DOI: 10.5061/dryad.x95x69pxd
title: The role of cytonuclear interactions in plant adaptation across a <em>Populus</em> hybrid zone

description: Co-adaptation of cytoplasmic and nuclear genomes is critical to
 physiological function for many species. Despite this understanding,
 hybridization can disrupt co-adaptation, leading to a mismatch between
 maternally-inherited cytoplasmic genomes and biparentally inherited
 nuclear genomes. Few studies have examined the consequences of cytonuclear
 interactions on physiological function across environments. Here, we
 quantify the degree of co-introgression between chloroplast and
 nuclear-chloroplast (N-cp) genes across repeated hybrid zones and their
 consequences to physiological function across environments. We use
 whole-genome resequencing and common garden experiments with clonally
 replicated genotypes sampled across the natural hybrid zone between
 Populus trichocarpa and P. balsamifera. We use geographic clines to test
 for co-introgression of the chloroplast genome with N-cp and
 non-interacting nuclear genes. Co-introgression of chloroplast and N-cp
 genes was limited, although contact zone-specific patterns suggest that
 local environments may influence co-introgression. Combining ancestry
 estimates with phenotypic data across common gardens revealed that
 mismatches between chloroplast and nuclear ancestry can influence
 physiological performance, but the strength and direction of these effects
 vary depending on the environment. Overall, this study highlights the
 importance of cytonuclear interactions to adaptation and the role of the
 environment in modifying the effect of those interactions.
description: ## The role of cytonuclear interactions in plant adaptation across a
 *Populus* hybrid zone This repository contains the code, data, and
 analyses used in the study of how chloroplast and nuclear genome
 interactions contribute to adaptation across *Populus* hybrid zones. It
 includes chloroplast genome assembly, phylogenetic inference,
 environmental association tests, geographic cline analyses, and trait
 modeling in common garden experiments.
 \-------------------------------------------------------------------------------- Code_Populus_CNI.zip #### 1. Chloroplast Genome Assembly and Phylogeny ##### 1.1 Assembly (NOVOPlasty) **Folder:** `1.1_Assembly_NOVO_Plasty/` – Input files for NOVOPlasty assembly. * **config_poplar** – Configuration file with assembly parameters: * Type: chloro * Genome range: 140,000–170,000 bp * k-mer: 33 * Variance detection: Enabled * Sequencing: Illumina PE, 151 bp reads, 300 bp insert, PCR-free **rbcL_seed.fasta** – FASTA file used to initiate assembly; region NC_009143.1:55716–57143 from *P. trichocarpa* chloroplast genome. **P_trichocarpa_plastid.fasta** – NC_009143.1 *P. trichocarpa* complete chloroplast genome used as reference. **Sequencing data:** Genomic libraries were sequenced on an S4 flow cell in 2x150bp format on an Illumina NovaSeq 6000 instrument with 64 samples per lane \-------------------------------------------------------------------------------- ##### 1.2 Phylogeny **Folder:** `1.2_Phylogeny/` – Alignment files, scripts, and results for chloroplast genome phylogenetic inference. **Software versions:** - MAFFT v7.481 - IQ-TREE v1.0 **Files:** * **Phylogeny_ALL.fasta** – Chloroplast genome sequences for all genotypes. * **Phylogeny_ALL_outputmafft.fasta** – MAFFT-aligned chloroplast genomes. * **Phylogeny_ALL_outputmafft2.fasta** – Same alignment as above, with *Salix* group labeled as outgroup. * **Phylogeny_ALL_outputmafft.fasta.contree** – Consensus tree from IQ-TREE analysis. * **iqtree.sh** – Shell script for IQ-TREE: ```` \```bash iqtree -s ALL_samples_Outgroups/Phylogeny_ALL_outputmafft2.fasta \ -m TVM+F+R2 \ -bb 1000 \ -redo \``` \- `-s` – Input alignment file \- `-m TVM+F+R2` – Substitution model \- `-bb 1000` – Ultrafast bootstrap (1000 replicates) \- `-redo` – Overwrite previous runs ```` \-------------------------------------------------------------------------------- ##### 1.3 Fixed Differences **Folder:** `1.3_Fixed_differences/` – Identifies fixed nucleotide and amino acid differences across chloroplast haplotypes. **Software:** R v4.3.1 **Packages:** - Biostrings - seqinr - dplyr - GenomicRanges - rtracklayer - readr **Files:** - **Fixed_differences.R** – Estimates fixed differences from alignment. - **Poplar_540_plus_Reference.fasta** – Alignment of all chloroplast genomes with reference sequence. \-------------------------------------------------------------------------------- #### 2. Environmental Influence on Chloroplast Ancestry **Folder:** `2_Environmental_influence_chloroplast_ancestry/` – Tests whether climate predicts chloroplast ancestry. **Software:** R v4.3.1 **Packages:** - ggplot2 - dplyr - logistf - gridExtra **Files:** - **Binomial_regressions.R** – Logistic regression: chloroplast ancestry ~ climate variables. - **Climate_chloroplast.xlsx** – Environmental, geographic, and ancestry variables per genotype: - **MAT** – Mean Annual Temperature (°C) - **TD** – Continentality (°C) - **MAP** – Mean Annual Precipitation (mm) - **PAS** – Precipitation as Snow (mm) - **CMD** – Climatic