Lipophilic arsenic compounds in the cultured green alga Chlamydomonas reinhardtii.

In this study, arsenic (As) speciation was investigated in the freshwater alga Chlamydomonas reinhardtii treated with 20 µg/L arsenate using fractionation as well as ICP-MS/ESI-MS analyses and was compared with the known As metabolite profile of wild-grown Saccharina latissima. While the total As accumulation in C. reinhardtii was about 85% lower than in S. latissima, the relative percentage of arsenolipids was significantly higher in C. reinhardtii (57.0% vs. 5.01%). As-containing hydrocarbons and phospholipids dominated the hydrophobic As profile in S. latissima, but no As-containing hydrocarbons were detectable in C. reinhardtii. Instead for the first time, an arsenoriboside-containing phytol (AsSugPhytol) was found to dominate the hydrophobic arsenicals of C. reinhardtii. Interestingly, this compound and its relatives had so far been only found in green marine microalgae, open sea plankton (mixed assemblage), and sediments but not in brown or red macroalgae. This compound family might therefore relate to differences in the arsenic metabolism between the algae phyla.

Antibiotic-Induced Treatments Reveal Stress-Responsive Gene Expression in the Endangered Lichen Lobaria pulmonaria

Antibiotics are primarily found in the environment due to human activity, which has been reported to influence the structure of biotic communities and the ecological functions of soil and water ecosystems. Nonetheless, their effects in other terrestrial ecosystems have not been well studied. As a result of oxidative stress in organisms exposed to high levels of antibiotics, genotoxicity can lead to DNA damage and, potentially, cell death. In addition, in symbiotic organisms, removal of the associated microbiome by antibiotic treatment has been observed to have a big impact on the host, e.g., corals. The lung lichen Lobaria pulmonaria has more than 800 associated bacterial species, a microbiome which has been hypothesized to increase the lichen's fitness. We artificially exposed samples of L. pulmonaria to antibiotics and a stepwise temperature increase to determine the relative effects of antibiotic treatments vs. temperature on the mycobiont and photobiont gene expression and the viability and on the community structure of the lichen-associated bacteria. We found that the mycobiont and photobiont highly reacted to different antibiotics, independently of temperature exposure. We did not find major differences in bacterial community composition or alpha diversity between antibiotic treatments and controls. For these reasons, the upregulation of stress-related genes in antibiotic-treated samples could be caused by genotoxicity in L. pulmonaria and its photobiont caused by exposure to antibiotics, and the observed stress responses are reactions of the symbiotic partners to reduce damage to their cells. Our study is of great interest for the community of researchers studying symbiotic organisms as it represents one of the first steps to understanding gene expression in an endangered lichen in response to exposure to toxic environments, along with dynamics in its associated bacterial communities.

The Bacterial Community of the Foliose Macro-lichen Peltigera frigida Is More than a Mere Extension of the Microbiota of the Subjacent Substrate.

Lichens host highly diverse microbial communities, with bacteria being one of the most explored groups in terms of their diversity and functioning. These bacteria could partly originate from symbiotic propagules developed by many lichens and, perhaps more commonly and depending on environmental conditions, from different sources of the surroundings. Using the narrowly distributed species Peltigera frigida as an object of study, we propose that bacterial communities in these lichens are different from those in their subjacent substrates, even if some taxa might be shared. Ten terricolous P. frigida lichens and their substrates were sampled from forested sites in the Coyhaique National Reserve, located in an understudied region in Chile. The mycobiont identity was confirmed using partial 28S and ITS sequences. Besides, 16S fragments revealed that mycobionts were associated with the same cyanobacterial haplotype. From both lichens and substrates, Illumina 16S amplicon sequencing was performed using primers that exclude cyanobacteria. In lichens, Proteobacteria was the most abundant phylum (37%), whereas soil substrates were dominated by Acidobacteriota (39%). At lower taxonomic levels, several bacterial groups differed in relative abundance among P. frigida lichens and their substrates, some of them being highly abundant in lichens but almost absent in substrates, like Sphingomonas (8% vs 0.2%), and others enriched in lichens, as an unassigned genus of Chitinophagaceae (10% vs 2%). These results reinforce the idea that lichens would carry some components of their microbiome when propagating, but they also could acquire part of their bacterial community from the substrates.

Neogene speciation and Pleistocene expansion of the genus Pseudephebe (Parmeliaceae, lichenized fungi) involving multiple colonizations of Antarctica.

