Dataset DOI: 10.5061/dryad.vq83bk472

Description of the data and file structure

Sequence assemblies, alignments, tree files, and log files accompanying the paper: "Phylogenomics illuminate complex evolutionary history of Bibionomorpha".

Files and variables

File: transcriptome_assemblies.zip

Description: Collection of transcriptome assemblies generated in the current study.

We used transcriptomes sequenced as part of the 1K Insect Transcriptome Evolution (1KITE) Project (Misof et al., 2014) together with the publicly available transcriptome and genome sequences from GenBank. 1KITE transcriptome samples were collected in RNAlater and were sequenced at BGI (Beijing Genomic Institute, China) with the Illumina HiSeq 2000 (Illumina, San Diego, CA, USA) platform, following the protocol of Misof et al. (2014) and Peters et al. (2017).

Raw transcriptome reads were then assembled using multi-assembler pipeline TransPi v 1.3.0 (Rivera-Vicéns et al., 2021) to obtain de novo consensus transcriptome assemblies from raw reads. Briefly, raw reads were first checked with FastQC v 0.11.9 (Andrews, 2010), followed by filtration and adapter removal via fastp v 0.24.0 (Chen, 2023). Processed reads were then assembled with rnaSPAdes v 3.15.3 (Bushmanova et al., 2019), Trans-ABySS v 2.0.1 (Robertson et al., 2010), SOAPdenovo-Trans v 1.03 (Xie et al., 2014), Velvet v 1.2.10/Oases v 0.2.09 (Zerbino & Birney, 2008; Schulz et al., 2012) using k-mer set C of Rivera-Vicéns et al. (2021), and also using Trinity v 2.15.2 (Grabherr et al., 2011). Resulting assemblies were reduced to consensus assemblies with EvidentialGene v 2019.05.14 (Gilbert, 2013; 2019) and then decontaminated using MCSC (Lafond-Lapalme et al., 2017) to remove transcripts that were putatively non-arthropod in origin.

File naming convention

(family name) _(genus name)_ (species epithet)_T_decont.fasta

e.g., Anisopodidae_Sylvicola_dubius_T_decont.fasta.

T_decont stands for "transcriptome", "decontaminated".

File: AHE_assemblies.zip

Description: Collection of AHE assemblies generated in the current study.

We also used anchored hybrid enrichment (AHE; Lemmon et al., 2012) data in addition to transcriptome data for phylogenetic analyses. DNA was extracted from ethanol-preserved samples using the OmniPrep™ for Tissue Kit (Cat. #786–395; G-Biosciences®, USA), following the manufacturer's instructions. We followed previously published methods of Young et al. (2016) for library construction, using the same probes and procedures. Final reads were then assembled using SOAPdenovo 2 v r241 (Luo et al., 2012).

Fine naming convention

(family name) _(genus name)_ (species epithet)_AHE.fasta

e.g., Canthyloscelidae_Synneuron_decipiens_AHE.fasta.

File: Analysis_n_scripts.zip

Description: Analysis files including alignments, tree files, logs, etc.

1. og_filtering

   |__ data

         |__ Bibionomorpha_OrthologousMatrix.txt

   |__functions

         |__ og_filter.R

         |__ threshold.R

   |__ result

         |__ og_Bibionomorpha.txt

         |__ og_Bibionomorpha_noOut.txt

   |__ og_filtering.R

   |__ pre_filtering.R

   This folder includes scripts and results to perform orthogroup (OG) filtering, as described in the main article. "data" folder contains output of OMA standalone v2.6.0 (Altenhoff et al, 2019) ran on high-quality reference genomes of Bibionomorpha, which was used as an input for the OG filtering pipeline. "function" folder includes R functions to perform OG filtering, and the "result" folder includes output of OG filtering. "og_Bibionomorpha_noOut.txt" (filtering ran excluding the outgroup, Drosophila melanogaster) was used for actual further downstream analyses. "og_filtering.R" script calls functions in the "function" folder and run the actual OG filtering. "pre_filtering.R" performs pre-filtering of OGs prior to OG filtering, by setting lenght threshold and taxonomic coverage for each OG. The resulting list of OGs were further filtered according to the procedure detailed in the main article, and those OG alignments were then used for further downstream analyses.

2. phylogenetic_analysis_genomic_concat

   |__ AA_Partitioned

   |__ AA_Partitioned_noAHE

   |__ AA_Subsample_noRF

         |__ AA_Mixture_Subsample_noRF

         |__ AA_Partitioned_Subsample_noRF

   |__ AA_Subsample_RF

         |__ AA_Mixture_Subsample_RF

         |__ AA_Partitioned_Subsample_RF

   |__ NT2_Partitioned

   |__ NT2_Partitioned_noAHE

   |__ NT2_Subsample_noRF

         |__ NT2_Mixture_Subsample_noRF

         |__ NT2_Partitioned_Subsample_noRF

   |__ NT2_Subsample_RF

         |__ NT2_Mixture_Subsample_RF

         |__ NT2_Partitioned_Subsample_RF

   This folder includes results of phylogenetic analyses done using IQ-TREE 3 v 3.0.1 (Wong et al., 2025) for concatenated genomic data. "AA" stands for amino acid alignment, and "NT2" stands for nucleotide alignment using only second codon position. "Partitioned" stands for partitioned analysis, and "Mixture" stands for either profile mixture (AA) or nucleotide mixture (NT2) analysis. "noAHE" stands for analyses done excluding the AHE data. "Subsample" stands for analyes done using subsampled data using genesortR (Mongiardino Koch, 2021; Mongiardino Koch & Thompson, 2021). "RF" and "noRF" stands for analyses done with or without using the topological filtering option implemented in genesortR.