Moisture Deficit (mm) - **RH** – Relative Humidity (%) - **Chlorotype** – Chloroplast haplotype (0 = *P. balsamifera*, 1 = *P. trichocarpa*) - **Transect** – Contact zone identifier - **Distance_along_transect** – Distance from reference point along transect (km) - **Latitude**, **Longitude** – Coordinates - **P_trichocarpa_Ancestry** – Proportion of *P. trichocarpa* nuclear ancestry \-------------------------------------------------------------------------------- #### 3. Geographic Clines ##### 3.1 Local Ancestry Inference **Folder:** `3.1_Local_Ancestry_inference/` – Scripts for estimating local ancestry per transect using whole genome resequencing data and LOTER. **Software:** - vcftools v0.1.17 - LOTER v1.0 (CLI) - datamash v1.8 - bash (GNU) **Files:** - **3.1.1_parsing_vcf_files.sh** – Extracts reference individuals, SNP positions, and per-individual VCFs for admixed genotypes. - **3.1.2_LOTER.sh** – Runs LOTER for each admixed individual and chromosome. - **3.1.3_diploid_matrix.sh** – Converts LOTER outputs to 0/1/2 diploid matrices per chromosome. - **3.1.4_combined_CHRs.sh** – Combines per-chromosome matrices into a genome-wide file. - **Admixed_IDs_all_transects**, **Balsam_IDs_all_transects**, **Tricho_IDs_all_transects** – ID lists for each ancestry group. \-------------------------------------------------------------------------------- ##### 3.2 Estimating Ancestry per Gene **Folder:** `3.2_Estimating_Ancestry_genes/` – Extracts ancestry for nuclear–chloroplast (N-cp) and non-interacting (non-N-cp) genes **Software:** R v4.3.1 **Packages:** - dplyr - data.table **Files:** - **Ancestry_N_cp_genes.R** – Estimates ancestry for N-cp genes. - **Ancestry_non_interac_genes.R** – Estimates ancestry for non-N-cp genes. \-------------------------------------------------------------------------------- ##### 3.3 Geographic Cline Modeling (HZAR) **Folder:** `3.3_HZAR/` – Geographic cline modeling with HZAR. **Software:** R v4.3.1 **Packages:** - MCMCpack - hzar - dplyr - parallel **Files:** - **3.3.1_Geo_clines_N_cps.R** – Fits clines for N-cp genes. - **3.3.2_Geo_clines_non_n_cp_genes.R** – Fits clines for non-N-cp genes. - **3.3.3_Mean_cline_param_non_n_cp.R** – Calculates mean cline parameters for non-N-cp genes. - **Data_frame_Ncp_clines_HZAR.csv** – Geographic and ancestry data for N-cp genes: **ID**, **Transect**, **Longitude**, **Latitude**, **Plasid_ID** (0 = *P. balsamifera*, 1 = *P. trichocarpa*), **Distance_along_transect**, ancestry columns for each N-cp gene (0= *P. balsamifera* homozygous, 0.5 heterozygous, 1 = *P. trichocarpa* homozygous). - **Data_frame_non_n_cp_genes_clines_HZAR.csv** – Geographic and ancestry data for non-N-cp genes: **ID**, **Transect**, **Longitude**, **Latitude**, **Plasid_ID**, **Distance_along_transect**, ancestry columns for each non-N-cp gene (0= *P. balsamifera* homozygous, 0.5 heterozygous, 1 = P. trichocarpa homozygous). \-------------------------------------------------------------------------------- ##### 3.4 Co-introgression Test **Folder:** `3.4_Co_introgression_test/` – Tests if N-cp genes have cline parameters more similar to the chloroplast cline. **Software:** R v4.3.1 **Packages:** - MCMCpack - hzar - dplyr - parallel **Files:** - **3.4.1_Co_introgression.R** – Performs co-introgression analysis. - **df_for_clines_genome_wide_chloroplast.xlsx** – Summary for all gene clines: **ID**, **Transect**, **TD**, **Distance_along_transect**, **non.N.cp_genes_mean_ancestry**, **Chlorotype** (0 = *P. balsamifera*, 1 = *P. trichocarpa*). \-------------------------------------------------------------------------------- **Additional file:** - **HaversineFormula.R** – Computes pairwise geographic distances between sampling sites. \-------------------------------------------------------------------------------- #### 4. Phenotypic Data – Linear Mixed Models **Folder:** `4_Phenotypic_data_LMM/` – Mixed-model analysis of phenotypic traits in common gardens. **Software:** R v4.3.1 **Packages:** - ggplot2 - lme4 - dplyr - sjPlot **Files:** - **4.1_Linear_Mixed_Models.R** – Fits linear mixed-effects models for phenotypic traits across gardens. - **4.2_Heritability.R** – Estimates broad-sense heritability of traits using linear mixed models. - **Phenotypic_Data_CNI.xlsx** – Trait, ancestry, and collection metadata. **Columns:** * **PLANT_ID** – Individual plant identifier * **Transect_SL** – Sampling contact zone name * **GARDEN** – Common garden site * **BLOCK**, **mBlock** – Garden block identifiers * **MAT** – Mean Annual Temperature (°C) * **MAP** – Mean Annual Precipitation (mm) * **TD** – Continentality (°C) * **RH** – Relative Humidity (%) * **CMD** – Climatic Moisture Deficit (mm) * **PAS** – Precipitation as Snow (mm) * **Nuclear_ancestry** – Proportion of *P. trichocarpa* nuclear ancestry * **Chloroplast_Ancestry** – Chloroplast haplotype (0 = *P. balsamifera*, 1 = *P. trichocarpa*) * **gsw** – Stomatal conductance (mmol m⁻² s⁻¹) * **PS2** – Photosystem II quantum efficiency * **ETR** – Electron transport rate (µmol m⁻² s⁻¹) * **Fs** – Steady-state fluorescence * **Fm** – Maximum fluorescence * **d13C** – Carbon isotope discrimination (‰) * **N** – Leaf nitrogen content (%)