Meeting of the

TREBOUXIA-WORKING

group

Trieste, Italy, 26-28 September 2016

Organizers:

Lucia Muggia

Stefano Martellos

Mauro Tretiach

Sponsorship and co-organizers:

Department of Life Sciences

University of Trieste

and

SIMBIOLIQUEN GVA Project

Dir. Eva Barreno Universitat de València

ABSTRACTS

General Information: As lichenologists, phycologists, and anyone else

interested in improving our understanding diversity in the lichenised green

algal genus Trebouxia, you are very welcome to join our first Workshop. This

meeting is dedicated to discussing and clarifying molecular phylogenetic and

morphological approaches to better understand and delimit the diversity of this

important lichen photobiont genus.

The workshop will be held in the city of Trieste, NE Italy, close to Venice, on

26-28 September 2016. There will be no congress fee.

Registration is compulsory. Please, confirm your participation to the

organizers by 31st May 2016 (e-mail to: lmuggia@units.it) by indicating in your

e-mail your data (name, family name, institution) and the type of your

contribution if any (poster or oral presentation are welcome) together with a

brief abstract (up to 2000 digits, spaces included) by 30th June 2016. Early

registration and any abstract submission is appreciated, to facilitate the pre-

publication of an attractive program.

Venue: The Workshop will take place at the University of Trieste, Department

of Life Science, building M, main lecture room, Via Licio Giorgieri 10, I 34127

Trieste, Italy (see maps below). The Department will provide lab facilities and

other seminar rooms for any additional needs during the workshop. The venue

can be easily reached by 15-20 min from the city centre (bus line n. 17 or n.

4), or from the train station (bus line n. 17/).

Website: http://www.units.it, website: http://dsv.units.it/en.

Trieste: The city is located at the very edge of NE Italy on the Adriatic Sea,

close to the border with Slovenia. Trieste enjoys a submediterranean climate,

conditioned by the mild effects of the sea and a peculiar cold, dry wind

blowing from ENE (“Bora”). Bora is not uncommon in winter, with maximum

speed of 120-140 km/h. While visitors can expect Mediterranean weather in

September, with plenty of sunshine and high temperatures, Trieste does

experience a fair amount of rainfall throughout the year, so that every season

can experience drops of temperatures and rain.

PROGRAM

Monday 26th September:

9.00-10.30: Opening and THEME 1 – Diversity and molecular phylogenetics of Trebouxia in lichens

9.00-9.15: Opening. Lucia Muggia

9.15-9.45: About the lifestyle of Trebouxia algae. Ondrej Peksa , Tereza Řídká, Lucie Vančurová,

Zuzana Vaiglová Z, Pavel Škaloud

9.45-10.15: Lichen photobionts at high elevations of the hyperarid Atacama Desert. Sergio Pérez-Ortega, Carmen Ascaso, Octavio Artieda, Jacek Wierzchos

10.15-10.45: Biodiversity and distribution in Austral Trebouxia taxa focused on Patagonia and Antarctica. Ulrike Ruprecht

10.45-11.15: coffee break

11.15-11.45: Assessing diversity and patterns of association in Trebouxia. Mattew P. Nelsen

11.45-12.15: Inferring a phylogenetic hypothesis for Trebouxia to better characterize diversity in this important photobiont genus. Leavitt Steven

12.15-12.45: Photobiont Diversity.org: genetic diversity of lichen photobionts and related

organisms. Heath O’ Brien

12.45-13.45: lunch break

13.45-16.15: THEME 2 – Species delimitation and association patterns in Trebouxia

13.45-14.15: Assessing species boundaries in the lichen photobiont Trebouxia. Anna Sadowska-Deś,

Francesco Dal Grande, Thorsten H. Lumbsch, Andreas Beck, Imke Schmitt

14.15-14.45: ITS2 secondary structure and morpho-genetic groups of species in the genus

Trebouxia. Andreas Beck

14.45-15.15 Identification, association and coevolutionary patterns of the photobionts associated

with Protoparmelia s.str. Garima Singh, Francesco Dal Grande, Pradeep K Divakar, Jürgen Otte, Ana

Crespo, Imke Schmitt

15.15-15.45: Interdisciplinary approaches to assess diversity and coexistence in Trebouxiophyceae:

ultraestructural, genomic, molecular and functional analyses. Eva Barreno and the “Lichen

Symbiogenesis team”

15.45-16.15: Patterns in photobiont association in Cetraria aculeata. Fernando Fernandez-Mendoza

16.15-16.45: coffee break

16.45-19.00: THEME 3 – Physiology and cell biology of cultured Trebouxia and Asterochloris

16.45-17.15: Trebouxiophyceae: a cell biology perspective. Myriam Catalá, Eva Barreno

17.15-17.45: About the role of Plant hormones in Trebouxia: data and hypothesis. Pedro Carrasco, Ernesto Hinojosa-Vidal, Francisco Marco, Fernando Martínez-Alberola, Eva Barreno

17.45-18.15: How does Trebouxia prepare for anabiosis? A metabolic approach to desiccation

tolerance. Andres Sadowsky

18.15-18.45: Tolerance to abiotic stress in Trebouxia and Asterochloris lichen microalgae:

physiological standpoints. Francisco Gasulla, Pedro Carrasco, Eva Barreno

Remaining available time for general discussion.

