Jouko Rikkinen

Discovery of Rare and Highly Toxic Microcystins from Lichen-Associated Cyanobacterium Nostoc sp. Strain IO-102-I

ABSTRACT The production of hepatotoxic cyclic heptapeptides, microcystins, is almost exclusively reported from planktonic cyanobacteria. Here we show that a terrestrial cyanobacterium Nostoc sp. strain IO-102-I isolated from a lichen association produces six different microcystins. Microcystins were identified with liquid chromatography-UV mass spectrometry by their retention times, UV spectra, mass fragmentation, and comparison to microcystins from the aquatic Nostoc sp. strain 152. The dominant microcystin produced by Nostoc sp. strain IO-102-I was the highly toxic [ADMAdda5]microcystin-LR, which accounted for ca. 80% of the total microcystins. We assigned a structure of [DMAdda5]microcystin-LR and [d-Asp3,ADMAdda5]microcystin-LR and a partial structure of three new [ADMAdda5]-XR type of microcystin variants. Interestingly, Nostoc spp. strains IO-102-I and 152 synthesized only the rare ADMAdda and DMAdda subfamilies of microcystin variants. Phylogenetic analyses demonstrated congruence between genes involved directly in microcystin biosynthesis and the 16S rRNA and rpoC1 genes of Nostoc sp. strain IO-102-I. Nostoc sp. strain 152 and the Nostoc sp. strain IO-102-I are distantly related, revealing a sporadic distribution of toxin production in the genus Nostoc. Nostoc sp. strain IO-102-I is closely related to Nostoc punctiforme PCC 73102 and other symbiotic Nostoc strains and most likely belongs to this species. Together, this suggests that other terrestrial and aquatic strains of the genus Nostoc may have retained the genes necessary for microcystin biosynthesis.

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.

Genetic Diversity of Nostoc Symbionts Endophytically Associated with Two Bryophyte Species

ABSTRACT The diversity of the endophytic Nostoc symbionts of two thalloid bryophytes, the hornwort Anthoceros fusiformis and the liverwort Blasia pusilla, was examined using the tRNALeu (UAA) intron sequence as a marker. The results confirmed that many different Nostocstrains are involved in both associations under natural conditions in the field. The level of Nostoc diversity within individual bryophyte thalli varied, but single DNA fragments were consistently amplified from individual symbiotic colonies. Some Nostocstrains were widespread and were detected from thalli collected from different field sites and different years. These findings indicate a moderate level of spatial and temporal continuity in bryophyte-Nostoc symbioses.

A new species of resinicolous Chaenothecopsis (Mycocaliciaceae, Ascomycota) from 20 million year old Bitterfeld amber, with remarks on the biology of resinicolous fungi

Chaenothecopsis bitterfeldensis sp. nov. is described and illustrated from Bitterfeld amber dating back to at least 20 million years ago. There has been no previous report of the sporing perfect stage of any Ascomycota in amber. The fungus also represents the first fossil record of resinicolous fungi and of the Mycocaliciaceae. The fossilised specimen contains over 20 fruiting bodies arising from an extensive mycelium. There are also hundreds of detached ascospores, some of which had germinated before the resin was transformed into amber. The taxonomy and palaeontology of the fossil fungus is discussed and stages in its preservation are described. Also some general aspects in the biology of resinicolous Mycocaliciaceae are reviewed. The striking similarity of C. bitterfeldensis to some extant species from East Asia suggests that the present distribution of these fungi is a relict of an ancient Laurasian range.

DNA barcoding: a tool for improved taxon identification and detection of species diversity

Recently it was decided that portions of rbcL and matK gene regions are approved and required standard barcode regions for land plants. Ideally, DNA barcoding can provide a fast and reliable way to identify species. Compiling a library of barcodes can be enhanced by the numerous specimens available in botanic gardens, museums and herbaria and in other ex situ conservation collections. Barcoding can strengthen ongoing efforts of botanic gardens and ex situ conservation collections to preserve Earth’s biodiversity. Our study aimed to detect the usability of the universal primers of the standard DNA barcode, to produce standard barcodes for species identification and to detect the discriminatory power of the standard barcode in a set of different groups of plant and fungal taxa. We studied Betula species originating from different parts of the world, and Salix taxa, bryophytes and edible and poisonous fungal species originating from Finland. In Betula and Salix, the standard DNA barcode regions, portions of matK and rbcL, were able to identify species to genus level, but did not show adequate resolution for species discrimination. Thus, supplementary barcode regions are needed for species identification. In Salix, the trnH-psbA spacer was also used, and it proved to have more resolution but, yet, not adequate levels of interspecific divergence for all studied taxa. In a set of bryophyte species, the rbcL gene region was found to possess adequate resolution for species discrimination for most genera studied. In bryophytes, matK failed to amplify properly. In fungi, the combination of ITS1 and ITS2 proved to be effective for species discrimination, although alignment difficulties were encountered. In general, closely related or recently diverged species are the greatest challenge, and the problem is most difficult in plants, both in terms of a suitable combination of barcoding regions and the universality of used primers.

