DOI: 10.5061/dryad.q83bk3jv4
title: Data from: Genomic analysis suggests that mitonuclear coevolution proceeds over rapid timescales in the Amazonian Pipra manakin complex

description: Mitonuclear coevolution is defined as reciprocal selection between the
 nuclear and mitochondrial genomes. It is necessary to maintain
 compatibility between nuclear- and mitochondrially-encoded products that
 interact during mitochondrial processes, including mitochondrial genome
 replication, transcription, and translation, and oxidative
 phosphorylation. Theory predicts that mitonuclear coevolution may play a
 crucial role in the early phases of speciation by generating strong
 genetic incompatibilities between recently diverged taxa that have evolved
 unique mitochondrial-mitonuclear haplotypes. However, the timescale over
 which mitonuclear coevolution proceeds remains unclear, making it
 difficult to definitively link this process with early speciation. Here,
 we test for expected genomic signals of mitonuclear coevolution across the
 Amazonian Pipra manakin complex, which includes recently and more deeply
 diverged avian lineages. Using dN/dS ratio analyses, we compared signals
 of positive selection in mitonuclear gene categories and functionally
 equivalent nuclear gene categories that do not participate in mitonuclear
 coevolution for each pair of Pipra lineages separately and for all the
 lineages simultaneously. For the ribosomal protein and aminoacyl tRNA
 synthetase (AARS) gene categories, we identified genomic patterns
 consistent with stronger positive selection in mitonuclear versus nuclear
 genes which is suggestive of mitonuclear coevolution having occurred
 across the Pipra complex. Significantly, we determined that expected
 genomic signals of mitonuclear coevolution could be identified between
 lineages that diverged as recently as 0.35-0.4 MYA. This time span is in
 keeping with the initial stages of avian speciation and suggests that
 mitonuclear coevolution may operate on a timescale that would allow it to
 play an important role during early speciation.
description: The methods employed in this study can be found in the linked
 2025 Molecular Ecology publication with additional information specified
 in the provided scripts and data files. To summarize, DNA was extracted
 from twenty-five <em>Pipra </em>manakin tissue samples and
 sequenced using a whole-genome sequencing methodology. The produced
 genetic data was processed and then mitonuclear and nuclear protein-coding
 gene sequences were obtained for each individual. Pairwise dN/dS
 comparisons and maximum likelihood codon evolution models were employed to
 assess positive selection in mitonuclear and functionally equivalent
 nuclear gene categories where stronger positive selection associated with
 mitonuclear gene categories is a putative signal of mitonuclear
 coevolution having occurred in the system. The scripts necessary to
 complete these analyses are included in this repository along with all
 required metatdata files.
description: # Genomic analysis suggests that mitonuclear coevolution proceeds over
 rapid timescales in the Amazonian Pipra manakin complex Dataset DOI:
 [10.5061/dryad.q83bk3jv4](10.5061/dryad.q83bk3jv4) ## Description of the
 data and file structure This repository is associated with:
 "Nikelski, E., & Weir, J. T. (2025). Genomic analysis suggests
 that mitonuclear coevolution proceeds over rapid timescales in the
 Amazonian *Pipra* manakin complex. (2025). *Molecular Ecology*. In Press.
 In this study, DNA was obtained from five genetically distinct *Pipra*
 manakin lineages (*Pipra filicauda, Pipra aureola, Pipra aureola borbae,
 Pipra fasciicauda scarlatina and Pipra fasciicauda calamae*) and sequenced
 using whole-genome sequencing. We then extracted protein-coding sequences
 for mitonuclear and functionally equivalent nuclear genes for each
 individual and assessed signals of positive selection in these gene
 categories using pairwise-lineage dN/dS comparisons and whole-system codon
 evolution models. We determined that signals of positive selection were
 stronger in mitonuclear gene categories which suggests that mitonuclear
 coevolution has occurred uniquely within each *Pipra* lineage and across
 the *Pipra* manakin complex generally. This repository contains the coding
 scripts and metadata files necessary to complete the analyses outlined in
 the above study. The files are split into six directories: 1.