20.00: Dinner (typical Trieste restaurant)

Tuesday 27th September:

9.00-13.00: THEME 4 – Genomic and NGS approaches to study Trebouxia diversity

9.00-9.30: Transcriptomic analysis of the lichen photobiont Trebouxia gelatinosa subjected to

dehydration and rehydration processes. Alice Montagner, Fabio Candotto Carniel, Marco Gerdol,

Elisa Banchi, Chiara Manfrin, Lucia Muggia, Alberto Pallavicini, Mauro Tretiach

9.30-10.00: Microalgal diversity inside a single lichen thallus revealed by HGS approaches (454

amplicon pyrosequencing assay). Patricia Moya, Arantxa Molins, Fernando Martínez-Alberola, Eva

Barreno

10.00-10.30: Exploring the possibility of multiple Trebouxia lineages in individual lichen thalli using

next-generation sequencing. Leavitt Steven

10.30-11.00: coffee break

11.30-11.30: Altitude and host genetic identity shape the algal microbiome of congeneric hosts: a

metabarcoding approach. Francesco Dal Grande, Miklós Bálint, Pradeep K Divakar, Jürgen Otte, Ana

Crespo, Imke Schmitt

11.30-13.00: THEME 5 – Morphological and phylogenetic species delimitation in Trebouxia

11.30-12.00: Morpho-genetic groups of species in the genus Trebouxia. Anna Voytsekhovich,

Andreas Beck

12.00-12.30: Species delimitation in Asterochloris, a close relative to the genus Trebouxia. Pavel

Škaloud, Ondrej Peksa, Jana Steinová, Tereza Řídka, Lucie Vančurová, Jiři Malíček

12.30-13.00: Time for general discussion.

13.00-14.00: lunch break

14.00-18.00 h: THEME 6 - Practical session: analyses of cultures. Andreas Beck, Pavel Škaloud and

Lucia Muggia will provide some cultured strain for microscopy observation. Discussion and exchange

of expertise of photobiont isolation and culture approaches will be the focus of this session.

Everyone who wish to share their own strain for microscopy analyses is welcome to bring them.

16.00-16.30: coffee break

16.30-18.00 h: THEME 6 - Practical session: analyses of cultures – Continue.

18.00-18.30: Time for general discussion.

19.30/20.00 h: Dinner (typical Trieste restaurant Scarpon)

Wednesday 28th September:

9.00-10.30: THEME 7 – Isolation and in vitro cultures of trebouxioid microalgae as crucial pieces for

species delimitation

9.00-09.30: Trouble with Trebouxia? Culture experiments and progress in understanding the

nutrient requirements of major symbiotic algae. Elfie Stocker-Wörgötter

9.30-10.00: Improved propagation method, rapid molecular identification and ultrastructural

characterization as a multidisciplinary approach for Trebouxia species delimitation. Patricia Moya,

Salvador Chiva, Arantxa Molins, Francisco García-Breijo, José Reig-Armiñana, Eva Barreno

10.00-10.30: Presentation of the website “symbioticgreenalage.com”. Lucia Muggia, Eva Barreno

10.30-11.00 h: coffee break

11.00-12.30: Schematic summary of the sessions, general discussion, project initiatives and

conclusions

Summarizing contributions by Lucia Muggia, Eva Barreno.

General discussion with open contributions from anybody else.

Outlooks for future, international project funding: Pedro Carrasco (Cost initiative and other European

funding) and open contributions from anybody else.

12.30-14.00: lunch break

THEME 1

Diversity and molecular phylogenetics of Trebouxia

in lichens

About the lifestyle of Trebouxia algae

Ondrej Peksa1, Tereza Řídká2, Lucie Vančurová2, Zuzana Vaiglová2, Pavel Škaloud2

1 The West Bohemian Museum in Pilsen, Kopeckého sady 2, CZ-30100 Plzeň, Czech Republic, email: opeksa@zcm.cz 2 Charles University in Prague, Faculty of Science, Department of Botany, Benátská 2, CZ-12801 Prague, Czech Republic

The detailed inventory of saxicolous lichens in central Europe revealed high diversity of Trebouxia photobionts and interesting differencies in a lifestyle of individual Trebouxia lineages. Many of them exhibit clear affiliation to specific environment and lichen community (guild). Their ecology seems to be a very good character useful in species delimitation. Moreover, the photobionts seem to get a various „degree of domestication“ – some Trebouxia lineages associate with sorediose lichens with high frequency, other live more likely in free-living state and associate with sexually reproducing lichen-forming fungi.

Lichen photobionts at high elevations of the hyperarid Atacama Desert Sergio PÉREZ-ORTEGA1, Carmen ASCASO2, Octavio ARTIEDA3, Jacek WIERZCHOS2 1Real Jardín Botánico (CSIC), Calle Claudio Moyano 1, E-28014 Madrid, Spain. 2Museo Nacional de Ciencias Naturales (CSIC), Calle Serrano 115-dpdo., E-28006 Madrid, Spain.

3Universidad de Extremadura, Avda. Virgen del Puerto, 2, E-10600 Plasencia, Spain The Atacama Desert, northern Chile is a hyperarid environment, where the scarce rainfall and high rate of evapotranspiration play primary roles in determining the presence of photosynthetic life. In addition to its extreme dryness, the Atacama holds records for the highest surface ultraviolet (UV) radiations and total solar irradiances ever measured on Earth, which have been reported from its high altitude “Altiplano” and pre-Andean area. We performed an elevational/climatic gradient survey (Altiplano-Monturaqui transit; AMT) in the Atacama Desert between 23º57’S/ 068º10’W and 23º04’S/ 067º28’W from c. 4500 m a.s.l. to c. 2850 m a.s.l. All rock boulders sampled for lichen colonization derived from Pliocene Patao and Tucucaro ignimbrite. Along the AMT (4500-2850 m), the mean annual precipitations range from ~160 mm/yr at the upper sites (ALTI, QD and MIS), to 27 mm/yr at the lower sites (TILO and MTQ). By means of the use of nrITS as barcode we identified the photobionts of all species of lichen-forming fungi found along the gradient. All species were associated with Trebouxia spp. In spite of the low diversity of lichen-forming fungi, at least four different Trebouxia lineages were found: 1) a lineage close to T. incrustata, 2) cf. T. vagua, 3) cf. T. cretacea, and 4) an undescribed lineage of photobionts so far found only in the Southern Hemisphere.

