Cyanobacteria produce a high variety of hepatotoxic peptides in lichen symbiosis.

Lichens are symbiotic associations between fungi and photosynthetic algae or cyanobacteria. Microcystins are potent toxins that are responsible for the poisoning of both humans and animals. These toxins are mainly associated with aquatic cyanobacterial blooms, but here we show that the cyanobacterial symbionts of terrestrial lichens from all over the world commonly produce microcystins. We screened 803 lichen specimens from five different continents for cyanobacterial toxins by amplifying a part of the gene cluster encoding the enzyme complex responsible for microcystin production and detecting toxins directly from lichen thalli. We found either the biosynthetic genes for making microcystins or the toxin itself in 12% of all analyzed lichen specimens. A plethora of different microcystins was found with over 50 chemical variants, and many of the variants detected have only rarely been reported from free-living cyanobacteria. In addition, high amounts of nodularin, up to 60 µg g(-1), were detected from some lichen thalli. This microcystin analog and potent hepatotoxin has previously been known only from the aquatic bloom-forming genus Nodularia. Our results demonstrate that the production of cyanobacterial hepatotoxins in lichen symbiosis is a global phenomenon and occurs in many different lichen lineages. The very high genetic diversity of the mcyE gene and the chemical diversity of microcystins suggest that lichen symbioses may have been an important environment for diversification of these cyanobacteria.

Estimating the Phanerozoic history of the Ascomycota lineages: combining fossil and molecular data.

The phylum Ascomycota is by far the largest group in the fungal kingdom. Ecologically important mutualistic associations such as mycorrhizae and lichens have evolved in this group, which are regarded as key innovations that supported the evolution of land plants. Only a few attempts have been made to date the origin of Ascomycota lineages by using molecular clock methods, which is primarily due to the lack of satisfactory fossil calibration data. For this reason we have evaluated all of the oldest available ascomycete fossils from amber (Albian to Miocene) and chert (Devonian and Maastrichtian). The fossils represent five major ascomycete classes (Coniocybomycetes, Dothideomycetes, Eurotiomycetes, Laboulbeniomycetes, and Lecanoromycetes). We have assembled a multi-gene data set (18SrDNA, 28SrDNA, RPB1 and RPB2) from a total of 145 taxa representing most groups of the Ascomycota and utilized fossil calibration points solely from within the ascomycetes to estimate divergence times of Ascomycota lineages with a Bayesian approach. Our results suggest an initial diversification of the Pezizomycotina in the Ordovician, followed by repeated splits of lineages throughout the Phanerozoic, and indicate that this continuous diversification was unaffected by mass extinctions. We suggest that the ecological diversity within each lineage ensured that at least some taxa of each group were able to survive global crises and rapidly recovered.

New resinicolous Chaenothecopsis species from China.

Four new resinicolous species of Chaenothecopsis are described from China: Chaenothecopsis perforata from exudate of Rhus chinensis (Anacardiaceae), C. pallida from exudate of Ailanthus altissima (Simaroubaceae), C. resinophila from exudate of Kalopanax septemlobus (Araliaceae) and C. hunanensis from resin of Pinus massoniana (Pinaceae). All the new species are compared with previously described resinicolous mycocalicioid taxa, and several new features in these species are presented. The newly described species cannot always be distinguished by any single character, but they all possess unique combinations of morphological, chemical and ecological features. Several aspects in the ecology and evolution of boreal and tropical resinicolous species are briefly discussed.

Convergent evolution of [D-Leucine(1)] microcystin-LR in taxonomically disparate cyanobacteria.

BACKGROUND: Many important toxins and antibiotics are produced by non-ribosomal biosynthetic pathways. Microcystins are a chemically diverse family of potent peptide toxins and the end-products of a hybrid NRPS and PKS secondary metabolic pathway. They are produced by a variety of cyanobacteria and are responsible for the poisoning of humans as well as the deaths of wild and domestic animals around the world. The chemical diversity of the microcystin family is attributed to a number of genetic events that have resulted in the diversification of the pathway for microcystin assembly. RESULTS: Here, we show that independent evolutionary events affecting the substrate specificity of the microcystin biosynthetic pathway have resulted in convergence on a rare [D-Leu(1)] microcystin-LR chemical variant. We detected this rare microcystin variant from strains of the distantly related genera Microcystis, Nostoc, and Phormidium. Phylogenetic analysis performed using sequences of the catalytic domains within the mcy gene cluster demonstrated a clear recombination pattern in the adenylation domain phylogenetic tree. We found evidence for conversion of the gene encoding the McyA(2) adenylation domain in strains of the genera Nostoc and Phormidium. However, point mutations affecting the substrate-binding sequence motifs of the McyA(2) adenylation domain were associated with the change in substrate specificity in two strains of Microcystis. In addition to the main [D-Leu(1)] microcystin-LR variant, these two strains produced a new microcystin that was identified as [Met(1)] microcystin-LR. CONCLUSIONS: Phylogenetic analysis demonstrated that both point mutations and gene conversion result in functional mcy gene clusters that produce the same rare [D-Leu(1)] variant of microcystin in strains of the genera Microcystis, Nostoc, and Phormidium. Engineering pathways to produce recombinant non-ribosomal peptides could provide new natural products or increase the activity of known compounds. Our results suggest that the replacement of entire adenylation domains could be a more successful strategy to obtain higher specificity in the modification of the non-ribosomal peptides than point mutations.

