NKFI K_124341 "Global patterns to molecular problems in lichenology" [Globális mintázatoktól a molekuláris problémákig a lichenológia területén]

HU NKFI (NKFI K_124341, 2017 - 2022, Futó projekt)
A projekt fő vezetője: 
Dr. Farkas Edit témavezető
A projektet vezető intézmény: 
MTA ÖK Ökológiai és Botanikai Intézet
Rövid leírás: 

A környezeti problémák megoldásában jelentős zuzmó-együtteseket kutatjuk. A zuzmók között vannak bioindikációra alkalmasak, amelyek kipusztulásukkal jelzik a kedvezőtlen változásokat, mások elszaporodásukkal reagálnak ezekre a hatásokra. A zuzmók változatos színű, felépítésű, teleptestű élőlények. Legalább két különböző rendszertani helyzetű partner alkotja őket, a fotoszintézisre képes algákat vagy cianobaktériumokat gombafonalak veszik körül. Ehhez a biológiai változatossághoz járul hozzá az a kémiai sokféleség, amit kb.1000-féle speciális szerves, az élővilágban egyedülálló
zuzmóvegyület alkot. A speciálisan csak rájuk jellemző anyagaik kémiai azonosításán alapuló revíziójuknak eredményeként pontosabban azonosíthatók lesznek, illetve eddig ismeretlen kémiai változatokat fedezhetünk fel. Molekuláris genetikai vizsgálatokkal igazoljuk néhány hazánkból leírt faj taxonómiai helyzetét és fajon belüli változatosságát. Mindezeknek természetvédelmi szempontból is jelentősége van. Speciális mikroszkópi diagnosztikai módszert dolgozunk ki. UV-tűrő tesztfajokat választunk ki a globális környezeti vizsgálatok hatásainak vizsgálatához.

A rangos folyóirat-publikációkon túl távlati célként gazdagon illusztrált határozókönyv és elterjedési atlasz megjelentetése szerepel (részben kiegészítő forrásból), ami segíti a további kutatásokat és alkalmazott területeket az ökológiától a monitorozásig, illetve oktatási célokat szolgál, kiváló kiindulópont a lichenológia tudományát, illetve a biológiai és kémiai diverzitást megismerni vágyó fiatalok, egyetemisták számára.

Célok: 

Revíziós vizsgálatokat folytatunk, hogy a Közép-Európában/a Kárpáti régióban élő zuzmótaxonok pontos elterjedését és azok globális környezeti változásokkal összefüggő változását megállapíthassuk. Számos taxont vizsgálunk, ahol a zuzmóanyagoknak kiemelt szerepe van azonosításukban. A globális változások monitorozásához főként a leveles és bokros telepű zuzmók azonosítása, a bioindikációhoz pedig az apró kéregtelepű zuzmók ismerete jelentős.
A módszerek fejlődése lehetővé teszi, hogy a kromatográfiás módszerek (HPTLC, HPLC) alkalmazásával ki tudjuk mutatni ezeknek az anyagoknak a jelenlétét és mennyiségét. A másutt nem kutatott endemikus fajok példányain molekuláris genetikai vizsgálatokkal igazoljuk taxonómiai pozíciójukat és fajon belüli változatosságukat. Revideáljuk az 1994-ben megjelent határozókulcsot. Digitális illusztrációs anyagot állítunk össze, a fajok múlt és jelenlegi elterjedését térképen ábrázoljuk. Célunk, hogy az elterjedési típusok megállapításával a környezeti változásokhoz köthetően a különböző területek biodiverzitására vonatkozó predikciókat tehessünk. Szatellit kutatásként a zuzmóanyagok lokalizációjának flluoreszcenciamikroszkóppal történő vizualizálhatóságát kutatjuk. UV-szűrő pigment tartalmú tesztzuzmóval kutatjuk a sugárzás és a kémiai diverzitás kapcsolatát.
Az új kémiai változatokról, az európai és világjelentőségű zuzmó- és zuzmólakó gombataxonokról rangos folyóiratpublikációkat jelentetünk meg, összegző tanulmányokat készítünk a különböző elterjedés-típusokról, azok kialakulásának lehetséges magyarázatáról. Az eredmények szintézise könyvkéziratot eredményez, aminek megjelentetését további források bevonásával tervezzük.