Biodiversity and distribution in Austral Trebouxia taxa focused on Patagonia and Antarctica Ulrike RUPRECHT University of Salzburg, Ecology & Evolution, Hellbrunnerstrasse 34, 5020 Salzburg, Austria Trebouxia as a widespread and cosmopolitan genus is an important photobiont partner of a high number of lichen species in the southern hemisphere. Several taxa are molecularly similar to the ones of the northern hemisphere, such as Trebouxia sp. URa3, a common species in the milder regions of Antarctica, which occurs also in Europe and the Arctic. However, there are more cosmopolitan taxa which are more heterogeneous and form subgroups in more extreme climate zones in Antarctica, e.g. T. jamesii. Some further distinct groups seem to be restricted to the southern hemisphere or are endemic to the most extreme areas in continental Antarctica, such as Trebouxia sp. URa1.

Assessing diversity and patterns of association in Trebouxia Mattew P. Nelsen Integrative Research Center - The Field Museum, Chicago, USA

Over the past 15+ years, the ITS has become the most-sequenced locus of Trebouxia algae. Sequencing efforts have revealed substantial diversity – far greater than was previously appreciated. Here we synthesize publicly available ITS sequence data and summarize our efforts to characterize OTU diversity, geographic richness and range of Trebouxia OTU’s, and patterns of association with lichen mycobionts. We assess how close we are as a community to capturing the diversity of Trebouxia algae, and emphasize research avenues that will further contribute to our understanding of this lineage of algae.

Inferring a phylogenetic hypothesis for Trebouxia to better characterize diversity in this important photobiont genus Steven LEAVITT Herbarium of Non-vascular Cryptogams, Department of Biology, Brigham Young University, 4143 LSB, Provo, UT 84602, 801 422-4879

The algal genus Trebouxia is comprised of diverse symbiotic algae, representing perhaps the most commonly associated lichen photobiont. Research into this important algal genus has provided vital perspective into symbiotic interactions in lichens, and symbiosis in general. However, in spite of the central role of Trebouxia in many lichen symbioses, a robust phylogenetic hypothesis of relationships has not yet been reconstructed for the genus. In this study, we assembled Trebouxia sequence data from over 1000 specimens (lichens and pure algal cultures) representing the currently known phylogenetic diversity in the genus. We generated sequence data from the nuclear ribosomal ITS, a fragment of the protein-coding rbcL chloroplast gene, and a fragment of the mitochondrial COX2 gene. Our phylogenetic reconstructions support four major clades previously recognized in Trebouxia and the monophyly of many of the candidate species level lineages. This study provides a key foundation for advancing our understanding of evolutionary processes, patterns of species interactions, and species delimitation in Trebouxia.

PhotobiontDiversity.org: genetic diversity of lichen photobionts and related organisms Heath O'BRIEN Cardiff University School of Medicine, Haydn Ellis Building, Maindy Road, Cardiff, CF24 4HQ Molecular phylogenetics has revolutionised our understanding of the diversity of Trebouxia and other lichen-associated algae over the last 20 years by revealing cryptic diversity that is belied by the simple, reduced morphology of these unicellular microbes. However, formal taxonomic classification has not kept pace with these new insights. This has given rise to a situation where various informal names have been assigned to the clades that emerge from phylogenetic analyses. Unfortunately, these names are not always applied consistently in different studies, which usually include only a small subset of sequences from earlier studies as reference points. This makes it difficult to compare results among studies and has resulted in contradictory conclusions being drawn about host specificity and geographic ranges of lichen associated algae simply because different levels of phylogenetic resolution have been examined. PhotobiontDiversity.org is an effort to rectify this situation by assembling a database of all available sequence data from lichen associated algae and cyanobacteria, along with geographic and host association information and to develop a unified phylogenetic framework and user interface that will provide consistent measures of phylogenetic diversity across disparate studies and enhance our understanding of the ecological forces shaping the genetic diversity of these enigmatic organisms.

THEME 2

Species delimitation and association patterns in

Trebouxia

Assessing species boundaries in the lichen photobiont Trebouxia Anna SADOWSKA-DEŚ1,2, Francesco DAL GRANDE2, H. Thorsten LUMBSCH3, Andreas BECK4,5, Imke SCHMITT1,2 1Department of Biological Sciences, Institute of Ecology, Evolution and Diversity, Goethe Universität, Max-von-Laue-Str. 13, D-60438 Frankfurt, Germany 2Biodiversity and Climate Research Centre (BiK-F), Senckenberg Gesellschaft für Naturforschung, Senckenberganlage 25, D-60325 Frankfurt, Germany 3 Science & Education, The Field Museum, 1400 S. Lake Shore Drive, Chicago, IL 60605, USA 4Department of Lichenology and Bryology, Botanische Staatssammlung München, Menzinger Straße 67, D-80638 München, Germany 5GeoBio-Center, Ludwig-Maximilians Universität München, Richard-Wagner-Str. 10, D-80333 München, Germany The objective of this work was to assess the species boundaries in the trebouxioid photobionts of the lichen-forming fungus Lasallia pustulata. We applied a multifaceted approach based on coalescent methods, phylogenetic relationships, and morphological evidence. Additionally, we analyzed species-to-species and species-to-community interactions based on Trebouxia putative species. Our investigation revealed that L. pustulata is associated with several species of Trebouxia. These algae may also associate with other lichen-forming fungi that exhibit various ecological requirements. The species delimitation method here presented is a promising tool to disentangle species diversity in the genus Trebouxia.

