Coexistence of different intrathalline symbiotic algae and bacterial biofilms in the foliose Canarian lichen Parmotrema pseudotinctorum

[EN] Parmotrema pseudotinctorum (des Abb.) Hale is a foliose lichen able to colonize large areas on rock surfaces in semiarid and warm localities in the Canary Islands. In this contribution, we investigate the phycobionts of this successful lichen under these extreme environmental conditions using ultrastructural and genetic methodologies. Two populations from La Gomera and La Palma islands were analyzed. After TEM analyses, three algal types were clearly distinguished in intrathalline symbiosis, provisionally named Ph1, Ph2, and Ph3. Two of them (Ph1 and Ph2) were Trebouxia showing a well visible pyrenoid corticolatype the chloroplast thylakoids being very different in both. The type Ph3 could be a taxon included in the genus Asterochloris. Our molecular approach consisted in sequencing two different DNA loci: a portion of the chloroplast psbA gene and nuclear ITS. Sequences of the psbA gene resulted in electrophoretograms showing double peaks when DNA extracted from the whole lichen thallus was used as template. Such double peaks were interpreted as single nucleotide polymorphisms (SNPs). This interpretation was confirmed by cloning. However, no intrathalline polymorphisms were detected among the nrITS sequences. Phylogenetic analyses on the basis of the psbA gene revealed three distinct clades. It is likely that these clades corresponded to the the three different morphotypes revealed by TEM. One of these clades, was closely related to T. corticola, other was related to Asterochloris glomerata and the third did not grouped with any specific taxa. These results are the first piece of evidence that algal coexistence may even be established between species of different genera of the Trebouxiophyceae (Asterochloris and Trebouxia at least). Moreover, the coexistence of several microalgal taxa evidenced in this study appears as a consistent character among the populations of this foliose lichen. Further isolation and cultivation of the three different algal types and physiological studies should shed light on the ecological plasticity of the entire holobiont. Along with such variety of intrathalline coexisting algae, another unexpected result was the observation of an almost continuous layer of bacterial-communities coating the lower cortex in all the studied samples of P. pseudotinctorum. The function of these biofilms in the lichen symbiosis remains to be elucidated. The existence of such particular symbiosis involving different algal species and bacteria could be explained by an increased fitness in particular habitats or under specific environmental conditions[ES] Parmotrema pseudotinctorum (des Abb.) Hale es un liquen foliáceo que coloniza roquedos volcánicos y es frecuente en localidades semiáridas y cálidas de las islas Canarias. En este trabajo, se investigan los ficobiontes de este liquen utilizando métodos ultraestructurales y genéticos. Se analizaron dos poblaciones de La Gomera y La Palma. Mediante análisis TEM, se han distinguido claramente tres tipos de algas en simbiosis intratalinas, llamados provisionalmente Ph1, Ph2 y Ph3. Dos de ellas (Ph1 y Ph2), pertenecientes al género Trebouxia, muestran un pirenoide bien visible del tipo corticola, pero los tilacoides del cloroplasto son muy diferentes en ambos. El tipo Ph3 podría ser un taxón incluido en el género Asterochloris. El enfoque molecular consistió en la secuenciación de dos loco diferentes de ADN: una porción del gen psbA del cloroplasto e ITS nuclear. Las secuencias del gen psbA proporcionaron electroforetogramas que mostraban dobles picos cuando se usó como patrón el ADN extraído de los talos completos. Estos dobles picos se interpretaron como polimorfismos de nucleótido simple (SNP). Esta interpretación se confirmó mediante técnicas de clonación. Sin embargo, no se detectaron polimorfismos intratalinos con las secuencias nrITS. Los análisis filogenéticos basados en las secuencias del gen psbA constataron la presencia de tres clados diferenciados. Es probable que éstos correspondan a los tres diferentes morfotipos puestos en evidencia por TEM. Uno de ellos está estrechamente relacionado con T. corticola, otro con Asterochloris glomerata, y el tercero no se agrupaba a ningún taxón especifico. Estos resultados son la primera evidencia de que en los líquenes la coexistencia de algas intratalinas se puede establecer incluso entre especies de difierentes géneros de Trebouxiophyceae (Asterochloris y Trebouxia al menos). Por otra parte, la coexistencia de varios taxones de microalgas, constatada en este estudio, puede considerarse como una sólida característica entre las poblaciones de este liquen foliáceo. Posteriores aislamientos y el cultivo de los tres tipos de algas, además de estudios fisiológicos, podrán explicar la plasticidad ecológica que muestra el holobionte. Otro resultado inesperado fue la observación de una capa casi continua de comunidades bacterianas que recubren el córtex inferior en todas las muestras estudiadas de P. pseudotinctorum. Está por determinar la función que estos biofilms puedan tener en las simbiosis liquénicas. La existencia de estos tipos de simbiosis tan particulares, que involucran tanto a diferentes especies de algas como a bacterias, podría explicarse por un incremento en la capacidad de colonizar hábitats peculiares o con condiciones ambientales específicasThis study was funded by the Spanish Ministry of Economy and Innovation (MINECO CGL2012-40058-C02-01/02), FEDER and the Generalitat Valenciana (PROMETEO 021/2013 GVA).Molins, A.; García-Breijo, F.; Reig Armiñana, J.; Del Campo, EM.; Casano, L.; Barreno Rodriguez, E. (2013). Coexistence of different intrathalline symbiotic algae and bacterial biofilms in the foliose Canarian lichen Parmotrema pseudotinctorum. Vieraea. Folia Scientarum Biologicarum Canariensium. 41:349-370. http://hdl.handle.net/10251/77348S3493704

