Life on top: cryptoendolithic ascomycetes and microalgae isolated from over 6000 m altitude

Rocks are among the oldest terrestrial niches hosting a multiplicity of life forms, of which diversity has been only partially uncovered. Endolithic metacommunities comprise all major groups of microorganisms, such as chemo-organotrophic, chemo-lithotrophic and phototrophic, represented by bacteria, microalgae and microfungi. Their diversity is often difficult to describe and may remain underestimated. Furthermore, knowledge about the diversity of microorganisms colonizing rocks in peculiar niches is even poorer due to the difficulty to retrieve environmental specimens. Here, we report the phylogenetic and phenotypic characterization of a few endolithic fungi and algae isolated from rock fragments collected at high elevation, i.e., on the top of two mountains over 6000 m altitude, Muztagh Ata (China) and Cerro Mercendario (Argentina). The identity of the strains was confirmed by sequencing the nuclear ITS and LSU, the plastidial rbcL loci and by morphological analysis. Three fungal strains belonging to the class Dothideomycetes and one algal strain belonging to the genus Trebouxia were isolated from Muztagh Ata, while six fungal strains belonging to the order Chaetothyriales and four algal strains belonging to the genus Myrmecia were isolated from Cerro Mercedario. The detected species diversity is discussed in an evolutionary and ecological context

Photobiont selectivity leads to ecological tolerance and evolutionary divergence in a polymorphic complex of lichenized fungi

Background and Aims The integrity and evolution of lichen symbioses depend on a fine-tuned combination of algal and fungal genotypes. Geographically widespread species complexes of lichenized fungi can occur in habitats with slightly varying ecological conditions, and it remains unclear howthis variation correlates with symbiont selectivity patterns in lichens. In an attempt to address this question, .300 samples were taken of the globally distributed and ecologically variable lichen-forming species complex Tephromela atra, together with closely allied species, in order to study genetic diversity and the selectivity patterns of their photobionts. \u2020 Methods Lichen thalli of T. atra and of closely related species T. grumosa, T. nashii and T. atrocaesia were collected from six continents, across 24 countries and 62 localities representing a wide range of habitats. Analyses of genetic diversity and phylogenetic relationships were carried out both for photobionts amplified directly from the lichen thalli and from those isolated in axenic cultures. Morphological and anatomical traits were studied with light and transmission electron microscopy in the isolated algal strains. \u2020 Key Results Tephromela fungal species were found to associate with 12 lineages of Trebouxia. Five new clades demonstrate the still-unrecognized genetic diversity of lichen algae. Culturable, undescribed lineageswere also characterized by phenotypic traits. Strong selectivity of the mycobionts for the photobionts was observed in six monophyletic Tephromela clades. Seven Trebouxia lineages were detected in the poorly resolved lineage T. atra sensu lato, where co-occurrence of multiple photobiont lineages in single thalli was repeatedly observed. \u2020Conclusions Lowselectivity apparently allows widespread lichen-forming fungi to establish successful symbioses with locally adapted photobionts in a broader range of habitats. This flexibility might correlate with both lower phylogenetic resolution and evolutionary divergence in species complexes of crustose lichen-forming fungi

