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Algal treatment of wastewater is one of the most interesting topics in recent years. It is a sustainable alternative for recovering nutrients and energy compared to conventional wastewater treatments, which require the expenditure of large amounts of energy and lead to the loss of valuable organic elements. The rationale behind the treatment of municipal wastewater by algal microorganisms is that these photosynthetic organisms not only gather from the surrounding environment the macro- and micronutrients they need for the formation of their organic structures, but they also entrap energy from solar radiation. This whole process is based on the use of wastewater as a free and sustainable growth medium. In this way, it is possible not only to contribute to the remediation of effluents that cannot be released as such into receiving water bodies but also to produce biomass with a higher energy content that can be further utilized for biochemical production.

The ecophysiological, cytomorphological, biochemical and molecular data presently available for the acidophilic red algal species Cyanidium caldarium, Cyanidioschyzon merolae and Galdieria sulphuraria are summarised. The taxonomic position of the three genera is discussed and emendements to the generic diagnosis are presented.

A wide range of genotoxicity assays are available to determine the DNA damage in plant cells caused by allelochemicals. However most of them, sometimes present some technical difficulties in interpretation of results. Acridine orange/ethidium bromide double staining assay is proposed as a rapid, inexpensive and easy-to-perform assay to investigate the apoptotic damage from phenols in plant cells. Our data support the validity of this assay and the hypothesis that phenols induce unrepairable severe genetic alterations due to apoptosis. The proapoptotic effects of Olive oil Mill wastewaters (OMWW, rich in polyphenols and catechol) were investigated on radish root cells. In 25% OMWW treated root cells, strong DNA fragmentations and hypersegmented nuclei were observed and increase in percentage of total apoptotic cells was registered during 24 h. The root cells treated with 12.5% OMWW showed the typical apoptotic hallmarks, but the percentage of total apoptotic cells decreased from 92% to 60% during 24 h, due to the significant decrease in early apoptotic cells and a concomitant increase in cell viability. The catechol at 10-4 to 10-3 M concentrations caused dose-dependent effect, nuclear damage increased during the 24 h and the root cells showed drastic chromatin disintegrations. A wide range of genotoxicity assays are available to determine the DNA damage in plant cells caused by allelochemicals. However most of them, sometimes present some technical difficulties in interpretation of results. Acridine orange/ethidium bromide double staining assay is proposed as a rapid, inexpensive and easy-to-perform assay to investigate the apoptotic damage from phenols in plant cells. Our data support the validity of this assay and the hypothesis that phenols induce unrepairable severe genetic alterations due to apoptosis. The proapoptotic effects of Olive oil Mill wastewaters (OMWW, rich in polyphenols and catechol) were investigated on radish root cells. In 25% OMWW treated root cells, strong DNA fragmentations and hypersegmented nuclei were observed and increase in percentage of total apoptotic cells was registered during 24 h. The root cells treated with 12.5% OMWW showed the typical apoptotic hallmarks, but the percentage of total apoptotic cells decreased from 92% to 60% during 24 h, due to the significant decrease in early apoptotic cells and a concomitant increase in cell viability. The catechol at 10-4 to 10-3 M concentrations caused dose-dependent effect, nuclear damage increased during the 24 h and the root cells showed drastic chromatin disintegrations.

The Cyanidiales is a group of asexual, unicellular red algae, which thrive in acidic and high temperature conditions around hot springs. These unicellular taxa have a relatively simple morphology and are currently classified into three genera, Cyanidium, Cyanidioschyzon and Galdieria. Little is known, however, about the biodiversity of Cyanidiales, their population structure and their phylogenetic relationships. Here we used a taxonomically broadly sampled three-gene data set of plastid sequences to infer a robust phylogenetic framework for the Cyanidiales. The phylogenetic analyses support the existence of at least four distinct Cyanidiales lineages: the Galdieria spp. lineage (excluding Galdieria maxima), the Cyanidium caldarium lineage, a novel monophyletic lineage of mesophilic Cyanidium spp. and the Cyanidioschyzon merolae plus Galdieria maxima lineage. Our analyses do not support the notion of a mesophilic ancestry of the Cyanidiales and suggest that these algae were ancestrally thermo-acidotolerant. We also used environmental polymerase chain reaction (PCR) for the rbcL gene to sample Cyanidiales biodiversity at five ecologically distinct sites at Pisciarelli in the Phlegrean Fields in Italy. This analysis showed a high level of sequence divergence among Cyanidiales species and the partitioning of taxa based on environmental conditions. Our research revealed an unexpected level of genetic diversity among Cyanidiales that revises current thinking about the phylogeny and biodiversity of this group. We predict that future environmental PCR studies will significantly augment known biodiversity that we have discovered and demonstrate the Cyanidiales to be a species-rich branch of red algal evolution.

