Bacillus cereus spores and cereulide in food-borne illness

B. cereus is a gram-positive bacterium that possesses two different forms of life:the large, rod-shaped cells (ca. 0.002 mm by 0.004 mm) that are able to propagate and the small (0.001 mm), oval shaped spores. The spores can survive in almost any environment for up to centuries without nourishment or water. They are insensitive towards most agents that normally kill bacteria: heating up to several hours at 90 ºC, radiation, disinfectants and extreme alkaline (≥ pH 13) and acid (≤ pH 1) environment. The spores are highly hydrophobic and therefore make them tend to stick to all kinds of surfaces, steel, plastics and live cells. In favorable conditions the spores of B. cereus may germinate into vegetative cells capable of producing food poisoning toxins. The toxins can be heat-labile protein formed after ingestion of the contaminated food, inside the gastrointestinal tract (diarrhoeal toxins), or heat stable peptides formed in the food (emesis causing toxin, cereulide). Cereulide cannot be inactivated in foods by cooking or any other procedure applicable on food. Cereulide in consumed food causes serious illness in human, even fatalities. In this thesis, B. cereus strains originating from different kinds of foods and environments and 8 different countries were inspected for their capability of forming cereulide. Of the 1041 isolates from soil, animal feed, water, air, used bedding, grass, dung and equipment only 1.2 % were capable of producing cereulide, whereas of the 144 isolates originating from foods 24 % were cereulide producers. Cereulide was detected by two methods: by its toxicity towards mammalian cells (sperm assay) and by its peculiar chemical structure using liquid-chromatograph-mass spectrometry equipment. B. cereus is known as one of the most frequent bacteria occurring in food. Most foods contain more than one kind of B. cereus. When randomly selected 100 isolates of B. cereus from commercial infant foods (dry formulas) were tested, 11% of these produced cereulide. Considering a frequent content of 103 to 104 cfu (colony forming units) of B. cereus per gram of infant food formula (dry), it appears likely that most servings (200 ml, 30 g of the powder reconstituted with water) may contain cereulide producers. When a reconstituted infant formula was inoculated with >105 cfu of cereulide producing B. cereus per ml and left at room temperature, cereulide accumulated to food poisoning levels (> 0.1 mg of cereulide per serving) within 24 hours. Paradoxically, the amount of cereulide (per g of food) increased 10 to 50 fold when the food was diluted 4 - 15 fold with water. The amount of the produced cereulide strongly depended on the composition of the formula: most toxin was formed in formulas with cereals mixed with milk, and least toxin in formulas based on milk only. In spite of the aggressive cleaning practices executed by the modern dairy industry, certain genotypes of B. cereus appear to colonise the silos tanks. In this thesis four strategies to explain their survival of their spores in dairy silos were identified. First, high survival (log 15 min kill ≤ 1.5) in the hot alkaline (pH >13) wash liquid, used at the dairies for cleaning-in-place. Second, efficient adherence of the spores to stainless steel from cold water. Third, a cereulide producing group with spores characterized by slow germination in rich medium and well preserved viability when exposed to heating at 90 ºC. Fourth, spores capable of germinating at 8 ºC and possessing the psychrotolerance gene, cspA. There were indications that spores highly resistant to hot 1% sodium hydroxide may be effectively inactivated by hot 0.9% nitric acid. Eight out of the 14 dairy silo tank isolates possessing hot alkali resistant spores were capable of germinating and forming biofilm in whole milk, not previously reported for B. cereus. In this thesis it was shown that cereulide producing B. cereus was capable of inhibiting the growth of cereulide non-producing B. cereus occurring in the same food. This phenomenon, called antagonism, has long been known to exist between B. cereus and other microbial species, e.g. various species of Bacillus, gram-negative bacteria and plant pathogenic fungi. In this thesis intra-species antagonism of B. cereus was shown for the first time. This brother-killing did not depend on the cereulide molecule, also some of the cereulide non-producers were potent antagonists. Interestingly, the antagonistic clades were most frequently found in isolates from food implicated with human illness. The antagonistic property was therefore proposed in this thesis as a novel virulence factor that increases the human morbidity of the species B. cereus, in particular of the cereulide producers.

