https://doi.org/10.5061/dryad.7d7wm382c

This data collection contains multiple groups of datafiles, all associated with physiological activity in Evernia mesomorpha (I and II) and other lichens (III). Order of listing reflects order of appearance in associated manuscript.

I. Ecophysiological data from measurements of Evernia mesomorpha, including gas-exchange datafiles, chlorophyll fluorescence imaging results, and associated supporting data (e.g. thallus masses and areas) and R script.

These files include the data files: Evernia_LRC.csv; Thallus_Masses.csv; Evernia_SPRUCE.csv; CNALHrecords_NA.csv; Vapor_IPAM.csv and the R script Evernia_Analyses_and_Figures.R

II. Transcriptome data and analyses for Evernia mesomorpha

These files include the data files: 

III. Ecophysiological data from measurements across 42 species

Description of the data and file structure

I. Evernia mesomorpha ecophysiology data

I.a) Evernia_LRC.csv

Data used for the construction of light-response curves for individual thalli of the lichen Evernia mesomorpha at different temperatures and hydration levels. Each light-response curve reflects the variation in instantaneous carbon balance (as determined by infra-red gas exchange, here denoted as Flux*) of the thallus at light levels (photon flux, here denoted as Qin) ranging from dark (effectively respiration) to saturating light. To reduce the effect of noise in the measurement low rates of gas exchange, 10 readings were taken in short succession for each measurement, and averaged in further analyses. Multiple replicate thalli were measured for each combination of conditions. This file is in the format output by the LICOR LI-6800 with an Aquatic Chamber. As the raw, compiled data is presented, some entries were later excluded because of operational error (for example data logged by mistake when stable conditions had not yet been reached). Many of the columns are of raw data, with detailed definitions available from https://www.licor.com/env/support/LI-6800/topics/folders-and-data-files.html

Fresh thalli (0.1- 0.3 g) of Evernia mesomorpha Nyl. were collected from a Picea mariana-dominated bog at Cedar Creek Ecosystem Reserve, Anoka, Minnesota (N 45.4233, W 93.1902 ) in September 2022. Prior to measurement, thalli were thawed on the lab bench (25ºC, ~50% RH) until room temperature and air dry. To achieve vapor hydration, thalli were placed in sealed containers (~700 cm3) over distilled water for 12 hours at low light (<15 μmol m-2 s-1). These conditions achieve complete equilibration with humid air within hours (13) (fig S1) and mimic natural nocturnal conditions. Assimilation and respiration rates were measured using a portable Infra-Red Gas Analyzer (LI-6800, LI-COR, Lincoln, NE, USA) equipped with an Aquatic Chamber (6800-18, internal volume of 20 cm3) that enables measurements under very high humidity conditions. High humidity in the measurement space is essential to minimize water loss from thalli during the course of measurements. Measurements were conducted at 6 temperature levels (5,10, 15, 20, 25 and 30ºC) in a controlled temperature room. Chamber conditions were RH target 95% (realized RH ~91%), 500 µmol/s flow rate and reference CO2 of 400 ppm, Light response curves consisting of a 4-5 min stabilization stage followed by ten repeated measurements at 2 second intervals were constructed from the following sequence of programmed PAR levels: 0, 1000, 2000, 2500, 1000, 500, 250, 100, 50 μmol m-2 s-1. Because of light attenuation by the chamber walls, realized PAR values were: 0, 544, 815, 1086, 1357, 544, 273, 136, 55, 27 μmol m-2 s-1. Following vapor hydrated measurements, thalli were brought to maximal internal liquid hydration using standardized protocols (10) and re-measured following the same instrumental settings as above.