Ecophysiological, morphological, ultrastructural, and phylogenetic methods were used to study nine strains of Galdieria isolated from different geographical regions in order to clarify the taxonomy and evolution of the genus and to understand the phylogeny of the Cyanidiales. The studied characters were often conserved in all of the isolates. Confocal laser scanning microscopy (CLSM) observations confirmed the presence of a single plastid and allowed its 3-D reconstruction. Phylogenetic analysis showed that all Galdieria isolates but one (G. maxima) form a strongly supported monophyletic lineage within the Cyanidiales. G. maxima is closely related to Cyanidioschyzon merolae and its taxonomic status needs to be revised. The Cyanidiales is an evolutionarily distinct group within the Rhodophyta. In spite of the uniform morphology among Galdieria strains, analysis of rbcL sequences showed moderate to high levels of sequence divergence within this genus. Apart from G. maxima, which is from Kunashir, Russia, the two other Russian isolates, G. daedala and G. partita grouped together with the remaining Galdieria taxa forming three separate clades. The Galdieria clades reflected the geographic site of origin of the isolates. On the basis of these data, we suggest that Galdieria (excluding G. maxima) represents a species complex. The limited sampling makes it difficult, however, to recommend changes to the existing classification system. We suggest that a broad and systematic study of Galdieria is required to understand fully the evolutionary history and taxonomy of this genus.

The Cyanidiophyceae are a group of unicellular organisms that diverged from ancestral red algae around 1.3 billion years ago. Present-day species are restricted to hot springs and geothermal habitats from around the world. Because of discontinuous geothermal environments, the distribution patterns and dispersal modes of the cyanidiophycean species are poorly understood. Iceland is the third largest island in the Atlantic Ocean and has intense underground volcanic activity that generates broad hydrothermal areas with different ecological conditions that are excellent for thermoacidophilic microfloral development. We analyzed populations to address the Icelandic cyanidiophycean biodiversity and dispersal. A global rbcL phylogeny showed two main populations inhabiting Iceland, Galdieria sulphuraria and G. maxima. Their areas of distribution are not completely superimposed because they coexisted only in New Zealand, Kamchatka (Russia), Japan, and Iceland. Because of the strong monophyly of Icelandic species with Japanese and Russian species, we hypothesized an origin and dispersion of Icelandic G. suphuraria and G. maxima from northeastern Asia. On the basis of network analysis of rbcL haplotypes, it is likely that the southwestern region of Iceland is the diversity center of both G. sulphuraria and G. maxima.