 **Whole_Genome_Sequencing_Processing:** This directory contains the coding
 files necessary to process whole-genome sequencing reads from the *Pipra*
 individuals utilized in this research project. The raw whole-genome
 sequencing reads are available on the NCBI SRA (BioProject: PRJNA1232061).
 2. **Mitochondrial_Analyses:** This directory contains the coding files
 necessary to conduct the mitochondrial genome analyses completed in this
 research project. In short, the coding scripts are designed to assemble a
 mitochondrial genome for each *Pipra* individual, calculate mitochondrial
 gene differentiation among all inter-lineage pairs of *Pipra* individuals
 and fit codon evolution models to whole-system alignments of mitochondrial
 genes. 3. **Pairwise_Lineage_Analyses:** This directory contains the
 coding files necessary to conduct pairwise inter- and intra-lineage dN/dS
 ratio comparisons of mitonuclear and nuclear gene categories. In short,
 the coding scripts are designed to create concatenated pairwise alignments
 of mitonuclear and nuclear gene categories for each inter- and
 intra-lineage pair of *Pipra* individuals, calculate a dN/dS ratio for all
 of these alignments and then perform a bootstrapped t-test procedure to
 determine whether mitonuclear dN/dS ratios are significantly higher than
 nuclear dN/dS ratios in inter-lineage comparisons. 4.
 **Codon_Evolution_Models:** This directory contains the coding files
 necessary to run codon evolution models on concatenated whole-system
 alignments of mitonuclear and nuclear gene categories. In short, the
 coding scripts are designed to create concatenated whole-system alignments
 of mitonuclear and nuclear gene categories that contain data from all
 *Pipra* individuals included in this study, fit codon evolution models to
 these alignments and process the outputs from these models. 5.
 **Supplemental_Analyses:** This directory contains the coding files
 necessary to run supplemental codon evolution models described in the
 publication. In short, the coding scripts are designed to run codon
 evolution models on two subsets of the data: one where a single high
 genetic coverage individual represents each lineage and one where a
 chimeric sequence based on sequence data from all individuals within a
 lineage represents each lineage. 6. **Metadata_Files:** This directory
 contains the metadata files necessary to complete the analyses contained
 within directories 1-5. The software utilized in coding scripts are: R,
 RStudio, fastp, Bowtie2, Samtools, BCFtools, VCFtools, Qualimap, AGAT,
 seqkit, faSplit, getOrganelle, MACSE2, MEGAX, MITOS2, R2DT, Paup4,
 Iqtree2, vcf2phylip and PAML4. All software is open source and free to
 access. ### Files and variables ### Directory: 6.Metadata\_Files.zip ####
 **File: All\_Samples\_Species\_Codes.txt** **Description:** This file
 contains a list of values ranging from "Species00" to
 "Species24" that were utilized during data processing in the
 associated study. #### **File: Aureola\_Bam.txt** **Description:** This
 file contains a list of pathways to the bam files associated with five
 Aureola individuals included in the associated study. #### **File:
 aureola\_pop.txt** **Description:** This file contains a list of the
 sample names associated with the five Aureola individuals included in the
 associated study.  #### **File: Aureola\_Sex.txt** **Description:** This
 file contains two variables. The first variable is a list of pathways to
 the bam files associated with five Aureola individuals included in the
 associated study as they appear in the Aureola_Bam.txt file. The second
 variable is the sexes of each Aureola individual where "M"
 indicates male and "F" indicates female. #### **File:
 Borbae\_Bam.txt** **Description:** This file contains a list of pathways
 to the bam files associated with five Borbae individuals included in the
 associated study. #### **File: borbae\_pop.txt** **Description:** This
 file contains a list of the sample names associated with the five Borbae
 individuals included in the associated study.  #### **File:
 Borbae\_Sex.txt** **Description:** This file contains two variables. The
 first variable is a list of pathways to the bam files associated with five
 Borbae individuals included in the associated study as they appear in the
 Borbae_Bam.txt file. The second variable is the sexes of each Borbae
 individual where "M" indicates male and "F" indicates
 female. #### **File: Calomae\_Bam.txt** **Description:** This file
 contains a list of pathways to the bam files associated with five Calamae
 individuals included in the associated study. #### **File:
 calomae\_pop.txt** **Description:** This file contains a list of the
 sample names associated with the five Calamae individuals included in the
 associated study.  #### **File: Calomae\_Sex.txt** **Description:** This
 file contains two variables. The first variable is a list of pathways to
 the bam files associated with five Calamae individuals included in the
 associated study as they appear in the Calamae_Bam.txt file. The second
 variable is the sexes of each Calamae individual where "M"
 indicates male and "F" indicates female. #### **File:
 Chiroxiphia\_Lancolata\_Ploidy.txt** **Description:** This file contains
 information relating to the ploidy of chromosomes associated with
 the *Chiroxiphia lancolata* reference genome used in the associated study.
