DOI: 10.5061/dryad.g4f4qrfzq
title: Data from: CAnDI: a new tool to investigate conflict in homologous gene trees and explain convergent trait evolution

description: Phenotypic convergence is found across the tree of life, and morphological
 similarities in distantly related species are often presumed to have
 evolved independently. However, clarifying the origins of traits has
 recently highlighted the complex nature of evolution, as apparent
 convergent features often share similar genetic foundations. Hence, the
 tree topology of genes that underlie such traits frequently conflicts with
 the overall history of species relationships. This conflict creates both a
 challenge for systematists and an exciting opportunity to investigate the
 rich, complex network of information that connects molecular trajectories
 with trait evolution. Here we present a novel conflict identification
 program named CAnDI (Conflict And Duplication Identifier), which enables
 the analysis of conflict in homologous gene trees rather than inferred
 orthologs. We demonstrate that the analysis of conflicts in homologous
 trees using CAnDI yields more comparisons than in ortholog trees in six
 datasets from across the eukaryotic tree of life. Using the carnivorous
 trap of Caryophyllales, a charismatic group of flowering plants, as a case
 study we demonstrate that analysing conflict on entire homolog trees can
 aid in inferring the genetic basis of trait evolution: by dissecting all
 gene relationships within homolog trees, we find genomic evidence that the
 molecular basis of the pleisiomorphic mucilaginous sticky trap was likely
 present in the ancestor of all carnivorous Caryophyllales. We also show
 that many genes whose evolutionary trajectories group species with similar
 trap devices code for proteins contributing to plant carnivory and
 identify a LATERAL ORGAN BOUNDARY DOMAIN transcription factor as a
 possible candidate for regulating sticky trap development. 
description: <strong>Comparison of whole homolog trees with ortholog
 trees for identifying gene tree conflict in six
 datasets</strong> We compiled six datasets of
 species trees, ortholog trees, and gene families (homolog trees) from
 published studies that broadly span the tree of life. We used the species
 relationships and homolog trees inferred in the initial studies as a basis
 for each conflict analysis. If available, we used rooted homolog trees
 from the original study. If the rooted homolog trees were not available,
 we used the extract_clades.py script from (Yang et al. 2015) to extract
 them.  All of the datasets, both homologs and
 orthologs, were analysed using CAnDI to count the total number of nodes
 assessed corresponding to each node in the species, where each node was
 assessed as either conflicting with the species tree or concordant with
 the species tree. Gene duplication nodes were not counted. 
 We extracted 12,172 rooted homologs from the unrooted homologs
 previously inferred for the plant family Amaranthaceae (AMAR) by
 (Morales-Briones et al. 2021), using a threshold of 40 ingroup taxa. The
 AMAR dataset did not have support values for homolog trees. The 12,699
 orthologs of the AMAR dataset with appropriate outgroup representation for
 rooting were from the original study where they were extracted using the
 monophyletic outgroup (MO) method (Yang and Smith, 2014). The Diatom
 (DIAT) dataset from (Parks et al. 2018) had duplications extracted with a
 minimum of 10 ingroup taxa, resulting in 18,914 homolog trees. The support
 values for this dataset were from a rapid bootstrap analysis; thus, we
 applied a cutoff of ≥70%. The 197 orthologs from the original study, that
 were extracted using the Rooted Tree (RT) method (Yang and Smith, 2014),
 were used for the ortholog comparison for the DIAT dataset. The Legumes
 dataset (LEGU), consisting of 8,038 homolog trees from (Koenen et al.
 2020), had been extracted in the original study, and support values on the
 nodes were from a rapid bootstrap analysis; thus, a cutoff of (≥70) was
 used. For the LEGU orthologs, we used the ortholog trees extracted with
 the RT method from the original study. To extract the homologs we used the
 same outgroup taxa, resulting in 1014 trees with outgroup representation.
 The Hymenoptera dataset (HYMN) was originally published by (Johnson et al.
 2013) but was subsequently re-analysed by (Smith et al. 2015), who
 identified 5,863 homolog trees and calculated rapid bootstrap support;
 therefore, we used a support cutoff of ≥70% for the HYMN dataset. To
 generate orthologs for the HYMN dataset, we used the RT method with a
 minimum cutoff of four taxa using the prune_from_rooted_tree.py script of
 (Morales-Briones et al. 2021), resulting in 6519 orthologs. The 6902
 homolog trees from the Ericales (ERIC) dataset from (Larson et al. 2020)
 were analysed using a support value of (SH-aLRT ≥ 80). Conflict analysis
 on ortholog trees from the ERIC dataset was conducted using the 382 rooted
 ortholog trees from the original study that had been extracted using the
 RT method.  The details of the carnivorous Caryophyllales (CARN) dataset,
 also used in this comparison, are described below.