ITS2 secondary structure and morpho-genetic groups of species in the genus Trebouxia Andrea BECK Department of Lichenology and Bryology, Botanische Staatssammlung München, Menzinger Str. 67, 80638 München, Germany

ITS2 secondary structures are a prominent and useful tool in taxon delimitation analyses in many groups of organisms. Here, it is demonstrated that these kinds of investigations are also helpful in delimitating species in Trebouxia and mainly add further support for the four morpho-genetic groups of species in the genus Trebouxia: arboricola-group including T. aggregata, T. arboricola, T. asymmetrica, T. crenulata, T. cretacea, T. decolorans, T. gigantea, T. jamesii, T. incrustata, T. showmanii, T. solaris, T. vagua; impressa-group including T. anticipata, T. gelatinosa, T. impressa, T. flava, T. potteri; corticola-group including T. corticola, T. galapagensis, T. higginsiae, T. usneae; and simplex-group including T. angustilobata, T. australis, T. brindabellae, T.simplex,T. suecica. These groups correspond also to individual, highly supported clades according to molecular phylogenetic analyses based on ITS and partial SSU nuclear rDNA data (Beck 2002; Helms 2003; Doering & Piercey-Normore 2009; Voytsekhovich & Beck 2015).

Identification, association and coevolutionary patterns of the photobionts associated with Protoparmelia s.str. Garima SINGH1,2, Francesco DAL GRANDE2, Pradeep K. DIVAKAR3, Jürgen OTTE2, Ana CRESPO3, Imke SCHMITT1,2 1Department of Biological Sciences, Institute of Ecology, Evolution and Diversity, Goethe Universität, Frankfurt am Main, Germany 2Senckenberg Biodiversity und Climate Research Centre, D-60325 Frankfurt, Germany 3Departamento de Biologia Vegetal II, Facultad de Farmacia, Universidad Complutense, Plaza de Ramón y Cajal s/n, Ciudad Universitaria, E-28040 Madrid, Spain. Species diversity and association patterns of symbiotic systems are a result of the interplay between evolutionary and environmental forces. Here we studied how macroclimate and evolution may shape the symbiont diversity and association patterns in Protoparmelia s.str., a small cosmopolitan genus of about 30 species occupying different macroclimates. We generated the multi-locus phylogenies followed by coalescent-based species delimitation of Protoparmelia s.str. and their green algal symbionts Trebouxia. Species delimitation approaches indicated that 23 Protoparmelia species associate with 20 Trebouxia species. We found that one-to-one fungal-algal relationships are more common in warmer climates, whereas one-to-many relationships are more common in cooler climates. Coevolutionary analyses suggest congruent fungal-algal phylogenies. We did not find any evidence for cospeciation even in highly selective associations. Instead host switch is a common evolutionary event in warm climates, whereas failure of the photobiont to diverge with its fungal host is a frequent event in cooler climates. We conclude that both fungus and alga displayed higher selectivity in the warmer regions as compared to the cooler regions and that different coevolutionary forces drive fungal-algal associations in different macrohabitats.

Interdisciplinary approaches to assess diversity and coexistence in Trebouxiophyceae: ultraestructural, genomic, molecular and functional analyses.

Eva BARRENO and “Lichen Symbiogenesis team”

Universitat de València, Inst. “Cavanilles” de Biodiversidad y Biología Evolutiva, Botánica, Fac. CC. Biológicas, Valencia, 46100 Burjassot, Spain.

Lichen thalli are complex biosystems resulting from cyclical symbiotic associations between different organisms (mycobiont, photobionts, bacteria, yeasts) integrated both morphologically and metabolically. The understanding of such complexity requires the implementation of diverse methodologies. A common objective of our research team is to study the identity, genetic diversity and functional features of lichen phycobionts and its role in the complexity of the thalli to gain insights on the knowledge of lichen symbiosis. Interdisciplinary approaches integrating ultrastructural, genomic, molecular and functional analyses have been used to investigate the huge diversity of phycobionts, especially those of the trebouxiophyceae, in both lichenized and cultured states. The combination of several molecular markers as well as ultrastructural techniques (TEM and CM), both in culture and in symbiotic states, should be utilized to add precision to the phylogenetic analyses and the circumscription of trebouxiophyceae taxa, as exemplified in the case of those microalgae found in Ramalina fraxinea or the new taxon Asterochloris mediterranea. We reported, for the first time, the coexistence lichens, through the design of new molecular markers (chloroplast, nuclear, mitochondrial). Now, this phenomenon has been detected in several lichens -even with the co-occurrence of different genera. Further, the use of 454 amplicon pyrosequencing assays allowed us the detection of 33 OTUs inside a single thallus of Ramalina farinacea, opening new perspectives on the adaptive mechanisms that provide the biological success of lichen symbioses. Lichen microalgae are poikilohydric organisms able to cope with diverse abiotic stresses. To understand the mechanisms involved in desiccation tolerance and other inducible responses against different stresses (light, ROS, Pb, oxidative pollutants, extreme temperatures, etc.) we have used as models Asterochloris erici, Trebouxia sp. TR9 and T. jamesii. The results of these investigations are summarized in several communications of Theme 3. The scarcity of genomic information about Trebouxia algae prompted us to generate a survey of the genomic sequences of Trebouxia sp. TR9 by NGS (454 and Solexa) using DNA and RNA. The assemblage of the DNA obtained sequences from the chloroplast, mitochondrial and nuclear genomes resulted in sizes of more than 300 Kb, 70 Kb and 59 Mb, respectively. Ab initio gene predictions calculated 9,500 nuclear genes. Gene annotations based on RNAseq data produced 13,887 genes and 21,076 isoforms. Results showed the absence of genome reductions and suggested the existence of overlapping UTRs. This study provides, for the first time, the organelle genomes and the draft nuclear genome of a Trebouxia species and may shed light on evolutionary trends in Trebouxiophyceae. (GVA_PROMETEOII/2013/021; MINECO_CGL2012-40058-C02-01; FEDER)

