DOI: 10.13020/3vst-gp16
title: Dataset supporting Coming out of the cold: Vanadium is an important player in N-fixation in warm tropical environments

description: Aim: This study aimed to investigate the presence of vanadium nitrogenase genes in a tropical cyanolichen and assess the impact of micronutrient supply (molybdenum and vanadium) and elevated carbon dioxide concentrations on nitrogen fixation.

Methods and Results: We obtained sequences of vnfN and vnfDG genes from tropical lichen specimens in Brazil and compared them to specimens from deciduous forests in the United States and boreal forests in Canada. Sequences were assembled with sequences from GenBank and aligned with MAFFT using the auto option and then manually corrected in Geneious Prime 2023.2.1. The dataset was then subjected to a maximum likelihood search using IQ-TREE web server with parametric bootstrapping using 1000 replicates under the best-fitted model. The tree inference results were submitted to FigTree 1.4.4 version. Our results revealed that tropical samples form a completely separate group from temperate and boreal samples, and, for the first time, vanadium nitrogenase genes were found in tropical cyanolichens.
We also counted the number of heterocysts on specimens exposed to different treatments to obtain a proxy for nitrogen fixation activity. Paradermal sections of lichen fragments were photographed, and the number of heterocysts and total cells (heterocysts + vegetative cells) was quantified. The results showed a significant influence of carbon dioxide concentrations and vanadium on heterocyst investment.

Conclusions: The findings suggest that vanadium nitrogenase is not an adaptive strategy to cold environments, as this isoform can be found in the tropics. Heterocyst investment, and likely nitrogen fixation, are controlled by carbon dioxide and vanadium. Finally, if we want accurate models of the N cycle, vanadium nitrogenase needs to be considered in the estimates.

The data have been released as part of the manuscript review process at Ecology.
description: 1. File List
 A. Filename: vnfN_assemblage.fasta
 Short description: File containing 25 samples (description + sequence) 

 B. Filename: vnfDG_assemblage.fasta
 Short description: File containing 39 samples (description + sequence)
 
 C. Filename: ecology_ressubmission_counting_data.csv
 Short description: Matrix of 133 rows (pictures) and 8 columns (treatment, period, lichen, heterocysts_frequency, total_cells, treatment_CO2, treatment_Mo, and treatment_V)

2. Relationship between files: The two FASTA files contain gene sequence data (vnfN and vnfDG) obtained from samples associated with the experimental treatments described in the ecological dataset. The file ecology_ressubmission_counting_data.csv provides quantitative and treatment-related metadata (e.g., CO₂, Mo, V treatments, heterocyst frequency, total cell counts) corresponding to the samples from which the sequences were derived. Together, these files link molecular data with ecological and experimental variables used in the analyses.

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METHODOLOGICAL INFORMATION
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1. Description of methods used for the collection/generation of data: 

Lichen transplants were maintained in a growth chamber(CONVIRON®, ATC60, Canada) at ~75% relative humidity, with day and night temperatures of 20°C and 17°C, respectively, a 10-hour photoperiod, and photosynthetic active radiation (PAR) of 10 µmol m−2 s−1. Specimens were acclimatized for four days before exposure to high CO2 concentration and micronutrients. The experiment then ran for 27 days, and heterocyst numbers were recorded on days 1 and 27. To evaluate how high CO2 concentrations and micronutrient supply affect heterocyst proportions, we assigned L. cyanescens specimens to six treatments: control, Mo, V, CO2, CO2Mo, and CO2V (n = 11 per treatment). Elevated CO2 treatments exposed specimens sampled in June 2020 to 800 ppm CO2, whereas ambient CO2 treatments exposed specimens sampled in July 2020 to laboratory CO2 concentrations (~450 ppm). We sprayed approximately 2 mL of either Mo (2.1 µM Na2MoO4.2H2O) or V(12.1 µM Na3VO4) onto specimens from the micronutrient treatments daily.
Heterocysts serve the sole role of fixing N, and their differentiation from vegetative cells is energetically costly, occurring only under reactive N limitation. Because heterocyst abundance correlates with Nase activity (Hitch and Millbank 1975), we used heterocyst counts as a cost-effective proxy for N-fixation rates instead of ARA. Heterocyst assessments followed an adapted protocol from Fávaro et al. (2021). We removed fragments (1 × 1 cm) from thallus margins, hydrated them with distilled water, and performed eight-μm paradermal sections beneath the upper cortex using a LEICA 133 CM1850 cryostat. At least four fragments were analyzed under an Olympus BX41 compound microscope at 1000x magnification (Appendix S1: Figure S1). Then, we selected five random microscope fields per specimen, photographed them, and counted heterocysts and vegetative cells in ImageJ. We calculated heterocyst proportion by dividing the number of heterocysts by the total number of cyanobacterial cells (heterocysts + vegetative cells), and assumed it to be representative of the thallus.
To verify whether L. cyanescens harbors genes encoding V-Nase (vnfDG and vnfN), we sequenced six randomly selected Brazilian specimens from our experiment and five additional specimens from the USA (Florida and North Carolina). The vnfDG genes encode subunits of the Nase enzyme, while vnfN is involved in assembly and regulation(Harwood 2020). All molecular procedures were performed at the Sumner Laboratory, Botanical Research Institute of Texas (BRIT). Genomic DNA was extracted from lichen fragments using the FastDNATM SPIN Kit (MP Biomedicals, USA) with minor modifications, and PCR amplification, clean-up, and Sanger sequencing followed Dal-Forno et al. (2013). We used primers from Darnajoux et al. (2019) and primers specifically designed for L. cyanescens (Appendix S1: Table S1). Newly generated sequences were assembled with GenBank sequences and aligned with MAFFT (auto option), then manually refined in Geneious Prime 2023.2.1. Phylogenetic relationships among vnf genes were inferred using samples from a tropical forest in Brazil, deciduous forests in the United States, and boreal forests in Canada, with free-living cyanobacteria included for comparison. We conducted a maximum-likelihood search using IQ-TREE with 1,000 bootstrap replicates under the best-fitted model. Trees were visualized in FigTree 1.4.4.