Rare earth elements (REEs) are essential components of modern technologies and are often challenging to acquire from natural resources. The demand for REEs is so high that there is a clear need to develop efficient and environmentally-friendly recycling methods. In the present study, freeze-dried cells of the extremophile Galdieria sulphuraria were employed to recover yttrium, cerium, europium, and terbium from quaternary-metal aqueous solutions. The biosorption capacity of G. sulphuraria freeze-dried algal biomass was tested at different pHs, contact times, and biosorbent dosages. All rare earths were biosorbed in a more efficient way by the lowest dose of biosorbent, at pH 4.5, within 30 min; the highest removal rate of cerium was recorded at acidic pH (2.5) and after a longer contact time, i.e., 360 min. This study confirms the potential of freeze-dried cells of G. sulphuraria as innovative ecological biosorbents in technological applications for sustainable recycling of metals from e-waste and wastewater. Rare earth elements (REEs) are essential components of modern technologies and are often challenging to acquire from natural resources. The demand for REEs is so high that there is a clear need to develop efficient and environmentally-friendly recycling methods. In the present study, freeze-dried cells of the extremophile Galdieria sulphuraria were employed to recover yttrium, cerium, europium, and terbium from quaternary-metal aqueous solutions. The biosorption capacity of G. sulphuraria freeze-dried algal biomass was tested at different pHs, contact times, and biosorbent dosages. All rare earths were biosorbed in a more efficient way by the lowest dose of biosorbent, at pH 4.5, within 30 min; the highest removal rate of cerium was recorded at acidic pH (2.5) and after a longer contact time, i.e., 360 min. This study confirms the potential of freeze-dried cells of G. sulphuraria as innovative ecological biosorbents in technological applications for sustainable recycling of metals from e-waste and wastewater.

The toxic effects of Triclosan (2,4,4'-trichloro-2'-hydroxydiphenyl ether) on morphology and sexual and asexual reproduction of C. ehrenbergii were examined. In addition, the genotoxic effects of Triclosan were evaluated on the same alga using the microgel electrophoresis test, also-called Comet assay. Increasing Triclosan concentrations in the range 0.125-5 mg L-1 did not affect size and shape of the cells but had relevant effects on both chloroplast morphology and dimension. Triclosan inhibited the vegetative growth of C. ehrenbergii at 0.5 mg L-1. The effects on sexual reproduction indicate that the number of C. ehrenbergii zygospores was significantly reduced by the application of 0.937 mg L-1. The Comet assay showed that Triclosan treatments led to a dose-dependent DNA damage of C. ehrenbergii; 0.25 mg L-1 caused significant genotoxic effects and higher concentrations irreversibly altered the DNA strands. These results suggest that C. ehrenbergii could represent a useful organism to evaluate the whole toxicity of pharmaceuticals and personal care products (PPCPs), giving valuable information for a risk assessment. (c) 2005 Elsevier B.V. All rights reserved.

We consider Pisciarelli (Campi Flegrei, Italy) as a model site for investigating the factors which influence the distribution of microalgae in hydrothermal environments. This hydrothermal system is liquid-dominant with very low pH values both in water and in the soil; spring water has a temperature of up to 92°C. At Pisciarelli, chemico-physical conditions are extremely variable in different sites, seasons and years. Five ecological stations were identified on the basis of temperature, pH and fluid vents conditions. Different algal communities have been observed according to their adaptability to these controlling factors. Cyanidiales were found to be largely diffused in the entire area, proving very successful at adapting to environmental changes. Chlamydomonas sp. and Pinnularia sp. were scattered in at least three of the five ecological stations. Some green acid-tolerant algae, such as Pseudococcomyxa simplex and Chlorella protothecoldes var. acidicola, were strongly influenced by temperature and pH fluctuations and mainly limited to one station. Dunaliella acidophila is confined to a narrow area, being able to live exclusively in restricted pH and temperature ranges. In some sampling sites the peculiar chemico-physical conditions together with silica oversaturation cause the formation of a thick silica crust around cell walls. Generally, algae could act as a nucleation site for neoforming minerals, offering in some cases an ideal substrate for the formation of a thick silica crust on their surface We consider Pisciarelli (Campi Flegrei, Italy) as a model site for investigating the factors which influence the distribution of microalgae in hydrothermal environments. This hydrothermal system is liquid-dominant with very low pH values both in water and in the soil; spring water has a temperature of up to 92°C. At Pisciarelli, chemico-physical conditions are extremely variable in different sites, seasons and years. Five ecological stations were identified on the basis of temperature, pH and fluid vents conditions. Different algal communities have been observed according to their adaptability to these controlling factors. Cyanidiales were found to be largely diffused in the entire area, proving very successful at adapting to environmental changes. Chlamydomonas sp. and Pinnularia sp. were scattered in at least three of the five ecological stations. Some green acid-tolerant algae, such as Pseudococcomyxa simplex and Chlorella protothecoldes var. acidicola, were strongly influenced by temperature and pH fluctuations and mainly limited to one station. Dunaliella acidophila is confined to a narrow area, being able to live exclusively in restricted pH and temperature ranges. In some sampling sites the peculiar chemico-physical conditions together with silica oversaturation cause the formation of a thick silica crust around cell walls. Generally, algae could act as a nucleation site for neoforming minerals, offering in some cases an ideal substrate for the formation of a thick silica crust on their surface.