 This file is necessary for alignment of sequence reads to the reference
 genome. #### **File: Chiroxiphia\_Lancolata\_Translation\_Table.txt**
 **Description:** This file contains information necessary to relate the
 names of the scaffolds within the *Chiroxiphia lancolata* reference genome
 and the chromosome associated with each of these scaffolds. This file
 contains two variables. The first variable is a list of scaffold names as
 they appear in the *Chiroxiphia lancolata* reference genome and the second
 variable is a list of chromosomes written as "chr#". ####
 **File: Combined\_pipra\_tree\_correctly\_rooted\_newick\_rooted.txt**
 **Description:** This file contains a rooted phylogenetic tree of
 twenty-five *Pipra* individuals in newick format. This tree is necessary
 to run the codon evolution models described in the associated study. ####
 **File: ETC\_Complex1\_SU.txt** **Description:** This file contains a list
 of nuclear genes associated with the mitonuclear ETS complex I gene
 category in the associated study. #### **File: ETC\_Complex2\_SU.txt**
 **Description:** This file contains a list of nuclear genes associated
 with the nuclear ETS complex II gene category in the associated study.
 #### **File: ETC\_Complex3\_SU.txt** **Description:** This file contains a
 list of nuclear genes associated with the mitonuclear ETS complex III gene
 category in the associated study. #### **File: ETC\_Complex4\_SU.txt**
 **Description:** This file contains a list of nuclear genes associated
 with the mitonuclear ETS complex IV gene category in the associated study.
 #### **File: ETC\_Complex5\_SU.txt** **Description:** This file contains a
 list of nuclear genes associated with the mitonuclear ETS complex V gene
 category in the associated study. #### **File: Filicauda\_Bam.txt**
 **Description:** This file contains a list of pathways to the bam files
 associated with five Filicauda individuals included in the associated
 study. #### **File: filicauda\_pop.txt** **Description:** This file
 contains a list of the sample names associated with the five Filicauda
 individuals included in the associated study.  #### **File:
 Filicauda\_Sex.txt** **Description:** This file contains two variables.
