Cyanobacteria and their toxins in lichen symbiosis

Lichens are symbiotic associations between a fungus (mycobiont) and a photosynthetic partner (photobiont) which may be a green alga or cyanobacterium (cyanobiont). In lichen symbiosis the mycobiont lives on sugars photosynthesized by the photobiont and, in cyanobacterial symbiosis, also nitrogen compounds are provided to the fungal host. Several cyanobacterial genera are known to associate with lichen forming fungi but by far the most common cyanobacterial genus in lichen symbioses is Nostoc. Lichen-symbiotic Nostoc is a diverse group including at least two distinct phylogenetic lineages which tend to associate with different groups of lichen mycobionts. Microcystins and nodularins are small, cyclic, hepatotoxic peptides responsible for poisonings of humans and animals. They are produced by aquatic, bloom forming cyanobacteria of several different genera and found in fresh and brackish waters around the world. The previously known microcystin producers of the genus Nostoc include the lichen associated cyanobacterium Nostoc sp. IO-102-I isolated from Finland, and some aquatic strains from Brazil, Finland, and India. While all producers of nodularin were previously thought to belong to the genus Nodularia, it has recently been shown that also some Nostoc strains isolated from cycad roots can produce nodularin. The aim of this study was to find out which cyanobacterial toxins are produced in lichen symbiosis and how widespread this production is, both from the geographical and lichen-symbiotic perspective. In addition I wanted to broaden the knowledge on lichen-symbiotic cyanobacteria and symbiont selectivity in lichen symbiosis. The study was based on the analysis of over 800 cyanolichen specimens collected from different parts of the world, mainly analysed with molecular biological methods and liquid chromatography-mass spectrometry. The results show that hepatotoxic microcystins are produced in situ in lichen symbioses by symbiotic cyanobacteria, and that these compounds are produced quite commonly in many different lichen genera all around the world. Also nodularin is produced in some lichens. The cyanobacterial toxins may act as grazing deterrents and provide some protection to the thallus. However the actual consequences to grazers and the faith of the toxins in the food chain remain unknown. The chemical and genetic diversity of microcystin production in lichens was remarkable. The evolution of this diversity may be related to genetic bottlenecks that commonly occur during the lifecycle of symbiotically dispersing cyanobacteria and the concurrent close association with the fungal hosts. The presently known distribution of toxin-producing cyanobacteria in lichens was found to concentrate into certain taxonomic groups within the Lobariaceae, Nephromataceae, and Peltigeraceae (Peltigerales, Ascomycota). The diversity of microcystin structures correlated with the genetic identity of Nostoc symbionts in different lichens, but also geographical patterns seemed to exist. Symbiont selection in the lichen genus Nephroma was found to be more specific locally than globally, and the identity of the cyanobiont to differ between bi- and tripartite members of the genus.Jäkälä on usean eri eliön muodostama symbioosi, jonka pääosakkaina ovat sieni (mykobiontti) ja yhteyttävä fotobiontti, joka voi olla joko viherlevä, syanobakteeri (syanobiontti) tai joissakin tapauksissa molemmat. Jäkäläsymbioosissa sieniosakas saa energiansa fotobiontin yhteyttämistuotteista, minkä lisäksi syanobiontti tarjoaa mykobiontille myös typpiyhdisteitä. Jäkäläsymbioosissa esiintyy useita eri syanobakteerisukuja, mutta selvästi yleisin suku on Nostoc. Jäkäläsymbioottiset Nostoc-syanobakteerit ovat hyvin monimuotoinen ryhmä, joka sisältää ainakin kaksi toisistaan eroavaa kehityslinjaa. Sieniosakkaiden on useimmiten todettu valikoivan symbioosikumppaninsa tietystä rajatusta joukosta, joka vaihtelee eri jäkäläsieniryhmien välillä. Mikrokystiinit ja nodulariinit ovat pieniä, rengasrakenteisia, maksamyrkyllisiä peptidejä, joiden tiedetään aiheuttaneen useita ihmisten ja eläinten myrkytystapauksia. Mikrokystiinien tutuimpia tuottajia ovat usean eri suvun makeassa ja murtovedessä elävät ja kukintoja muodostavat syanobakteerit. Nostoc-sukuun kuuluvia tunnettuja mikrokystiinin tuottajia ovat suomalaisesta jäkälästä eristetty kanta Nostoc sp. IO-102-I, sekä vesiympäristöistä eristetyt brasilialainen, intialainen ja suomalainen kanta. Perinteisesti vain Nodularia-syanobakteerisuvun edustajien on ajateltu tuottavan nodulariinia, mutta hyvin äskettäin myös käpypalmun (Cycas) juuresta eristetystä Nostoc-kannasta löydettiin tätä myrkkyä. Tässä tutkimuksessa halusin selvittää tuotetaanko syanobakteerimyrkkyjä myös jäkäläsymbioosissa, sekä kuinka yleistä niiden tuotanto maantieteellisestä ja jäkäläsystemaattisesta näkökulmasta on. Tämän lisäksi halusin laajentaa tietämystä sekä jäkäläsyanobionteista yleensä, että jäkäläsymbioosin osakkaiden valinnan tarkkuudesta. Tutkimus perustuu yli 800 eri puolilta maailmaa kerättyyn syanojäkälänäytteeseen, joita on pääasiassa analysoitu molekyylibiologisin menetelmin sekä nestekromatografi-massaspektrometrin avulla. Tuloksemme osoittavat että symbioottinen syanobakteeri tuottaa maksamyrkyllisiä mikrokystiinejä myös jäkäläsymbioosissa, ja että näitä yhdisteitä esiintyy melko yleisesti eri puolilla maailmaa ja useissa eri jäkäläryhmissä. Lisäksi muutamista jäkälänäytteistä löydettiin nodulariinia. Havaitsemamme mikrokystiineihin liittyvä geneettinen ja kemiallinen monimuotoisuus oli huomattavaa. Syy monimuotoisuuden kehittymiseen saattaa liittyä symbioottisesti leviävän syanobakteerin elinkierrossa säännöllisesti esiintyvään geneettiseen pullonkaulailmiöön sekä samanaikaiseen läheiseen vuorovaikutussuhteeseen sieni-isännän kanssa. Myrkkyjä tuottavat syanobiontit vaikuttaisivat olevan yleisimpiä tietyissä heimojen Lobariaceae, Nephromataceae ja Peltigeraceae (Peltigerales, Ascomycota) ryhmissä. Mikrokystiinirakenteiden vaihtelu noudattaa pääasiassa Nostoc-symbionttien kahta kehityslinjaa, mutta myös maantieteellisiä eroja esiintyy. On mahdollista että syanobakteerimyrkyt toimivat karkotteina ja tarjoavat suojaa jäkälänsyöjiä vastaan, mutta näiden myrkkyjen todelliset vaikutukset jäkäliä syöviin eläimiin ja niiden kohtalo ravintoketjussa kaipaavat vielä lisätutkimuksia. Jäkäläsuvussa Nephroma syanobionttien havaittiin eroavan kaksi- ja kolmebionttisten jäkälälajien välillä, ja lisäksi symbiontin valinta oli löyhempää maailmanlaajuisessa mittakaavassa kuin paikallisesti

