An Exception to the Rule? Could Photobiont Identity Be a Better Predictor of Lichen Phenotype than Mycobiont Identity?

With rare exceptions, the shape and appearance of lichen thalli are determined by the fungal partner; thus, mycobiont identity is normally used for lichen identification. However, it has repeatedly been shown in recent decades that phenotypic data often does not correspond with fungal gene evolution. Here, we report such a case in a three-species complex of red-fruited Cladonia lichens, two of which clearly differ morphologically, chemically, ecologically and in distribution range. We analysed 64 specimens of C. bellidiflora, C. polydactyla and C. umbricola, mainly collected in Europe, using five variable mycobiont-specific and two photobiont-specific molecular markers. All mycobiont markers exhibited very low variability and failed to separate the species. In comparison, photobiont identity corresponded better with lichen phenotype and separated esorediate C. bellidiflora from the two sorediate taxa. These results can be interpreted either as an unusual case of lichen photomorphs or as an example of recent speciation, in which phenotypic differentiation precedes the separation of the molecular markers. We hypothesise that association with different photobionts, which is probably related to habitat differentiation, may have triggered speciation in the mycobiont species.

Choosing the Right Life Partner: Ecological Drivers of Lichen Symbiosis.

Lichens are an iconic example of symbiotic systems whose ecology is shaped by the requirements of the symbionts. Previous studies suggest that fungal (mycobionts) as well as photosynthesizing (phycobionts or cyanobionts) partners have a specific range of acceptable symbionts that can be chosen according to specific environmental conditions. This study aimed to investigate the effects of climatic conditions and mycobiont identity on phycobiont distribution within the lichen genera Stereocaulon, Cladonia, and Lepraria. The study area comprised the Canary Islands, Madeira, Sicily, and the Aeolian Islands, spanning a wide range of climatic conditions. These islands are known for their unique and diverse fauna and flora; however, lichen phycobionts have remained unstudied in most of these areas. In total, we genetically analyzed 339 lichen samples. The phycobiont pool differed significantly from that outside the studied area. Asterochloris mediterranea was identified as the most abundant phycobiont. However, its distribution was limited by climatic constraints. Other species of Asterochloris and representatives of the genera Chloroidium, Vulcanochloris, and Myrmecia were also recovered as phycobionts. The selection of symbiotic partners from the local phycobiont pool was driven by mycobiont specificity (i.e., the taxonomic range of acceptable partners) and the environmental conditions, mainly temperature. Interestingly, the dominant fungal species responded differently in their selection of algal symbionts along the environmental gradients. Cladonia rangiformis associated with its phycobiont A. mediterranea in a broader range of temperatures than Stereocaulon azoreum, which favors other Asterochloris species along most of the temperature gradient. Stereocaulon vesuvianum associated with Chloroidium spp., which also differed in their temperature optima. Finally, we described Stereocaulon canariense as a new endemic species ecologically distinct from the other Stereocaulon species on the Canary Islands.

Anaerobic microbial corrosion of carbon steel under conditions relevant for deep geological repository of nuclear waste.

We examined microbial corrosion of carbon steel in synthetic bentonite pore water inoculated with natural underground water containing microorganisms over a period of 780-days under sterile and anaerobic conditions. Corrosion behaviour was determined using the mass loss method, SEM-EDS analysis and Raman spectroscopy, while qualitative and quantitative changes in the microbial community were analysed using molecular-biological tools (16S rDNA amplicon sequencing and qPCR analysis, respectively). Corrosion rates were significantly higher in the biotic environment (compared with an abiotic environment), with significant localisation of corrosion attacks of up to 1 mm arising within 12-months. Nitrate reducing bacteria, such as Pseudomonas, Brevundimonas and Methyloversatilis, dominated the microbial consortium, the high abundance of Methyloversatilis correlating with periods of highest localised corrosion penetrations, suggesting that this bacterium plays an important role in microbially influenced corrosion. Our results indicate that nitrate-reducing bacteria could represent a potential threat to waste canisters under nuclear repository conditions.