2. Methods for processing the data: 
We used dummy coding (0, 1) to represent the presence or absence of each treatment factor: CO2 (ambient/450 ppm = 0, elevated/800 ppm = 1), V (absent = 0, present = 1), and Mo (absent = 0, present = 1). Treatments served as explanatory variables in all models, with heterocyst proportion or number of total cells as response variables. We analyzed these response variables at three stages: (1) at the beginning of the experiment to assess any pre-existing differences between treatments, (2) at the end to check absolute differences, and (3) as the net change (delta) throughout the experiment to assess the magnitude of treatment-induced changes. 

3. Instrument- or software-specific information needed to interpret the data:
Genomic DNA was extracted using the FastDNA™ SPIN Kit (MP Biomedicals). Sequence alignment was performed with MAFFT, and alignments were manually refined in Geneious Prime 2023.2.1. Phylogenetic analyses were conducted using IQ-TREE with 1,000 bootstrap replicates under the best-fit substitution model. Phylogenetic trees were visualized using FigTree 1.4.4. Statistical analyses were conducted in R version 4.4.3 (R Core Team 2025), and graphs were created with the ggplot2 (Wickham 2016). 

4. Standards and calibration information, if appropriate: NA

5. Environmental/experimental conditions:
Lichen transplants were maintained in a growth chamber(CONVIRON®, ATC60, Canada) at ~75% relative humidity, with day and night temperatures of 20°C and 17°C, respectively, a 10-hour photoperiod, and photosynthetic active radiation (PAR) of 10 µmol m−2 s−1. Specimens were acclimatized for four days before exposure to high CO2 concentration and micronutrients. The experiment then ran for 27 days, and heterocyst numbers were recorded on days 1 and 27. To evaluate how high CO2 concentrations and micronutrient supply affect heterocyst proportions, we assigned L. cyanescens specimens to six treatments: control, Mo, V, CO2, CO2Mo, and CO2V (n = 11 per treatment). Elevated CO2 treatments exposed specimens sampled in June 2020 to 800 ppm CO2, whereas ambient CO2 treatments exposed specimens sampled in July 2020 to laboratory CO2 concentrations (~450 ppm). We sprayed approximately 2 mL of either Mo (2.1 µM Na2MoO4.2H2O) or V(12.1 µM Na3VO4) onto specimens from the micronutrient treatments daily.

6. Describe any quality-assurance procedures performed on the data:
Sequence data were quality-checked by trimming low-quality regions, verifying sequence length and alignment accuracy, and assessing phylogenetic support using bootstrap values. Counting data were inspected for transcription errors, missing values, and consistency among treatments.

7. People involved with sample collection, processing, analysis and/or submission:
 Sample collection: A.F., L.C.O.L.
 Processing: A.F., M.D.F.
 Analysis: A.F., M.D.F., D.S.
 Submission: A.F., M.D.F., D.S., L.C.O.L., F. F. C.

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DATA-SPECIFIC INFORMATION FOR: vnfN_assemblage.fasta
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1. Number of variables: 25

2. Number of cases/rows: 50

3. Missing data codes: NA
 
4. Variable List:
Each sequence entry starts with a line that begins with the "&gt;" symbol. It indicates the sequence identifier.
Each variable contains a brief description (sample code/accession number, species, and sequence)

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DATA-SPECIFIC INFORMATION FOR: vnfDG_assemblage.fasta
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1. Number of variables: 39

2. Number of cases/rows: 78

3. Missing data codes: NA

4. Variable List
Each sequence entry starts with a line that begins with the "&gt;" symbol. It indicates the sequence identifier.
Each variable contains a brief description (sample code/accession number, species, and sequence)

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DATA-SPECIFIC INFORMATION FOR: counting_data.csv
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1. Number of variables: 7

2. Number of cases/rows: 716

3. Missing data codes: NA

4. Variable List
treatment (categorical data): Conditions to which specimens were exposed (natural = no micronutrient addition and forest CO2 concentrations; control = no micronutrient addition and lab ambient CO2 concentrations; CO2 = no micronutrient addition and high CO2 concentrations; Mo = Molybdenum addition and lab ambient CO2 concentrations; V = vanadium addition and lab ambient CO2 concentrations; CO2Mo = Molybdenum addition and high CO2 concentrations; CO2V = Vanadium addition and high CO2 concentrations).
period (categorical data): When the data was collected (single = sample from the natural environment; didn't go through the experimental conditions; beginning = first days of experiment, after acclimatation; end = end of the experiment, after being exposed to all experimental conditions)
lichen (categorical data): Code given to a certain specimen so we could differentiate them.
heterocysts_frequency (continuous data): Number of heterocysts divided by the number of total cells
total_cells (continuous data): Number of heterocysts plus the number of vegetative cells found in the image
treatment_CO2 (continuous data): Binary code used to report if CO2 was present or not
treatment_Mo (continuous data): Binary code used to report if Mo was present or not
treatment_V (continuous data): Binary code used to report if V was present or not