 <strong>Investigating the relationship of conflict with
 trait distribution in the carnivorous Caryophyllales (CARN)
 dataset</strong> We used a minimum threshold of
 four ingroup taxa to extract 6,006 rooted homolog trees from the unrooted
 homolog trees inferred by Walker et al. (Walker et al. 2017). We term this
 the carnivorous Caryophyllales (CARN) dataset. Information regarding this
 and other datasets as well as all supplementary material is available on
 DRYAD (doi:10.5061/dryad.w3r2280xt). The 1237 orthologs we used for the
 CARN dataset were those from the original study, which were extracted
 using one-to-one orthology with 100% gene occupancy required. All trees in
 the CARN dataset had SH-aLRT support values; thus, we used a support
 cutoff of ≥80%. We then used CAnDI to identify all homolog trees in the
 CARN dataset that contained relationships (bipartition) of interest and
 the number of times the relationship of interest occurred in a single
 homolog tree. We identified trees with the bipartition
 <em>Drosophyllum</em> + <em>Drosera</em> and all
 trees with the bipartition <em>Drosophyllum</em> +
 <em>Ancistrocladus</em>. We also used CAnDI to identify all
 bipartitions that contained only <em>Nepenthes
 ampullaria</em>, <em>Nepenthes alata,</em> and
 <em>Drosophyllum lusitanicum</em>. The same procedure was
 performed to identify all instances in homolog trees where
 Ancistrocladaceae (Ancistrocladus) was sister to Nepenthaceae
 (<em>N. alata</em> and <em>N. ampullaria</em>).
 For each group of taxa investigated, we compared the number of
 bipartitions that contained only those taxa against the number of
 instances where an additional species was also included in the bipartition
 to eliminate any bias caused by differences in gene recovery in different
 samples. <strong>Investigating gene function in
 the CARN dataset</strong> Annotations for the
 CARN dataset were inferred using the coding sequences of the genome of
 <em>Arabidopsis thaliana</em> (TAIR10; Downloaded from Ensembl
 Plants on Jan 27<sup>th</sup>, 2021). The best blast hit
 (e-value 1e-3) to the transcriptome of<em> Nepenthes
 alata</em> from each homolog cluster was used to predict the
 function of each cluster. Using this approach, 137 out of the 146 clusters
 supporting the monotypic family Drosophyllaceae sister to the genus
 <em>Drosera</em> could be annotated (9 clusters failed to
 return a blast hit against <em>A</em><em>.
 thaliana</em>. These 137 annotations represented 132 distinct
 Arabidopsis genes. Gene ontology analyses with PANTHER16.0 were performed
 on this list of 132 <em>A. thaliana</em> genes using the
 PANTHER Overrepresentation Test (Released 20210224) and the GO Ontology
 database (DOI: 10.5281/zenodo.5228828 Released 2021-08-18) and either the
 “GO biological process complete” or the “GO molecular function complete”
 dataset. The Fisher’s exact test was performed with False Discovery Rate
 correction for each analysis. A <em>Chitinase-like protein 1
 </em>[homolog to <em>Arabidopsis CTL1</em>; labelled
 cluster2518 by Walker et al. (Walker et al. 2017)] was further
 investigated since this gene is associated with carnivory. We spot-checked
 the annotation for this gene by testing the blast annotation of other
 homologs within cluster2518 against the NCBI RefSeq non-redundant protein
 database to ensure that this was not the result of a spurious hit. All
 sequences from cluster2518 matched a Chitinase gene. We then performed the
 reverse mapping feature with CAnDI (“-r”) and mapped the coalescent-based
 species tree topology for the CARN dataset to the cluster2518 homolog
 tree. <strong>Plant
 material</strong> <em>Drosera
 admirabilis</em>, <em>Drosera aliciae</em> and
 <em>Drosera coccicaulis</em> were purchased from Wack’s Wicked
 plants (UK), grown in ambient conditions (natural light, with temperature
 kept between 18-25°C) and watered from the base with distilled water.
 Tissues were harvested from emerging young leaves (unfurled primordia
 &lt;1cm long, trichomes absent or not yet able to secrete mucilage),
 mature leaves representing fully developed sticky traps densely covered
 with trichomes (stalked glandular hairs) secreting mucilage droplets (Fig.
 S5) and inflorescences and immediately frozen in liquid nitrogen. For each
 species tissues were harvested from three distinct individuals to
 constitute three biological replicates for the gene expression analysis.
 All tissue samples were stored at -80°C until RNA extraction.
 <strong>Gene expression analysis</strong>
 Frozen tissues (from three distinct individuals for each species,
 constituting three biological replicates) were ground to a fine powder
 using a mortar and pestle. RNA was extracted using the Spectrum Plant
 Total RNA kit (Sigma-Aldrich) and retro-transcribed using SuperscriptIII
 reverse transcriptase (Invitrogen) following the manufacturer’s
 instructions. Quantitative real time PCR was performed using the Luna
 Universal qPCR Mastermix (NEBiolabs) and the Light Cycler 480 system
 (Roche). For each species and tissue type, three independent biological
 replicates were used (i.e., RNA extracted from three distinct individuals)
 and three technical replicates were performed for each condition (i.e.,
 three repeats of the qRT-PCR reaction assessing gene expression for a
 given tissue type, of a given biological replicate for each species).