Balanced gene losses, duplications and intensive rearrangements led to an unusual regularly sized genome in Arbutus unedo chloroplasts

Completely sequenced plastomes provide a valuable source of information about the duplication, loss, and transfer events of chloroplast genes and phylogenetic data for resolving relationships among major groups of plants. Moreover, they can also be useful for exploiting chloroplast genetic engineering technology. Ericales account for approximately six per cent of eudicot diversity with 11,545 species from which only three complete plastome sequences are currently available. With the aim of increasing the number of ericalean complete plastome sequences, and to open new perspectives in understanding Mediterranean plant adaptations, a genomic study on the basis of the complete chloroplast genome sequencing of Arbutus unedo and an updated phylogenomic analysis of Asteridae was implemented. The chloroplast genome of A. unedo shows extensive rearrangements but a medium size (150,897 nt) in comparison to most of angiosperms. A number of remarkable distinct features characterize the plastome of A. unedo: five-fold dismissing of the SSC region in relation to most angiosperms; complete loss or pseudogenization of a number of essential genes; duplication of the ndhH-D operon and its location within the two IRs; presence of large tandem repeats located near highly re-arranged regions and pseudogenes. All these features outline the primary evolutionary split between Ericaceae and other ericalean families. The newly sequenced plastome of A. unedo with the available asterid sequences allowed the resolution of some uncertainties in previous phylogenies of Asteridae

A multi-tool approach to assess microalgal diversity in lichens. isolation, Sanger sequencing, HTS and ultrastructural correlations