Several columns have been added to describe the specimens being measured. These are:

• Round - Round of measurements
• Temperature - Measurement temperature (ºC)
• Thallus - Replicate thallus (within round)
• Hydration - Hydration treatment: vapor (extended exposure to high humidity) or liquid (gentle spraying to water-holding capacity)
• Discard - Record to omit due to operational error (y = to be discarded)
• Other columns most relevant to data analyses:
• Flux* - Carbon flow from aquatic chamber µmol s-1
• Qin - Flux incident on aquatic sample µmol m-2 s-1

I.b) Thallus_Masses.csv

This file contains the masses (wet and dry) and area for the Evernia mesomorpha thalli used in light-response curves in I.a

• Round - Round of measurements
• Thallus - Replicate thallus (within round)
• Temperature - Measurement temperature (ºC)
• Hydration - Hydration treatment: vapor (12hr exposure to high humidity) or liquid (gentle spraying to water-holding capacity over 15min)
• Mass_start - Mass in mg at start of gas-exchange measurements
• Mass_end - Mass in mg at end of gas-exchange measurements
• Mass_dry - Mass in mg after oven-drying
• Area - Thallus area in cm3 (gently flattened while hydrated)

I.c) Evernia_SPRUCE.csv

Gas-exchange measurements of Evernia mesomorpha thalli. To reduce the effect of noise in the measurement low rates of gas exchange, 10 readings were taken in short succession for each measurement, and averaged in further analyses. Each thallus was measured under 4 conditions (dark and vapor-hydrated, saturating light and vapor hydrated, dark and liquid hydrated, saturating light and liquid hydrated). Vapor hydration was achieved by 12hrs exposure to saturating humidity conditions in a sealed container over distilled water. Liquid hydration was achieved by repeated gentle spraying with water over 15+ minutes. Multiple replicate thalli were measured for each combination of conditions. This file is in the format output by the LICOR LI-6800 with the Bryophyte Chamber attachment. All measurements made at ~6ºC in a controlled temperature room. Many of the columns are of raw data, with detailed definitions available from https://www.licor.com/env/support/LI-6800/topics/folders-and-data-files.html

Several columns have been added to describe the specimens being measured. These are:

• encl - Source enclosure at SPRUCE experiment
• ziptie2019 - transplant ziptie identifier
• transplant - transplant replicate number
• condition - treatment condition: dark (dark and vapor hydrated), light (saturating light and vapor hydrated), dark_liquid (dark and liquid hydrated) and light_liquid (light and liquid hydrated)

Other columns most relevant to data analyses:

• A - Assimilation rate (scaled to thallus area) µmol m-2 s-1
• Qin - Flux incident on sample µmol m-2 s-1

I.d) CNALHrecords_NA.csv

Records of the lichen Evernia mesomorpha Nyl. from North America, obtained from the Consortium of North America Lichen Herbaria.

Columns have been trimmed from this file compared to the output from the CNALH platform to reduce file size while retaining all relevant information. Categories follow DarwinCore nomenclature.

Column names

• id - Specimen id in CNALH system
• institutionCode- Herbarium code of specimen storage location
• basisOfRecord - type of record (specimen, observation,etc)
• occurrenceID - CNALH occurrence id, when available
• catalogNumber - Accession number, when available
• otherCatalogNumbers - additional associated catalog numbers, when available
• higherClassification - Systematic classification of taxon, from Kingdom to Genus
• scientificName - Genus and species epithet
• scientificNameAuthority - Taxon name authority
• recordedBy - Primary collector
• recordNumber - Collection number if available
• eventDate - Collection date
• country - Country of collection
• stateProvince - State or Province of collection
• decimalLatitude - Latitude
• decimalLongitude - Longitude

I.e) Vapor_IPAM.csv

This file contains the Chlorophyll fluorescence data for Supplementary Figure 1. Photosynthetic efficiency (Fv/Fm), chlorophyll fluorescence measurements were performed using a red‐light IMAGING‐PAM m‐series chlorophyll fluorometer (Walz, Effeltrich, Germany). The E. mesomorpha thallus was placed in a saturating humidity chamber and a Saturating pulse was applied every 10 minutes for 2 hours to record activation of Fv/Fm, followed by lower frequency measurements until values saturated. Liquid hydration was then achieved by repeatedly gently spraying with liquid water and allowing the thalli to drain over 15min.  Fv/Fm was automatically calculated in ImagingWinGigE version 2.56p. Three regions of interest (ROI) were selected to provide an average activation.