Three Chlamydomonas strains were isolated from the soils of a hot spring located in the Campi Flegrei Caldera (Naples, Italy). Ecophysiological, morpho-cytological and molecular features were used to characterize these isolates and to compare them with chlamydomonax acidophila strains from algal culture collections. The strains were collected from three points of the volcanic site, differing in their physico-chemical conditions. Among the examined Chlamydomonas strains, only the isolates from Campi Flegrei could grow optimally at pH values ≤3.0. These isolates also showed a high tolerance to desiccation and high temperatures, not evidenced by the other Chlamydomonas strains included in the study. 18S rDNA phylogeny indicates that the isolates from Campi Flegrei are closely related to Chlamydomonas pitschmannii and two strains isolated in Canada and Europe, that have been designated as Chlamydomonas acidophila. A Chlamydomonas acidophila strain isolated from the type locality in Japan is less closely related according to its molecular phylogeny, and can also be discerned by light and electron microscopy. Moreover, vegetative cells and sporangia of Chlamydomonas acidophila from Japan showed a median trilaminar structure not observed in the other strains. Our results show that Chlamydomonas pitschmannii could represent a hitherto unknown extremophilic Chlamydomonas species. Three Chlamydomonas strains were isolated from the soils of a hot spring located in the Campi Flegrei Caldera (Naples, Italy). Ecophysiological, morpho-cytological and molecular features were used to characterize these isolates and to compare them with chlamydomonax acidophila strains from algal culture collections. The strains were collected from three points of the volcanic site, differing in their physico-chemical conditions. Among the examined Chlamydomonas strains, only the isolates from Campi Flegrei could grow optimally at pH values

The lanthanides are among the rare earth elements (REEs), which are indispensable constituents of modern technologies and are often challenging to acquire from natural resources. The demand for REEs is so high that there is a clear need to develop efficient and environmentally-friendly recycling methods. In the present study, living cells of the extremophile Galdieria sulphuraria were used to remove four REEs, Yttrium, Cerium, Europium, and Terbium, from single- and quaternary-metal aqueous solutions. Two different strains, SAG 107.79 and ACUF 427, were exposed to solutions buffered at pH 2.5, 3.5, 4.5, and 5.5. Our data demonstrated that the removal performances were strain and pH dependent for all metal ions. At lower pH, ACUF 427 outperformed SAG 107.79 considerably. By increasing the pH of the solutions, there was a significant surge in the aqueous removal performance of both strains. The same trend was highlighted using quaternary-metal solutions, even if the quantities of metal removed were significantly lower. The present study provided the first insight into the comparative removal capacity of the Galdieria sulphuraria strains. The choice of the appropriate operational conditions such as the pH of the metal solutions is an essential step in developing efficient, rapid, and straightforward biological methods for recycling REEs.

During the last year, science has been focusing on the research of antivirally active compounds overall after the SARS-CoV-2 pandemic, which caused a great amount of deaths and the downfall of the economy in 2020. Photosynthetic organisms such as microalgae are known to be a reservoir of bioactive secondary metabolites; this feature, coupled with the possibility of achieving very high biomass levels without excessive energetic expenses, make microalgae worthy of attention in the search for new molecules with antiviral effects. In this work, the antiviral effects of microalgae against some common human or animal viruses were considered, focusing our attention on some possible effects against SARS-CoV-2. We summed up the data from the literature on microalgae antiviral compounds, from the most common ones, such as lectins, polysaccharides and photosynthetic pigments, to the less known ones, such as unidentified proteins. We have discussed the effects of a microalgae-based genetic engineering approach against some viral diseases. We have illustrated the potential antiviral benefits of a diet enriched in microalgae.