THEME 3

Physiology and cell biology of cultured

Trebouxia and

Asterochloris

Trebouxiophyceae: a cell biology perspective Myriam CATALÁ1,2, Eva BARRENO3

1 Department of Biology and Geology, Physics and Inorganic Chemistry, Higher School of Science and Technology, Rey Juan Carlos University, C/ Tulipán s/n E-28933 Móstoles, Madrid, Spain 2Environmental Toxicology Area, National Centre of Environmental Health, National Institute of Health Carlos III, Ctra. Majadahonda – Pozuelo E-28220 Majadahonda (Madrid) 3 ICBIBE, Dpto. Botánica y Geología, Universitat de València, Facc. CC.Biológicas, C/ Dr. Moliner 50. 46100-Burjassot (Valencia), Spain. Lichens are complex biological entities. The use of genomic and molecular taxonomy tools has revealed their complex taxonomy and ecology, especially regarding the mycobiont. However, the understanding of lichens as living beings also needs the thoughtful study of the photosynthetic symbionts whose individual characteristics determine species ecophysiology. Cellular Theory states that cells are the most basic functional and morphological unit of life. This perspective needs a specific focus on individual cells and the integration of a plethora of research techniques. From cell isolation to electron microscopy or high throughput technologies, cell biology combines routinely almost every biological tool to grasp a global idea of cell complexity. With the conviction that all eukaryotic cells share fundamental morphological, physiological and biochemical traits due to a common evolutionary origin, our team postulated a key role of NO in lichen cell communication (intra and interspecific) associated with abiotic stress tolerance. Our studies in the model species Ramalina farinacea have confirmed a key role of NO in lichen rehydration for modulation of lipid peroxidation damage using in vivo confocal microscopy with functional intracellular probes. We have demonstrated that trebouxiophyceae phycobionts also release NO despite they depend on the mycobiont’s. We have reported the physiological strategies against oxidative stress of the two different microalgae coexisting in R. farinacea providing phenotypical plasticity in different environments and demonstrated that thallus dependence on NO and oxidative stress response upon rehydration relies on the predominant phycobiont. The isolation and culture of Trebouxia and Asterochloris strains point to complex population dynamics with aggregation patterns probably related with reproduction strategies. Finally, using enzyme inhibitors of animal and plant NO generating enzymes we are revealing NO synthesis mechanisms in the heterogeneous thalli. (GVA, PROMETEOII/2013/021; MINECO, CGL2012-40058-C02-01; FEDER)

About the role of Plant hormones in Trebouxia: data and hypothesis Pedro CARRASCO1, Ernesto HINOJOSA2, Francisco MARCO3, Fernando MARTINEZ-ALBEROLA2, Eva BARRENO2

2Universitat de València, Inst. “Cavanilles” de Biodiversidad y Biología Evolutiva, Botánica, Fac. CC. Biológicas, Valencia, 46100 Burjassot, Spain. 3Universitat de València, Dept. Biología Vegetal, Fac. CC. Biológicas, Valencia, 46100 Burjassot, Spain. 1Universitat de València, Dept. Bioquímica y Biología Vegetal, Fac. CC. Biológicas, Valencia, 46100 Burjassot, Spain. Tolerance to stress in plants is a coordinated response of multiple stress-related genes, which also cross-talk with other components of stress-signaling transduction pathways. Abscisic acid (ABA) is the critical phytohormone that controls a series of downstream stress responses and integrates signaling from saline, thermal and drought stress conditions. Hormones like salicylic acid (SA) or indolacetic acid (IAA) are also involved in signal transduction and communication among cells in plants. Lichens are entities resulting from cyclical symbiotic associations between different organisms (fungi, algae and bacteria) intertwined both morphologically and metabolically. In Ramalina farinacea two Trebouxia taxa (T. jamesii and T. sp. TR9) coexist. These algae are able to withstand severe abiotic stresses from aerial environment, including osmotic and oxidative stress due to desiccation. In the genome of Trebouxia sp. TR9, sequenced and annotated by our group, we have identified genes directly implicated in ABA biosynthesis and ABA-mediated responses, IAA and SA biosynthesis and signaling. We have determined by mass spectrometry the endogenous concentrations of these phytohormones in Trebouxia TR9, under control and salt stress conditions. On the other hand, our results do not evidence the presence of another plant hormone, jasmonic acid (JA), mostly involved in plant responses to biotic stress. We used mass spectrometry to measure ABA, IAA and SA levels under a wide range of salt concentrations. Respect to ABA, although involved in salt response, our results seem to point to a completely different behavior between land plants and Trebouxia sp. TR9. About the role of IAA and SA, we suggest that both hormones could be involved in signal transduction from T. sp. TR9 to other symbiotic organisms in the complex lichen thalli. (GVA_PROMETEOII/2013/021; MINECO_CGL2012-40058-C02-01; FEDER)

Tolerance to abiotic stress in Trebouxia and Asterochloris lichen microalgae: physiological standpoints Francisco GASULLA1, Pedro CARRASCO2, Eva BARRENO1

