen extracts and their effectiveness in the evaluation of biological activities related to neurodegenerative diseases and metabolic syndrome.

UV photo-degradation of the secondary lichen substance parietin: A multi-spectroscopic analysis in astrobiology perspective.

The cortical anthraquinone yellow-orange pigment parietin is a secondary lichen substance providing UV-shielding properties that is produced by several lichen species. In our work, the secondary metabolite has been extracted from air-dried thalli of Xanthoria parietina. The aims of this study were to characterize parietin absorbance through UV-VIS spectrophotometry and with IR spectroscopy and to evaluate its photodegradability under UV radiation through in situ reflectance IR spectroscopy to understand to what extent the substance may have a photoprotective role. This allows us to relate parietin photo-degradability to the lichen UV tolerance in its natural terrestrial habitat and in extreme environments relevant for astrobiology such as Mars. Extracted crystals were UV irradiated for 5.59 h under N2 flux. After the UV irradiation, we assessed relevant degradations in the 1614, 1227, 1202, 1160 and 755 cm-1 bands. However, in light of Xanthoria parietina survivability in extreme conditions such as space- and Mars-simulated ones, we highlight parietin UV photo-resistance and its relevance for astrobiology as photo-protective substance and possible bio-hint.

Thallus hydrophobicity: A low-cost method for understanding lichen ecophysiological responses to environmental changes.

Premise: Methods to evaluate lichen thalli hydrophobicity have previously been described, but only recently has hydrophobicity been shown to be an important functional trait related to water regulation dynamics that could be used to predict future climate change effects. We describe a novel protocol to measure lichen thallus hydrophobicity that aims to be an easier and more affordable approach. Methods and Results: Our protocol requires only a micropipette, distilled water, a tripod, and a smartphone or camera. Hydrophobicity is inferred from multiple metrics associated with the absorption times of standardized droplets (initial and total absorption time). We used a data set of 93 lichen taxa with different growth forms and from different biomes and demonstrated that this method is well suited for capturing different levels of hydrophobicity, including very hydrophilic species. Conclusions: Our results show that this new protocol to measure lichen hydrophobicity is a rapid and low-cost method to assess an ecophysiologically based functional trait that can be used with almost no limitations, including in different climates, lichen species, and growth forms.

Premisa: En el pasado se han descrito métodos para evaluar la hidrofobicidad de los talos liquénicos, sin embargo, no fue hasta recientemente que se demostró la importancia de esta propiedad como rasgo funcional en relación a la dinámica de regulación hídrica de los talos, permitiendo utilizarla como herramienta para predecir futuros efectos del cambio climático. Describimos un nuevo protocolo para medir la hidrofobicidad de los talos liquénicos que pretende ser más fácil y asequible. Métodos y Resultados: Nuestro protocolo requiere solamente de una micropipeta, agua destilada, un trípode y un teléfono o una cámara. La hidrofobicidad es inferida a partir de múltiples métricas asociadas a los tiempos de absorción de gotas de un volumen estandarizado (tiempo de absorción inicial y tiempo de absorción total). Utilizamos datos de 93 taxones de líquenes con diferentes formas de crecimiento y provenientes de distintos biomas, lo que permitió demostrar que este método es adecuado para capturar diferentes niveles de hidrofobicidad, incluyendo a especies altamente hidrofílicas. Conclusiones: Nuestros resultados muestran que la medición de la hidrofobicidad de los talos es un método rápido y de bajo costo que permite evaluar un rasgo funcional basado en la ecofisiología de líquenes y que puede ser utilizado prácticamente sin limitaciones, incluyendo en diferentes climas, especies y formas de crecimiento.

Patterns of Endemism in Lichens: Another Paradigm-Shifting Example in the Lichen Genus Xanthoparmelia from Macaronesia.

