DOI: 10.5061/dryad.msbcc2fzv
title: Gene expression responses to thermal shifts in the endangered lichen Lobaria pulmonaria

description: Anthropogenic climate change has led to unprecedented shifts in
 temperature across many ecosystems. In a context of rapid environmental
 changes, acclimation is an important process as it may influence the
 capacity of organisms to survive under novel thermal conditions.
 Mechanisms of acclimation could involve upregulation of stress response
 genes involved in protein folding, DNA damage repair and the regulation of
 signal transduction genes, along with a simultaneous downregulation of
 genes involved in growth or cell cycle, in order to maintain cellular
 functions and equilibria. We transplanted Lobaria pulmonaria lichens
 originating from different forests to determine the relative effects of
 long-term acclimation and genetic factors on the variability in expression
 of mycobiont and photobiont genes. We found a strong response of mycobiont
 and photobiont to high temperatures, regardless of sample origin. The
 green-algal photobiont had an overall lower response than the mycobiont.
 The gene expression of both symbionts was also influenced by acclimation
 to transplantation sites and by genetic factors. Lobaria pulmonaria seems
 to have evolved powerful molecular pathways to deal with environmental
 fluctuations and stress and can acclimate to new habitats by
 transcriptomic convergence. Although L. pulmonaria has the molecular
 machinery to counteract short-term thermal stress, survival of lichens
 like L. pulmonaria depends mostly on their long-term positive carbon
 balance, which can be compromised by warmer temperatures and reduced
 precipitation, and both these outcomes have been predicted for Central
 Europe in connection with global climate change.
description: We established reciprocal transplants between four Austrian
 sites: Koralpe (K), Kleinsölk Valley (S) Tamischbach Canyon (T) and Raab
 Canyon (R), which received additional transplants from six Swiss sites
 (Table 1) (Fig 1B, Fig S1). In early summer 2015, Silke Werth (SW) set up
 reciprocal transplants between four ‘recipient’ sites in Austria (Lowland;
 Southern, Central and Northern Alps) differing in elevation and
 macroclimatic regime (K, S, R, T) (Fig 1B, Fig S1, Table S1). In 2015,
 lichen lobes originating from two sites in the Swiss Jura mountains were
 transplanted to the recipient sites in Austria, and the transplantation
 was repeated in 2016 with lobes from two additional Swiss Jura Mountain
 sites. Furthermore, in early summer 2016, additional reciprocal
 transplants were made between the Austrian sites. In each case, parts of
 large thalli were collected from Austria (A) or Switzerland (W), split
 into four lobes, and each lobe was transplanted to a different recipient
 site. After acclimatization to the recipient sites over at least three
 vegetation periods, the transplants were harvested on two consecutive days
 in August of 2019 during a time period with constant weather conditions.
 They were grown under controlled, cold (4 °C) conditions with medium light
 (150-200 PAR) for 24 hours to adjust to the lab conditions so that the
 results would not simply reflect the prevailing local weather or light
 conditions during sampling. At a temperature of 4 °C, we took three
 samples of marginal tissue (ca. 25 mm2) from a given lobe of each fully
 hydrated lichen to include lichen lobes originating from the two Origin
 Sites (W and A) that were transplanted to each of the four transplantation
 sites (K, R, S, T). The samples were immediately flash-frozen in liquid
 nitrogen and stored at -80 °C until further processing. We transferred the
 lichen lobes into a growth chamber at 15°C and we kept them there for two
 hours and took another three samples of the lichen lobes exposed to this
 temperature. Lastly, we increased the temperature to 25 °C and kept the
 lichen lobes for two more hours at this temperature before collecting
 three additional samples from each lobe (Fig 1B) RNA extraction was
 performed according to the manufacturer’s protocol using the RNeasy Plant
 Mini Kit (Qiagen, Stockach, Germany). After elution of extracted RNA in
 RNase-free H2O, RNA concentration and quality were checked using first a
 NanoDrop ND-1000 UV/Vis-Spectrophotometer (Thermo Scientific, Carlsbad CA,
 USA) and then a Qubit 2.0 fluorometer with the DNA HS assay kit (Qiagen,
 Stockach, Germany). First, RNA concentration and quality were checked
 using a high-sensitivity RNA chip (Agilent Bioanalyzer). RNA sequencing
 was performed using an adapted version of the prime-seq protocol