Eredmények: 

Project report - NKFI K 124341
First year [01.09.2017_31.08.2018]
The part time contracts with assitant researcher fellows (Katalin Veres, Nóra Varga) and research assistants (Krisztina Szabó, Dóra Smahajcsik) were established.
The works on the project have been started according to the main aims.
Revisions
were started in various taxonomic groups (Cladonia – furcata/rangiformis, subulata/rei, pyxidata aggr., Lepraria – 12 species, Xanthoparmelia – usnic acid containing species, pokornyi/ryssolea and some other taxa) on the basis of morphology (LM) and lichen secondary chemistry. During the analysis of c. 800 specimens (from herbaria BP, DE, EGR, JPU, SZE, SZO, VBI) several (c. 50) lichen secondary substances known from earlier studies and 13 metabolites (LSMs) not extracted earlier from Hungarian lichen specimens were identified by HPTLC.
Posters has been presented on Lepraria [Farkas et al. 2017, 2018]. Papers on revisions of cetrelioid and leprarioid species are under preparation.
For a more detailed morphological investigation and analysis of localization of fluorescent lichen substances a modern SMZ18 NIKON stereo microscope was purchased. The digital camera unit is planned to be ordered in the second year of the project.
Rare and protected species
were investigated and their distribution studied. The results were presented as posters on protected species and Cladina species in region Balaton-felvidék and as a journal publication on Solorina saccata [Farkas et al.2017, Sinigla et al.2018a, b].
Further lichenicolous species were identified form Hungary and the Carpathian Basin. The manuscripts of journal publications are under preparation.
Results were presented
in Hungarian and international scientific meetings as posters.
Although it was planned, at last we had to make the decision not to participate in IMC11 congress because of its high costs, however our participation in the 2nd Lichen Genomics Workshop (Graz, Austria:2-5.11.2017) was considered very important and useful.
Molecular genetic studies were initiated.
Taxa and methodology for additional studies by molecular genetic methods were discussed. Analysis of Cladonia magyarica was selected as first priority. Since this species is legally protected in Hungary, permission for collection and scientific investigation were requested and received.
The background and plans of molecular genetic research was presented as oral presentation during 2nd Lichen Genomics Workshop [Farkas 2017]. First DNA extractions have been carried out in the Molecular Taxonomic Laboratory of the Hungarian Natural History Museum, Budapest. Specimens from lowland steppe and rocky grassland habitats were collected, samples of apothecial and sterile parts were selected for isolation.
Several DNA extraction methods and protocols were tested and compared. PCR reaction for ITS region was optimized and products were sequenced using the Sanger sequencing method. Further samples, genes and primers are going to be tested according to the most recent literature data to combine our previous results for a multigene analysis. The methods, applied parameters were consulted with colleagues R. Pino-Bodas and S. Stenroos in Finnish Museum of Natural History, Helsinki.
A field experiment was established
to justify the production of UV-protecting LSMs after acetone rinsing in the experimental field of the National Botanical Garden, Vácrátót (6 repeated investigations from 2018 to 2020 in each spring and autumn). The preliminary studies for this experiment have already been started and supported also by OTKA K81232. It was essential to know if the selected species Cladonia foliacea survives the acetone treatment [Farkas et al. 2017a, b, Veres et al. 2018]. Its vitality was analysed by chlorophyll fluorescence measurements (Fv/Fm values).
The first data show differences between control and acetone rinsed samples, as well as between samples collected from the lowland and the mountain site, however a further more detailed evaluation is necessary.
Currently a manuscript is under preparation on the results of chlorophyll fluorescence analysis (Fv/Fm measurements) of the preliminary studies.
We had 6 posters and 4 oral presentations in the following conferences:
XXth Symposium of Baltic Mycologists and Lichenologists, Gdansk, Poland, 25–29.09.2017
4th Conference on Cryptogams, Eger, Hungary, 1.11–30.12.2017
12th International Conference Advances in research on the flora and vegetation of the Carpato ‐ Pannonian region, Debrecen, Hungary, 23–25.02.2018
11. Magyar Ökológus Kongresszus [11th Congress of Hungarian Ecologists] Nyíregyháza, Hungary, 28–30.08.2018
2 papers have been published (Lőkös et al. 2018, Sinigla et al. 2018) and further 5 manuscripts are near to submission.