Modern techniques in detection, identification and quantification of bacteria and peptides from foods

Standards have been placed to regulate the microbial and preservative contents to assure that foods are safe to the consumer. In a case of a food-related disease outbreak, it is crucial to be able to detect and identify quickly and accurately the cause of the disease. In addition, for every day control of food microbial and preservative contents, the detection methods must be easily performed for numerous food samples. In this present study, quicker alternative methods were studied for identification of bacteria by DNA fingerprinting. A flow cytometry method was developed as an alternative to pulsed-field gel electrophoresis, the golden method . DNA fragment sizing by an ultrasensitive flow cytometer was able to discriminate species and strains in a reproducible and comparable manner to pulsed-field gel electrophoresis. This new method was hundreds times faster and 200,000 times more sensitive. Additionally, another DNA fingerprinting identification method was developed based on single-enzyme amplified fragment length polymorphism (SE-AFLP). This method allowed the differentiation of genera, species, and strains of pathogenic bacteria of Bacilli, Staphylococci, Yersinia, and Escherichia coli. These fingerprinting patterns obtained by SE-AFLP were simpler and easier to analyze than those by the traditional amplified fragment length polymorphism by double enzyme digestion. Nisin (E234) is added as a preservative to different types of foods, especially dairy products, around the world. Various detection methods exist for nisin, but they lack in sensitivity, speed or specificity. In this present study, a sensitive nisin-induced green fluorescent protein (GFPuv) bioassay was developed using the Lactococcus lactis two-component signal system NisRK and the nisin-inducible nisA promoter. The bioassay was extremely sensitive with detection limit of 10 pg/ml in culture supernatant. In addition, it was compatible for quantification from various food matrices, such as milk, salad dressings, processed cheese, liquid eggs, and canned tomatoes. Wine has good antimicrobial properties due to its alcohol concentration, low pH, and organic content and therefore often assumed to be microbially safe to consume. Another aim of this thesis was to study the microbiota of wines returned by customers complaining of food-poisoning symptoms. By partial 16S rRNA gene sequence analysis, ribotyping, and boar spermatozoa motility assay, it was identified that one of the wines contained a Bacillus simplex BAC91, which produced a heat-stable substance toxic to the mitochondria of sperm cells. The antibacterial activity of wine was tested on the vegetative cells and spores of B. simplex BAC91, B. cereus type strain ATCC 14579 and cereulide-producing B. cereus F4810/72. Although the vegetative cells and spores of B. simplex BAC91 were sensitive to the antimicrobial effects of wine, the spores of B. cereus strains ATCC 14579 and F4810/72 stayed viable for at least 4 months. According to these results, Bacillus spp., more specifically spores, can be a possible risk to the wine consumer.