 The first variable is a list of pathways to the bam files associated with
 five Filicauda individuals included in the associated study as they appear
 in the Filicauda_Bam.txt file. The second variable is the sexes of each
 Filicauda individual where "M" indicates male and "F"
 indicates female. #### **File:
 five\_individual\_chimeric\_tree\_rooted.txt** **Description:** This file
 contains a rooted phylogenetic tree of five individuals each representing
 a chimeric sequence of each *Pipra* lineage in newick format. This tree is
 necessary to run the supplemental codon evolution models described in the
 associated study. #### **File: five\_individual\_tree\_rooted.txt**
 **Description:** This file contains a rooted phylogenetic tree of five
 high-coverage individuals each representing one *Pipra* lineage* *in
 newick format. This tree is necessary to run the supplemental codon
 evolution models described in the associated study. #### **File:
 Functional\_Group.txt** **Description:** This file contains a list of
 abbreviations for the nine mitonuclear and functionally equivalent nuclear
 gene categories investigated for signals of positive selection in the
 associated study. #### **File:
 Functional\_Kappa\_Omega\_Values\_Rooted\_Tree\_Full.txt**
 **Description:** This file contains the metadata necessary to run
 iterations of codon evolution models with various starting parameter
 values on whole-system concatenated alignments of mitonuclear and nuclear
 gene categories. This file is broken into three variables. The first
 variable consists of a list of abbreviations for the nine mitonuclear and
 nuclear gene categories that models were fit to. The second variable
 consists of a list of starting values for the Kappa parameter in a codon
 evolution model. The third variable consists of a list of starting values
 for the Omega parameter in a codon evolution model. So, using this file,
 each codon evolution model was specified a gene category, a Kappa starting
 value and a Omega starting value. #### **File:
 Illumina\_PCR\_FREE\_Tagmentation.fasta.txt** **Description:** This file
 contains the fasta sequences of the adaptors added to whole-genome
 sequencing reads during library preparation that need to be trimmed as
 part of sequence read processing. #### **File:
 Lepidothrix\_coronata\_Bam.txt** **Description:** This file contains the
 pathway to a bam file for a *Lepidothrix coronata* individual utilized as
 an outgroup when constructing lineage-specific *Pipra* phylogenies in the
 associated study. #### **File: Lepidothrix\_coronata\_Sex.txt**
 **Description:** This file contains two variables. The first variable is
 the pathway to a bam file for a *Lepidothrix coronata* individual utilized
 as an outgroup when constructing lineage-specific *Pipra* phylogenies in
 the associated study as it appears in the Lepidothrix_coronata_Bam.txt
 file. The second variable is the sex *Lepidothrix coronata* individual
 where "M" indicates male and "F" indicates female.
 #### **File: LTM\_Mitonuclear\_Gene\_Codes.txt** **Description:** This
 file contains the metadata necessary to extract mitonuclear and nuclear
 protein-coding sequences from whole-genome *Pipra* fasta files based on
 annotations associated with the *Chiroxiphia lancolata* reference genome.
 This file consists of three variables. The first variable is a list of
 mitonuclear and nuclear gene names as they appear in the *Chiroxiphia
 lancolata* reference genome annotation file. The second variable is a list
 of mitonuclear and nuclear gene names as they are commonly referred to in
 literature. The third variable is a list of the mRNA accession codes
 associated with the *Chiroxiphia lancolata* reference genome for each
 mitonuclear and nuclear gene. #### **File:
 Mito\_Kappa\_Omega\_Values\_Full.txt** **Description:** This file contains
 the metadata necessary to run iterations of codon evolution models with
 various starting parameter values on whole-system concatenated alignments
 of protein-coding mitochondrial genes. This file is broken into three
 variables. The first variable consists of a list of the thirteen
 mitochondrial genes that models were fit to. The second variable consists
 of a list of starting values for the Kappa parameter in a codon evolution
 model. The third variable consists of a list of starting values for the
 Omega parameter in a codon evolution model. So, using this file, each
 codon evolution model was specified a mitochondrial gene, a Kappa starting
 value and a Omega starting value. #### **File:
 Mitochondrial\_Gene\_codes.csv** **Description:** This file contains a
 list of the thirty-seven mitochondrial genes found in the mitochondrial
 genome of bilaterian animals in .csv format. #### **File:
 Mitochondrial\_gene\_codes.txt** **Description:** This file contains a
 list of the thirty-seven mitochondrial genes found in the mitochondrial
 genome of bilaterian animals in .txt format. #### **File:
 Mitochondrial\_Protein\_Coding\_Gene\_Codes.txt** **Description:** This
 file contains a list of the thirteen protein-coding mitochondrial genes
 found in the mitochondrial genome of bilaterian animals in .txt format.