Project report - NKFI K 124341
Second year [01.09.2018_31.08.2019]
The works on the project has been continued according to the main aims.
Revisions based on HPTLC analysis of lichen secondary metabolites (LSMs) were continued and carried out in further groups (Xanthoparmelia – brown species not containing usnic acid; Cladonia chlorophaea group; various Cladonia species; Cladonia magyarica forms sensu Gallé). Manuscripts were prepared on earlier studied groups (Cetrelia, Lepraria). Additional specimens and fields were analysed: α-alectoronic and β-alectoronic, furthermore α- and β-collatolic acids were identified in Cetrelia chicitae applying anysaldehide for visualization of the TLC plate; comparative morphometric studies on the soredia of the 4 Cetrelia species were carried out, IUCN red list categories of the species were established. A form described by Gyelnik is lectotypified (Farkas et al. 2019, Cryptogamie, Mycologie – submitted, revised – minor revision).
Identification key for species of Lepraria s.l. was compiled.
Various Cladonia species (c. 180 specimens) collected in Hungary during the last decades were identified using HPTLC. Fresh collections from 34 coenological sampling sites (Mt. Bakony) containing Cladina species and urban collections (on roof of high buildings in Debrecen) of Cladonia and Xanthoparmelia specimens were analysed (Matus et al. 2018, 2019a, 2019b).
Albanian Cladonia species (152 specimens of 15 species) were identified as a contribution to a joint publication with Spanish colleagues. Short characterization of species was published with distribution maps for each species, chemical variability for many of the species was discussed (Burgaz et al. 2019).
Chemical diversity within Cladonia chlorophaea species group was unknown in Hungarian specimens– 5 species were differentiated: morphometric studies are carried out – based on parameters of primary thalli, podetia, soredia – to compare chemically different species.
Lichenicolous fungi living on Cladonia species were studied in details and several of them were identified for the first time from Hungary. Most of the lichenicolous fungi on frequent Cladonia species (e.g., C. pyxidata, C. foliacea) appear as black dots, macroscopically impossible to differentiate them though these species are taxonomically very diverse belonging to ascomycetes, basidiomycetes or hyphomycetes. Seven of the most frequent taxa (like Didymocyrtis sp., Lichenoconium sp and others) found on podetia or primary thallus were presented on a poster illustrating their characteristic propagules.
Brown Xanthoparmelia species contains stenosporic, divaricatic and gyrophoric acids and terpenoids in different combinations. The preliminary results of HPTLC studies suggest that the morphological characters might have priority in identifications over chemical differences during identification process.
Two posters were prepared on the above topics for the 18th Congress of European Mycologists by the titles "Morphologically similar, cryptic taxa of macrolichens of conservation importance in the Carpathian Basin" and "Cladoniicolous fungi in the Carpathian Basin". (Farkas et al. 2019, Varga et al. 2019).
New records of Solenopsora candicans, S. cesatii, S. grisea, S. marina, S. olivacea subsp. olbiensis and S. olivacea subsp. olivacea from calcareous rocks, and S. liparina from ultramafic rocks in the Balkan Peninsula and adjacent islands were recorded. Their distribution, ecology and key morphological features are discussed in a paper published together with Slovakian colleagues (Fackovcova et al. 2019). The taxa S. candicans, S. cesatii, S. liparina, S. marina and S. olivacea subsp. olbiensis are reported for the first time from Albania, and S. cesatii from Bulgaria due to our contribution.
Cyphelium, Brodoa collected in Transsylvanian alps „Kelemen havasok” ( Eastern Carpathians) were identified after HPTLC studies.
35 lichen species and 5 lichenicolous fungi were presented from a microclimatically special habitat – considered as a refugium for cryptogams - in Jablanica Mts. (North Macedonia) on a poster for 13th Symposium on the Flora of Southeastern Serbia and Neighboring Regions (Farkas et al. 2019).
Despite of the intensive floristical investigations of the Balkan Peninsula in the last decades, there are many undiscovered or unpublished taxa, especially among microscopical fungi invading lichen thalli. In higher altitudes in the alpine regions cryptogamic organisms like lichens are conspicuous both in diversity and biomass. Our results show that several lichenicolous taxa seem to be new for the Midžor region (Serbia) as it was presented on a poster prepared for the 7th Balkan Botanical Congress (Varga et al. 2018).