 (step-by-step protocol: dx.doi.org/10.17504/protocols.io.s9veh66), but
 with adjustments as follows. Raw fastq files were processed using zUMIs
 software (Parekh et al. 2018).The normalized counts (vst) were used to
 perform principal components analysis (PCA) (R version 3.6.3). The Adonis
 test (a randomization/Monte Carlo permutation test was utilized to test
 for significant relationships between gene expression variation in samples
 exposed to different temperatures and from different transplantation
 sites. Differential expression analysis was carried out by DESeq2 in R. We
 used the Wald test for hypothesis testing when comparing differentially
 express transcripts between two groups, and only transcripts with a
 log2-fold change &gt;1.5 and adjusted Benjamini-Hochberg p-value
 &lt;0.0001 were considered differentially expressed. We used the
 adjusted Benjamini-Hochberg p-value and such low p-value levels to reduce
 the false positive rate. The study design allowed us to identify
 transcripts that were up and downregulated in the lichen thalli, among the
 three temperature treatments (acclimation effects), among the two origins
 (genetic effects), or among the four transplantation sites
 (acclimatization effects). We made three Wald test comparations for the
 different temperatures: 1) 4 °C vs 25 °C 2) 4 °C vs 15 °C 3) 15 °C vs 25
 °C. Six different Wald test comparations between the transplantation
 sites: 1) K vs S 2) K vs R 3) K vs T 4) S vs R 5) S vs T 6) T vs R. In
 addition, we made a Wald test comparison between the two L. pulmonaria
 samples origin: A vs W. These Wald test comparations were made for the
 mycobiont and for the photobiont. While DESeq2 is useful for conducting
 Wald pairwise comparisons, the Weighted Gene Co-Expression Network
 Analysis (WGCNA) (Langfelder & Horvath 2008) package in R allows
 to examine patterns of gene expression across all treatments
 simultaneously. WGCNA was used to generate a correlation network to
 identify clusters of similarly expressed transcripts that clustered into
 gene modules, using a cutoff of minimum 30 transcripts per module. A
 module’s so-called ‘eigengene’ summarizes the overall module expression
 profile. Modules with highly correlated eigengenes were merged using a
 threshold value by the ‘dynamicMergeCut’ WGCNA function in R and the
 clusters were summarized by the eigengene. Then we related the modules to
 the explanatory variables as temperature treatment (acclimation effects),
 transplantation site (acclimatization effects), and origin site (genetic
 effects) using eigengene network methodology. A heatmap was created to
 visualize all significant correlations between modules and treatments in
 WGCNA. Then we identified transcripts with high significance for each of
 the explanatory variables for the mycobiont and the photobiont separately.
 Gene ontologies of the Lobaria pulmonaria reference genome hosted at
 DOE-JGI, https://mycocosm.jgi.doe.gov/LobpulSc1/LobpulSc1.home.html (date
 accessed: 17/07/2020) were used to match the differentially expressed (DE)
 transcripts with protein and GO-term information. Lists of gene ontology
 (GO) terms were generated for the differentially expressed transcripts
 found with DESeq2. When there was no GO term information available, we
 annotated the respective transcript manually by homology searches via the
 translated nucleotide BLAST algorithm (blastx) (Altschul et al. 1997)
 against the Uniprot database (https://www.uniprot.org/blast/). We searched
 for homology of L. pulmonaria mycobiont transcript sequences to protein
 sequences available for several whole-genome sequenced species of fungi.
 Then we searched for homology of the S. reticulata photobiont’s transcript
 sequences to protein sequences available for algae. We used the target
 databases‘ fungi’ and ‘algae’ with default algorithm parameters (expect
 threshold=10, matrix=auto, filtering=none, gapped=yes, hits=250) for the
 Blast searches. We considered a locus homologous if the e-value returned
 was smaller than 1.0 * 10-5. We only used this method for the
 differentially expressed transcripts found with DESeq2 analysis, if we did
 not find any GO term information available for those transcripts. The GO
 term gene information for the WGCNA is presented in the Supporting
 Information (Tables S1- S2).
description: We upload all the gene data counts needed for our
 analysis We included the R script with our
 analysis We included all the DE genes list from the
 Wald test comparing variables. We also included the list of the total
 genes found in the modules of the WGCNA. The data with
 M is from the mycobiont and the P is from the photobiont