It has long been assumed that lichen-forming fungi have very large distribution ranges, and that endemic species are rare in this group of organisms. This is likely a consequence of the "everything small is everywhere" paradigm that has been traditionally applied to cryptogams. However, the description of numerous endemic species over the last decades, many of them in oceanic islands, is challenging this view. In this study, we provide another example, Xanthoparmelia ramosae, a species that is described here as new to science on the basis of morphological, chemical, and macroclimatic data, and three molecular markers (ITS rDNA, nuLSU rDNA, and mtSSU). The new species is endemic to the island of Gran Canaria but clusters into a clade composed exclusively of specimens collected in Eastern Africa, a disjunction that is here reported for the first time in lichen-forming fungi. Through the use of dating analysis, we have found that Xanthoparmelia ramosae diverged from its closely related African taxa in the Pliocene. This result, together with the reproductive strategy of the species, points to the Relict theory as a likely mechanism behind the disjunction, although the large gap in lichenological knowledge in Africa makes this possibility hard to explore any further.

Freezing temperature effects on photosystem II in Antarctic lichens evaluated by chlorophyll fluorescence.

This study explores and compares the limits for photosynthesis in subzero temperatures of six Antarctic lichens: Sphaerophorus globosus, Caloplaca regalis, Umbilicaria antarctica, Pseudephebe minuscula, Parmelia saxatilis and Lecania brialmontii combining linear cooling and chlorophyll fluorescence methods. The results revealed triphasic S-curves in the temperature response of the maximum quantum yield (FV/FM) and effective quantum yield of photosystem II (ΦPSII) for all species. All investigated species showed a high level of cryoresistance with critical temperatures (Tc) below -20 °C. However, record low Tc temperatures have been discovered for L. brialmotii (-54 °C for FV/FM and -40 °C for ΦPSII) and C. regalis (-52 °C for FV/FM and -38 °C for ΦPSII). Additionally, the yield differentials (FV/FM - ΦPSII) in functions of temperature revealed one or two peaks, with the larger one occurring for temperatures below -20 °C for the above-mentioned species. Finally, Kautsky kinetics were measured and compared at different temperatures (20 °C, 10 °C, 0 °C and -10 °C and then -10 °C after 1 h of incubation). This research serves as a foundation for further developing investigations into the biophysical mechanisms by which photosynthesis is carried out at subzero temperatures.

Phylogeny of the genus Loxospora s.l. (Sarrameanales, Lecanoromycetes, Ascomycota), with Chicitaea gen. nov. and five new combinations in Chicitaea and Loxospora.

Loxospora is a genus of crustose lichens containing 13 accepted species that can be separated into two groups, based on differences in secondary chemistry that correlate with differences in characters of the sexual reproductive structures (asci and ascospores). Molecular phylogenetic analyses recovered these groups as monophyletic and support their recognition as distinct genera that differ in phenotypic characters. Species containing 2'-O-methylperlatolic acid are transferred to the new genus, Chicitaea Guzow-Krzem., Kukwa & Lendemer and four new combinations are proposed: C.assateaguensis (Lendemer) Guzow-Krzem., Kukwa & Lendemer, C.confusa (Lendemer) Guzow-Krzem., Kukwa & Lendemer, C.cristinae (Guzow-Krzem., Lubek, Kubiak & Kukwa) Guzow-Krzem., Kukwa & Lendemer and C.lecanoriformis (Lumbsch, A.W. Archer & Elix) Guzow-Krzem., Kukwa & Lendemer. The remaining species produce thamnolic acid and represent Loxospora s.str. Haplotype analyses recovered sequences of L.elatina in two distinct groups, one corresponding to L.elatina s.str. and one to Pertusariachloropolia, the latter being resurrected from synonymy of L.elatina and, thus, requiring the combination, L.chloropolia (Erichsen) Ptach-Styn, Guzow-Krzem., Tønsberg & Kukwa. Sequences of L.ochrophaea were found to be intermixed within the otherwise monophyletic L.elatina s.str. These two taxa, which differ in contrasting reproductive mode and overall geographic distributions, are maintained as distinct, pending further studies with additional molecular loci. Lectotypes are selected for Lecanoraelatina, Pertusariachloropolia and P.chloropoliaf.cana. The latter is a synonym of Loxosporachloropolia. New primers for the amplification of mtSSU are also presented.