Specialist taxa restricted to threatened habitats contribute significantly to the regional diversity of Peltigera (Lecanoromycetes, Ascomycota) in Estonia

The widespread cyanolichen genus Peltigera comprises many insufficiently known poorly delimited and/ or undescribed species. Phylogenetic analysis of 252 Peltigera specimens from a wide range of habitat types in Estonia revealed 31 putative taxa (OTUs). Multivariate analysis revealed habitat-specific segregation between the Peltigera species along a gradient from humid eutrophic forests to dry oligotrophic forests and grasslands and along a soil pH gradient from alkaline soils of alvar grasslands to acidic soils of conifer forests. The diversity of Peltigera was the highest on roadsides and dunes and the lowest in alvar habitats which, however, supported the unique assemblage of undescribed Peltigera taxa. Deciduous broad-leaved forests, too, included several undescribed or rare and red-listed species. The results demonstrate that in Estonia many Peltigera species have narrow habitat requirements and are at present threatened by habitat loss and degradation.Peer reviewe

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.Peer reviewe

Use of Naturally Available Reference Targets to Calibrate Airborne Laser Scanning Intensity Data

We have studied the possibility of calibrating airborne laser scanning (ALS) intensity data, using land targets typically available in urban areas. For this purpose, a test area around Espoonlahti Harbor, Espoo, Finland, for which a long time series of ALS campaigns is available, was selected. Different target samples (beach sand, concrete, asphalt, different types of gravel) were collected and measured in the laboratory. Using tarps, which have certain backscattering properties, the natural samples were calibrated and studied, taking into account the atmospheric effect, incidence angle and flying height. Using data from different flights and altitudes, a time series for the natural samples was generated. Studying the stability of the samples, we could obtain information on the most ideal types of natural targets for ALS radiometric calibration. Using the selected natural samples as reference, the ALS points of typical land targets were calibrated again and examined. Results showed the need for more accurate ground reference data, before using natural samples in ALS intensity data calibration. Also, the NIR camera-based field system was used for collecting ground reference data. This system proved to be a good means for collecting in situ reference data, especially for targets with inhomogeneous surface reflection properties

Peltigera (Ascomycota) living in open and shady environments depend on different Nostoc photobionts

Species of Peltigera (lichen-forming Ascomycota) establish obligate symbioses with several monophyletic groups (phylogroups) of Nostoc cyanobacteria. Some of these fungi are strict specialists and only associate with one Nostoc phylogroup throughout their range, while others are generalists and associate with many different Nostoc phylogroups. We sampled 20 Peltigera taxa, mostly representing the Peltigera section Peltigera, from grassland and forest habitats across Europe. Mycobionts’ identities were confirmed using fungal ITS sequences. The Nostoc cyanobionts were identified and grouped based on single nucleotide differences in the trnL region and on the phylogenetic analysis of the rbcLX region. Our data confirmed that some Peltigera species clearly prefer open habitats, while others are largely confined to shady forest habitats. The two habitat preferences are consistently paired with association with different Nostoc groups. As a specific example, two ecologically versatile species, Peltigera canina and P. didactyla, both growing in open and shady habitats, consistently associated with different groups of Nostoc when growing in different habitats. As associations between specific mycobionts and cyanobionts are influenced by habitat conditions, the diversity of both taxon groups should be considered in the context of habitat management and conservation planning