Galdieria is a photosynthetic unicellular protist, inhabiting thermoacidic environments around the world. The synchronicity of these thermoacidophilic algae with their extreme habitats makes them unable to thrive in different ecological conditions. The genetic structure of Galdieria populations has not yet been studied. In this report, the level of genetic diversity and struc- ture of five Galdieria populations from Iceland were assessed through both random amplified polymorphic DNA (RAPD) markers, and a partial calmodulin gene fragment (previously used to assess the population structure of these extremophilic algae). The level of population differentiation from both the RAPD and CaM markers was estimated using PLP, percentage of polymorphic loci; H, the expected heterozygosity; I, Shannon’s information index, Ks, Kst. The migration ability of the Galdieria populations was suggested by the high level of genetic variations scored within each Galdieria population and by the small number of polymorphisms detected between the different Icelandic populations. The low genetic diversity between the two species was highlighted by RAPD and calmodulin markers, suggesting for the first time an interspecific genetic flow between species as strategy to evolve in stressful environments. Galdieria is a photosynthetic unicellular protist, inhabiting thermoacidic environments around the world. The synchronicity of these thermoacidophilic algae with their extreme habitats makes them unable to thrive in different ecological conditions. The genetic structure of Galdieria populations has not yet been studied. In this report, the level of genetic diversity and structure of five Galdieria populations from Iceland were assessed through both random amplified polymorphic DNA (RAPD) markers, and a partial calmodulin gene fragment (previously used to assess the population structure of these extremophilic algae). The level of population differentiation from both the RAPD and CaM markers was estimated using PLP, percentage of polymorphic loci; H, the expected heterozygosity; I, Shannon’s information index, Ks, Kst. The migration ability of the Galdieria populations was suggested by the high level of genetic variations scored within each Galdieria population and by the small number of polymorphisms detected between the different Icelandic populations. The low genetic diversity between the two species was highlighted by RAPD and calmodulin markers, suggesting for the first time an interspecific genetic flow between species as strategy to evolve in stressful environments.

Heterotrophic components of biofilms on wood artefacts were studied at the Conservation Laboratory for Wood Artefacts of the University Suor Orsola Benincasa of Naples, Italy. The aim of the study was to add new information on the micro-habitats represented by biofilms formed by wood-dwelling organisms. Light and electron microscopy of histological features of woods used to make the artefacts showed that the woods belonged to species of lime (Tilia sp.), poplar (Populus sp.) and pear (Pyrus sp.). A Denaturing Gradient Gel Electrophoresis analysis performed on heterotrophic microorganisms colonizing the artefacts led to identify four species of bacteria, namely Bacillus cereus, B. mycoides, B. subtilis and Microbacterium oleivorans, and seven species of fungi, namely Alternaria alternata, Aspergillus fumigans, A. versicolor, Cladosporium cladosporioides, C. oxysporum, Fusarium oxysporum and Penicillium chrysogenum. Based on its morphological features, an insect found on some artefacts was identified as the xylophagous beetle Nicobium castaneum (Anobiidae). The influence of wood type and environmental conditions on the diversity of microorganisms was discussed.