 #### **File: Mitogenome\_Metadata.csv** **Description:** This file
 contains the metadata associated with the mitogenomes assembled from
 whole-genome sequencing data for each *Pipra* individual. It consists of
 three variables named "Species", "Sample" and
 "Mitogenome". The variable "Species" contains a list
 of letters indicating the *Pipra* lineage (A = Aureola, B = Borbae, C =
 Calamae, F = Filicauda, S = Scarlatina). The variable "Sample"
 contains a list of the sample names associated with the twenty-five
 *Pipra* individuals included in the associated study. The variable
 "Mitogenome" contains the names of the mitogenomes assembled for
 each *Pipra* individuals. Some individuals possessed multiple mitogenomes
 that differed by a few base pairs. #### **File: MN\_AARS2.txt**
 **Description:** This file contains a list of nuclear genes associated
 with the mitonuclear AARS gene category in the associated study. ####
 **File: MN\_MitoRib.txt** **Description:** This file contains a list of
 nuclear genes associated with the mitonuclear ribosomal protein gene
 category in the associated study. #### **File: NUC\_AARS1.txt**
 **Description:** This file contains a list of nuclear genes associated
 with the nuclear AARS gene category in the associated study. #### **File:
 Nuc\_Rib.txt** **Description:** This file contains a list of nuclear genes
 associated with the nuclear ribosomal protein gene category in the
 associated study. #### **File: Pipra\_Comparisons\_Breakdown.csv**
 **Description:**  This file summarizes all the inter-lineage *Pipra*
 comparisons completed in the associated study. It consists of two
 variables named "Species_1" and "Species_2". The
 variable "Species_1" contains a list of *Pipra* lineages
 representing the first member of an inter-lineage pair. The variable
 "Species_2" contains a list of *Pipra* lineages representing the
 second member of an inter-lineage pair. #### **File:
 Pipra\_Mitogenome\_List.txt** **Description:** This file contains the
 metadata associated with the finalized mitogenomes assembled for each
 *Pipra* individual included in the associated study. It consists of three
 variables named "Species", "Sample" and
 "Mitogenome". The variable "Species" contains a list
 of letters indicating the *Pipra* lineage (A = Aureola, B = Borbae, C =
 Calamae, F = Filicauda, S = Scarlatina). The variable "Sample"
 contains a list of the sample names associated with the twenty-five
 *Pipra* individuals included in the associated study. The variable
 "Mitogenome" contains the names of the finalized mitogenomes
 assembled for each *Pipra* individual. #### **File:
 Pipra\_Paml\_Comparisons\_headers.txt** **Description:** This file
 contains the metadata necessary to calculate pairwise inter- and
 intra-lineage dN/dS ratios for all mitonuclear and nuclear gene categories
 for all combinations of the twenty-five *Pipra* individuals included in
 the associated study. It consists of ten variables named
 "Comparison", "Comparison_number",
 "Sample_1", "Individual_1", "Species_1",
 "Code_1", "Sample_2", "Individual_2",
 "Species_2" and "Code_2". The variable
 "Comparison" contains a list of the different inter- and
 intra-lineage comparisons completed in the associated study written as
 "Lineage1_Lineage2_Comparison". The variable
 "Comparison_number" contains a list of values that indicate the
 number of each comparison within its inter and intra-lineage comparison
 category. A total of twenty-five and ten comparisons were completed for
 each inter- and intra-lineage comparison category respectively. The
 variable "Sample_1" includes a list of names associated with the
 sequence data of each *Pipra* individual that represent the first member
 of each inter- or intra-lineage comparison. The variable
 "Individual_1" includes a list of samples names associated with
 each *Pipra* individual that represent the first member of each inter- or
 intra-lineage comparison. The variable "Species_1" includes a
 list of lineage names associated with each *Pipra* individual that
 represent the first member of each inter- or intra-lineage comparison. The