 <em>Actin</em> and <em>eIF4A</em> homologs were
 used as reference genes as established by Arai and collaborators (Arai et
 al. 2021). The primer efficiencies were 100% for
 <em>Actin</em>, 93% for <em>eIF4</em> and 109% for
 <em>LBD4-like</em> homolog genes. Primers were designed to
 match gene regions conserved between the three different species of
 <em>Drosera</em>. Primer sequences are given in the
 Supplementary Spreadsheet 3. The gene expression was calculated relative
 to the housekeeping gene actin and the common base method, which accounts
 for the measured efficiency of each primer pair, was used to calculate
 relative expression levels (Ganger et al. 2017). Welch’s t-tests or ANOVA
 followed by Tukey’s HSD post-hoc tests were conducted in R [https://www.r-project.org, (R Core Team 2017)] and used to test for the likelihood that LBD4-like average expression levels were equivalent across tissues.
description: # CAnDI: a new tool to investigate conflict in homologous gene trees and
 explain convergent trait evolution
 [https://doi.org/10.5061/dryad.g4f4qrfzq](https://doi.org/10.5061/dryad.g4f4qrfzq) Here we present a novel conflict identification program named CAnDI (Conflict And Duplication Identifier), which enables the analysis of conflict in homologous gene trees rather than inferred orthologs. We demonstrate that the analysis of conflicts in homologous trees using CAnDI yields more comparisons than in ortholog trees in six datasets from across the eukaryotic tree of life. Using the carnivorous trap of Caryophyllales, a charismatic group of flowering plants, as a case study we demonstrate that analysing conflict on entire homolog trees can aid in inferring the genetic basis of trait evolution: by dissecting all gene relationships within homolog trees, we find genomic evidence that the molecular basis of the pleisiomorphic mucilaginous sticky trap was likely present in the ancestor of all carnivorous Caryophyllales. We also show that many genes whose evolutionary trajectories group species with similar trap devices code for proteins contributing to plant carnivory and identify a *LATERAL ORGAN BOUNDARY DOMAIN* transcription factor as a possible candidate for regulating sticky trap development. ## Description of the data and file structure Our manuscript did not generate new sequencing data but instead re-analyse publicly available data. We described the source of those data below.  Our manuscript describe a new software, CAnDI (Conflict And Duplication Identifier) and we give below the link to access all codes, manual and related information via github. In the folder CarnivoryDataset.zip we also provide: * Subfolder 'CarnivoryHomologs': contains the extracted homolog trees used in the paper from the carnivorous plant dataset. * 'CarnivorySpeciesTree.tre': the species tree used for mapping * Subfolder 'SearchResultsCarnivory': contains the results from the search. In this subfolder, the file names begin with the genera being compared using the CAnDI search function. Files with \_nosup\_ in the name are the genes identified as conflicting, regardless of statistical support for the relationship. Files with \_withsup\_ in the name are the genes that had statistical support for the relationship in question. Files with \_annotation at the end have the BLAST identified Arabidopsis annotation. ## Sharing/Access information We used six datasets of species trees, ortholog trees, and gene families (homolog trees) from published studies that broadly span the tree of life. Data was derived from the following sources: * CARN: Walker J.F., Yang Y., Moore M.J., Mikenas J., Timoneda A., Brockington S.F., Smith S.A. 2017. Widespread paleopolyploidy, gene tree conflict, and recalcitrant relationships among the carnivorous Caryophyllales. Am. J. Bot. 104:858–867.DIAT: Parks M.B., Nakov T., Ruck E.C., Wickett N.J., Alverson A.J. 2018. Phylogenomics reveals an extensive history of genome duplication in diatoms (Bacillariophyta). Am. J. Bot. 105:330-347. * LEGU: Koenen E.J.M., Ojeda D.I., Steeves R., Migliore J., Bakker F.T., Wieringa J.J., Kidner C., Hardy O.J., Pennington R.T., Bruneau A., Hughes C.E. 2020. Large-scale genomic sequence data resolve the deepest divergences in the legume phylogeny and support a nearsimultaneous evolutionary origin of all six subfamilies. New Phytol. 225:1355–1369. * ERIC: Larson D.A., Walker J.F., Vargas O.M., Smith S.A. 2020. A consensus phylogenomic approach highlights paleopolyploid and rapid radiation in the history of Ericales. Am. J. Bot. 107:773–789. * HYMN: Johnson B.R., Borowiec M.L., Chiu J.C., Lee E.K., Atallah J., Ward P.S. 2013. