DOI: 10.5061/dryad.5tb2rbp9f
title: Complex polyploids: Origins, genomic composition, and role of introgressed alleles

description: Introgression allows polyploid species to acquire new genomic content from
 diploid progenitors or from other unrelated diploid or polyploid lineages,
 contributing to genetic diversity and facilitating adaptive allele
 discovery. In some cases, high levels of introgression elicit the
 replacement of large numbers of alleles inherited from the
 polyploid's ancestral species, profoundly reshaping the
 polyploid's genomic composition. In such complex polyploid, it is
 often difficult to determine which taxa were the progenitor species and
 which taxa provided additional introgressive blocks through subsequent
 hybridization. Here, we use population-level genomic data to reconstruct
 the phylogenetic history of Betula pubescens (downy birch), a tetraploid
 species often assumed to be of allopolyploid origin and which is known to
 hybridize with at least four other birch species. This was achieved by
 modeling polyploidization and introgression events under the multispecies
 coalescent and then using an approximate Bayesian computation (ABC)
 rejection algorithm to evaluate and compare competing polyploidization
 models. We provide evidence that B. pubescens is the outcome of an
 autoploid genome doubling event in the common ancestor of B. pendula and
 its extant sister species, B. platyphylla, that took place approximately
 178,000-188,000 generations ago. Extensive hybridization with B. pendula,
 B. nana, and B. humilis followed in the aftermath of autopolyploidization,
 with the relative contribution of each of these species to the B.
 pubescens genome varying markedly across the species' range.
 Functional analysis of B. pubescens loci containing alleles introgressed
 from B. nana identified multiple genes involved in climate adaptation,
 while loci containing alleles derived from B. humilis revealed several
 genes involved in the regulation of meiotic stability and pollen viability
 in plant species.
description: Analysis based on 269 newly sequenced <em>B.
 pubescens</em> individuals collected from twenty-tree locations
 along the species' range. Additional accessions were obtained from
 the European Nucleotide Archive (ENA) for <em>B. lenta</em>,
 <em>B. nana</em>, <em>B. occidentalis</em>,
 <em>B. pendula</em>, <em>B. platyphylla</em>,
 <em>B. populifolia</em>, <em>B. pubescens</em>,
 <em>Alnus glutinosa</em>, and <em>A. incana</em>,
 at www.ebi.ac.uk, under
 the PRJEB14544 accession code. For the newly collected samples, individual
 libraries were generated using custom probes designed for targeted exome
 capture and sequenced on paired-end mode (150 bp) on an Illumina NovaSeq
 6000 platform.
description: # Supplementary data for: Complex Polyploids: Origins, Genomic
 Composition, and Role of Introgressed Alleles #### J.L. Leal, Pascal
 Milesi, Eva Hodková, Qiujie Zhou, Jennifer James, D. Magnus Eklund, Tanja
 Pyhäjärvi, Jarkko Salojärvi, and Martin Lascoux ####
 https://doi.org/10.5061/dryad.5tb2rbp9f --- Scripts used for analysis of
 complex polyploids are available at:
 https://github.com/LLN273/Complex-Polyploids ## General description **A.
 Analysis of B. pubescens genomic data** Data used to produce Fig. 2a, Fig.
 2b, Fig. S7, and Fig. S8. 01_MSAs_BEFORE_POLARIZATION.tzr.gz
 02_MSAs_AFTER_POLARIZATION.tzr.gz 03_IQTREE_RESULTS.tzr.gz
 04_Polyploid_pairing_profiles.tzr.gz **B. STRUCTURE analysis:** Results
 from STRUCTURE analysis (used to produce Fig. 2c, Fig. S9, and Fig. S10).
 05_STRUCTURE_analysis.tzr.gz **C. DAPC analysis:** Input data used to
 produce Fig. 2d, and Fig. S11. 06_DAPC_adegenet_analysis.tzr.gz **D.
 Simulated datasets and polyploidization model testing:** Data used to
 produce Fig. 3, Fig. 4, Table S2, Fig. S12, and Fig. S13.
 07_SIMULATION_DATA_AND_POLYPLOIDIZATION_MODEL_TESTING.tzr.gz **E.
 Demographic modeling:** Input data used to produce Fig. 5, Table S3, and
 Table S4. 08_Demographic_modeling.tzr.gz **F. Analysis of B. pubescens
 alleles of B. nana or B. humilis origin: geographic distribution &
 functional analysis:** Data used to produce Fig. 6, Fig. 7, and Table 1.
 09_nana_humilis_alleles_geographic_distribution.tzr.gz ## Detailed
 description #### A. Analysis of B. pubescens genomic data A.1
 Multiple-sequence alignments (MSAs) for different samples and individual
 gene families, BEFORE polarization. Data provided for 269 B. pubescens
 specimens, 25 B. pendula autotetraploids, and 2 triploid hybrids.. MSAs
 are provided both in fasta (.fa) and nexus (.nxs) format. Actual pipeline
 was run using MSAs in fasta format. MSAs can be opened using an alignment
 viewer, for example, AliView (https://ormbunkar.se/aliview/). Produced
 using scripts in https://github.com/LLN273/Complex-Polyploids
 [01_Read_mapping_and_variant_calling and 03_Generate_MSAs], based on the
 raw Illumina libraries available in The European Nucleotide Archive (ENA)
 at www.ebi.ac.uk under the PRJEB64873 project accession code. Folders:
 01_MSAs_BEFORE_POLARIZATION/fasta A.2 MSAs for different samples and
 individual gene families, AFTER polarization of the focal polyploid
 sequence. MSAs are provided both in fasta (.fasta) and nexus (.nxs)
 format. Actual pipeline was run using MSAs in fasta format. Produced using
 scripts in
 https://github.com/LLN273/Complex-Polyploids/tree/main/04_Polarize_polyploid Input data: MSAs described in A.1 above # 2.A Reference sequence used during polarization: B. pendula Folders: 02_MSAs_AFTER_POLARIZATION/A_Bpubescens_POLARIZED.1_REFSEQ.PENDULA_FINLAND_LOIMAA.LOIMAA # 2.B Reference sequence used during polarization: B. nana Folders: 02_MSAs_AFTER_POLARIZATION/B_Bpubescens_POLARIZED.2_REFSEQ.NANA_FINLAND_ENONTEKIO.A002_2 # 2.C Reference sequence used during polarization: B. platyphylla Folders: 02_MSAs_AFTER_POLARIZATION/C_Bpubescens_POLARIZED.3_REFSEQ.PLATYPHYLLA_RUSSIA.A001_1 # 2.D Reference sequence used during polarization: B. humilis Folders: 02_MSAs_AFTER_POLARIZATION/D_Bpubescens_POLARIZED.4_REFSEQ.HUM02 A.3 IQTREE2 output files, for different samples and polarization geometries. Multiple output files (.iqtree and .log). Can be opened with a text editor. Produced using script 01_IQ-TREE2_SINGLEGenes_query.sh in https://github.com/LLN273/Complex-Polyploids/tree/main/05_Phylogenetic_Analysis Input data: MSAs described in A.2 Folders: 03_IQTREE_RESULTS/A_Bpubescens_POLARIZED.1_REFSEQ.PENDULA_FINLAND_LOIMAA.LOIMAA 03_IQTREE_RESULTS/B_Bpubescens_POLARIZED.2_REFSEQ.NANA_FINLAND_ENONTEKIO.A002_2 03_IQTREE_RESULTS/C_Bpubescens_POLARIZED.3_REFSEQ.PLATYPHYLLA_RUSSIA.A001_1 03_IQTREE_RESULTS/D_Bpubescens_POLARIZED.4_REFSEQ.HUM02 A.4 Observed polyploid pairing profiles, for different SAMPLES and polarization geometries. Produced using scripts in https://github.com/LLN273/Complex-Polyploids/tree/main/05_Phylogenetic_Analysis Input data: MSAs described in A.3 Folders: 04_Polyploid_pairing_profiles/A_Bpubescens_POLARIZED.1_REFSEQ.PENDULA_FINLAND_LOIMAA.LOIMAA 04_Polyploid_pairing_profiles/B_Bpubescens_POLARIZED.2_REFSEQ.NANA_FINLAND_ENONTEKIO.A002_2 04_Polyploid_pairing_profiles/C_Bpubescens_POLARIZED.3_REFSEQ.PLATYPHYLLA_RUSSIA.A001_1 04_Polyploid_pairing_profiles/D_Bpubescens_POLARIZED.4_REFSEQ.HUM02 File description: sister_ID_analysis_COUNTS-XXXXX.txt lists number of times (and normalized frequency) polarized focal polyploid sequence pairs with different species/clades included in the MSA sister_ID_analysis_summary_-XXXXX.txt polyploid pairing profile (same information as above for selected species and clades). A.5 Observed polyploid pairing profiles, for different POPULATIONS and polarization geometries. Used to produce Fig. 2a, Fig. 2b, Fig. S7, and Fig. S8. Input data: polyploid pairing profiles listed A.4 Folders: 04_Polyploid_pairing_profiles/SUMMARY_TABLES_Observed_Pairing_Profiles_per_population #### B. STRUCTURE analysis Input data used to produce Fig. 2c, Fig. S9, and Fig. S10. Figures produced using scripts in https://github.com/LLN273/Complex-Polyploids/tree/main/06_Prepare_data_for_STRUCTURE_and_adegenet_analyses and https://github.com/LLN273/Complex-Polyploids/tree/main/07_STRUCTURE_analysis Folders: 05_STRUCTURE_analysis/15_NANA_SILVER_WHITE_HUMILIS_PLATYPHYLLA_ReducedPEND_ReducedSW_10kSNP_SHORT/ Files (all files can be opened using a text editor): mainparams STRUCTURE input file (key parameters file) extraparams STRUCTURE input file (extra parameters file) XXX.str input data file seed.txt and strdataset files produced by structure; can be ignored Folders: 05_STRUCTURE_analysis/15_NANA_SILVER_WHITE_HUMILIS_PLATYPHYLLA_ReducedPEND_ReducedSW_10kSNP_SHORT/STRUCTURE_RESULTS Files (all files can be opened using a text editor): str_KXX_repYY_f results from STRUCTURE analysis, computed for K=XX , replicate YY [K=2...9; 16 replicates for each K] #### C. DAPC analysis Input data used in DAPC analysis used to produce Fig. 2d, and Fig. S11. Compressed VCF file produced using scripts in https://github.com/LLN273/Complex-Polyploids/tree/main/06_Prepare_data_for_STRUCTURE_and_adegenet_analyses Compressed VCF file can be viewed using the bash command "zcat myfile.VCF.gz" DAPC plots produced using scripts in https://github.com/LLN273/Complex-Polyploids/tree/main/08_DAPC_adegenet Folders: 06_DAPC_adegenet_analysis/INPUT_VCF_FILE/ #### D. Simulated datasets and polyploidization model testing D.1 Generation of simulated MSAs Data used to produce Fig. 3, Fig. 4, Table S2, Fig. S12, and Fig. S13. Simulated gene trees and MSAs; 25 replicates were generated; 50 gene families were generated for each replicate. For each replicate, we provide the raw data obtained after running Simphy and INDELible. Trees can be displayed using, for example, TreeGraph2 (http://treegraph.bioinfweb.info/). MSAs can be opened using an alignment viewer, for example, AliView (https://ormbunkar.se/aliview/). D.1.1 Gene trees generated by Simphy, after fixing tree tips. Produced using scripts 01_simphy_ILS_birch.sh and 02_fix_tree_tips_lowILS_MAIN.sh in https://github.com/LLN273/Complex-Polyploids/tree/main/09_Model_Testing_1_Polyploidization_and_hybridization_simulations Folders: 07_SIMULATION_DATA_AND_POLYPLOIDIZATION_MODEL_TESTING/01_simphy_ILS/A_modILS_birch_CLEAN Files: g_treesXXXX.trees simulated gene trees, after fixing tips A_modILS_birch.command, A_modILS_birch.db, A_modILS_birch.params extra files produced by simphy (can be ignored) l_trees.trees, s_tree.trees, and Rplots.pdf extra files produced by simphy and our home scripts (can be ignored) 02a_fix_tree_tips_birch.R R script used to fix tree tips after running Simphy D.1.2 Contains gene trees after ILS correction and MSAs produced by INDELible. Produced using scripts 03_adjust_ILS_NEW_MAIN.sh and 04_INDELIble.sh in https://github.com/LLN273/Complex-Polyploids/tree/main/09_Model_Testing_1_Polyploidization_and_hybridization_simulations Input data: gene trees produced in D.1.1 Folders: 07_SIMULATION_DATA_AND_POLYPLOIDIZATION_MODEL_TESTING/01_simphy_ILS/A_modILS_birch_CLEAN_reducedILS_40perct Files: g_treesXXXX.trees simulated gene trees, after ILS correction data_XXXX_TRUE.fasta MSAs generated by INDELible A_modILS_birch.command, A_modILS_birch.db, A_modILS_birch.params extra files produced by simphy (can be ignored) l_trees.trees, s_tree.trees extra files produced by simphy (can be ignored) LOG.txt, trees.txt, control.txt extra files produced by INDELible (can be ignored) D.2 Results from polyploidization model evaluation Simulated polyploid pairing profiles estimated using an approximate Bayesian computation (ABC) framework based on nine polyploidization models [AAAA, AAHH, AANH, AANN, PPHH, PPNH, PPNN, PPPP, PPyPPy]. ABC results also available for models that include homoeologous exchange (HE). For each polyploidization model, ABC was carried out using priors sampled from distributions centered on model parameters initially estimated using simulated annealing (SA), based on observed polyploid pairing profiles for seven B. pubescens populations [Arctic, Central Asia, JOK, LT, Spain, SVsouth, UA] Produced using scripts in https://github.com/LLN273/Complex-Polyploids/tree/main/10_Model_Testing_2_Model_evaluation Input data: simulated MSAs computed in D.1.2 Folders: 07_SIMULATION_DATA_AND_POLYPLOIDIZATION_MODEL_TESTING/03_ABC_Final_optimization Files (all files can be opened using a text editor): summary_ABC_1000_simulations.txt ABC-simulated polyploid pairing profiles for 1,000 independent runs L2-norm_ABC_1000_simulations.txt L2 norm distance computed for each ABC run, using observed polyploid pairing profiles as a reference ABC_PRIORS_PPPP_SVsouth (folder) ABC priors for the twelve model parameters (HO, H1, ..., H9, ILSreduction, platy_pend_gene_flow) #### E. Demographic modeling Input data used to produce Fig. 5, Table S3, and Table S4. Demographic modeling results obtained using the scripts in https://github.com/LLN273/Complex-Polyploids/tree/main/11_Demographic_modeling Folders: 08_Demographic_modeling/INPUT_DATA/ Files: [Note: all files can be opened using a text editor, apart from compressed VCF files which can be viewed using the bash command "zcat myfile.VCF.gz"] MASTER_SingleRuns_ALLsamples_GOODonly_SILVER_WHITE_PLATYPHYLLA.vcf.gz input VCF file Bpendula.annotation-targetGenes_exons_CLEAN.bed B. pendula annotation file covering list of exons include in targeted exome probes Bpendula.annotation-targetGenes_introns_CLEAN.bed B. pendula annotation file covering list of introns include in targeted exome probes 00_samples_SILVER_WHITE_PLATYPHYLLA_divergenceTime_XXXX_4_8.args List of samples included in analysis, with B. pubescens samples collected from XXXX population presence_matrix_annotated_genes_pendula_XXXX-Samples_4.txt List of loci containing alleles exclusively of B. pendula and/or B. platyphylla ancestry; computed separately for each B. pubescens population Nana-Finland-Enontekio.A002-2_snps-CLEAN.vcf.gz VCF file listing variants observed in B. nana Finland-Enontekio.A002-2 sample; used to estimate ancestral B. pendula allele Populifolia-Canada.A005-11_snps-CLEAN.vcf.gz VCF file listing variants observed in B. populifolia Canada.A005-11 sample; used to estimate ancestral B. pendula allele Occidentalis-Canada-Alberta.A009-17_snps-CLEAN.vcf.gz VCF file listing variants observed in B. occidentalis Canada-Alberta.A009-17 sample; used to estimate ancestral B. pendula allele 00_samples_ancestral_state_PENDULA_8.txt List of B. pendula samples included in analysis 00_samples_ancestral_state_PLATYPHYLA_8.txt List of B. platyphylla samples included in analysis 10_simulation_birch_3pop_divTime_model_YYY.est fastsimcoal2 est input file for model YYY 10_simulation_birch_3pop_divTime_model_YYY.tpl fastsimcoal2 tpl input file for model YYY 16_PAR_ending.txt file required when running script 16_fastsimcola26_confidence_intervals_MAIN.sh in https://github.com/LLN273/Complex-Polyploids/tree/main/11_Demographic_modeling #### F. Analysis of B. pubescens alleles of B. nana or B. humilis origin: geographic distribution & functional analysis Data used to produce Fig. 6, Fig. 7, and Table 1. Produced using scripts in https://github.com/LLN273/Complex-Polyploids/tree/main/12_nana_humilis_alleles_geographic_distribution Folders: 09_nana_humilis_alleles_geographic_distribution Files (all files can be opened using a text editor): db_hum_pop_ALL.txt Fraction of B. pubescens individuals in a population containing an allele of B. humilis origin db_nana_pop_ALL.txt Fraction of B. pubescens individuals in a population containing an allele of B. nana origin db_pend_pop_ALL.txt Fraction of B. pubescens individuals in a population containing an allele of B. pendula origin ID_genes-XXXX_summary_ALT1.txt List of loci containing a. B. pubescens allele of XXXX origin, for each B. pubescens sample, after polarizing the focal B. pubescens sequence (four polarizing geometries tested: ALT1, ALT2, ALT3, and ALT4) presence_matrix_after_kmeans_annotated_genes.txt GO annotation for B. pubescens geneset of interest presence_matrix_after_kmeans_hum_ALLgenes.txt Fraction of B. pubescens individuals in a population containing an allele of B. humilis origin (all genes); after k-means cluster analysis presence_matrix_after_kmeans_hum_annotated_0.65_NEW2.txt Fraction of B. pubescens individuals in a population containing an allele of B. humilis origin (only genes where fraction &gt; 0.65 is observed in at least one population); after k-means cluster analysis presence_matrix_after_kmeans_nana_ALLgenes.txt Fraction of B. pubescens individuals in a population containing an allele of B. nana origin (all genes); after k-means cluster analysis presence_matrix_after_kmeans_nana_annotated_0.65_NEW2.txt Fraction of B. pubescens individuals in a population containing an allele of B. nana origin (only genes where fraction &gt; 0.65 is observed in at least one population); after k-means cluster analysis