We decided on multigene approach for molecular studies of the planned taxa. Most probably next generation sequencing will not be available for our studies – there was insufficient information available about this method during the planning of this project. The role and possibility for application of PKS genes is also under consideration since it is not leading to equally useful results in all groups of lichens. However markers (nuc ITS, IGS, EF1a, Rpb2 partial) – including those for photobionts (originally not planned) ¬ were selected on the basis of relevant literature, primers were bought accordingly. PCR reactions of DNA isolated from Cladonia magyarica specimens of Hungarian lowland and mountain sites have been processed. DNA was isolated from Vezdaea species and Xanthoparmelia mougeotii, samples are ready for further investigations .
From the combined phylogenetic analysis of multi-locus sequence data of the Lecanoraceae including two nuclear protein-coding markers (RPB2 and RPB1), the internal transcribed spacer and a fragment of the mitochondrial small subunit, it was found that the originally monotypic eastern Asian genus Verseghya is positioned within the Verseghya-Lecidella-Pyrrhospora clade of the Lecanoraceae and includes one more taxon Verseghya thysanophora widely distributed in Northern Hemisphere. Validation of one name as Polyozosia perpruinosa Fröberg ex S. Y. Kondr., L. Lőkös et Farkas is also proposed (Kondratyuk et al. 2019).
For testing diagnostic possibilities for visualisation of LSMs, NIKON SMZ18 research stereo microscope with photographic unit has been bought and ready to work with.
LSM localization investigations have been started on common foliose and fruticose lichens. Microscopic studies were carried out on usnic acid and atranorin containing cortical layers of various Usnea and Cladonia species. Specimens with lichenicolous fungi were also included in the study.
A cooperation has been developed with Dr. László Márk (University of Pécs, Medical School) for further examinations by a more sensitive method MALDI-MS imaging. The work covering a great number of taxa will hopefully increase our chances for valuable publications.
The preliminary studies for the ongoing acetone rinsing experiment justified that the selected species Cladonia foliacea survives the acetone treatment. Its vitality was analysed by chlorophyll fluorescence measurements and found that the long-term tolerance of this species is higher than any other species checked earlier under more humid conditions (Farkas et al. 2019, submitted).
Significant seasonal differences were found in lichen secondary metabolites (usnic acid, fumarprotocetraric acid) analysed by HPTLC and HPLC-PDA between summer and winter collected thalli (Farkas et al. 2019, submitted).
Data of the first year basically show sligthly decreasing differences between control and acetone rinsed samples in the re-production of LSMs, while Fv/Fm values indicate good condition of the thalli, though results of fumarprotocetraric acid are less clear and further masurements (from samples of spring and autumn 2019) are necessary for the interpretations. First results are summarised in a short publication (manuscript under preparation).
The effect of seasonal variation and different micro-environmental conditions on the metabolic activity of five terricolous lichen species, representing different growth forms were explored in temperate semiarid grasslands. The results of two years chlorophyll fluorescence analysis and background parameters (air temperature, photosynthetically active radiation, soil temperature and vapour pressure deficit) were analysed and summarised in a detailed paper submitted recently (Veres et al. 2019, submitted). The results direct attention to the importance of investigating more than one species, representing different growth forms, to find the response of lichens to changing environment in space and time.
Relation of distribution and climatic parameters is investigated on selected species. Concerning to prediction of lichen distribution due to global climatic change a cooperation has been built within the institute – species were selected for further studies.
Submitted papers mentioned in the report and listed among publications are indicated with their status, pdf will be deposited in the repository "mtmt" upon acceptance.