PLANiTS: a curated sequence reference dataset for plant ITS DNA metabarcoding

DNA metabarcoding combines DNA barcoding with high-throughput sequencing to identify different taxa within environmental communities. The ITS has already been proposed and widely used as universal barcode marker for plants, but a comprehensive, updated and accurate reference dataset of plant ITS sequences has not been available so far. Here, we constructed reference datasets of Viridiplantae ITS1, ITS2 and entire ITS sequences including both Chlorophyta and Streptophyta. The sequences were retrieved from NCBI, and the ITS region was extracted. The sequences underwent identity check to remove misidentified records and were clustered at 99% identity to reduce redundancy and computational effort. For this step, we developed a script called 'better clustering for QIIME' (bc4q) to ensure that the representative sequences are chosen according to the composition of the cluster at a different taxonomic level. The three datasets obtained with the bc4q script are PLANiTS1 (100\u2009224 sequences), PLANiTS2 (96\u2009771 sequences) and PLANiTS (97\u2009550 sequences), and all are pre-formatted for QIIME, being this the most used bioinformatic pipeline for metabarcoding analysis. Being curated and updated reference databases, PLANiTS1, PLANiTS2 and PLANiTS are proposed as a reliable, pivotal first step for a general standardization of plant DNA metabarcoding studies. The bc4q script is presented as a new tool useful in each research dealing with sequences clustering. Database URL: https://github.com/apallavicini/bc4q; https://github.com/apallavicini/PLANiTS

Highly heterogeneous mycobiota shape fungal diversity in two globally distributed lichens

Lichens are multi-kingdom symbioses in which fungi, algae and bacteria interact to develop a stable selection unit. In addition to the mycobiont forming the symbiosis, fungal communities associated with lichens represent the lichen mycobiota. Because lichen mycobiota diversity is still largely unknown, we aimed to characterize it in two cosmopolitan lichens, Rhizoplaca melanophthalma and Tephromela atra. The mycobiota were investigated across a broad distribution using both a culture-dependent approach and environmental DNA metabarcoding. The variation of the mycobiota associated with the two lichen species was extremely high, and a stable species-specific core mycobiota was not detected with the methods we applied. Most taxa were present in a low fraction of the samples, and no fungus was ubiquitously present in either lichen species. The mycobiota are thus composed of heterogeneous fungi, and some taxa are detectable only by culture-dependent approaches. We suspect that lichens act as niches in which these fungi may exploit thallus resources and only a few may establish more stable trophic relationships with the major symbiotic partners

Environmental DNA assessment of airborne plant and fungal seasonal diversity

Environmental DNA (eDNA) metabarcoding and metagenomics analyses can improve taxonomic resolution in biodiversity studies. Only recently, these techniques have been applied in aerobiology, to target bacteria, fungi and plants in airborne samples. Here, we present a nine-month aerobiological study applying eDNA metabarcoding in which we analyzed simultaneously airborne diversity and variation of fungi and plants across five locations in North and Central Italy. We correlated species composition with the ecological characteristics of the sites and the seasons. The most abundant taxa among all sites and seasons were the fungal genera Cladosporium, Alternaria, and Epicoccum and the plant genera Brassica, Corylus, Cupressus and Linum, the latter being much more variable among sites. PERMANOVA and indicator species analyses showed that the plant diversity from air samples is significantly correlated with seasons, while that of fungi varied according to the interaction between seasons and sites. The results consolidate the performance of a new eDNA metabarcoding pipeline for the simultaneous amplification and analysis of airborne plant and fungal particles. They also highlight the promising complementarity of this approach with more traditional biomonitoring frameworks and routine reports of air quality provided by environmental agencies

Enforced fungal-algal symbioses in alginate spheres

The thallus structure of the lichen symbiosis provides a fungal shelter for the growth of algal partners. The long-living thallus also provides a habitat for other fungi, but experimental studies, which could inform us about the details of their interactions have hardly been conducted. We present a new approach by embedding axenically cultured strains of fungi together with algae in alginate spheres, which allows easy transfer of co-cultures on solid media. As the growth rates of the organisms are differentially triggered by the underlying medium, alginate embedding can help to adjust optimal parameters for stable culture of the combined symbionts. In our experiments, direct contacts between hyphae and algae and the formation of layered structures were observed in a fungus that is living as a commensal in the host lichen without visible symbiotic structures. The growth of primary lichen symbionts cannot be accelerated by alginate embedding so far, but our approach could artificially enforce symbiotic interactions that are not normally observed in nature

Cross Taxon Congruence Between Lichens and Vascular Plants in a Riparian Ecosystem