Genetic diversity of green algal and cyanobacterial photobionts in Nephroma (Peltigerales)

Abstract Genetic diversity of green algal and cyanobacterial photobionts in Nephroma was examined by using nucleotide sequences of the ribosomal gene cluster. The lichens studied included both bipartite and tripartite species. There was very little variation in green algal-ITS sequences of N. arcticum and N. expallidum. Almost identical sequences were obtained from all thalli analysed and also from two tripartite Peltigera species. On the basis of SSU rDNA data the green algal photobionts of N. arcticum are closely related to the primary photobiont of P. britannica, and also to an endophytic alga of Ginkgo biloba. The SSU rDNA region of lichen-forming cyanobacteria was rather variable. A phylogenetic analysis indicated that the Nostoc specimens formed a monophyletic group and the strains were divided into two main groups. One clade included only cyanobionts of lichens, including those of all bipartite Nephroma species. The second group was genetically more heterogeneous and included mainly cyanobionts of terricolous cyanolichens, including those of both tripartite Nephroma species studied. The distinction between bi- and tripartite Nephroma species is significant as the mycobionts of tripartite species are not monophyletic. It implies that within Nephroma, evolutionary transitions between symbiosis types cannot have been achieved simply via an acquisition or loss of the green algal photobiont. As the Nostoc symbionts of bi- and tripartite species belong to different phylogenetic groups, an evolutionary change in green algal association has required a concurrent change in cyanobiont composition.

A phylogenetic study of Nephroma (lichen-forming Ascomycota)

The phylogeny of Nephroma was studied by nucleotide sequences of the mitochondrial ribosomal small subunit (mtSSU rDNA) and the internal transcribed spacers of the nuclear ribosomal repeat (ITS), together with chemical characters. The biological material included both bipartite and tripartite species and all Nephroma species native to northern Europe. Phylogenetic analyses demonstrated that all Nephroma species form a monophyletic group and that Peltigera constitutes the sister group to Nephroma. The two gene regions revealed qualitatively similar relationships within Nephroma and chemical characters had a minor impact on tree topologies. The results demonstrated that tripartite Nephroma species do not form a monophyletic group within the genus, this being in agreement with previous findings from bi- and tripartite Peltigera species. The results also indicated that N. resupinatum does not form a monophyletic group with all other bipartite Nephroma species but form a sister group to the studied Nephroma taxa. Furthermore, N. helveticum s. lat. is highly variable and seems to represent aggregates of closely related taxa. Also N. laevigatum and N. resupinatum are genetically variable. All European Nephroma species can be rapidly and accurately identified on the basis of their fungal ITS sequences. This will prove useful in ecological and environmental studies.

Fossilised Anzia (Lecanorales, lichen-forming Ascomycota) from European Tertiary amber

Anzia electra sp. nov. is described and illustrated from Baltic amber dating back approx. 40 Myr. The diagnostic features of the fossilised species include the small, narrow lobed foliose thallus, two-layered medulla, lack of chondroidal axis, moniliform spongiostratum, and rhizines born singly at the margins of spongiostratum cushions. The fossil species demonstrates that all distinguishing features in the thallus morphology of Anzia sect. Anzia have remained stable for tens of millions of years. Hence the divergence of anzioid lichens must have occurred in the distant past, most probably before the Tertiary. The fossil also provides the first hard evidence that the disjunct-Laurasian distributions of some modern lichens represent relicts of a formerly wider range. The close similarity of the fossil to extant species in East Asia and eastern North America suggests that the present range of Anzia sect. Anzia is highly relict.

Genotype variability of Nostoc symbionts associated with three epiphytic Nephroma species in a boreal forest landscape

Abstract We studied lichen photobiont diversity patterns of three epiphytic Nephroma species in a 900-ha boreal forest landscape using cyanobacterial tRNALeu (UAA) intron sequences. Our aim was to investigate if there was a link between lichen species identity, reproductive strategy, and photobiont selectivity. We show high photobiont specificity and selectivity within the forest landscape: only five closely related tRNALeu (UAA) intron genotypes were found from 232 Nephroma thalli. Two Nostoc genotypes were shared by N. bellum and N. resupinatum, while N. parile associated with two different genotypes. One genotype was only found from some specimens of N. resupinatum. On a single tree trunk all thalli of an individual lichen species usually housed the same photobiont strain, and the lichen species that mainly dispersed with fungal diaspores (N. bellum and N. resupinatum) usually shared identical photobionts. Both patterns were attributed to a founder effect presumably caused by relatively low colonization rates between trees. The photobiont spectrum of the symbiotically dispersing N. parile indicates that it maintained its own cyanobacterial symbionts. Our study shows that mycobionts of the diverse Nephroma guild do not exchange their cyanobacterial photobionts at random, and that reproductive strategy is reflected in species photobiont choice.