Assessment and control of Bacillus cereus emetic toxin in food

Despite of improving levels of hygiene, the incidence of registered food borne disease has been at the same level for many years: there were 40 to 90 epidemics in which 1000-9000 persons contracted food poisoning through food or drinking water in Finland. Until the year 2004 salmonella and campylobacter were the most common bacterial causes of food borne diseases, but in years 2005-2006 Bacillus cereus was the most common. Similar developement has been published i.e. in Germany already in the 1990´s. One reason for this can be Bacillus cereus and its emetic toxin, cereulide. Bacillus cereus is a common environmental bacterium that contaminates raw materials of food. Otherwise than salmonella and campylobacter, Bacillus cereus is a heat resistant bacterium, capable of surviving most cooking procedures due to the production of highly thermo resistant spores. The food involved has usually been heat treated and surviving spores are the source of the food poisoning. The heat treatment induces germination of the spore and the vegetative cells then produce toxins. This doctoral thesis research focuses on developing methods for assessing and eliminating risks to food safety by cereulide producing Bacillus cereus. The biochemistry and physiology of cereulide production was investigated and the results were targeted to offer tools for minimizing toxin risk in food during the production. I developed methods for the extraction and quantitative analysis of cereulide directly from food. A prerequisite for that is knowledge of the chemical and physical properties of the toxin. Because cereulide is practically insoluble in water, I used organic solvents; methanol, ethanol and pentane for the extraction. For extraction of bakery products I used high temperature (100C) and pressure (103.4 bars). Alternaties for effective extraction is to flood the plain food with ethanol, followed by stationary equilibration at room temperature. I used this protocol for extracting cereulide from potato puree and penne. Using this extraction method it is also possible also extract cereulide from liquid food, like milk. These extraction methods are important improvement steps for studying of Bacillus cereus emetic food poisonings. Prior my work, cereulide extraction was done using water. As the result, the yield was poor and variable. To investigate suspected food poisonings, it is important to show actual toxicity of the incriminated food. Many toxins, but not cereulide, inactivate during food processing like heating. The next step is to identify toxin by chemical methods. I developed with my colleague Maria Andesson a rapid assay for the detection of cereulide toxicity, within 5 to 15 minutes. By applying this test it is possible to rapidly detect which food was causing the food poisoning. The chemical identification of cereulide was achieved using mass spectrometry. I used cereulide specific molecular ions, m/z (+/-0.3) 1153.8 (M+H+), 1171.0 (M+NH4+), 1176.0 (M+Na+) and 1191.7 (M+K+) for reliable identification. I investigated foods to find out their amenability to accumulate cereulide. Cereulide was formed high amounts (0.3 to 5.5 microg/g wet wt) when of cereulide producing B. cereus strains were present in beans, rice, rice-pastry and meat-pastry, if stored at non refrigerated temperatures (21-23C). Rice and meat pastries are frequently consumed under conditions where no cooled storage is available e.g. picnics and outdoor events. Bacillus cereus is a ubiquitous spore former and is therefore difficult to eliminate from foods. It is therefore important to know which conditions will affect the formation of cereulide in foods. My research showed that the cereulide content was strongly (10 to 1000 fold differences in toxin content) affected by the growth environment of the bacterium. Storage of foods under nitrogen atmosphere (> 99.5 %) prevented the production of cereulide. But when also carbon dioxide was present, minimizing the oxygen contant (< 1%) did not protect the food from formation of cereulide in preliminary experiments. Also food supplements affected cereulide production at least in the laboratory. Adding free amino acids, leucine and valine, stimulated cereulide production 10 to 20 fold. In peptide bonded form these amino acids are natural constituents in all proteins. Interestingly, adding peptide bonded leucine and valine had no significant effect on cereulide production. Free amino acids leucine and valine are approved food supplements and widely used as flawour modifiers in food technology. My research showed that these food supplements may increase food poisoning risk even though they are not toxic themselves.

Food and Indoor Air Isolated Bacillus Non-Protein Toxins: : Structures, Physico-Chemical Properties and Mechanisms of Effects on Eukaryotic Cells

We report here the structures and properties of heat-stable, non-protein, and mammalian cell-toxic compounds produced by spore-forming bacilli isolated from indoor air of buildings and from food. Little information is available on the effects and occurrence of heat-stable non-protein toxins produced by bacilli in moisture-damaged buildings. Bacilli emit spores that move in the air and can serve as the carriers of toxins, in a manner similar to that of the spores of toxic fungi found in contaminated indoor air. Bacillus spores in food cause problems because they tolerate the temperatures applied in food manufacture and the spores later initiate growth when food storage conditions are more favorable. Detection of the toxic compounds in Bacillus is based on using the change in mobility of boar spermatozoa as an indicator of toxic exposure. GC, LC, MS, and nuclear magnetic resonance NMR spectroscopy were used for purification, detection, quantitation, and analysis of the properties and structures of the compounds. Toxicity and the mechanisms of toxicity of the compounds were studied using boar spermatozoa, feline lung cells, human neural cells, and mitochondria isolated from rat liver. The ionophoric properties were studied using the BLM (black-lipid membrane) method. One novel toxin, forming ion channels permeant to K+ > Na+ > Ca2+, was found and named amylosin. It is produced by B. amyloliquefaciens isolated from indoor air of moisture-damaged buildings. Amylosin was purified with an RP-HPLC and a monoisotopic mass of 1197 Da was determined with ESI-IT-MS. Furthermore, acid hydrolysis of amylosin followed by analysis of the amino acids with the GS-MS showed that it was a peptide. The presence of a chromophoric polyene group was found using a NMR spectroscopy. The quantification method developed for amylosin based on RP-HPLC-UV, using the macrolactone polyene, amphotericin B (MW 924), as a reference compound. The B. licheniformis strains isolated from a food poisoning case produced a lipopeptide, lichenysin A, that ruptured mammalian cell membranes and was purified with a LC. Lichenysin A was identified by its protonated molecules and sodium- and potassium- cationized molecules with MALDI-TOF-MS. Its protonated forms were observed at m/z 1007, 1021 and 1035. The amino acids of lichenysin A were analyzed with ESI-TQ-MS/MS and, after acid hydrolysis, the stereoisomeric forms of the amino acids with RP-HPLC. The indoor air isolates of the strain of B. amyloliquefaciens produced not only amylosin but also lipopeptides: the cell membrane-damaging surfactin and the fungicidal fengycin. They were identified with ESI-IT-MS observing their protonated molecules, the sodium- and potassium-cationized molecules and analysing the MS/MS spectra. The protonated molecules of surfactin and fengycin showed m/z values of 1009, 1023, and 1037 and 1450, 1463, 1493, and 1506, respectively. Cereulide (MW 1152) was purified with RP-HPLC from a food poisoning strain of B. cereus. Cereulide was identified with ESI-TQ-MS according to the protonated molecule observed at m/z 1154 and the ammonium-, sodium- and potassium-cationized molecules observed at m/z 1171, 1176, and 1192, respectively. The fragment ions of the MS/MS spectrum obtained from the protonated molecule of cereulide at m/z 1154 were also interpreted. We developed a quantification method for cereulide, using RP-HPLC-UV and valinomycin (MW 1110, which structurally resembles cereulide) as the reference compound. Furthermore, we showed empirically, using the BLM method, that the emetic toxin cereulide is a specific and effective potassium ionophore of whose toxicity target is especially the mitochondria.