1Universitat de València, Inst. “Cavanilles” de Biodiversidad y Biología Evolutiva, Botánica, Fac. CC. Biológicas, Valencia, 46100 Burjassot, Spain. 2Universitat de València, Dpto. Bioquímica y Biología Vegetal, Fac. CC. Biológicas, Valencia, 46100 Burjassot, Spain. Lichen microalgae are poikilohydric organisms able to undergo under diverse abiotic stresses like salt, light irradiance, heavy metals, etc., including a continuous series of desiccation-rehydration cycles. Each rehydration cycle causes a reactive oxygen species (ROS) burst, contributed greatly by microalgae’ photosynthetic machinery. However little is known about the induction of cellular responses in these phycobionts. The lichen phycobiont Asterochloris erici is able to survive rapid desiccation through several constitutive protection mechanisms, such as a powerful antioxidant system, late embryogenesis abundant proteins and others. However, we have demonstrated that desiccation also triggers physiological responses that contribute to anhydrobiosis adaption, among them the activation of an alternative energy dissipation mechanisms in the photosystem II or the modification of lipid membrane composition. In these induced responses the phospholipase D pathway seems to play a key role through the activation of mitogen activated protein kinases cascades and the subsequent phosphorylation of proteins. To understand the mechanisms involved in desiccation tolerance and other inducible responses against different stresses (light, ROS, Pb, oxidative pollutants, extreme temperatures, etc.) we have used as models the lichen Ramalina farinacea and its isolated phycobionts, Trebouxia sp. TR9 and T. jamesii (TR1), which are coexisting in the thalli. These algae show distinct physiological responses to acute photooxidative stress (through the ROS propagator CuHP), irradiance or incubation with Pb. TR9 exhibits lower decay in photosynthesis and photosynthetic pigments than jamesii. Similarly, antioxidant enzymes like glutathione reductase, superoxide dismutase, ascorbate peroxidase and catalase were only induced in TR9, and the stress-related protein HSP70 decreased in TR1 but increased in TR9. On the other hand, TR9 immobilized Pb extracellularly while the intracellular accumulation was three times higher in TR1. The better physiological performance of TR9 under oxidative conditions may reflect its greater capacity to undertake key metabolic adjustments, including increased non-photochemical quenching, higher antioxidant protection and the induction of repair mechanisms. We also find out remarkable differences in composition, structure and physicochemical features of the cell walls and EPS between TR1 and TR9 that could account for the differential responses to stress conditions. Lastly, to investigate the genetic basis of other physiological features of this algal taxon, we generated a survey of the genomic sequences of Trebouxia sp. TR9 by NGS, and detected several genes that can support CO2 concentrating mechanisms. To corroborate this surveillance 13C discrimination experiments were performed. The 13C discrimination values calculated in TR9 were similar to C4 plants (-15.84) and in TR1 to those of C3 plants (-21.7). GVA_PROMETEOII/2013/021; MINECO_CGL2012-40058-C02-01; FEDER)

THEME 4

Genomic and NGS approaches to study Trebouxia diversity

Transcriptomic analysis of the lichen photobiont Trebouxia gelatinosa subjected to dehydration and rehydration processes Alice MONTAGNER1, Fabio CANDOTTO CARNIEL1,2, Marco GERDOL1, Elisa BANCHI1, Chiara MANFRIN1, Lucia MUGGIA1, Alberto PALLAVICINI1, Mauro TRETIACH1 1 Dipartimento di Scienze della Vita, Università degli Studi di Trieste, via L. Giorgieri, 10, 34127 Trieste, Italy 2 Institute of Botany, University of Innsbruck, Sternwartestraße, 15, 6020 Innsbruck, Austria All Trebouxia species, the most common lichen-forming genus of aero-terrestrial green algae, are desiccation tolerant. This remarkable adaptation is, however, poorly known on its molecular bases. We applied a transcriptomic approach to investigate changes in the gene expression of T. gelatinosa induced by changes in the cell water status. The mRNA of three sample groups (control, dehydrated, rehydrated) was subjected to Illumina massive sequencing (2×100 bp), that produced a total of 243×106 fragments. From this pool 13,648 protein-coding transcripts could be annotated and used for the subsequent gene expression analysis. During the dehydration/rehydration cycle c. 92% of the total protein-coding transcripts displayed a stable expression, suggesting that the desiccation tolerance of T. gelatinosa mostly relies on constitutive mechanisms. Components of the photosynthetic apparatus, the ROS-scavenging system, expansins, and Desiccation Related Proteins (DRP) were those most affected by changes in the cell water status. The assembled T. gelatinosa transcriptome here presented is a valuable standard reference for future RNA-seq based gene expression studies and represents a new tool to investigate the mechanisms of desiccation tolerance in aero-terrestrial green algae and lichen photobionts.

Microalgal diversity inside a single lichen thallus revealed by HTS approaches (454 amplicon pyrosequencing assay) Patricia MOYA*, Arantxa MOLINS*, Fernando MARTÍNEZ-ALBEROLA, Eva BARRENO *These two authors contributed equally in this work Dpto. Botánica, ICBIBE, Fac. CC. Biológicas, Universitat de València, C/ Dr. Moliner 50. 46100-Burjassot, Valencia, Spain. Current literature has revealed that in lichens the intrathalline coexistence of different microalgal lineages or taxa is a more common event than previously thought. In Ramalina farinacea, the microalgae Trebouxia sp. TR9 and T. jamesii (TR1) always coexist even in populations located at great distances far from each other. Most of the studies dealing with molecular phycobiont diversity within entire thalli are based mainly on the Sanger methodology which may underestimate the potential heterogeneity of the microalgae. The aim of this study was to design an accurate protocol (prior to lichen handling) to allow the detection of the vast number of microalgal genotypes that remain concealed when using traditional molecular techniques by using HGS approaches (454 pyrosequencing assay). In order to analyze the epiphytic algae, a thallus of R. farinacea was separated into two sections and washed following different procedures. It was then joined and divided into sections to analyze the intrathalline algal diversity and distribution. To validate the sensitivity of the proposed technique, we included three algal cultures and an equimolar mix. A total of 64955 raw sequences were obtained which allowed the detection of 37 OTUs inside a single thallus including different genera: 26 Trebouxia, one Asterochloris and other additional algae (3 Chlorophyceae, 4 Trebouxiophyceae and 3 unknown OTUs). Our study corroborates the results obtained with traditional techniques, but revealed much higher intrathalline microalgal diversity, and a feasible differential phycobiont distribution. Several additional OTUs were detected in middle and low abundances, giving insights into an “algal rare biosphere”. These additional OTUs may also be important components of thalli communities and have to be scrutinized from an ecological perspective. Moreover, this diversity should not be underestimated in phylogenetic and ecophysiological studies. GVA_PROMETEOII/2013/021; MINECO_CGL2012-40058-C02-01; FEDER)