• Time: Time exposed to ~100% RH (min)
• FvFm.1: FvFm for ROI 1
• FvFm.2: FvFm for ROI 2
• FvFm.3: FvFm for ROI 3
• Hydration: Vapor = Measurement taken during vapor hydration (i.e. 100% RH), Liquid = Measurement taken after lichen sprayed with liquid water.

I.f) Evernia_Analyses_and_Figures.R

Annotated R script for the analysis of the preceding files and generation of figures 1, S1 and S2 of the manuscript.

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II. Transcriptomic data and analyses

The metatranscriptomes of 5 dry, 5 water vapor, and 5 liquid water hydrated E. mesomorpha thalli were sequenced on a NovaSeq 6000. The following contains all scripts to identify which symbiont each transcript originated from, assign function to each gene, and perform differential expression and GO enrichment analysis. 

II.a) transdecoder_ORF_IDs.zip

To identify which symbiont each transcript originated from, open reading frames (ORFs) were computed from the full Trinity assembly using the Transdecoder plugin (TransDecoder.LongOrfs) and coding regions were subsequently predicted (TransDecoder.Predict). The output of these commands were used to filter the longest splicing isoform per trinity assembled gene (Using the Transcript_Taxa_Identification.R script). This was done so that the best E-value blast result over that isoform was used as the taxonomic assignment for that gene. Identifiers have been edited to separate all fields. With the following UNIX command: path/to/transdecoder.pep | cut -c 2- | tr ‘::’ '\t' | cut -f1,3,5,13 | awk -F '[\t ]' '{print $1, $2, $3, $4}' > transdecoder_ORF_IDs.txt.

File has no header.

• Col 1: Trinity assembled gene
• Col 2: Trinity assembled isoform
• Col 3: G-number, which refers to the original transcript for which the ORF comes from
• Col 4: isoform length

II.b) Trinity.gene.counts.zip

To estimate transcript abundance and create an expression matrix, the Trinity utility align_and_estimate_abundance.pl with the -est-method Salmon was used to align each sample file to the combined Trinity assembly. This file contains the estimated transcripts for each gene.

Rows: Trinity assembled genes

Columns:

• dry_1: raw expression summarized across the gene for sample dry_1
• dry_2: raw expression summarized across the gene for sample dry_2
• dry_3: raw expression summarized across the gene for sample dry_3
• dry_4: raw expression summarized across the gene for sample dry_4
• dry_5: raw expression summarized across the gene for sample dry_5
• liquid_1: raw expression summarized across the gene for sample liquid_1
• liquid_2: raw expression summarized across the gene for sample liquid_2
• liquid_3: raw expression summarized across the gene for sample liquid_3
• liquid_4: raw expression summarized across the gene for sample liquid_4
• liquid_5: raw expression summarized across the gene for sample liquid_5
• vapor_1: raw expression summarized across the gene for sample vapor_1
• vapor_2: raw expression summarized across the gene for sample vapor_2
• vapor_3: raw expression summarized across the gene for sample vapor_3
• vapor_4: raw expression summarized across the gene for sample vapor_4
• vapor_5: raw expression summarized across the gene for sample vapor_5

II.c) longest_isoform_blastp.zip

To identify which symbiont each transcript originated from, open reading frames (ORFs) were computed from the full Trinity assembly using the Transdecoder plugin (TransDecoder.LongOrfs) and coding regions were subsequently predicted (TransDecoder.Predict). Predicted coding regions were blastp searched against the NCBI non-redundant (nr) database using the blastp algorithm with the addition of the staxid output option to include taxonomic identifiers. This file contains search results from the longest splicing isoform, including the evalue and TaxID tag for each blast hit. 

File has no header.