[EN] Lichen thalli represent the most conspicuous examples of fungal-algal interactions. Studies that describe phycobiont diversity within entire thalli are based mainly on Sanger sequencing. In some lichen species, this technique could underestimate the intrathalline coexistence of multiple microalgae. In this study different multi-tool approaches were applied to two lichen taxa, Circinaria hispida and Flavoparmelia soredians, to detect algal coexistence. Here, we combined Sanger sequencing, a specific polymerase chain reaction (PCR) primer, 454-pyrosequencing, phycobiont isolation and ultrastructural characterization. Furthermore, we compared pyrenoid ultrastructural features of lichenized phycobionts with microalgae isolated in culture. An improved methodology was used to isolate and propagate phycobionts which, in combination with fast genetic identification, resulted in a considerable reduction in time and cost to complete the process. This isolation method, coupled with a specific PCR primer, allowed for the detection of coexisting algae in C. hispida (four Trebouxia lineages). 454-pyrosequencing detected only a fraction of such diversity, while Sanger sequencing identified only the primary phycobiont. Ultrastructural features of the isolated algae were observed by transmission electron microscopy; the maintenance of the pyrenoid characteristics suggested the existence of different Trebouxia lineages. In F. soredians a single Trebouxia lineage was identified using all these approaches. In cases of lichens with algal coexistence, a combination of different molecular and ultrastructural approaches may be required to reveal the underlying algal diversity within a single thallus. The approach proposed in this study provides information about the relationship between molecular and ultrastructural data, and represents an improvement in the delimitation of taxonomic features which is needed to recognize intrathalline Trebouxia diversity.Supported by the Ministerio de Economia y Competitividad (MINECO, Spain) (CGL2016-79158-P) and Excellence in Research (Generalitat Valenciana, Spain) (PROMETEOII/2013/021 and PROMETEO/2017/039). We wish to thank the technicians (Maria Teresa Minguez and Nuria Cebrian) of the Servicio de Microscopia Electronica, SCSIE and Jardi Botanic (Universitat de Valencia) who helped us to perform the TEM process, and Santiago Catala for the pyrosequencing analyses. Daniel Sheerin revised the English manuscript.Molins, A.; Moya, P.; García-Breijo, F.; Reig-Armiñana, J.; Barreno, E. (2018). A multi-tool approach to assess microalgal diversity in lichens. isolation, Sanger sequencing, HTS and ultrastructural correlations. The Lichenologist. 50(1):123-138. https://doi.org/10.1017/S0024282917000664S12313850

Whole genome alignment of the <i>Arbutus unedo</i> chloroplast genome with other asterid chloroplast genomes obtained with MultiPipMaker [32] taking that of <i>Nicotiana tabacum</i> as the reference.

<p> Sequence identity is shown by red (75–100%), green (50–75%), and white (<50%). Positions of some genes in <i>N. tabacum</i> are indicated as a guide (genes encoding proteins and rRNAs are indicated as yellow and red arrows, respectively). The taxonomic classification is indicated on the left (AP: Apiales, AS: Asterales, GE: Gentianales, LA: Lamiales, SO: Solanales, ER: Ericales).</p

Genes found in the <i>Arbutus unedo</i> chloroplast genome.

a<p>Gene containing two introns.</p>b<p>Gene containing a single intron.</p>c<p>Two gene copies in the IRs.</p>d<p>Gene whose transcripts are trans-spliced.</p

Comparison of the lengths of LSC, SSC and IR regions among Asteridae.

<p>Accession numbers of the corresponding genomes are indicated in Table S2.</p

Gene map and alignment of the LSC region of three ericalean species in relation to <i>Nicotiana tabacum</i>.

<p>(A) Gene map of the LSC region in the chloroplast genome of <i>Nicotiana tabacum</i>. (B) Gene alignment of the LSC region of <i>Ardisia polysticta</i>, <i>Camellia sinensis</i>, <i>Arbutus unedo</i>, <i>Vaccinium macrocarpon</i> belonging to Ericales and <i>Nicotiana tabacum</i> belonging to Solanales. MAUVE multiple alignment <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079685#pone.0079685-Darling1" target="_blank">[33]</a> implemented in Geneious <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079685#pone.0079685-Kearse1" target="_blank">[23]</a>. Colored outlined blocks surround regions of the genome sequence that aligned with part of another genome. The coloured bars inside the blocks are related to the level of sequence similarities. Lines link blocks with homology between two genomes. Accession numbers of the corresponding genomes are indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079685#pone.0079685.s006" target="_blank">Table S2</a>.</p

Genes having cis-spliced introns in the <i>Arbutus unedo</i> cpDNA and the lengths of exons and introns.

<p>Genes having cis-spliced introns in the <i>Arbutus unedo</i> cpDNA and the lengths of exons and introns.</p

Phylogram based on sequence analysis of 83 chloroplast genes from 57 plant species (Table S2).

<p>Asterisks indicate nodes with values of 0.1 and 100 for bootstrap values and posterior probabilities, respectively. The scale bar indicates substitutions/site. The current taxonomic classifications are indicated on the right (<i>i</i>.<i>s</i>., <i>incertae sedis</i>).</p

Codon-anticodon recognition pattern and codon usage for the chloroplast genome of <i>Arbutus unedo</i>.

*<p>Numerals indicate the frequency of usage of each codon in 17,947 codons in 73 potential protein-coding genes.</p