   Each folder includes input alignment file (*.fasta), partition information file (if applicable, *.nex), .iqtree file listing model selection results (only for Partitioned and Partitioned_noAHE folders), resulting phylogeny from 3 replicate runs (.runtrees), and the final tree with the best log-likelihood (.treefile). AA Mixture folders additionally include file listing site-specific frequency (.sitefreq) and profile mixture specification (udm_hogenom_0064_lclr_iqtree.nex; from Schrempf et al., 2020). 

3. phylogenetic_analysis_genomic_MSC

   |__ AA

   |__ gene_tree_variance

   |__ NT2

   |__ NT12

   This folder includes results of phylogenetic analyses done using wASTRAL  v1.22.3.7 (Zhang & Mirarab, 2022) implemented in ASTER (Zhang et al., 2025).  "AA" stands for amino acid alignment, and "NT2" stands for nucleotide alignment using only second codon position, and  "NT12" stands for nucleotide alignment using first and second codon positions. "AA", "NT2", and "NT12" each contains input ".trees" file including list of gene trees inferred using IQ-TREE 3  v 3.0.1, and "tree" file for the multi-species coalescent tree inferred using wASTRAL. 

   "gene_tree_variance" includes imputed gene trees for AA, NT2, and NT12 (.trees files) obtained using tripVote v1.2 (Mai & Mirarab, 2022), and results of gene tree topological-dispersion calculation ("dist_TV_result.txt") done using R package TreeDist v 2.9.2 (Smith, 2022), as detailed in the main article.

4. phylogenetic_analysis_mitochondrial

   |__ AA_Mixture

   |__ AA_Partitioned

   |__ NT_codon12_Mixture

   |__ NT_codon12_Partitioned

   |__ NT_codon123_Partitioned

   |__ Tree_TopoTest

         |__ AA_Mix_treetest

         |__ AA_Partitioned_treetest

         |__ NT12_Mixture_treetest

         |__ NT12_Partitioned_treetest

         |__ NT123_Partitioned_treetest

   This folder includes results of phylogenetic analyses done using IQ-TREE 3 v 3.0.1 (Wong et al., 2025) for mitochondrial data. Naming convention for files in each foler is identical to "2. phylogenetic_analysis_genomic_concat", except for the "Tree_TopoTest" folder. "Tree_TopoTest" folder includes result of tree topology tests done for each alignment, which is listed in the .iqtree file.

5. phylogenetic_network

   This folder includes results of phylogenetic network analyses done for AA gene trees using MSCquartets v 3.2 (Rhodes et al., 2021). "AA_MSCquartet_alpha.R" is a script used for the analysis. "combined_AA_bibio.trees" includes list of AA gene trees which was used as an input, and Network_.tree/nexml includes phylogenetic networks inferred using specific alpha value threshold, and ToB_.tree includes tree of blobs inferred using specific alpha value threshold.

6. BBB

   This folder includes results of fossil-based age distribution estimation using rootBBB v 0.2 (Silvestro et al., 2021; Carlisle et al., 2023). "extant_species.txt" includes list of number of extant species for each clade of interest, and "fossil_counts.txt" includes number of fossil species per time bin for each clade of interest. These two files were used as an input for rootBBB, and the resulting MCMC log files are named as "(clade name)_ mcmc _(seed number)_f0.95_qvar.log".

7. MCMCtree

   |__ AA_Sub_RF_Mixture

        |__ BSS_1

        |__ BSS_2

   |__ NT2_Sub_RF_Mixture

        |__ BSS_1

        |__ BSS_2

   This folder includes results of phylogenetic dating done using IQ2MC (Demotte et al., 2025) and MCMCtree (Yang, 2007) pipeline implemented in IQ-TREE 3 v 3.0.1 and PAML v 4.10.8.iq2mc. We generated two timetrees for "AA_Sub_RF_Mixture" and "NT2_Sub_RF_Mixture" trees. Each folder includes input and output of the IQ2MC pipeline: .fa is an input alignment, .treefile is an input tree topology, and profile mixture specification (only for AA) file "udm_hogenom_0064_lclr_iqtree.nex". The outputs of IQ2MC pipeline are: .iqtree file reporting summary of the analysis, dummy.phy file which is a dummy alignment file that is fed to MCMCtree, .ctl file for run specification for MCMCtree, calculated hessian matrix (.hessian), and input tree file for MCMCtree (.nwk file). 

   BSS_1 and BSS_2 includes MCMCtree results done using two different fossil calibration sets. Inputs from IQ2MC pipeline is contained in each folder, but with different calibration prior specification for .nwk file. MCMC logs are printed in .log file, with run report listed in .out file. Resulting timetree is summarized in a file "FigTree.tre". 

References

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