Fossil Usnea and similar fruticose lichens from Palaeogene amber

Fruticose lichens of the genus Usnea Dill. ex Adans. (Parmeliaceae), generally known as beard lichens, are among the most iconic epiphytic lichens in modern forest ecosystems. Many of the c. 350 currently recognized species are widely distributed and have been used as bioindicators in air pollution studies. Here we demonstrate that usneoid lichens were present in the Palaeogene amber forests of Europe. Based on general morphology and annular cortical fragmentation, one fossil from Baltic amber can be assigned to the extant genus Usnea. The unique type of cortical cracking indirectly demonstrates the presence of a central cord that keeps the branch intact even when its cortex is split into vertebrae-like segments. This evolutionary innovation has remained unchanged since the Palaeogene, contributing to the considerable ecological flexibility that allows Usnea species to flourish in a wide variety of ecosystems and climate regimes. The fossil sets the minimum age for Usnea to 34 million years (late Eocene). While the other similar fossils from Baltic and Bitterfeld ambers cannot be definitely assigned to the same genus, they underline the diversity of pendant lichens in Palaeogene amber forests.Peer reviewe

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).Peer reviewe

Relationships between mycobiont identity, photobiont specificity and ecological preferences in the lichen genus Peltigera (Ascomycota) in Estonia (northeastern Europe)

We studied the genotype diversity of cyanobacterial symbionts in the predominately terricolous cyanolichen genus Peltigera (Peltigerales, Lecanoromycetes) in Estonia. Our sampling comprised 252 lichen specimens collected in grasslands and forests from different parts of the country, which represented all common Peltigera taxa in the region. The cyanobacteria were grouped according to their tRNA(Leu) (UAA) intron sequences, and mycobiont identities were confirmed using fungal ITS sequences. The studied Peltigera species associated with 34 different "Peltigera-type" Nostoc trnL genotypes. Some Peltigera species associated with one or a few trnL genotypes while others associated with a much wider range of genotypes. Mycobiont identity was the primary factor that determined the presence of the specific Nostoc genotype within the studied Peltigera thalli. However, the species-specific patterns of cyanobiont selectivity did not always reflect phylogenetic relationships among the studied fungal species but correlated instead with habitat preferences. Several taxa from different sections of the genus Peltigera were associated with the same Nostoc genotype or with genotypes in the same habitat, indicating the presence of functional guild structure in the photobiont community. Some Nostoc trnL genotypes were only found in the Peltigera species of moist and mesic forest environments, while another set of Nostoc genotypes was typically found in the Peltigera species of xeric habitats. Some Nostoc trnL genotypes were only found in the Peltigera taxa that are common on alvars and may have specialized to living in this unusual and threatened habitat type. (C) 2018 Elsevier Ltd and British Mycological Society. All rights reserved.Peer reviewe

Symbionts and changing environment: Lichen diversity and photobiont associations in tropical mountain ecosystems

Epiphytes comprise a significant component of biodiversity and biomass in tropical forests. They are ecologically important in intercepting and retaining moisture, providing habitat and food for invertebrates, and contributing fixed nitrogen into the ecosystem. Lichens are mutualistic symbioses between lichen-forming fungi (mycobionts) and algae and/or cyanobacteria (photobionts). Most lichen mycobionts are specific in their photobiont choice and the local availability of compatible photobionts may limit their ability to disperse into new habitats. The aims of this study are to 1) provide the first account of lichen symbiont diversity in tropical mountains, with focus on changes along topographic gradients, and 2) elucidate the effects of human induced environmental change to lichen symbiotic organisms, including the effects of expansion of agricultural and other disturbed ecosystems, and changing climate. The results will be a significant contribution to understanding tropical biodiversity since so far very few studies deploying modern molecular biological methods have included lichens from East Africa. Lichens, bryophytes, and free-living cyanobacteria and green algae, along the natural environmental gradient of the southern slope of Kilimanjaro including all main ecosystem types. The sampling is focused on study plots established by the KiLi project . The collected specimens will be studied microscopically, with chemical analyses, and molecular biology methods. So far we have sampled several plots within the natural savanna, maize fields, grassland, and Chagga homegardens (3–5 sampled plots each ecosystem type). The specimens have been studied microscopically. The preliminary results show, that clear differences exist in lichen biota between different plot types: lichen abundance seems to depend especially on presence/absence of woody plants, lichen species on the climate, and lichen diversity on substrate variability. In all studied plots lichens mainly occur epiphytically on shrubs and trees.Non peer reviewe