• Col 1: Trinity assembled gene
• Col 2: Trinity assembled isoform
• Col 3: G-number, which refers to the original transcript for which the ORF comes from
• Col 4: M-umber, the ORF identified on the transcript
• Col 5: E-value
• Col 6: TaxID

II.d) esearch_TaxIDs.zip

The Taxonomizr R package was used to identify taxonomy from staxid; however, some Tax IDs were not found using this method. For those IDs that returned NA, Tax IDs were searched in the Enterez database to link taxonomic data to Tax ID. This file contains the taxonomic information for TaxIDs that were not found with the Taxonomizr search with the esearch command using Enterez Direct. The file has no header, and the columns correspond to the following:

• Col1: TaxID
• Col2: superkingdom
• Col3: phylum
• Col4: class
• Col5: order
• Col6: family
• Col7: genus
• Col8: species

II.e) longest_isoform_gene_identification.zip

Functional annotation of transcripts was performed by using predicted coding regions (open reading frames (ORFs) computed from the full Trinity assembly using the Transdecoder plugin (TransDecoder.LongOrfs) and coding regions predicted (TransDecoder.Predict)) and blastp searching against the Uniprot database (release 2023_01) with a E-value threshold of 0.001. This file contains the results of this  blastp search for the longest splicing isoform. File contains the evalue, Uniprot Gene ID and Uniprot Organism ID for each blast hit.

File has no header.

• Col 1: Trinity assembled gene
• Col 2: Trinity assembled isoform
• Col 3: G-number, which refers to the original transcript for which the ORF comes from
• Col 4: M-umber, the ORF identified on the transcript
• Col 5: E-value
• Col 6: Uniprot gene
• Col 7: Uniprot organism

II.f) best_evalue_gene_ids.zip

This file is constructed in the R script Gene_Function_Identification.R, and it contains the gene function assignment for each gene. Functional annotation of transcripts was performed by using predicted coding regions and blastp searching against the Uniprot database (release 2023_01) with a E-value threshold of 0.001. Gene functions were inferred by assigning the best E-value Viridiplantae match for the lichen-forming algae assigned genes, Ascomycete match for the lichen-forming fungus assigned genes, Basidiomycete match for the lichen-associated basidiomycete genes, and Bacteria for the lichen-associated bacteria genes. 

• iso: Trinity assembed isoform
• Evalue: the evalue of the blastp search result
• Gene: the uniprot gene
• Organism: the uniprot organism from which that gene comes from
• LinkID: the uniprot ID that links organism to NCBI taxonomy
• TaxonomyValue: The taxonomy of the organism from which the gene comes from.
• Biont: the "symbiont" from which each isoform has been assigned. Mycobiont = primary lichen-forming fungus. Photobiont = primary lichen-forming alga. Bacteriabiont = lichen-associated bacteria. Basidiobiont = lichen-assocated basidiomycetes.

II.g) gene_count_matrix_taxa.zip

This file is the output of Transcript_Taxa_Identification.R. This file contains the estimated transcript abundance for each gene generated using the Trinity utility align_and_estimate_abundance.pl and abundance_estimates_to_matrix.pl and the -est-method Salmon combined with the taxonomic assignment for each gene.

• Gene: Trinity assembled gene
• dry_1: raw expression summarized across the gene for sample dry_1
• dry_2: raw expression summarized across the gene for sample dry_2
• dry_3: raw expression summarized across the gene for sample dry_3
• dry_4: raw expression summarized across the gene for sample dry_4
• dry_5: raw expression summarized across the gene for sample dry_5
• liquid_1: raw expression summarized across the gene for sample liquid_1
• liquid_2: raw expression summarized across the gene for sample liquid_2
• liquid_3: raw expression summarized across the gene for sample liquid_3
• liquid_4: raw expression summarized across the gene for sample liquid_4
• liquid_5: raw expression summarized across the gene for sample liquid_5
• vapor_1: raw expression summarized across the gene for sample vapor_1
• vapor_2: raw expression summarized across the gene for sample vapor_2
• vapor_3: raw expression summarized across the gene for sample vapor_3
• vapor_4: raw expression summarized across the gene for sample vapor_4
• vapor_5: raw expression summarized across the gene for sample vapor_5
• iso: Trinity assembled isoform for which the taxonomic information is derived i.e. the longest splicing isoform for the Trinity assembled gene
• TaxID: the TaxID
• superkingdom: the super kingdom of the taxonomic assignment
• phylum: the phylum of the taxonomic assignment
• class: the class of the taxonomic assignment
• order: the order of the taxonomic assignment
• family: the family of the taxonomic assignment
• genus: the genus of the taxonomic assignment
• species: the species of the taxonomic assignment

II.h) Fungal_isoforms.zip

This file contains the isoforms that were assigned to the lichen-forming fungus in the Differential_Expression&GO_analysis.R script. These are any isoform for which the best blast against the NCBI nr database was a Lecanoromycete. 