 variable "Code_1" includes a list of codes associated with the
 sequence data of each *Pipra* individual that represent the first member
 of each inter- or intra-lineage comparison. The variable
 "Sample_2" includes a list of names associated with the sequence
 data of each *Pipra* individual that represent the second member of each
 inter- or intra-lineage comparison. The variable "Individual_2"
 includes a list of samples names associated with each *Pipra* individual
 that represent the second member of each inter- or intra-lineage
 comparison. The variable "Species_2" includes a list of lineage
 names associated with each *Pipra* individual that represent the second
 member of each inter- or intra-lineage comparison. The variable
 "Code_2" includes a list of codes associated with the sequence
 data of each *Pipra* individual that represent the second member of each
 inter- or intra-lineage comparison. #### **File:
 Pipra\_Paml\_Comparisons.txt** **Description:** This file is identical to
 the file "Pipra_Paml_Comparisons_headers.txt", but it lacks a
 header. #### **File: Population\_Names.txt** **Description:** This file
 contains a list of the five *Pipra* lineages included in the associated
 study. #### **File: SAMPLES\_LIST.txt** **Description:** This file
 contains a list of sample names associated with the twenty-five *Pipra*
 individuals included in the associated study. #### **File:
 Scarlatina\_Bam.txt** **Description:** This file contains a list of
 pathways to the bam files associated with five Scarlatina individuals
 included in the associated study. #### **File: scarlatina\_pop.txt**
 **Description:** This file contains a list of the sample names associated
 with the five Scarlatina individuals included in the associated study. 
 #### **File: Scarlatina\_Sex.txt** **Description:** This file contains two
 variables. The first variable is a list of pathways to the bam files
 associated with five Scarlatina individuals included in the associated
 study as they appear in the Scarlatina_Bam.txt file. The second variable
 is the sexes of each Scarlatina individual where "M" indicates
 male and "F" indicates female. ## Code/software ### Directory:
 1.Whole\_Genome\_Sequencing\_Processing #### **File:
 1.Pipra\_Sequence\_Processing.R** **Description:** This file contains the
 code necessary to process whole-genome sequencing reads from the 25
 *Pipra* individuals included in the associated study. The majority of the
 scripts contained in this file are written in R, but some command-line
 linux commands may be included where using this language is more
 convenient. The coding language used will be indicated throughout the file
 which can be opened with any text editing software. The general steps
 completed in this file include read trimming, read alignment, genotype
 calling, genotype filtering and mitonuclear and nuclear gene sequence
 isolation. The following metadata files are necessary to complete the
 steps contained in this code:  *
 Chiroxiphia_Lancolata_Translation_Table.txt,  *
 Chiroxiphia_Lancolata_Ploidy.txt *
 Illumina_PCR_FREE_Tagmentation.fasta.txt * SAMPLES_LIST.txt *
 Aureola_Bam.txt * Aureola_Sex.txt * Borbae_Bam.txt * Borbae_Sex.txt *
 Calomae_Bam.txt * Calomae_Sex.txt * Filicauda_Bam.txt * Filicauda_Sex.txt
 * Scarlatina_Bam.txt * Scarlatina_Sex.txt * Population_Names.txt *
 aureola_pop.txt * borbae_pop.txt * calomae_pop.txt * filicauda_pop.txt *
 scarlatina_pop.txt * LTM_Mitonuclear_Gene_Codes.txt ### Directory:
 2.Mitochondrial\_Analyses #### **File:
 1.Pipra\_Mitochondrial\_Genome\_Assembly\_Pairwise\_Analyses.R**
 **Description:** This file contains the code necessary to isolate
 mitochondrial genomes from the 25 *Pipra* individuals included in the
 associated study and complete pairwise mitochondrial gene differentiation
 analyses. The majority of the scripts contained in this file are written
 in R, but some command-line linux commands may be included where using
 this language is more convenient. The language will be indicated
 throughout the file which can be opened with any text editing software.