The life cycle and genetic structure of the red alga Furcellaria lumbricalis on a salinity gradient

The life cycle and genetic diversity of the red alga Furcellaria lumbricalis (Hudson) Lamouroux were investigated in 15 populations in northern Europe. The occurrence of different life cycle phases and seasonality of reproduction were studied in four brackish populations in the northern Baltic Sea. Furthermore, a new method, based on genome screening with ISSR markers combined with a restriction-ligation method, was developed to discover microsatellite markers for population genetic analyses. The mitochondrial DNA cox2-3 spacer sequence and four microsatellite markers were used to examine the genetic diversity and differentiation of red algal populations in northern Europe. In addition, clonality and small-scale genetic structure of one Irish and four Baltic Sea populations were studied with microsatellite markers. It was discovered that at the low salinities of the northern Baltic Sea, only tetrasporophytes and males were present in the populations of F. lumbricalis and that winter was the main season for tetrasporangial production. Furthermore, the population occurring at the lowest salinity (3.6 practical salinity units, psu) did not produce spores. The size of the tetraspores was smaller in the Baltic Sea populations than that in the Irish population, and there were more deformed spores in the Baltic Sea populations than in the Irish populations. Studies with microsatellite markers indicated that clonality is a common phenomenon in the Baltic Sea populations of F. lumbricalis, although the proportion of clonal individuals varied among populations. Some genetic divergence occurred within locations both in Ireland and in the northern Baltic Sea. Even though no carpogonia were detected in the field samples during the study, the microsatellite data indicated that sexual reproduction occurs at least occasionally in the northern Baltic Sea. The genetic diversity of F. lumbricalis was highest in Brittany, France. Since no variation was discovered in the mtDNA cox2-3 spacer sequence, which is generally regarded as an informative phylogeographic marker in red algae, it can be assumed that the studied populations probably share the same origin.

Should I stay or should I go? : Reproductive and dispersal strategies of site-specific liverwort species

The area of intensively managed forests, in which required conditions for several liverwort species are seldom found, has expanded over the forest landscape during the last century. Liverworts are very sensitive to habitat changes, because they demand continuously moist microclimate. Consequently, about third of the forest liverworts have been classified as threatened or near threatened in Finland. The general objective of this thesis is to increase knowledge of the reproductive and dispersal strategies of the substrate-specific forest bryophytes. A further aim was to develop recommendations for conservation measures for species inhabiting unstable and stable habitats in forest landscape. Both population ecological and genetic methods have been applied in the research. Anastrophyllum hellerianum inhabits spatially and temporally limited substrate patches, decaying logs, which can be considered as unstable habitats. The results show that asexual reproduction by gemmae is the dominant mode of reproduction, whereas sexual reproduction is considerably infrequent. Unlike previously assumed, not only spores but also the asexual propagules may contribute to long-distance dispersal. The combination of occasional spore production and practically continuous, massive gemma production facilitates dispersal both on a local scale and over long distances, and it compensates for the great propagule losses that take place preceding successful establishment at suitable sites. However, establishment probability of spores may be restricted because of environmental and biological limitations linked to the low success of sexual reproduction. Long-lasting dry seasons are likely to result in a low success of sexual reproduction and decreased release rate of gemmae from the shoots, and consequent fluctuations in population sizes. In the long term, the substratum limitation is likely to restrict population sizes and cause local extinctions, especially in small-sized remnant populations. Contrastingly, larger forest fragments with more natural disturbance dynamics, to which the species is adapted, are pivotal to species survival. Trichocolea tomentella occupies stable spring and mesic habitats in woodland. The relatively small populations are increasingly fragmented with a high risk for extinction for extrinsic reasons. The results show that T. tomentella mainly invests in population persistence by effective clonal growth via forming independent ramets and in competitive ability, and considerably less in sexuality and dispersal potential. The populations possess relatively high levels of genetic diversity regardless of population size and of degree of isolation. Thus, the small-sized populations inhabiting stable habitats should not be neglected when establishing conservation strategies for the species and when considering the habitat protection of small spring sites. Restricted dispersal capacity, also on a relatively small spatial scale, is likely to prevent successful (re-)colonization in the potential habitat patches of recovering forest landscapes. By contrast, random short-range dispersal of detached vegetative fragments within populations at suitable habitat seems to be frequent. Thus, the restoration actions of spring and streamside habitats close to the populations of T. tomentella may contribute to population expansion. That, in turn, decreases the harmful effects of environmental stochasticity.