The Lichen Genus Sticta (Lobariaceae, Peltigerales) in East African Montane Ecosystems.

The lichen flora of Africa is still poorly known. In many parts of the tropics, recent studies utilizing DNA methods have revealed extraordinary diversity among various groups of lichenized fungi, including the genus Sticta. In this study, East African Sticta species and their ecology are reviewed using the genetic barcoding marker nuITS and morphological characters. The studied regions represent montane areas in Kenya and Tanzania, including the Taita Hills and Mt. Kilimanjaro, which belong to the Eastern Afromontane biodiversity hotspot. Altogether 14 Sticta species are confirmed from the study region, including the previously reported S. fuliginosa, S. sublimbata, S. tomentosa, and S. umbilicariiformis. Sticta andina, S. ciliata, S. duplolimbata, S. fuliginoides, and S. marginalis are reported as new to Kenya and/or Tanzania. Sticta afromontana, S. aspratilis, S. cellulosa, S. cyanocaperata, and S. munda, are described as new to science. The abundance of new diversity detected and the number of taxa represented by only few specimens show that more comprehensive sampling of the region may be needed to reveal the true diversity of Sticta in East Africa. More generally, our results highlight the need for further taxonomic studies of lichenized fungi in the region.

Species identity and behavior of cave-dwelling tree hyraxes of the Kenyan coast.

We surveyed tree hyrax populations living in forests, limestone rocky formations, and caves in coastal Kenya to identify the species and estimate the threat-level populations are in. Tree hyrax vocalizations were recorded in three different habitats with passive acoustic monitoring (PAM) for a total of 84 h in January and February 2022. We also observed tree hyrax behavior with thermal imaging camera and photographed individuals. Tree hyraxes in coastal Kenya are vocally active throughout the night, with most calls emitted between 23.00 and 04.00. We identified four different calls: snort, hac, hac ping-pong, and wheeze. Their calling range is between 220 and 15,000 Hz. Calls of tree hyraxes from the coast of Kenya were compared with calls stored by the Oxford Brookes University's Nocturnal Primate Research Group and identified as eastern tree hyrax, previously recorded from Tanzania. Here, we present what are, to our knowledge, the first photographs of live D. validus from Kenya. These tree hyraxes live in social groups. Due to strong pressure from humans, conservation measures are necessary to prevent the extinction of these isolated D. validus populations in Kenya.

Chaenothecopsis (Mycocaliciales, Ascomycota) from exudates of endemic New Zealand Podocarpaceae.

The order Mycocaliciales (Ascomycota) comprises fungal species with diverse, often highly specialized substrate ecologies. Particularly within the genus Chaenothecopsis, many species exclusively occur on fresh and solidified resins or other exudates of vascular plants. In New Zealand, the only previously known species growing on plant exudate is Chaenothecopsisschefflerae, found on several endemic angiosperms in the family Araliaceae. Here we describe three new species; Chaenothecopsismatai Rikkinen, Beimforde, Tuovila & A.R. Schmidt, C.nodosa Beimforde, Tuovila, Rikkinen & A.R. Schmidt, and C.novae-zelandiae Rikkinen, Beimforde, Tuovila & A.R. Schmidt, all growing on exudates of endemic New Zealand conifers of the Podocarpaceae family, particularly on Prumnopitystaxifolia. Phylogenetic analyses based on ribosomal DNA regions (ITS and LSU) grouped them into a distinct, monophyletic clade. This, as well as the restricted host range, suggests that all three taxa are endemic to New Zealand. Copious insect frass between the ascomata contain ascospores or show an early stage of ascomata development, indicating that the fungi are spread by insects. The three new species represent the first evidence of Chaenothecopsis from any Podocarpaceae species and the first from any gymnosperm exudates in New Zealand.