Endolichenic Fungi: A Promising Medicinal Microbial Resource to Discover Bioactive Natural Molecules-An Update.

Lichens are some of the most unique fungi and are naturally encountered as symbiotic biological organisms that usually consist of fungal partners (mycobionts) and photosynthetic organisms (green algae and cyanobacteria). Due to their distinctive growth environments, including hot deserts, rocky coasts, Arctic tundra, toxic slag piles, etc., they produce a variety of biologically meaningful and structurally novel secondary metabolites to resist external environmental stresses. The endofungi that live in and coevolve with lichens can also generate abundant secondary metabolites with novel structures, diverse skeletons, and intriguing bioactivities due to their mutualistic symbiosis with hosts, and they have been considered as strategically significant medicinal microresources for the discovery of pharmaceutical lead compounds in the medicinal industry. They are also of great importance in the fundamental research field of natural product chemistry. In this work, we conducted a comprehensive review and systematic evaluation of the secondary metabolites of endolichenic fungi regarding their origin, distribution, structural characteristics, and biological activity, as well as recent advances in their medicinal applications, by summarizing research achievements since 2015. Moreover, the current research status and future research trends regarding their chemical components are discussed and predicted. A systematic review covering the fundamental chemical research advances and pharmaceutical potential of the secondary metabolites from endolichenic fungi is urgently required to facilitate our better understanding, and this review could also serve as a critical reference to provide valuable insights for the future research and promotion of natural products from endolichenic fungi.

Contribution to the Study of Lichenicolous Fungi from Northwest Iberian Peninsula (León and Lugo Provinces).

We have found 117 taxa of lichenicolous fungi in the studied area. In this paper, we describe five taxa: Arthonia boomiana on Nephromopsis chlorophylla, Lawalreea burgaziana on Platismatia glauca, Pronectria scrobiculatae on Lobarina scrobiculata, Trichonectria parmeliellae on Parmeliella testacea and Trichonectria rubefaciens ssp. cryptoramalinae on Ramalina. Furthermore, the next records are interesting chorologically from the Iberian Peninsula: Arthophacopsis parmeliarum, Catillaria lobariicola, Lichenopuccinia poeltii, Myxotrichum bicolor, Nanostictis christiansenii, Niesslia lobariae, Opegrpaha sphaerophoricola, Pronectria fragmospora, Rhymbocarpus aggregatus, R. neglectus, and Tremella cetrariicola.

Influence of Orographic Factors on the Distribution of Lichens in the Franz Josef Land Archipelago.

During a geobotanical study of the Franz Josef Land archipelago, 111 lichen species were recorded on 130 sample plots. The significance of orographic factors in the distribution of lichens was assessed using principal component analysis (PCA) and multiple regression analysis. It was found that the absolute altitude and distance from the glacier are of the greatest importance for crustose lichens, while for fruticose lichens, the most critical factors were the slope exposure and steepness. Along the altitudinal gradient, the number of species decreased (from 88 to 25). The highest number of species (90) was recorded at distances of 0.1 to 1.0 km from the glacier edge, which is explained by the unstable species composition of areas recently released from under the glacier. The number of species in all groups generally decreased (from 81 to 52) with increasing slope steepness. With an increasing heat supply of slopes (on a gradient from northern to southern), the number of species steadily increased in all groups (from 39 to 75). The low sum of the explained variance values for the first two PCA components (21%) characterizes the specificity of the natural environment of polar deserts, where there is no leading environmental factor.

Lichen - air quality association rule mining for urban environments in the tropics.