A method based on the use of electromagnetic radiation (EMR) in the radiofrequency band was experimented to eliminate heterotrophic organisms from biofilms encrusting linen canvasses used for paintings. Artificial biofilms were used for exposure to EMR. Biofilms were grown on fragments of canvasses sampled from the backside of a painting. The fragments, after sterilization, were inoculated with several heterotrophic microorganisms previously isolated from other canvasses used for paintings and identified by molecular analyses as the bacteria Bacillus mycoides and Bacillus subtilis, and the microfungi Aspergillus versicolor, Cladosporium cladosporioides, Cladosporium oxysporum and Fusarium oxysporum. The newly formed biofilms resulted to contain the same microorganisms used for the inocula. They were exposed to EMR obtained by an amplitude-modulating radiofrequency sine wave with a train of rectangular pulses with 200 ns repetition time and 10% duty cycle. The exposure protocol consisted of three applications of two hours each, every other day. Denaturing gradient gel electrophoresis analyses carried out on the EMR-treated biofilms showed that all the microorganisms disappeared after the third application. The amplitude of the adopted EMR was not high enough to cause any significant increase in the temperature of the substrates. Therefore, the methodology showed to be non-invasive for the artefacts and its exposure levels not hazardous for operators and the environment compared to other techniques adopted in the field of conservation of cultural heritage. Among the latter techniques, the most common one is based on the use of biocides that, although effective, present problems in all the above-described areas.

The RADiation sensitive52 (RAD52) protein catalyzes the pairing between two homologous DNA sequences&rsquo, double-strand break repair and meiotic recombination, mediating RAD51 loading onto single-stranded DNA ends, and initiating homologous recombination and catalyzing DNA annealing. This article reports the characterization of RAD52 homologs in the thermo-acidophilic Cyanidiophyceae whose genomes have undergone extensive sequencing. Database mining, phylogenetic inference, prediction of protein structure and evaluation of gene expression were performed in order to determine the functionality of the RAD52 protein in Cyanidiophyceae. Its current function in Cyanidiophytina could be related to stress damage response for thriving in hot and acidic environments as well as to the genetic variability of these algae, in which, conversely to extant Rhodophyta, sexual mating was never observed.

Phlegrean Fields is a large volcanic area situated southwest of Naples (Italy), including both cave and thermoacidic habitats. These extreme environments host the genus Cyanidium; the species C. chilense represents a common phototrophic micro- organism living in anthropogenic caves. With a view to provide a comprehensive characterization for a correct taxonomic classification, morpho-ultrastructural investigations ofC. chilense from Syb’s cave (Phlegren Fields) was herein car-ried out and compared with the thermoacidophilic C. caldarium. The biofilm was also analyzed to define the roleofC. chilensein the establishment of a bio film within caveenvironments. Despite the peculiar ecological and molecular divergences,C. chilenseandC. caldariumshared all the maindiacritic features, suggesting morphological convergence within the genus; cytological identity was found amongC. chilensestrains geographically distant and adapted to different substrates, such as the porous yellow tuff of Sybil cave and calcyte, magnesite and basaltic rocks from other caves. C. chilense is generally dominant in all biofilms, developing monospecific islets, developing both super ficially or betweenfungal hyphae and coccoid cyanobacteria. Extracellular polymeric substances (EPS) were recorded in C. chilense bio filmsfrom Sybil cave, confirming the role of EPS in facilitating cellsadhesion to the surface, creating a cohesive network of inter-connecting biofilm cells

The allelochemical stress on Zea mays was analyzed by using walnut husk washing waters (WHWW), a by-product of Juglans regia post-harvest process, which possesses strong allelopathic potential and phytotoxic effects. Oxidative damage and cell-programmed death were induced by WHWW in roots of maize seedlings. Treatment induced ROS burst, with excess of H2O2 content. Enzymatic activities of catalase were strongly increased during the first hours of exposure. The excess in malonildialdehyde following exposure to WHWW confirmed that oxidative stress severely damaged maize roots. Membrane alteration caused a decrease in NADPH oxidase activity along with DNA damage as confirmed by DNA laddering. The DNA instability was also assessed through sequence-related amplified polymorphism assay, thus suggesting the danger of walnut processing by-product and focusing the attention on the necessity of an efficient treatment of WHWW.