Reconstruction of structural evolution in the trnL intron P6b loop of symbiotic Nostoc (Cyanobacteria).

In this study we reconstruct the structural evolution of the hyper-variable P6b region of the group I trnLeu intron in a monophyletic group of lichen-symbiotic Nostoc strains and establish it as a useful marker in the phylogenetic analysis of these organisms. The studied cyanobacteria occur as photosynthetic and/or nitrogen-fixing symbionts in lichen species of the diverse Nephroma guild. Phylogenetic analyses and secondary structure reconstructions are used to improve the understanding of the replication mechanisms in the P6b stem-loop and to explain the observed distribution patterns of indels. The variants of the P6b region in the Nostoc clade studied consist of different combinations of five sequence modules. The distribution of indels together with the ancestral character reconstruction performed enables the interpretation of the evolution of each sequence module. Our results indicate that the indel events are usually associated with single nucleotide changes in the P6b region and have occurred several times independently. In spite of their homoplasy, they provide phylogenetic information for closely related taxa. Thus we recognize that features of the P6b region can be used as molecular markers for species identification and phylogenetic studies involving symbiotic Nostoc cyanobacteria.

You eat what you find - Local patterns in vegetation structure control diets of African fungus-growing termites.

Fungus-growing termites and their symbiotic Termitomyces fungi are critically important carbon and nutrient recyclers in arid and semiarid environments of sub-Saharan Africa. A major proportion of plant litter produced in these ecosystems is decomposed within nest chambers of termite mounds, where temperature and humidity are kept optimal for the fungal symbionts. While fungus-growing termites are generally believed to exploit a wide range of different plant substrates, the actual diets of most species remain elusive. We studied dietary niches of two Macrotermes species across the semiarid savanna landscape in the Tsavo Ecosystem, southern Kenya, based on carbon (C) and nitrogen (N) stable isotopes in Termitomyces fungus combs. We applied Bayesian mixing models to determine the proportion of grass and woody plant matter in the combs, these being the two major food sources available for Macrotermes species in the region. Our results showed that both termite species, and colonies cultivating different Termitomyces fungi, occupied broad and largely overlapping isotopic niches, indicating no dietary specialization. Including laser scanning derived vegetation cover estimates to the dietary mixing model revealed that the proportion of woody plant matter in fungus combs increased with increasing woody plant cover in the nest surroundings. Nitrogen content of fungus combs was positively correlated with woody plant cover around the mounds and negatively correlated with the proportion of grass matter in the comb. Considering the high N demand of large Macrotermes colonies, woody plant matter seems to thus represent a more profitable food source than grass. As grass is also utilized by grazing mammals, and the availability of grass matter typically fluctuates over the year, mixed woodland-grasslands and bushlands seem to represent more favorable habitats for large Macrotermes colonies than open grasslands.

Habitat preferences, estimated abundance and behavior of tree hyrax (Dendrohyrax sp.) in fragmented montane forests of Taita Hills, Kenya.

We studied a previously almost unknown nocturnal mammal, an apparently undescribed species of tree hyrax (Dendrohyrax sp.) in the moist montane forests of Taita Hills, Kenya. We used thermal imaging to locate tree hyraxes, observe their behavior, and to identify woody plants most frequently visited by the selective browsers. We also documented acoustic behavior in forest fragments of different sizes. Data on calling type and frequency were analyzed together with lidar data to estimate population densities and to identify forest stand characteristics associated with large populations. Viable populations were found only in the largest forest fragments (> 90 ha), where tree hyraxes preferred most pristine forest stands with high, multilayered canopies. The estimated population sizes in smaller forest fragments were very limited, and hyraxes were heard to call only during late night and early morning hours, presumably in order to avoid detection. While we frequently recorded tree hyrax songs in the largest forest fragments, we almost never heard songs in the small ones. All remaining subpopulations of the Taita tree hyrax are under threat of human disturbance and further habitat deterioration. Conservation efforts should include protection of all remaining habitat patches, but also reforestation of former habitat is urgently needed.

Chaenothecopsis khayensis, a new resinicolous calicioid fungus on African mahogany.

The new species Chaenothecopsis khayensis (Ascomycota, Mycocaliciaceae) is described from Ghana, western Africa, on the resin of Khaya anthotheca and K. ivorensis. The species is distinctive in forming asci without crosiers and in possessing ascospores that are faintly longitudinally striate. Analysis of large subunit rDNA gene sequences positioned this species within a clade corresponding to the Mycocaliciales and identified its closest relative as Sphinctrina leucopoda. Chaenothecopsis khayensis occurs commonly on resin exuding from trees damaged by the larvae of the mahogany shoot borer (Hypsipyla sp.), and we discuss the possible ecological relationship between the fungus and these moths.