There are significant gaps in air quality monitoring across many low- and middle-income countries, which can be filled by bioindicators like lichen. This study examined the links between lichen and air quality across urban environments in Nigeria. Lichen surveys and air quality monitoring were carried out across four major cities focusing on NO2, SO2, PM2.5, and PM10. Association rule mining was used to identify robust rules defining the association between lichen and air quality categories. For the maximal frequent set with Lichen in the antecedent, 9 and 5 rules were identified by A priori and Eclat, respectively. These indicated that three genera: Diorygma, Pyxine, and Physcia are the most commonly associated lichen with poor air quality particularly NO2 and SO2. This showed that these lichens are viable indicators of long-term air quality due to their consistent occurrence across the rules from different algorithms.

Parietin, the Vibrant Natural Dye in Xanthoria parietina.

The use of natural dyes in several areas is regulated by current European and non-European legislation, due to various problems with synthetic dyes. The analysis revealed that the lichen studied: Xanthoria parietina has potential natural dye sources and provides bright colors for extraction solvents. Furthermore, dyed wool and toile fabric have good fastness properties in ammonia fermentation and boiling water, both with and without mordants. The sample dyes with Xanthoria parietina were characterized by several analytical techniques: high-performance liquid chromatography with diode array detection (HPLC-DAD) and electrospray ionization with tandem mass spectrometry (HPLC-ESI-Q-ToF). As compounds from Xanthoria parietina form a complex with mordants and tissues, it is impossible to identify the molecules responsible for coloring using chromatographic techniques. However, we have evaluated the dyeing power of their major molecule, parietin. To further confirm the coloring power of the isolated parietin molecule, we performed a dye test with pure parietin. Thus, CIALAB analyses have shown parietin is the molecule responsible for the coloring obtained by Xanthoria parietina. The utilization of parietin derived from lichens facilitates the development of sustainable dyes for textile coloring, presenting an environmentally friendly alternative to synthetic dyes while simultaneously enriching lichen biodiversity.

Steep declines in radioactive caesium after 30 years of monitoring alpine plants in mountain areas of central Norway.

The Chernobyl accident exposed large areas of northern Europe to radiocaesium (137Cs). We investigated temporal and spatial variation in concentrations of radiocaesium among five functional groups of alpine plants at two mountain areas in central Norway over a 31-year period from 1991 to 2022. Average concentrations of radiocaesium were initially high in lichens and bryophytes at around 4600-6400 Bq/kg dry weight during 1991-1994 but then decreased dramatically over three decades to current concentrations of <200 Bq/kg for all plant groups in 2019-2022. The effective half-life of radiocaesium was estimated to be 4-6 years in lichens and mosses, 7-13 years in herbaceous plants, and 22-30 years in woody plants, which were less than the physical half-life of 30.2 years. Concentrations of radiocaesium were greater at the nutrient-poor site than at the nutrient-rich site, probably due to greater deposition levels at higher elevations and the geographical pattern of the deposition. Functional groups of plants differed with higher concentrations among non-vascular than vascular plants. Common heather Calluna vulgaris was unusual among woody plants with high concentration of radiocaesium, especially in the new shoots. Our new estimates of concentrations and dynamics of radiocaesium for alpine plants in natural environments will be useful for modelling herbivore exposure and evaluating potential impacts on wildlife and human health.

Metatranscriptomics reveals diversity of symbiotic interaction and mechanisms of carbon exchange in the marine cyanolichen Lichina pygmaea.