Promiscuity in Lichens Follows Clear Rules: Partner Switching in Cladonia Is Regulated by Climatic Factors and Soil Chemistry.

Climatic factors, soil chemistry and geography are considered as major factors affecting lichen distribution and diversity. To determine how these factors limit or support the associations between the symbiotic partners, we revise the lichen symbiosis as a network of relationships here. More than one thousand thalli of terricolous Cladonia lichens were collected at sites with a wide range of soil chemical properties from seven biogeographical regions of Europe. A total of 18 OTUs of the algal genus Asterochloris and 181 OTUs of Cladonia mycobiont were identified. We displayed all realized pairwise mycobiont-photobiont relationships and performed modularity analysis. It revealed four virtually separated modules of cooperating OTUs. The modules differed in mean annual temperature, isothermality, precipitation, evapotranspiration, soil pH, nitrogen, and carbon contents. Photobiont switching was strictly limited to algae from one module, i.e., algae of similar ecological preferences, and only few mycobionts were able to cooperate with photobionts from different modules. Thus, Cladonia mycobionts generally cannot widen their ecological niches through photobiont switching. The modules also differed in the functional traits of the mycobionts, e.g., sexual reproduction rate, presence of soredia, and thallus type. These traits may represent adaptations to the environmental conditions that drive the differentiation of the modules. In conclusion, the promiscuity in Cladonia mycobionts is strictly limited by climatic factors and soil chemistry.

Reproductive and dispersal strategies shape the diversity of mycobiont-photobiont association in Cladonia lichens.

Ecological preferences, partner compatibility, or partner availability are known to be important factors shaping obligate and intimate lichen symbioses. We considered a complex of Cladonia species, traditionally differentiated by the extent of sexual reproduction and the type of vegetative propagules, to assess if the reproductive and dispersal strategies affect mycobiont-photobiont association patterns. In total 85 lichen thalli from 72 European localities were studied, two genetic markers for both Cladonia mycobionts and Asterochloris photobionts were analyzed. Variance partitioning analysis by multiple regression on distance matrices was performed to describe and partition variance in photobiont genetic diversity. Asexually reproducing Cladonia in our study were found to be strongly specific to their photobionts, associating with only two closely related Asterochloris species. In contrast, sexually reproducing lichens associated with seven unrelated Asterochloris lineages, thus being photobiont generalists. The reproductive mode had the largest explanatory power, explaining 44% of the total photobiont variability. Reproductive and dispersal strategies are the key factors shaping photobiont diversity in this group of Cladonia lichens. A strict photobiont specialisation observed in two studied species may steer both evolutionary flexibility and responses to ecological changes of these organisms, and considerably limit their distribution ranges.

Thermally enhanced in situ bioremediation of groundwater contaminated with chlorinated solvents - A field test.

In situ bioremediation (ISB) using reductive dechlorination is a widely accepted but relatively slow approach compared to other technologies for the treatment of groundwater contaminated by chlorinated ethenes (CVOCs). Due to the known positive kinetic effect on microbial metabolism, thermal enhancement may be a viable means of accelerating ISB. We tested thermally enhanced ISB in aquifers situated in sandy saprolite and underlying fractured granite. The system comprised pumping, heating and subsequent injection of contaminated groundwater aiming at an aquifer temperature of 20-30°C. A fermentable substrate (whey) was injected in separate batches. The test was monitored using hydrochemical and molecular tools (qPCR and NGS). The addition of the substrate and increase in temperature resulted in a rapid increase in the abundance of reductive dechlorinators (e.g., Dehalococcoides mccartyi, Dehalobacter sp. and functional genes vcrA and bvcA) and a strong increase in CVOC degradation. On day 34, the CVOC concentrations decreased by 87% to 96% in groundwater from the wells most affected by the heating and substrate. On day 103, the CVOC concentrations were below the LOQ resulting in degradation half-lives of 5 to 6days. Neither an increase in biomarkers nor a distinct decrease in the CVOC concentrations was observed in a deep well affected by the heating but not by the substrate. NGS analysis detected Chloroflexi dechlorinating genera (Dehalogenimonas and GIF9 and MSBL5 clades) and other genera capable of anaerobic metabolic degradation of CVOCs. Of these, bacteria of the genera Acetobacterium, Desulfomonile, Geobacter, Sulfurospirillum, Methanosarcina and Methanobacterium were stimulated by the substrate and heating. In contrast, groundwater from the deep well (affected by heating only) hosted representatives of aerobic metabolic and aerobic cometabolic CVOC degraders. The test results document that heating of the treated aquifer significantly accelerated the treatment process but only in the case of an abundant substrate.

Assessment of biodegradation potential at a site contaminated by a mixture of BTEX, chlorinated pollutants and pharmaceuticals using passive sampling methods - Case study.

The present study describes a pilot remediation test of a co-mingled plume containing BTEX, chlorinated pollutants and pharmaceuticals. Remediation was attempted using a combination of various approaches, including a pump and treat system applying an advanced oxidation process and targeted direct push injections of calcium peroxide. The remediation process was monitored intensively and extensively throughout the pilot test using various conventional and passive sampling methods, including next-generation amplicon sequencing. The results showed that the injection of oxygen-saturated treated water with residual hydrogen peroxide and elevated temperature enhanced the in situ removal of monoaromatics and chlorinated pollutants. In particular, in combination with the injection of calcium peroxide, the conditions facilitated the in situ bacterial biodegradation of the pollutants. The mean groundwater concentration of benzene decreased from 1349µg·L-1 prior to the test to 3µg·L-1 within 3months after the calcium peroxide injections; additionally, monochlorobenzene decreased from 1545µg·L-1 to 36µg·L-1, and toluene decreased from 143µg·L-1 to 2µg·L-1. Furthermore, significant degradation of the contaminants bound to the soil matrix in less permeable zones was observed. Based on a developed 3D model, 90% of toluene and 88% of chlorobenzene bound to the soil were removed during the pilot test, and benzene was removed almost completely. On the other hand, the psychopharmaceuticals were effectively removed by the employed advanced oxidation process only from the treated water, and their concentration in groundwater remained stagnant due to inflow from the surroundings and their absence of in situ degradation. The employment of passive sampling techniques, including passive diffusion bags (PDB) for volatile organic pollutants and their respective transformation products, polar organic compound integrative samplers (POCIS) for the pharmaceuticals and in situ soil microcosms for microbial community analysis, was proven to be suitable for monitoring remediation in saturated zones.

Pharmaceuticals, benzene, toluene and chlorobenzene removal from contaminated groundwater by combined UV/H2O2 photo-oxidation and aeration.

This study was performed to test the feasibility of several decontamination methods for remediating heavily contaminated groundwater in a real contaminated locality in the Czech Republic, where a pharmaceuticals plant has been in operation for more than 80 years. The site is polluted mainly by recalcitrant psychopharmaceuticals and monoaromatic hydrocarbons, such as benzene, toluene and chlorobenzene. For this purpose, an advanced oxidation technique employing UV radiation with hydrogen peroxide dosing was employed, in combination with simple aeration pretreatment. The results showed that UV/H2O2 was an efficient and necessary step for degradation of the pharmaceuticals; however, the monoaromatics were already removed during the aeration step. Characterization of the removal mechanisms participating in the aeration revealed that volatilization, co-precipitation and biodegradation contributed to the process. These findings were supported by bacterial metabolite analyses, phospholipid fatty acid analysis, qPCR of representatives of the degradative genes and detailed characterization of the formed precipitate using Mössbauer spectroscopy and scanning electron microscopy. Further tests were carried out in a continuous arrangement directly connected to the wells already present in the locality. The results documented the feasibility of combination of the photo-reactor employing UV/H2O2 together with aeration pretreatment for 4 months, where the overall decontamination efficiency ranged from 72% to 99% of the pharmaceuticals. We recorded even better results for the monoaromatics decontamination except for one month, when we encountered some technical problems with the aeration pump. This demonstrated the necessity of using the aeration step.

Combined nano-biotechnology for in-situ remediation of mixed contamination of groundwater by hexavalent chromium and chlorinated solvents.

The present report describes a 13month pilot remediation study that consists of a combination of Cr(VI) (4.4 to 57mg/l) geofixation and dechlorination of chlorinated ethenes (400 to 6526µg/l), achieved by the sequential use of nanoscale zerovalent iron (nZVI) particles and in situ biotic reduction supported by whey injection. The remediation process was monitored using numerous techniques, including physical-chemical analyses and molecular biology approaches which enabled both the characterization of the mechanisms involved in pollutant transformation and the description of the overall background processes of the treatment. The results revealed that nZVI was efficient toward Cr(VI) by itself and completely removed it from the groundwater (LOQ 0.05mg/l) and the subsequent application of whey resulted in a high removal of chlorinated ethenes (97 to 99%). The persistence of the reducing conditions, even after the depletion of the organic substrates, indicated a complementarity between nZVI and the whey phases in the combined technology as the subsequent application of whey phase partially assisted the microbial regeneration of the spent nZVI by promoting its reduction into Fe(II), which further supported remediation conditions at the site. Illumina sequencing and the detection of functional vcrA and bvcA genes documented a development in the reducing microbes (iron-reducing, sulfate-reducing and chlororespiring bacteria) that benefited under the conditions of the site and that was probably responsible for the high dechlorination and/or Cr(VI) reduction. The results of this study demonstrate the feasibility and high efficiency of the combined nano-biotechnological approach of nZVI and whey application in-situ for the removal of Cr(VI) and chlorinated ethenes from the groundwater of the contaminated site.

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.

Leptochlorella corticola gen. et sp. nov. and Kalinella apyrenoidosa sp. nov.: two novel Chlorella-like green microalgae (Trebouxiophyceae, Chlorophyta) from subaerial habitats.

The diversity of green microalgae in subaerial habitats remains largely unexplored and a number of new genus- and species-level lineages have been discovered recently. The traditional green algal genus, Chlorella, which accommodated coccoid unicellular green algal species with globular to oval cells, reproducing entirely by autospores, has been found to be polyphyletic. In this study, we provide a detailed characterization of two strains of microalgae isolated from tree bark in the Mediterranean. These algae share the general Chlorella-like morphology and their 18S rRNA and rbcL gene sequences place them in the Trebouxiophyceae. Strain CAUP H8401 forms an independent trebouxiophycean lineage, together with three previously published 18S rRNA gene environmental sequences of undescribed microalgae, which were retrieved from profoundly different habitats. In contrast, strain CAUP H7902 is related to Kalinella bambusicola in the Watanabea clade of the Trebouxiophyceae on the basis of its 18S rRNA gene sequence. This relationship is also supported by the rbcL gene sequence, acquired from the type strain of K. bambusicola. The investigated strains are described as representatives of a novel species in a new genus, Leptochlorella corticola gen. et sp. nov., and a novel species, Kalinella apyrenoidosa sp. nov., according to the International Code of Nomenclature for Algae, Fungi and Plants.

Age, sun and substrate: triggers of bacterial communities in lichens.

Bacterial communities colonize the surfaces of lichens in a biofilm-like manner. The overall structure of the bacterial communities harboured by the lichens shows similarities, in particular the dominance of not yet cultured Alphaproteobacteria. Parameters causing variation in abundance, composition and spatial organization of the lichen-associated bacterial communities are so far poorly understood. As a first step, we used a microscopic approach to test the significance of both lichen-intrinsic and extrinsic environmental factors on the bacterial communities associated with 11 lichen samples, belonging to six species. Some of these species have thalli with a distinct age gradient. A statistically significant effect can be attributed to the age of the thallus parts, which is an intrinsic factor: growing parts of the lichens host bacterial communities that significantly differ from those of the ageing portions of the thalli. The substrate type (rock, tree, understory) and (at a lower extent) the exposition to the sun also affected the bacterial communities. Interestingly, the abundance of bacterial cells in the lichens was also influenced by the same structure-triggering factors. No effect on the composition with main bacterial groups was attributed to different lichen species, differentiated thallus parts or thallus growth type. Our results are important for the experimental designs in lichen-bacterial ecology.