Woodpeckers can act as dispersal vectors for fungi, plants, and microorganisms.

Bird-mediated dispersal is presumed to be important in the dissemination of many different types of organisms, but concrete evidence remains scarce. This is especially true for biota producing microscopic propagules. Tree-dwelling birds, such as woodpeckers, would seem to represent ideal dispersal vectors for organisms growing on standing tree trunks such as epiphytic lichens and fungi. Here, we utilize bird natural history collections as a novel source of data for studying dispersal ecology of plants, fungi, and microorganisms. We screened freshly preserved specimens of three Finnish woodpecker species for microscopic propagules. Samples were taken from bird feet, and chest and tail feathers. Propagules were extracted using a sonication-centrifugation protocol, and the material obtained was studied using light microscopy. Diverse biological material was recovered from all specimens of all bird species, from all positions sampled. Most abundant categories of discovered biological material included bryophyte fragments, fungal spores, and vegetative propagules of lichens. Also, freshwater diatoms, bryophyte spores, algal cells, testate amebae, rotifers, nematodes, pollen, and insect scales were identified. The method developed here is applicable to living specimens as well, making it a versatile tool for further research. Our findings highlight the potential of bird-mediated dispersal for diverse organisms and showcase the use of natural history collections in ecological research.

Complex Interaction Networks Among Cyanolichens of a Tropical Biodiversity Hotspot.

Interactions within lichen communities include, in addition to close mutualistic associations between the main partners of specific lichen symbioses, also more elusive relationships between members of a wider symbiotic community. Here, we analyze association patterns of cyanolichen symbionts in the tropical montane forests of Taita Hills, southern Kenya, which is part of the Eastern Afromontane biodiversity hotspot. The cyanolichen specimens analyzed represent 74 mycobiont taxa within the order Peltigerales (Ascomycota), associating with 115 different variants of the photobionts genus Nostoc (Cyanobacteria). Our analysis demonstrates wide sharing of photobionts and reveals the presence of several photobiont-mediated lichen guilds. Over half of all mycobionts share photobionts with other fungal species, often from different genera or even families, while some others are strict specialists and exclusively associate with a single photobiont variant. The most extensive symbiont network involves 24 different fungal species from five genera associating with 38 Nostoc photobionts. The Nostoc photobionts belong to two main groups, the Nephroma-type Nostoc and the Collema/Peltigera-type Nostoc, and nearly all mycobionts associate only with variants of one group. Among the mycobionts, species that produce cephalodia and those without symbiotic propagules tend to be most promiscuous in photobiont choice. The extent of photobiont sharing and the structure of interaction networks differ dramatically between the two major photobiont-mediated guilds, being both more prevalent and nested among Nephroma guild fungi and more compartmentalized among Peltigera guild fungi. This presumably reflects differences in the ecological characteristics and/or requirements of the two main groups of photobionts. The same two groups of Nostoc have previously been identified from many lichens in various lichen-rich ecosystems in different parts of the world, indicating that photobiont sharing between fungal species is an integral part of lichen ecology globally. In many cases, symbiotically dispersing lichens can facilitate the dispersal of sexually reproducing species, promoting establishment and adaptation into new and marginal habitats and thus driving evolutionary diversification.

Diversity of Leptogium (Collemataceae, Ascomycota) in East African Montane Ecosystems.

Tropical mountains and especially their forests are hot spots of biodiversity threatened by human population pressure and climate change. The diversity of lichens in tropical Africa is especially poorly known. Here we use the mtSSU and nuITS molecular markers together with morphology and ecology to assess Leptogium (Peltigerales, Ascomycota) diversity in the tropical mountains of Taita Hills and Mt. Kasigau in Kenya and Mt. Kilimanjaro in Tanzania. The sampled habitats cover a wide range of ecosystems from savanna to alpine heath vegetation and from relatively natural forests to agricultural environments and plantation forests. We demonstrate that Leptogium diversity in Africa is much higher than previously known and provide preliminary data on over 70 putative species, including nine established species previously known from the area and over 60 phylogenetically, morphologically, and/or ecologically defined Operational Taxonomic Units (OTUs). Many traditional species concepts are shown to represent morphotypes comprised of several taxa. Many of the species were only found from specific ecosystems and/or restricted habitats and are thus threatened by ongoing habitat fragmentation and degradation of the natural environment. Our results emphasize the importance of molecular markers in species inventories of highly diverse organism groups and geographical areas.

Microbial Communities of Cladonia Lichens and Their Biosynthetic Gene Clusters Potentially Encoding Natural Products.