To date, the main source for phycocyanin production is the thermophilic cyanobacterium Spirulina platensis, although latest researches are exploring the possibility to exploit the thermoacidophilic Cyanidiophyceae (Rhodophyta) to this purpose. Galdieria phlegrea is a polyextremophilic red alga belonging to Cyanidiophyceae, colonizing, along with G. sulphuraria, acidic and thermal environments and distinguishable from this latter on the base of molecular and ecophysiological traits. In the present paper, a characterization of C-phycocyanin from two strains of G. phlegrea with different geographic provenance (Phlegrean Fields Naples, Italy; Diyadin, Turkey) was provided under autotrophic and heterotrophic conditions. The results showed different optimal pHs and thermostability between the strains and between autotrophic and heterotrophic cells. What is intriguing, a pre-heating at 70 °C of heterotrophic cells from the Italian strain resulted in a highly heat-resistant of C-phycocyanin. We hypothesized that the different features of this pigment, currently widely used for various applicative purposes, are related to the habitat from which the microalga comes from.

The nomenclatural history of the genus Cyanidium (Tilden) Geitler is long and complex, due to uncertainties regarding its taxonomic position. In the course of the last two centuries, Cyanidium has been assigned to Cyanophyta, Chlorophyta, and finally to Rhodophyta (Ott & Sechback 1994, Albertano et al. 2000). On the basis of morphological, ultrastructural and reproductive characters, Merola et al. (1981) instituted two new genera, Galdieria and Cyanidioschyzon, strictly related to Cyanidium, and assigned all three genera to the new class Cyanidiophyceae. Later, in the treatment proposed by Ott & Sechback (1994), all species belonging to Galdieria or Cyanidioschyzon were transferred to Cyanidium. In the most recent taxonomic proposal by Yoon et al. (2014), the tripartite generic architecture has been confirmed, even though a different taxonomic hierarchy was applied at the higher ranks.

Campi Flegrei is a large volcanic area situated northwest of Naples, Italy. Two archeological sites, the Sybil's Cave and the Piscina Mirabilis, are artificial caves dug in the yellow tuff and used during antiquity for various purposes. This paper describes for the first time the algal biodiversity of these caves and determines whether environmental factors such as light intensity and humidity are influential in species distribution. A total of twenty-two algal species were identified by molecular methods (18S rDNA); the largest group was Cyanobacteria (eleven species), followed by algae Chlorophyta (six), Rhodophyta (two) and Bacillariophyta (two). Cluster analysis of algal distribution in the caves in relation to light and humidity showed no relevant differences in algal distribution between the two caves. Three different algal groups were identified. The first one includes strains strictly dependent on low humidity, a second cluster was mainly associated with sites where humidity is not a severe constraint, and a third group, mainly represented by filamentous cyanobacteria, is probably dependent on high humidity, since it was detected only at Piscina Mirabilis.

Background Cyanidiales are unicellular extremophilic red algae that inhabit acidic and high temperature sites around hot springs and have also adapted to life in endolithic and interlithic habitats. Comparative genomic analysis of Cyanidioschyzon merolae and Galdieria sulphuraria predicts that the latter may be more broadly distributed in extreme environments because its genome contains membrane transporters involved in the uptake of reduced carbon compounds that are absent from C. merolae. Analysis of an endolithic site in the Phlegrean Fields near Naples, Italy is consistent with this prediction showing this population to be comprised solely of the newly described lineage Galdieria-B and C. merolae to be limited to humid habitats. Here, we conducted an environmental PCR survey of another extreme environment in Tuscany, Italy and contrasted Cyanidiales population structure at endolithic and interlithic habitats in Naples and Tuscany. Results We find a second Galdieria lineage (Galdieria-A) in endolithic and interlithic habitats in Tuscany but surprisingly Cyanidium was also present at these sites. The photoautotrophic Cyanidium apparently survives below the rock surface where sufficient light is available for photosynthesis. C. merolae is absent from all endolithic and interlithic sites in Tuscany. Population genetic analyses of a partial calmodulin gene fragment suggest a recent establishment or recurrent gene flow between populations in Tuscany, whereas the highly structured Galdieria-B population in Naples likely originated from 2–3 founder events. We find evidence of several recombination events across the calmodulin gene, potentially indicating the presence of sexual reproduction in the Tuscany populations. Conclusion Our study provides important data regarding population structure in extreme endolithic environments and insights into how Cyanidiales may be established in and adapt to these hostile environments.