Despite that congruence across taxa has been proved as an effective tool to provide insights into the processes structuring the spatial distribution of taxonomic groups and is useful for conservation purposes, only a few studies on cross-taxon congruence focused on freshwater ecosystems and on the relations among vascular plants and lichens. We hypothesized here that, since vascular plants could be good surrogates of lichens in these ecosystems, it would be possible to assess the overall biodiversity of riparian habitats using plant data only. In this frame, we explored the relationship between (a) species richness and (b) community composition of plants and lichens in a wetland area located in central Italy to (i) assess whether vascular plants are good surrogates of lichens and (ii) to test the congruence of patterns of species richness and composition among plants and lichens along an ecological gradient. The general performance of plant species richness per se, as a biodiversity surrogate of lichens, had poor results. Nonetheless, the congruence in compositional patterns between lichens and vascular plants varied across habitats and was influenced by the characteristics of the vegetation. In general, we discussed how the strength of the studied relationships could be influenced by characteristics of the data (presence/absence vs. abundance), by the spatial scale, and by the features of the habitats. Overall, our data confirm that the more diverse and structurally complex the vegetation is, the more diverse are the lichen communities it hosts

Arable plant communities as a surrogate of crop rhizosphere microbiota

Soil microbiota is a crucial component of agroecosystem biodiversity, enhancing plant growth and providing important services in agriculture. However, its characterization is demanding and relatively expensive. In this study, we evaluated whether arable plant communities can be used as a surrogate of bacterial and fungal communities of the rhizosphere of Elephant Garlic (Allium ampeloprasum L.), a traditional crop plant of central Italy. We sampled plant, bacterial, and fungal communities, i.e., the groups of such organisms co-existing in space and time, in 24 plots located in eight fields and four farms. At the plot level, no correlations in species richness emerged, while the composition of plant communities was correlated with that of both bacterial and fungal communities. As regards plants and bacteria, such correlation was mainly driven by similar responses to geographic and environmental factors, while fungal communities seemed to be correlated in species composition with both plants and bacteria due to biotic interactions. All the correlations in species composition were unaffected by the number of fertilizer and herbicide applications, i.e., agricultural intensity. Besides correlations, we detected a predictive relationship of plant community composition towards fungal community composition. Our results highlight the potential of arable plant communities to be used as a surrogate of crop rhizosphere microbial communities in agroecosystems

Solution Equilibrium Studies of Anticancer Ruthenium(II)-η6-p-cymene Complexes of Pyridinecarboxylic Acids

Stoichiometry and stability of antitumor ruthenium(II)-η6-p-cymene complexes of picolinic acid and its 6-methyl and 6-carboxylic acid derivatives were determined by pH-potentiometry, 1H NMR spectroscopy and UV–Vis spectrophotometry in aqueous solution in the presence or absence of coordinating chloride ions. The picolinates form exclusively mono-ligand complexes in which they can coordinate via the bidentate (O,N) mode and a chloride or a water molecule is found at the third binding site of the ruthenium(II)-η6-p-cymene moiety depending on the conditions. [Ru(η6-p-cymene)(L)(H2O/Cl)] species are predominant at physiological pH in all studied cases. Hydrolysis of the aqua complex or the chlorido/hydroxido co-ligand exchange results in the formation of the mixed-hydroxido species [Ru(η6-p-cymene)(L)(OH)] in the basic pH range. There is no indication for the decomposition of the mono-ligand complexes during 24 h in the ruthenium(II)-η6-p-cymene-picolinic acid system between pH 3 and 11; however, a slight dissociation with a low reaction rate was found in the other two systems leading to the appearance of the dinuclear trihydroxido-bridged species [Ru2(η6-p-cymene)2(OH)3]+ and free ligands at pH > 10. The replacement of the chlorido by an aqua ligand in [Ru(η6-p-cymene)(L)Cl] was also monitored and equilibrium constants for the exchange process were determined