Lichens have been widely used in traditional medicine, especially by indigenous communities worldwide. However, their slow growth and difficulties in the isolation of lichen symbionts and associated microbes have hindered the pharmaceutical utilisation of lichen-produced compounds. Advances in high-throughput sequencing techniques now permit detailed investigations of the complex microbial communities formed by fungi, green algae, cyanobacteria, and other bacteria within the lichen thalli. Here, we used amplicon sequencing, shotgun metagenomics, and in silico metabolomics together with compound extractions to study reindeer lichens collected from Southern Finland. Our aim was to evaluate the potential of Cladonia species as sources of novel natural products. We compared the predicted biosynthetic pathways of lichen compounds from isolated genome-sequenced lichen fungi and our environmental samples. Potential biosynthetic genes could then be further used to produce secondary metabolites in more tractable hosts. Furthermore, we detected multiple compounds by metabolite analyses, which revealed connections between the identified biosynthetic gene clusters and their products. Taken together, our results contribute to metagenomic data studies from complex lichen-symbiotic communities and provide valuable new information for use in further biochemical and pharmacological studies.

Microcystin production in the tripartite cyanolichen Peltigera leucophlebia.

We show that the cyanobacterial symbionts of a tripartite cyanolichen can produce hepatotoxic microcystins in situ. Microcystins were detected with high-performance liquid chromatography mass spectrometry both from cephalodia of the tripartite cyanolichen Peltigera leucophlebia and from a symbiotic Nostoc strain isolated from the same lichen specimen. Genetic identities of symbiotic Nostoc strains were studied by amplifying and sequencing the 16S rRNA gene. Also, the presence of the microcystin synthetase gene mcyE was confirmed by sequencing. Three highly toxic microcystins were detected from the lichen specimen. Several different Nostoc 16S rRNA haplotypes were present in the lichen sample but only one was found in the toxin-producing cultures. In culture, the toxin-producing Nostoc strain produced a total of 19 different microcystin variants. In phylogenetic analysis, this cyanobacterium and related strains from the lichen thallus grouped together with a previously known microcystin-producing Nostoc strain and other strains previously isolated from the symbiotic thalloid bryophyte Blasia pusilla. Our finding is the first direct evidence of in situ production of microcystins in lichens or plant-cyanobacterial symbioses. Microcystins may explain why cyanolichens and symbiotic bryophytes are not among the preferred food sources of most animal grazers.

Caste-specific nutritional differences define carbon and nitrogen fluxes within symbiotic food webs in African termite mounds.

Fungus-growing termites of the genus Macrotermes cultivate symbiotic fungi (Termitomyces) in their underground nest chambers to degrade plant matter collected from the environment. Although the general mechanism of food processing is relatively well-known, it has remained unclear whether the termites get their nutrition primarily from the fungal mycelium or from plant tissues partly decomposed by the fungus. To elucidate the flows of carbon and nitrogen in the complicated food-chains within the nests of fungus-growing termites, we determined the stable isotope signatures of different materials sampled from four Macrotermes colonies in southern Kenya. Stable isotopes of carbon revealed that the termite queen and the young larvae are largely sustained by the fungal mycelium. Conversely, all adult workers and soldiers seem to feed predominantly on plant and/or fungus comb material, demonstrating that the fungal symbiont plays a different nutritional role for different termite castes. Nitrogen stable isotopes indicated additional differences between castes and revealed intriguing patterns in colony nitrogen cycling. Nitrogen is effectively recycled within the colonies, but also a presently unspecified nitrogen source, most likely symbiotic nitrogen-fixing bacteria, seems to contribute to nitrogen supply. Our results indicate that the gut microbiota of the termite queen might be largely responsible for the proposed nitrogen fixation.

Crustose lichens with lichenicolous fungi from Paleogene amber.

Lichens, symbiotic consortia of lichen-forming fungi and their photosynthetic partners have long had an extremely poor fossil record. However, recently over 150 new lichens were identified from European Paleogene amber and here we analyse crustose lichens from the new material. Three fossil lichens belong to the extant genus Ochrolechia (Ochrolechiaceae, Lecanoromycetes) and one fossil has conidiomata similar to those produced by modern fungi of the order Arthoniales (Arthoniomycetes). Intriguingly, two fossil Ochrolechia specimens host lichenicolous fungi of the genus Lichenostigma (Lichenostigmatales, Arthoniomycetes). This confirms that both Ochrolechia and Lichenostigma already diversified in the Paleogene and demonstrates that also the specific association between the fungi had evolved by then. The new fossils provide a minimum age constraint for both genera at 34 million years (uppermost Eocene).