Widespread geographic distributions in lichens have been usually explained by the high dispersal capacity of their tiny diaspores. However, recent phylogenetic surveys have challenged this assumption and provided compelling evidence for cryptic speciation and more restricted distribution ranges in diverse lineages of lichen-forming fungi. To evaluate these scenarios, we focus on the fungal genus Pseudephebe (Parmeliaceae) which includes amphitropical species, a distribution pattern whose origin has been a matter of debate since first recognized in the nineteenth century. In our study, a six-locus dataset and a broad specimen sampling covering almost all Earth's continents is used to investigate species delimitation in Pseudephebe. Population structure, gene flow and dating analyses, as well as genealogical reconstruction methods, are employed to disentangle the most plausible transcontinental migration routes, and estimate the timing of the origin of the amphitropical distribution and the Antarctic populations. Our results demonstrate the existence of three partly admixed phylogenetic species that diverged between the Miocene and Pliocene, and whose Quaternary distribution has been strongly driven by glacial cycles. Pseudephebe minuscula is the only species showing an amphitropical distribution, with populations in Antarctica, whereas the restricted distribution of P. pubescens and an undescribed Alaskan species might reflect the survival of these species in European and North American refugia. Our microevolutionary analyses suggest a Northern Hemisphere origin for P. minuscula, which could have dispersed into the Southern Hemisphere directly and/or through "mountain-hopping" during the Pleistocene. The Antarctic populations of this species are sorted into two genetic clusters: populations of the Antarctic Peninsula were grouped together with South American ones, and the Antarctic Continental populations formed a second cluster with Bolivian and Svalbard populations. Therefore, our data strongly suggest that the current distribution of P. minuscula in Antarctica is the outcome of multiple, recent colonizations. In conclusion, our results stress the need for integrating species delimitation and population analyses to properly approach historical biogeography in lichen-forming fungi.

High levels of endemism and local differentiation in the fungal and algal symbionts of saxicolous lecideoid lichens along a latitudinal gradient in southern South America.

Saxicolous, lecideoid lichenized fungi have a cosmopolitan distribution but, being mostly cold adapted, are especially abundant in polar and high-mountain regions. To date, little is known of their origin or the extent of their trans-equatorial dispersal. Several mycobiont genera and species are thought to be restricted to either the Northern or the Southern Hemisphere, whereas others are thought to be widely distributed and occur in both hemispheres. However, these assumptions often rely on morphological analyses and lack supporting molecular genetic data. Also unknown is the extent of regional differentiation in the southern polar regions. An extensive set of lecideoid lichens (185 samples) was collected along a latitudinal gradient at the southern end of South America. Subantarctic climate conditions were maintained by increasing the elevation of the collecting sites with decreasing latitude. The investigated specimens were placed in a global context by including Antarctic and cosmopolitan sequences from other studies. For each symbiont three markers were used to identify intraspecific variation (mycobiont: ITS, mtSSU, RPB1; photobiont: ITS, psbJ-L, COX2). For the mycobiont, the saxicolous genera Lecidea, Porpidia, Poeltidea and Lecidella were phylogenetically re-evaluated, along with their photobionts Asterochloris and Trebouxia. For several globally distributed species groups, the results show geographically highly differentiated subclades, classified as operational taxonomical units (OTUs), which were assigned to the different regions of southern South America (sSA). Furthermore, several small endemic and well-supported clades apparently restricted to sSA were detected at the species level for both symbionts.

The evolution of fungal substrate specificity in a widespread group of crustose lichens.

Lichens exhibit varying degrees of specialization with regard to the surfaces they colonize, ranging from substrate generalists to strict substrate specialists. Though long recognized, the causes and consequences of substrate specialization are poorly known. Using a phylogeny of a 150-200 Mya clade of lichen fungi, we asked whether substrate niche is phylogenetically conserved, which substrates are ancestral, whether specialists arise from generalists or vice versa and how specialization affects speciation/extinction processes. We found strong phylogenetic signal for niche conservatism. Specialists evolved into generalists and back again, but transitions from generalism to specialism were more common than the reverse. Our models suggest that for this group of fungi, 'escape' from specialization for soil, rock and bark occurred, but specialization for wood foreclosed evolution away from that substrate type. In parallel, speciation models showed positive diversification rates for soil and rock dwellers but not other specialists. Patterns in the studied group suggest that fungal substrate specificity is a key determinant of evolutionary trajectory for the entire lichen symbiosis.

ITS2 metabarcoding analysis complements lichen mycobiome diversity data.

Lichen thalli harbor complex fungal communities (mycobiomes) of species with divergent trophic and ecological strategies. The complexity and diversity of lichen mycobiomes are still largely unknown, despite surveys combining culture-based methods and high-throughput sequencing (HTS). The results of such surveys are strongly influenced by the barcode locus chosen, its sensitivity in discriminating taxa, and the depth to which public sequence repositories cover the phylogenetic spectrum of fungi. Here, we use HTS of the internal transcribed spacer 2 (ITS2) to assess the taxonomic composition and diversity of a well-characterized, alpine rock lichen community that includes thalli symptomatically infected by lichenicolous fungi as well as asymptomatic thalli. Taxa belonging to the order Chaetothyriales are the major components of the observed lichen mycobiomes. We predict sequences representative of lichenicolous fungi characterized morphologically and assess their asymptomatic presence in lichen thalli. We demonstrated the limitations of metabarcoding in fungi and show how the estimation of species diversity widely differs when ITS1 or ITS2 are used as barcode, and particularly biases the detection of Basidiomycota. The complementary analysis of both ITS1 and ITS2 loci is therefore required to reliably estimate the diversity of lichen mycobiomes.

ITS1 metabarcoding highlights low specificity of lichen mycobiomes at a local scale.

As self-supporting and long-living symbiotic structures, lichens provide a habitat for many other organisms beside the traditionally considered lichen symbionts-the myco- and the photobionts. The lichen-inhabiting fungi either develop diagnostic phenotypes or occur asymptomatically. Because the degree of specificity towards the lichen host is poorly known, we studied the diversity of these fungi among neighbouring lichens on rocks in an alpine habitat. Using a sequencing metabarcoding approach, we show that lichen mycobiomes clearly reflect the overlap of multiple ecological sets of taxa, which differ in their trophic association with lichen thalli. The lack of specificity to the lichen mycobiome is further supported by the lack of community structure observed using clustering and ordination methods. The communities encountered across samples largely result from the subsampling of a shared species pool, in which we identify three major ecological components: (i) a generalist environmental pool, (ii) a lichenicolous/endolichenic pool and (iii) a pool of transient species. These taxa majorly belong to the fungal classes Dothideomycetes, Eurotiomycetes and Tremellomycetes with close relatives in adjacent ecological niches. We found no significant evidence that the phenotypically recognized lichenicolous fungi influence the occurrence of the other asymptomatic fungi in the host thalli. We claim that lichens work as suboptimal habitats or as a complex spore and mycelium bank, which modulate and allow the regeneration of local fungal communities. By performing an approach that minimizes ambiguities in the taxonomic assignments of fungi, we present how lichen mycobiomes are also suitable targets for improving bioinformatic analyses of fungal metabarcoding.

Characterization of microsatellite loci in the lichen-forming fungus Cetraria aculeata (Parmeliaceae, Ascomycota).

PREMISE OF THE STUDY: Polymorphic microsatellite markers were developed for the lichen species Cetraria aculeata (Parmeliaceae) to study fine-scale population diversity and phylogeographic structure. METHODS AND RESULTS: Using Illumina HiSeq and MiSeq, 15 fungus-specific microsatellite markers were developed and tested on 81 specimens from four populations from Spain. The number of alleles ranged from four to 13 alleles per locus with a mean of 7.9, and average gene diversities varied from 0.40 to 0.73 over four populations. The amplification rates of 10 markers (CA01-CA10) in populations of C. aculeata exceeded 85%. The markers also amplified across a range of closely related species, except for locus CA05, which did not amplify in C. australiensis and C. "panamericana," and locus CA10 which did not amplify in C. australiensis. CONCLUSIONS: The identified microsatellite markers will be used to study the genetic diversity and phylogeographic structure in populations of C. aculeata in western Eurasia.

Ecological Specialization of Two Photobiont-Specific Maritime Cyanolichen Species of the Genus Lichina.