II.i) Algal_isoforms.zip

This file contains the isoforms that were assigned to the lichen-forming alga in the Differential_Expression&GO_analysis.R script. These are any isoform for which the best blast against the NCBI nr database was in the Trebouxiophyceae.

II.j) Bacidio_isoforms.zip

This file contains the isoforms that were assigned to the lichen-associated basidiomycetes in the Differential_Expression&GO_analysis.R script. These are any isoform for which the best blast against the NCBI nr database was a Basidiomycete.

II.k) Bacteria_isoforms.zip

This file contains the isoforms that were assigned to the lichen-associated bacteria in the Differential_Expression&GO_analysis.R script. These are any isoform for which the best blast against the NCBI nr database was Bacteria

II.l) E.meso_E.prun_blast.out.zip

All genes with a best BLAST search E-value in the class Lecanoromycete were filtered and assigned to the lichen-forming fungus transcriptome (6177 genes). To ensure no genes were erroneously assigned to the lichen-forming fungus (e.g. other ascomycetes living in/on the E. mesomorpha lichen), assigned lichen-forming fungus isoforms were BLAST searched against the E. prunastri genome (Meiser et al. 2017). Any genes for which there was no BLAST match were removed from the lichen-forming fungus transcriptome (387). This file contains the tblastn search of all lichen-forming fungus isoforms against the Evernia prunastri genome (Meiser et al. 2017). File has no header, but columns are as follows:

• qseqid: query or source (gene) sequence id, in this case the lichen-forming fungus isoform
• sseqid: subject or target (reference genome) sequence id
• pident: percentage of identical positions
• length: alignment length (sequence overlap)
• mismatch: number of mismatches
• gapopen: number of gap openings
• qstart: start of alignment in query
• qend: end of alignment in query
• sstart: start of alignment in subject
• send: end of alignment in subject
• evalue: expect value
• bitscore: bitscore

II.m) uniprot2go.zip

Gene Ontology enrichment analysis was performed using the topGO R package. Because no curated databases of GO terms exist for the component organisms of the E. mesomorpha symbiosis, custom annotation databases were created from all genes associated with the lichen-forming fungus, alga, and lichen-associated bacteria. GO terms were retrieved by mapping gene identities to GO terms using UniProt’s mapping tool (www.uniprot.org/id-mapping). This file contains GO terms for each Uniprot gene function.

• Uniprot_ID: Uniprot gene_organism
• GO: The Gene Ontology of that gene.

II.n) uniprot_accession.zip

Functional annotation of transcripts was performed by using predicted coding regions and blastp searching against the Uniprot database (release 2023_01) with a E-value threshold of 0.001. Gene functions were inferred by assigning the best E-value Viridiplantae match for the lichen-forming algae assigned genes, Ascomycete match for the lichen-forming fungus assigned genes, Basidiomycete match for the lichen-associated basidiomycete genes, and Bacteria for the lichen-associated bacteria genes. To obtain taxonomic data for each uniprot gene_organism, the following SQL command was performed on the provided Trinotate boilerplate SQL database “Trinotate.sqlite”. 

.headers on
.mode csv
.output uniprot_accession.csv

SELECT UniprotIndex.Accession, UniprotIndex.LinkID, TaxonomyIndex.TaxonomyValue FROM UniprotIndex JOIN TaxonomyIndex ON UniprotIndex.LinkID = TaxonomyIndex.NCBITaxonomyAccession;

.quit

Columns:

Accession: The gene and organism from which that gene comes, in uniprot gene_organism format.
LinkID: ID that links Uniprot organism to NCBI taxonomy
TaxonomyValue:  NCBI Taxonomy Accession

II.o) Transcript_Taxa_Identification.R

R script for taxon identification from transcripts.

II.p) Gene_Function_Identification.R

R script for gene function identifications.

II.q) Differential_Expression_GO_analysis

R script for analysis of differentially expressed transcripts based on Gene Ontology.