 The general steps completed in this file include isolation of
 mitochondrial genomes from whole-genome sequencing data, annotation of
 mitochondrial genomes, isolation and alignment of mitochondrial gene
 sequences and analysis of mitochondrial gene differentiation. The
 following metadata files are necessary to complete the steps contained in
 this code: * SAMPLES_LIST.txt * Mitochondrial_Gene_codes.txt *
 Mitochondrial_Gene_codes.txt * Mitogenome_Metadata.csv *
 Pipra_Mitogenome_List.csv * Mitochondrial_Gene_codes.csv #### **File:
 2.Pipra\_Mitochondrial\_Phylogeny\_Codon\_Evolution\_Models.R**
 **Description:** This file contains the code necessary to run codon
 evolution models on alignments of 13 protein-coding mitochondrial genes
 that contain genetic information from the 25 *Pipra* individuals included
 in the associated study. The majority of the scripts contained in this
 file are written in R, but some command-line linux commands may be
 included where using this language is more convenient. The language will
 be indicated throughout the file which can be opened with any text editing
 software. The general steps completed in this file include running codon
 evolution models on protein-coding mitochondrial gene sequences and
 isolating parameter values from M0, M1a, M2a, M7 and M8 models with the
 highest log-likelihood values. The following metadata files are necessary
 to complete the steps contained in this code:  *
 Mitochondrial_Protein_Coding_Gene_Codes.txt *
 Combined_pipra_tree_correctly_rooted_newick_rooted.txt *
 Mito_Kappa_Omega_Values_Full.txt ### Directory:
 3.Pairwise\_Lineage\_Analyses #### **File:
 1.Pipra\_Pairwise\_DNDS\_Analyses.R** **Description:** This file contains
 the code necessary to calculate a pairwise dN/dS ratio for all mitonuclear
 and nuclear gene categories for all inter- and intra-lineage pairs of
 *Pipra* individuals. The majority of the scripts contained in this file
 are written in R, but some command-line linux commands may be included
 where using this language is more convenient. The language will be
 indicated throughout the file which can be opened with any text editing
 software. The general steps completed in this file include creating
 concatenated pairwise alignments for all gene categories for all inter-
 and intra- lineage pairs of 25 *Pipra* individuals, calculating pairwise
 dN/dS ratios for all of these combinations, processing raw pairwise dN/dS
 ratio data and consolidating all of this information into figures for
 publication. The following metadata files are necessary to complete the
 steps contained in this code:  * Pipra_Paml_Comparisons_headers.txt *
 LTM_Mitonuclear_Gene_Codes.txt * MN_AARS2.txt * MN_MitoRib.txt *
 Nuc_AARS1.txt * Nuc_Rib.txt * ETC_Complex1_SU.txt * ETC_Complex2_SU.txt *
 ETC_Complex3_SU.txt * ETC_Complex4_SU.txt * ETC_Complex5_SU.txt *
 Functional_Group.txt * Pipra_Paml_Comparisons.txt *
 Pipra_Paml_Comparisons_headers.txt #### **File:
 2.Pipra\_Pairwise\_DNDS\_Analyses\_Bootstrapped\_Ttest.R**
 **Description:** This file contains the code necessary to run bootstrapped
 t-tests comparing the mean pairwise dN/dS ratios of functionally
 equivalent mitonuclear and nuclear gene categories for all inter-lineage
 comparisons of *Pipra*. The majority of the scripts contained in this file
 are written in R, but some command-line linux commands may be included
 where using this language is more convenient. The language will be
 indicated throughout the file which can be opened with any text editing
 software. The general steps completed in this file include running a
 series of bootstrapped t-tests for each gene category for each
 inter-lineage comparison, consolidating the results from these
 bootstrapped t-tests and determining significance. The following metadata
 files are necessary to complete the steps contained in this code:  *
 Pipra_Paml_Comparisons_headers.txt * Pipra_Comparsions_Breakdown.csv ###
 Directory: 4.Codon\_Evolution\_Models #### **File:
