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.

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.

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).

Diversity and ecological adaptations in Palaeogene lichens.

Lichens are highly specialized symbioses between heterotrophic fungi and photoautotrophic green algae or cyanobacteria. The mycobionts of many lichens produce morphologically complex thalli to house their photobionts. Lichens play important roles in ecosystems and have been used as indicators of environmental change. Here we report the finding of 152 new fossil lichens from European Palaeogene amber, and hence increase the total number of known fossil lichens from 15 to 167. Most of the fossils represent extant lineages of the Lecanoromycetes, an almost exclusively lichen-symbiotic class of Ascomycota. The fossil lichens show a wide diversity of morphological adaptations that attached epiphytic thalli to their substrates, helped to combine external water storage with effective gas exchange and facilitated the simultaneous reproduction and dispersal of both partners in symbiosis. The fossil thallus morphologies suggest that the climate of European Palaeogene amber forests was relatively humid and most likely temperate.

Evolution of the tRNALeu (UAA) Intron and Congruence of Genetic Markers in Lichen-Symbiotic Nostoc.

The group I intron interrupting the tRNALeu UAA gene (trnL) is present in most cyanobacterial genomes as well as in the plastids of many eukaryotic algae and all green plants. In lichen symbiotic Nostoc, the P6b stem-loop of trnL intron always involves one of two different repeat motifs, either Class I or Class II, both with unresolved evolutionary histories. Here we attempt to resolve the complex evolution of the two different trnL P6b region types. Our analysis indicates that the Class II repeat motif most likely appeared first and that independent and unidirectional shifts to the Class I motif have since taken place repeatedly. In addition, we compare our results with those obtained with other genetic markers and find strong evidence of recombination in the 16S rRNA gene, a marker widely used in phylogenetic studies on Bacteria. The congruence of the different genetic markers is successfully evaluated with the recently published software Saguaro, which has not previously been utilized in comparable studies.

Alectorioid Morphologies in Paleogene Lichens: New Evidence and Re-Evaluation of the Fossil Alectoria succini Mägdefrau.

One of the most important issues in molecular dating studies concerns the incorporation of reliable fossil taxa into the phylogenies reconstructed from DNA sequence variation in extant taxa. Lichens are symbiotic associations between fungi and algae and/or cyanobacteria. Several lichen fossils have been used as minimum age constraints in recent studies concerning the diversification of the Ascomycota. Recent evolutionary studies of Lecanoromycetes, an almost exclusively lichen-forming class in the Ascomycota, have utilized the Eocene amber inclusion Alectoria succinic as a minimum age constraint. However, a re-investigation of the type material revealed that this inclusion in fact represents poorly preserved plant remains, most probably of a root. Consequently, this fossil cannot be used as evidence of the presence of the genus Alectoria (Parmeliaceae, Lecanorales) or any other lichens in the Paleogene. However, newly discovered inclusions from Paleogene Baltic and Bitterfeld amber verify that alectorioid morphologies in lichens were in existence by the Paleogene. The new fossils represent either a lineage within the alectorioid group or belong to the genus Oropogon.

The genetic basis for O-acetylation of the microcystin toxin in cyanobacteria.

Microcystins are a family of cyclic peptide toxins produced by cyanobacteria. They are responsible for the toxicosis and death of wild and domestic animals throughout the world. They display extensive variation in amino acid composition and functional group chemistry. O-acetylated microcystins are frequently produced by free-living and symbiotic strains of the genus Nostoc. Here, we show that the production of acetylated microcystins is catalyzed by an acetyl-coenzyme A-dependent O-acetyltransferase (McyL) encoded in the 57 kb microcystin synthetase gene cluster of Nostoc sp. 152. Phylogenetic analysis demonstrates that McyL belongs to a family of enzymes that inactivate antibiotics through O-acetylation. The McyL enzyme has a relaxed substrate specificity, allowing the preparation of semisynthetic microcystins. This study sheds light on the evolutionary origins and genetic diversity of an important class of enzymes involved in antibiotic resistance.

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.

Geographic mosaic of symbiont selectivity in a genus of epiphytic cyanolichens.

In symbiotic systems, patterns of symbiont diversity and selectivity are crucial for the understanding of fundamental ecological processes such as dispersal and establishment. The lichen genus Nephroma (Peltigerales, Ascomycota) has a nearly cosmopolitan distribution and is thus an attractive model for the study of symbiotic interactions over a wide range of spatial scales. In this study, we analyze the genetic diversity of Nephroma mycobionts and their associated Nostoc photobionts within a global framework. The study is based on Internal Transcribed Spacer (ITS) sequences of fungal symbionts and tRNA(L) (eu) (UAA) intron sequences of cyanobacterial symbionts. The full data set includes 271 Nephroma and 358 Nostoc sequences, with over 150 sequence pairs known to originate from the same lichen thalli. Our results show that all bipartite Nephroma species associate with one group of Nostoc different from Nostoc typically found in tripartite Nephroma species. This conserved association appears to have been inherited from the common ancestor of all extant species. While specific associations between some symbiont genotypes can be observed over vast distances, both symbionts tend to show genetic differentiation over wide geographic scales. Most bipartite Nephroma species share their Nostoc symbionts with one or more other fungal taxa, and no fungal species associates solely with a single Nostoc genotype, supporting the concept of functional lichen guilds. Symbiont selectivity patterns within these lichens are best described as a geographic mosaic, with higher selectivity locally than globally. This may reflect specific habitat preferences of particular symbiont combinations, but also the influence of founder effects.

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.

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.

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.