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III. Multi-species survey of ecophysiology

III.a) CrossTaxaSurvey.csv

This file "CrossTaxaSurvey.csv" contains the summarized gas-exchange data (assimilation and respiration rates in vapor and liquid) for lichens across a wide range of lineages and traits, including associated categorical traits and classification. Hydration conditions and gas-exchange measurement conditions were the same as with Evernia mesomorpha (see above). Larger thalli (macrolichens and microlichens attached to larger rocks) were measured using the Bryophyte Chamber (6800-24, internal volume of 193 cm3) and smaller thalli (mostly microlichens) were measured using the Aquatic Chamber (6800-18, internal volume of 20 cm3). To reduce rates of water loss during measurement (and thus maintain constant hydration levels over the course of measurement periods) with large thalli, low temperature and high humidity conditions were applied: 6ºC, RH target 95% (realized RH ~91%), 500 µmol/s flow rate and reference CO2 of 410 ppm. For light measurements, the head light was set at a PAR of 800 μmol/m2s and the color ratio of r90b10. With the smaller thalli, measurements were conducted at room temperature ~26ºC in the chamber, RH target 95% (realized RH ~91%), 500 µmol/s flow rate and reference CO2 of 400 ppm. For light measurements, the head light was set at a PAR of 500 μmol m-2 s-1 and the color ratio of r90b10 for all microlichen specimens, with additional light response curves conducted for at least 1 specimen/species. When Asat exceeded a PAR of 500 μmol m-2 s-1 (1 species, Roccella), an additional measurement at saturating light levels (1000 μmol m-2 s-1) was made. Gas-exchange measurements were recorded following stabilization of IRGA readings for at least two minutes (typical time, 3-5 minutes), at which point 10 replicate measurements at 1-2 second intervals were logged to obtain a mean value. Dark measurements always preceded light measurements.

• Taxon-Categorical- Informal taxon name used in measurements
• Type- Categorical- Instrumentation used: while all thalli were measured using a LI-6800 Infrared Gas Analyzer, "Macro" thalli were measured in the associated "Bryophyte" chamber, whereas "Micro" were measured in the "Aquatic" chamber. See methods for more details.
• Replicate- Numeric- Thallus replicate number (typically 5 replicates/taxon)
• Phot_V- Numeric- Net CO2 exchange rate (micromol m-2 s-1) in a saturating vapor atmosphere and saturating light
• Resp_V- Numeric- Net CO2 exchange rate (micromol m-2 s-1) in a saturating vapor atmosphere and dark
• Phot_L- Numeric- Net CO2 exchange rate (micromol m-2 s-1) after liquid hydration to water-holding capacity and saturating light
• Resp_L- Numeric- Net CO2 exchange rate (micromol m-2 s-1) after liquid hydration to water-holding capacity and dark
• Rprop- Numeric- Calculated ratio of vapor:liquid respiration rates Resp_V/Resp_L
• Aprop- Numeric- Calculated ratio of vapor:liquid gross assimilation rates (Phot_V-Resp_V)/(Phot_L-Resp_L)
• Avgross- Numeric- Calculated gross CO2 exchange rate (micromol m-2 s-1) in a saturating vapor atmosphere and saturating light
• Algross- Numeric- Calculated gross CO2 exchange rate (micromol m-2 s-1) after liquid hydration to water-holding capacity and saturating light
• Taxon2- Categorical- Formal taxon name of primary lichen-forming fungus (genus and species)
• GrowthForm- Categorical- Growth form of lichen thallus
• Photobiont- Categorical- Genus of primary lichen-forming alga in thallus, from literature
• Photo.Order- Categorical- Order of primary lichen-forming alga
• Substrate- Categorical- Growth context of lichen thallus (e.g. Epiphyte, Saxicolous, etc)
• Family- Categorical- Family of primary lichen-forming fungus
• Order- Categorical- Order of primary lichen-forming fungus
• Class- Categorical- Class of primary lichen-forming fungus
• State- Categorical- Location of collection of thallus (United States 2-letter state abbreviation)

Code/Software

R Scripts for analysis of the data are included, and listed above at the end of each thematic section