Exploring the possibility of multiple Trebouxia lineages in individual lichen thalli using next-generation sequencing Steven LEAVITT Integrative Research Center, Science & Education, The Field Museum, 1400 S. Lake Shore Drive, Chicago, IL 60605, USA Microbial symbionts are instrumental to the ecological and long-term evolutionary success of their hosts, and the central role of symbiotic interactions is increasingly recognized across the vast majority of life. Lichens provide an iconic group for investigating patterns in species interactions; however, the iconic perspective of lichen thallus comprised of a single mycobiont associating with a single photobiont has recently been challenged. The occurrence of multiple photobiont species-level lineages occurring in individual lichen thalli - intrathalline photobiont plurality - has been detected across a number of lichens groups. In this study, we implement a shotgun sequencing approach to characterize potential intrathalline photobiont plurality in members of Lecanoraceae and Parmeliaceae. We demonstrate that high throughput sequencing provides a powerful tool for assessing intrathalline algal diversity, in addition to generating phylogenomic datasets for inferring evolutionary relationships.

Altitude and host genetic identity shape the algal microbiome of congeneric hosts: a metabarcoding approach. Francesco DAL GRANDE1, Miklós BALINT1, Pradeep K. DIVAKAR2, Jürgen OTTE1, Ana CRESPO2, Matthias SCHLEUNING1, Imke SCHMITT1,3 1Senckenberg Biodiversity and Climate Research Centre (BiK-F), Senckenberganlage 25, D-60325 Frankfurt, Germany 2Universidad Complutense de Madrid, Facultad de Farmacia, Biologia Vegetal II, E-28040 Madrid, Spain 3Department of Biological Sciences, Institute of Ecology, Evolution and Diversity, Goethe Universität, Max-von-Laue-Str. 13, D-60438 Frankfurt, Germany Reshuffling symbiont-host combinations is a strategy used by symbiotic organisms to adapt to changing environments. Ecological contrasts are thus expected to drive changes in photobiont communities, but their reactions are not well understood. Here we used Illumina MiSeq sequencing of the ITS2 barcode to study altitudinal variation in photobiont diversity associated with two ecologically segregated, closely-related congeneric lichen-forming fungi, sympatric in the limit of their distribution. In particular, we examined the effects of macro-climatic variation and host genetic identity on intrathalline photobiont diversity, community structure, and strength of biotic interactions. Our results suggest that environmental changes on fungal–algal symbioses may be mediated by the population genetic processes of the fungal hosts and by the degree of specificity of the associations. Finally, we showed that the NGS approach provides superior power to interrogate photobiont genetic variation compared to the traditional Sanger sequencing technology.

THEME 5

Morphological and phylogenetic

species delimitation in Trebouxia

Morpho-genetic groups of species in the genus Trebouxia Anna VOYTSEKHOVICH¹, Andreas BECK² 1Department of Lichenology and Bryology, M.G. Kholodny Institute of Botany, National Academy of Sciences of Ukraine, Tereschenkivska str. 2, Kyiv 01601, Ukraine 2Department of Lichenology and Bryology, Botanische Staatssammlung München, Menzinger Str. 67, 80638 München, Germany Today the genus Trebouxia officially includes 26 species, however it still requires further taxonomical analysis of existing data. The high morphological variability in culture and lack of good identification keys lead to significant errors and inaccuracies in the identification of these algae. Nowadays molecular phylogeny and comparison of molecular data with the sequences of authentic strains available in GeneBank is the most accurate method of photobionts identification. Based on the results of cultural studies of 26 authentic strains of Trebouxia obtained from the collections of cultures SAG (Germany), CCAP (Scotland), UTEX (USA), and taking into account all available data, we distinguish four morpho-genetic groups of species in the genus Trebouxia. Group «arboricola» includes T. aggregata, T. arboricola, T. asymmetrica, T. crenulata, T. cretacea, T. decolorans, T. gigantea, T. jamesii, T. incrustata, T. showmanii, T. solaris, T. vagua; group «impressa» includes T. anticipata, T. gelatinosa, T. impressa, T. flava, T. potteri; group «corticola» consists of T. corticola, T. galapagensis, T. higginsiae, T. usneae; group «simplex» includes T. angustilobata, T. australis, T. brindabellae, T.simplex, T. suecica. According to molecular phylogenetic research of ITS and partial LSU nuclear rDNA, these groups correspond with a particular clade with high support. Rappresentative Trebouxia strains selected from the different groups, were analyzed and show to have, within the same group, similar dimensional characteristics, very similar ultrastructure of the pyrenoids, and the similar ratio of pigment content, which plays an important role in ecophysiology and geographical distribution of Trebouxia species.

Species delimitation in Asterochloris, a close relative to the genus Trebouxia Pavel ŠKALOUD1, Ondrej PEKSA2, Jana STEINOVÁ1, Tereza ŘÍDKÁ1, Lucie VANČUROVÁ1, Jiri MALÍČEK1 1Charles University in Prague, Faculty of Science, Department of Botany, Benátská 2, CZ-12801 Prague, Czech Republic 2The West Bohemian Museum in Pilsen, Kopeckého sady 2, CZ-30100 Plzeň, Czech Republic The genus Asterochloris represents one of the most common, widespread, and diverse taxa of lichen photobionts. Genetic investigations revealed the existence of numerous, morphologically cryptic lineages, with only a few of them being traditionally described as distinct taxa. According to our long-term investigations and metadata analyses, we are now able to clearly delimit each of these species-level lineages based on genetic, morphological, and ecological data, as well as with respect to their mycobiont partners. We hope that the similar approach can be applied to precisely delimit particular species within the genus Trebouxia.