Project report - NKFI K 124341
Third year [01.09.2019_31.08.2020]
The works on the project have been continued according to the main aims.
Laboratory work (HPTLC analysis of lichen secondary metabolites (LSMs), microscopical studies) were possible in limited time due to COVID-19 situation. Additional specimens and fields were analysed to improve former manuscripts.
Field studies, morphological and chemical investigations of brown Xanthoparmelia species were carried out to clarify our concept about the priority of morphological characters over differences in LSM production.
Morphometric studies are carried out to compare chemically different species within Cladonia chlorophaea species group.
New localities of Parmelia submontana in Hungary were discovered (abstract submitted for MMK7, 2020 – cancelled meeting), journal publication manuscript was prepared. Parmeliella triptophylla was revealed as new species to Hungary, new localities of further species (Cladonia mitis for Hungary, Multiclavula mucida and Xanthoparmelia protomatrae for Romania) were discovered (manuscript submitted/published – Papp et al. 2020).
20 numbers (61–80) of Lichenes Delicati Exsiccati Editae (LDEE5) containing collections from the Carpathians and various exotic sites were prepared and published in honour and memory of Dr Antonín Vězda born 100 years ago (Farkas 2020). A further 20 numbers (81–100) including specimens from natural and urban localities are compiled according to former plans, publication of LDEE6 is under preparation for Acta Bot. Hung. 63(1–2), 2021.
ITS sequences have been gained from DNA isolated from Vezdaea species, samples are studied for further sequences. A manuscript is under preparation.
LSM localization investigations have been carried out on common foliose and fruticose lichens. Microscopical studies were continued on usnic acid and atranorin containing cortical layers of various Usnea and Cladonia species. For detection of zeorin, Leucodermia leucomelos
was compared to Physcia and Phaeophyscia species by various methods and chemical reagents.
In a cooperation with Dr László Márk (University of Pécs, Medical School) terpenoid containing taxa (Peltigera spp., Pseudocyphellaria spp.) were analysed by MALDI-MS (manuscript under preparation).
The preliminary studies for the ongoing acetone rinsing experiment justified that the selected species Cladonia foliacea survives the acetone rinsing treatment (Farkas et al. 2020).
Significant seasonal differences were found in lichen secondary metabolites (usnic acid, fumarprotocetraric acid) analysed by HPTLC and HPLC-PDA between summer and winter collected thalli (Farkas et al. 2020).
According to the first results of the long-term light protection experiment, the LSMs might decrease the damaging effects of the radiation in the photosynthetic system of the symbiotic algae. It seems so, that the amount of usnic acid is increasing towards a saturation value at a supposedly lower level than the original control value. The re-production of fumarprotocetraric acid is uncertain under current experimental arrangements (abstract submitted for MMK7, 2020 – cancelled meeting; manuscript is under preparation). Samples were recollected from the experimental field after 2.5 and 3 years and prepared for HPLC analysis.
A paper on the results of two years chlorophyll fluorescence analysis and background parameters (air temperature, photosynthetically active radiation, soil temperature and vapour pressure deficit) was published (Veres et al. 2020).
Results on investigation of environmental variables affecting terricolous lichen communities on different scales in a duneland ecosystem were summarised (abstract submitted for MMK7, 2020 – cancelled meeting; a manuscripts is submitted (Lichenologist, major revision) and
another manuscript is under preparation – both by Veres et al.).
The distribution and habitat preference of three protected Cladonia species were compared in the Balaton Uplands region. It was established that while C. mitis is the most frequent, C. rangiferina has the narrowest requirements in a series of environmental variables (Sinigla et al. – Lichenologist, major revision).
The cryptogamic communities and the environmental conditions on flatroofs in the city of Debrecen (East Hungary) were studied (a publication is under preparation - major revision for Biologia Futura by Aszalósné Balogh et al. 2020).
Concerning to prediction of lichen distribution due to global climatic changes selected species and climatic and other environmental parameters are under study in a cooperation within the institute.
The current PI was accepted as a symposium organiser on “Progress in classical and genomic aspects of lichen secondary chemistry” for IAL9 international symposium, the major scientific meeting of the special field of lichenology (https://doity.com.br/ial9/blog/symposia).
Registration fee was transferred and accepted for the postponed meeting for 1–6 August 2021. (Fortunately air ticket and accommodation were not arranged yet for the 2020 meeting, therefore unnecessary costs were not spent.) A lecture was in preparation for IAL9, 2020 (cancelled meeting) on the development of analytical methods in the study of LSMs, abstracts of further 3 posters were also submitted for the meeting: 1) on the knowledge on extreme tolerance of Cladonia foliacea, a temperate continental grassland lichen species, 2) various and changing distribution patterns of lichens in the Carpathian Basin, 3) on the checklist of lichenicolous fungi in Hungary in preparation.
7 abstracts (1 lecture, 6 posters) were submitted for IAL9, 2020 (1+3) and MMK7, 2020 (3). Further planned participations in AFVK13, 2020 (currently planned for 2021), 2020 and 5th Conference on Cryptogams, 2020 (currently planned for 25–26 August 2021) were impossible either, because also these meetings were postponed.
Five of six earlier submitted papers have been published, one has been expected acceptance after minor revision since 18.05.2020, and seven further journal publications are under preparation.
Submitted papers mentioned in the report and listed among publications are indicated with their status, pdf will be deposited in the repository “mtmt” upon acceptance.

Kapcsolódó publikációk: 

2020

Farkas, E., Biró, B., Csintalan, Zs., Veres, K. (2020): Acetone rinsing tolerance of the lichen species Cladonia foliacea is considerable Lichenologist 52, 325-327

2019

Burgaz, A. R., Luna-González, S., Gutiérrez-Larruga, B., Pino-Bodas, R., Lőkös, L. & Farkas, E. (2019): Diversity of Albanian Cladoniaceae. Bot. Complut.43: pp. 15–40.
Fačkovcová, Z., Lőkös, L., Farkas, E. & Guttová, A. (2019): New records of the lichen genus Solenopsora A. Massal in the Balkan Peninsula and adjacent islands. Herzogia 32(1): pp. 101–110.