Lichens are exemplar symbioses based upon carbon exchange between photobionts and their mycobiont hosts. Historically considered a two-way relationship, some lichen symbioses have been shown to contain multiple photobiont partners; however, the way in which these photobiont communities react to environmental change is poorly understood. Lichina pygmaea is a marine cyanolichen that inhabits rocky seashores where it is submerged in seawater during every tidal cycle. Recent work has indicated that L. pygmaea has a complex photobiont community including the cyanobionts Rivularia and Pleurocapsa. We performed rRNA-based metabarcoding and mRNA metatranscriptomics of the L. pygmaea holobiont at high and low tide to investigate community response to immersion in seawater. Carbon exchange in L. pygmaea is a dynamic process, influenced by both tidal cycle and the biology of the individual symbiotic components. The mycobiont and two cyanobiont partners exhibit distinct transcriptional responses to seawater hydration. Sugar-based compatible solutes produced by Rivularia and Pleurocapsa in response to seawater are a potential source of carbon to the mycobiont. We propose that extracellular processing of photobiont-derived polysaccharides is a fundamental step in carbon acquisition by L. pygmaea and is analogous to uptake of plant-derived carbon in ectomycorrhizal symbioses.

Modelling the carbon balance in bryophytes and lichens: Presentation of PoiCarb 1.0, a new model for explaining distribution patterns and predicting climate-change effects.

PREMISE: Bryophytes and lichens have important functional roles in many ecosystems. Insight into their CO2 -exchange responses to climatic conditions is essential for understanding current and predicting future productivity and biomass patterns, but responses are hard to quantify at time scales beyond instantaneous measurements. We present PoiCarb 1.0, a model to study how CO2 -exchange rates of these poikilohydric organisms change through time as a function of weather conditions. METHODS: PoiCarb simulates diel fluctuations of CO2 exchange and estimates long-term carbon balances, identifying optimal and limiting climatic patterns. Modelled processes were net photosynthesis, dark respiration, evaporation and water uptake. Measured CO2 -exchange responses to light, temperature, atmospheric CO2 concentration, and thallus water content (calculated in a separate module) were used to parameterize the model's carbon module. We validated the model by comparing modelled diel courses of net CO2 exchange to such courses from field measurements on the tropical lichen Crocodia aurata. To demonstrate the model's usefulness, we simulated potential climate-change effects. RESULTS: Diel patterns were reproduced well, and the modelled and observed diel carbon balances were strongly positively correlated. Simulated warming effects via changes in metabolic rates were consistently negative, while effects via faster drying were variable, depending on the timing of hydration. CONCLUSIONS: Reproducing weather-dependent variation in diel carbon balances is a clear improvement compared to simply extrapolating short-term measurements or potential photosynthetic rates. Apart from predicting climate-change effects, future uses of PoiCarb include testing hypotheses about distribution patterns of poikilohydric organisms and guiding conservation strategies for species.

Circadian clock- and temperature-associated genes contribute to overall genomic differentiation along elevation in lichenized fungi.

Circadian regulation is linked to local environmental adaptation, and many species with broad climatic niches display variation in circadian genes. Here, we hypothesize that lichenizing fungi occupying different climate zones tune their metabolism to local environmental conditions with the help of their circadian systems. We study two species of the genus Umbilicaria occupying similar climatic niches (Mediterranean and the cold temperate) in different continents. Using homology to Neurospora crassa genes, we identify gene sets associated with circadian rhythms (11 core, 39 peripheral genes) as well as temperature response (37 genes). Nucleotide diversity of these genes is significantly correlated with mean annual temperature, minimum temperature of the coldest month and mean temperature of the coldest quarter. Furthermore, we identify altitudinal clines in allele frequencies in several non-synonymous substitutions in core clock components, for example, white collar-like, frh-like and various ccg-like genes. A dN/dS approach revealed a few significant peripheral clock- and temperature-associated genes (e.g. ras-1-like, gna-1-like) that may play a role in fine-tuning the circadian clock and temperature-response machinery. An analysis of allele frequency changes demonstrated the strongest evidence for differentiation above the genomic background in the clock-associated genes in U. pustulata. These results highlight the likely relevance of the circadian clock in environmental adaptation, particularly frost tolerance, of lichens. Whether or not the fungal clock modulates the symbiotic interaction within the lichen consortium remains to be investigated. We corroborate the finding of genetic variation in clock components along altitude-not only latitude-as has been reported in other species.