Damp building moulds: Assessment of sensitization in patients and studies into mechanisms of airway inflammation using experimental models

Exposure to water-damaged buildings and the associated health problems have evoked concern and created confusion during the past 20 years. Individuals exposed to moisture problem buildings report adverse health effects such as non-specific respiratory symptoms. Microbes, especially fungi, growing on the damp material have been considered as potential sources of the health problems encountered in these buildings. Fungi and their airborne fungal spores contain allergens and secondary metabolites which may trigger allergic as well as inflammatory types of responses in the eyes and airways. Although epidemiological studies have revealed an association between damp buildings and health problems, no direct cause-and-effect relationship has been established. Further knowledge is needed about the epidemiology and the mechanisms leading to the symptoms associated with exposure to fungi. Two different approaches have been used in this thesis in order to investigate the diverse health effects associated with exposure to moulds. In the first part, sensitization to moulds was evaluated and potential cross-reactivity studied in patients attending a hospital for suspected allergy. In the second part, one typical mould known to be found in water-damaged buildings and to produce toxic secondary metabolites was used to study the airway responses in an experimental model. Exposure studies were performed on both naive and allergen sensitized mice. The first part of the study showed that mould allergy is rare and highly dependent on the atopic status of the examined individual. The prevalence of sensitization was 2.7% to Cladosporium herbarum and 2.8% to Alternaria alternata in patients, the majority of whom were atopic subjects. Some of the patients sensitized to mould suffered from atopic eczema. Frequently the patients were observed to possess specific serum IgE antibodies to a yeast present in the normal skin flora, Pityrosporum ovale. In some of these patients, the IgE binding was partly found to be due to binding to shared glycoproteins in the mould and yeast allergen extracts. The second part of the study revealed that exposure to Stachybotrys chartarum spores induced an airway inflammation in the lungs of mice. The inflammation was characterized by an influx of inflammatory cells, mainly neutrophils and lymphocytes, into the lungs but with almost no differences in airway responses seen between the satratoxin producing and non-satratoxin producing strain. On the other hand, when mice were exposed to S. chartarum and sensitized/challenged with ovalbumin the extent of the inflammation was markedly enhanced. A synergistic increase in the numbers of inflammatory cells was seen in BAL and severe inflammation was observed in the histological lung sections. In conclusion, the results in this thesis imply that exposure to moulds in water damaged buildings may trigger health effects in susceptible individuals. The symptoms can rarely be explained by IgE mediated allergy to moulds. Other non-allergic mechanisms seem to be involved. Stachybotrys chartarum is one of the moulds potentially responsible for health problems. In this thesis, new reaction models for the airway inflammation induced by S. chartarum have been found using experimental approaches. The immunological status played an important role in the airway inflammation, enhancing the effects of mould exposure. The results imply that sensitized individuals may be more susceptible to exposure to moulds than non-sensitized individuals.