 1.Pipra\_Phylogeny\_Codon\_Evolution\_Models.R** **Description:** This
 file contains the code necessary to create whole-system concatenated
 alignments for mitonuclear and nuclear gene categories containing genetic
 information from the 25 *Pipra* individuals included in the associated
 study and to run codon evolution models on this data. The majority of the
 scripts contained in this file are written in R, but some command-line
 linux commands may be included where using this language is more
 convenient. The language will be indicated throughout the file which can
 be opened with any text editing software.The general steps completed in
 this file include creating a phylogeny of all 25 *Pipra* samples, creating
 whole-system alignments of mitonuclear and nuclear genes, concatenating
 alignments to make whole-system concatenated gene category alignments,
 running codon evolution models on concatenated alignments and isolating
 parameter values from M0, M1a, M2a, M7 and M8 models with the highest
 log-likelihood values. The following metadata files are necessary to
 complete the steps contained in this code: * Lepidothrix_coronata_Bam.txt
 * Lepidothrix_coronata_Sex.txt * Chiroxiphia_Lancolata_Ploidy.txt *
 Combined_pipra_tree_correctly_rooted_newick_rooted.txt *
 LTM_Mitonuclear_Gene_Codes.txt * All_Samples_Species_Codes.txt *
 MN_AARS2.txt * MN_MitoRib.txt * Nuc_AARS1.txt * Nuc_Rib.txt *
 ETC_Complex1_SU.txt * ETC_Complex2_SU.txt * ETC_Complex3_SU.txt *
 ETC_Complex4_SU.txt * ETC_Complex5_SU.txt * SAMPLES_LIST.txt *
 Functional_Kappa_Omega_Values_Rooted_Tree_Full.txt ### Directory:
 5.Supplemental\_Analyses #### **File:
 1.Pipra\_Phylogeny\_Codon\_Evolution\_Models\_5\_High\_Coverage\_Individuals.R** **Description:** This file contains the code necessary to create concatenated alignments for mitonuclear and nuclear gene categories that include data from the five *Pipra* samples (one from each lineage) with the highest genetic coverage and to run codon evolution models on this data. The majority of the scripts contained in this file are written in R, but some command-line linux commands may be included where using this language is more convenient. The language will be indicated throughout the file which can be opened with any text editing software. The general steps completed in this file include creating a five individual phylogeny, creating concatenated alignments for mitonuclear and nuclear gene categories based on sequence data from high-coverage samples, running codon evolution models on this subset of the data and isolating parameter values from M0, M1a, M2a, M7 and M8 models with the highest log-likelihood values. The following metadata files are necessary to complete the steps contained in this code: * five_individual_tree_rooted.txt * Functional_Kappa_Omega_Values_Rooted_Tree_Full.txt #### **File: 2.Pipra\_Phylogeny\_Codon\_Evolution\_Models\_5\_Chimeric\_Individuals.R** **Description:** This file contains the code necessary to create concatenated alignments for mitonuclear and nuclear gene categories that include data from chimeric sequences created for each *Pipra* lineage (one sequence per lineage) and to run codon evolution models on this data. The majority of the scripts contained in this file are written in R, but some command-line linux commands may be included where using this language is more convenient. The language will be indicated throughout the file which can be opened with any text editing software. The general steps completed in this file include creating chimeric concatenated gene sequences for each *Pipra* lineage for each gene category, running codon evolution models based on this subset of the data and isolating parameter values from M0, M1a, M2a, M7 and M8 models with the highest log-likelihood values. The following metadata files are necessary to complete the steps contained in this code: * five_individual_chimeric_tree_rooted.txt * Functional_Kappa_Omega_Values_Rooted_Tree_Full.txt