All fungi in the class Lichinomycetes are lichen-forming and exclusively associate with cyanobacteria. Two closely related maritime species of the genus Lichina (L. confinis and L. pygmaea) show similar distribution ranges in the Northeast Atlantic, commonly co-occurring at the same rocky shores but occupying different littoral zones. By means of 16S rRNA and phycocyanin operon markers we studied a) the phylogenetic relationships of cyanobionts associated with these species, b) the match of divergence times between both symbionts, and c) whether Lichina species differ in photobiont association and in how geography and ecology affect selectivity. The cyanobionts studied are closely related to both marine and freshwater strains of the genus Rivularia. We found evidence of a high specificity to particular cyanobiont lineages in both species: Lichina pygmaea and L. confinis incorporate specific lineages of Rivularia that do not overlap at the haplotype nor the OTU levels. Dating divergences of the fungal and cyanobacterial partners revealed an asynchronous origin of both lineages. Within each fungal species, selectivity varied across the studied area, influenced by environmental conditions (both atmospheric and marine), although patterns were highly correlated between both lichen taxa. Ecological speciation due to the differential association of photobionts to each littoral zone is suspected to have occurred in marine Lichina.

Pleistocene expansion of the bipolar lichen Cetraria aculeata into the Southern hemisphere.

Many boreal and polar lichens occupy bipolar distributional ranges that frequently extend into high mountains at lower latitudes. Although such disjunctions are more common among lichens than in other groups of organisms, the geographic origin of bipolar lichen taxa, and the way and time frame in which they colonized their ranges have not been studied in detail. We used the predominantly vegetative, widespread lichen Cetraria aculeata as a model species. We surveyed the origin and history of its bipolar pattern using population genetics, phylogenetic and genealogical reconstruction methods. Cetraria aculeata originated in the Northern Hemisphere and dispersed southwards during the Pleistocene. The genetic signal suggests a Pleistocene dispersive burst in which a population size expansion concurred with the acquisition of a South-American range that culminated in the colonization of the Antarctic.

Alphaproteobacterial communities in geographically distant populations of the lichen Cetraria aculeata.

Lichen symbioses were recently shown to include diverse bacterial communities. Although the biogeography of lichen species is fairly well known, the patterns of their bacterial associates are relatively poorly understood. Here we analyse the composition of Alphaproteobacteria in Cetraria aculeata, a common lichen species that occurs at high latitudes and various habitats. Using clone libraries we show that most of the associated Alphaproteobacteria belong to Acetobacteraceae, which have also been found previously in other lichen species of acidic soils and rocks in alpine habitats. The majority of alphaproteobacterial sequences from C. aculeata are very similar to each other and form a single clade. Data from C. aculeata reveal that alphaproteobacterial communities of high latitudes are depauperate and more closely related to each other than to those of extrapolar habitats. This agrees with previous findings for the fungal and algal symbiont in this lichen. Similar to the algal partner, the composition of lichen alphaproteobacterial communities is affected by environmental parameters.

Extreme phenotypic variation in Cetraria aculeata (lichenized Ascomycota): adaptation or incidental modification?

BACKGROUND AND AIMS: Phenotypic variability is a successful strategy in lichens for colonizing different habitats. Vagrancy has been reported as a specific adaptation for lichens living in steppe habitats around the world. Among the facultatively vagrant species, the cosmopolitan Cetraria aculeata apparently forms extremely modified vagrant thalli in steppe habitats of Central Spain. The aim of this study was to investigate whether these changes are phenotypic plasticity (a single genotype producing different phenotypes), by characterizing the anatomical and ultrastructural changes observed in vagrant morphs, and measuring differences in ecophysiological performance. METHODS: Specimens of vagrant and attached populations of C. aculeata were collected on the steppes of Central Spain. The fungal internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GPD) and the large sub-unit of the mitochondrial ribosomal DNA (mtLSUm), and the algal ITS and actin were studied within a population genetics framework. Semi-thin and ultrathin sections were analysed by means of optical, scanning electron and transmission electron microscopy. Gas exchange and chlorophyll fluorescence were used to compare the physiological performance of both morphs. KEY RESULTS AND CONCLUSIONS: Vagrant and attached morphs share multilocus haplotypes which may indicate that they belong to the same species in spite of their completely different anatomy. However, differentiation tests suggested that vagrant specimens do not represent a random sub-set of the surrounding population. The morphological differences were related to anatomical and ultrastructural differences. Large intercalary growth rates of thalli after the loss of the basal-apical thallus polarity may be the cause of the increased growth shown by vagrant specimens. The anatomical and morphological changes lead to greater duration of ecophysiological activity in vagrant specimens. Although the anatomical and physiological changes could be chance effects, the genetic differentiation between vagrant and attached sub-populations and the higher biomass of the former show fitness effects and adaptation to dry environmental conditions in steppe habitats.