Bacteria colonizing paper machines

Bacteria growing in paper machines can cause several problems. Biofilms detaching from paper machine surfaces may lead to holes and spots in the end product or even break the paper web leading to expensive delays in production. Heat stable endospores will remain viable through the drying section of paper machine, increasing the microbial contamination of paper and board. Of the bacterial species regularly found in the end products, Bacillus cereus is the only one classified as a pathogen. Certain B. cereus strains produce cereulide, the toxin that causes vomiting disease in food poisonings connected to B. cereus. The first aim of this thesis was to identify harmful bacterial species colonizing paper machines and to assess the role of bacteria in the formation of end product defects. We developed quantitative PCR methods for detecting Meiothermus spp. and Pseudoxanthomonas taiwanensis. Using these methods I showed that Meiothermus spp. and Psx. taiwanensis are major biofoulers in paper machines. I was the first to be able to show the connection between end product defects and biofilms in the wet-end of paper machines. I isolated 48 strains of primary-biofilm forming bacteria from paper machines. Based on one of them, strain K4.1T, I described a novel bacterial genus Deinobacterium with Deinobacterium chartae as the type species. I measured the transfer of Bacillus cereus spores from packaging paper into food. To do this, we constructed a green fluorescent protein (GFP) labelled derivative of Bacillus thuringiensis and prepared paper containing spores of this strain. Chocolate and rice were the recipient foods when transfer of the labelled spores from the packaging paper to food was examined. I showed that only minority of the Bacillus cereus spores transferred into food from packaging paper and that this amount is very low compared to the amount of B. cereus naturally occurring in foods. Thus the microbiological risk caused by packaging papers is very low. Until now, the biological function of cereulide for the producer cell has remained unknown. I showed that B. cereus can use cereulide to take up K+ from environment where K+ is scarce: cereulide binds K+ ions outside the cell with high affinity and transports these ions across cell membrane into the cytoplasm. Externally added cereulide increased the growth rate of cereulide producing strains in medium where potassium was growth limiting. In addition, cereulide producing strains outcompeted cereulide non-producing B. cereus in potassium deficient environment, but not when the potassium concentration was high. I also showed that cereulide enhances biofilm formation of B. cereus.

Taxonomy and phylogeny of the manna lichens and allied species (Megasporaceae)

This dissertation is focused on the taxonomy, phylogeny, and ecology of the vagrant, erratic and allied terricolous and saxicolous species of the genera Aspicilia A. Massal. and Circinaria Link (Megasporaceae), particularly those traditionally referred to as manna lichens . The group has previously been defined on the basis of few morphological characters. The phylogeny of the family Megasporaceae is inferred from the combined dataset of nuLSU and mtSSU sequences. Five genera Aspicilia, Circinaria, Lobothallia, Megaspora, and Sagedia are recognized. Lobothallia is sister of the four other genera, while Aspicilia and Sagedia form the next clade. All these genera have small asci with eight spores. Circinaria is a sister genus of Megaspora, and these two have in common asci with (1 4) 6 8 large spores. Circinaria forms a monophyletic group and sphaerothallioid species form a monophyletic group within Circinaria. The presence of certain morphological characters such as pseudocyphellae, thickness of cortex and medulla layers, as well as ecological differences in sphaerothallioid species distinguish it from some other crustose species, especially those containing aspicilin and characterised by thin cortex and medulla layers, conidium length c. 6 12 µm and absence of pseudocyphellae. If sphaerothallioid species are accepted as a distinct genus, the rest of the Circinaria species would remain as a paraphyletic assemblage. Currently, the genus Circinaria includes all the sphaerothallioid species and its generic position is confirmed and accepted. Thus, it is proposed as a correct generic name also for the manna lichens described originally in other genera. Phylogeny at the species level was studied using nrITS sequence data. Traditionally, morphological characters have been used for the recognition of species. They were re-evaluated in the light of molecular data. Since characters such as vagrant, erratic and crustose growth forms proved to be misleading for the recognition of some species, a combination of several characters (including molecular data) is recommended. Vagrant growth form seems to have evolved several times among the distantly related lineages and even within a single population. The reasons behind the high plasticity in the external morphology of the sphaerothallioid Circinaria remain, however, unknown. Six new species are recognized: Aspicilia tibetica, Circinaria arida, C. digitata nom provis., C. gyrosa nom. provis., C. rogeri nom. provis., and C. rostamii nom. provis. Based on an analysis of nrITS dataset, three new erratic, vagrant and crustose species were also recognized, but these require additional study. The results also reveal that C. elmorei and C. hispida are not monophyletic as currently understood. In addition, 13 new combinations in the genus Circinaria are proposed.