THEME 7

Isolation and in vitro cultures of

trebouxioid microalgae as

crucial pieces for species delimitation

Trouble with Trebouxia?? Culture experiments and progress in understanding the nutrient requirements of major symbiotic algae Elfie STOCKER-WÖRGÖTTER Institute of Plant Sciences, Karl-Franzens University of Graz, Holteigasse 6, 8010 Graz, Austria The genus Trebouxia is well known to represent a major photobiont (symbiotic photosynthetic partner) within lichen thalli. In 1980, Trebouxiaceae were, regarding their different mode of reproduction, split into Trebouxia and Pseudotrebouxia (later named Asterochloris) and merged again into Trebouxia. Nowadays, Asterochloris (since the late 1990s) is an own genus again, it is thought to be mainly restricted to associations with lichens in two closely related families, Cladoniaceae and Stereocaulaceae and a few others. John et al. ( 2002) continued to position Trebouxia in the large, traditional, though demonstrably artificial order, the Chlorococcales, due to lack of any consensus in considering the results of recent molecular studies. Referring to former studies, Trebouxia appears to be closely related to the free-living genus Pleurastrum. The question if Trebouxia occurs only in lichens and/or also free-living in the natural environment has occupied many lichenologists over several decades; since Vernon Ahmadjian raised this topic as one of high relevance in the seventieth of the 20th century. Trebouxia is the most widespread photobiont in lichens, occurring in many different lichen orders. They are not obligatory associated with single species of lichenized fungi as thought in early investigations, since they can be grown independently in culture. Meanwhile, it is also known that a given lichen fungus may form associations with different species of Trebouxia. In the very beginning, culturing Trebouxia independently had an important impact on the acceptance of lichens as a two-partner-consortium. Nowadays, culture experiments have “evolved” to an important tool for correctly determining species of Trebouxia and confirm their relationships to other algal groups by molecular studies. The presentation will highlight a few historical details about “culturing Trebouxia”, how they influenced modern culture experiments and show new, improved strategies (e.g. temperature, light regimes, nutrient media compositions, bioreactors) to obtain pure, optimized cultures in larger scale for genetic and genomic approaches. Trebouxia algae are found to have exceptional wide ecological amplitudes and adaptations to extreme environmental conditions (influence on the fitness of the lichen symbiosis), such as tolerance of low temperatures, desiccation tolerance of lichens in semiarid habitats (species of the genus Xanthoparmelia), tolerance of low intensities of light, etc.

Improved propagation method, rapid molecular identification and ultrastructural characterization as a multidisciplinary approach for Trebouxia species delimitation Patricia MOYA1, Salvador CHIVA1, Arantxa MOLINS1, Francisco GARCÍA-BREIJO2, José REIG-ARMIÑANA1, Eva BARRENO1

1 Dpto. Botánica, ICBIBE, Fac. CC. Biológicas, Universitat de València, C/ Dr. Moliner 50. 46100-Burjassot, Valencia, Spain. 2 Dpto. Ecosistemas Agroforestales, Universidad Politécnica de Valencia, Camino de Vera s/n, 46022-Valencia, Spain. The detection of new Trebouxia lineages is increasing dramatically, arising the need for an overall revision of taxonomic concepts to identify and delimit species boundaries. Multidisciplinary standardized approaches should be established in the lichenological community to avoid duplicating names. An improved method for rapid propagation and molecular identification was designed to easily separate and identify the predominant microalgal diversity. Moreover, the coexistence of several phycobionts needs to be studied using comparative ultrastructural analyses in symbiotic and culture states, as some taxonomic features remain fairly stable both within the lichen thalli and in culture, and this would allow us to identify and distinguish them from other co-occurring species. The ability to correlate molecular and ultrastructural data represents a necessary improvement in clarifying the taxonomy of Trebouxia diversity both in the symbiotic and culture states. Buellia zoharyi can be considered a model lichen for setting the boundaries when several Trebouxia lineages coexist. We have selected populations covering the entire range (from the Canary Islands, Mediterranean surrounding areas, and western Asia) of the species in the field. Phycobiont phylogenetic analyses were made using both chloroplast (LSU rDNA) and nuclear (nrDNA ITS) molecular markers. In addition, ultrastructural microscopic techniques were used to characterize each of the microalgae found. Our results evidence the presence throughout all populations of at least four different Trebouxia lineages. Furthermore, phycobiont coexistence events recovered in all the analyzed populations is strengthened by the presence of at least three lineages of co-occuring microalgae. The integration of diverse techniques has led to considering symbiotic interactions more complex events, and to deal with lichen symbioses from a multifaceted point of view to efficiently reveal the hidden diversity of intrathalline microalgae inside a single thallus (phycobiont coexistence). (GVA, PROMETEOII/2013/021; MINECO, CGL2012-40058-C02-01; FEDER)

Accommodations: Trieste offers a wide selection of hotels and B&B of

different classes in the city centre. We recommend booking early.

(http://www.booking.com/city/it/trieste). A short list of suggested hotels and B&B is given here below:

Hotels Albero Nascosto (***), Via F. Venezian 18, I 34124 Trieste. Phone +39-040-300188, fax +39-040-3186666; info@alberonascosto.it; www.alberonascosto.it Al Viale (***), Via Nordio 5, I 34125 Trieste. Phone +39-040-3480838, fax +39-040-3482708. info@hotelalviale.it; www.hotelalviale.it Continentale (****), Via S. Nicolò 25, I 34121 Trieste. Phone +39-040-631717; info@continentalehotel.com; www.continentalehotel.com. Filoxenia (***), Via Mazzini 3, I 34121 Trieste. Phone +39-040-3481644 fax +39-040-661371. info@filoxenia.it; www.filoxenia.it

B&B Advantage Accomodation, Via Lazzaretto Vecchio 13, I 34123 Trieste. Phone: +39 3284920428, www.accomodationtrieste.it Ai Moretti, Piazza Venezia 1, I 34123 Trieste. Phone: +39 328 7692696 info@aimoretti.it, www.aimoretti.it Decò, Via Scala al Belvedere 2, I 34135 Trieste. Phone: +39 3292080021 Palazzo Panfilli, Via della Geppa 2, 34132 Trieste. www.palazzopanfilli.it

Location of the University of Trieste

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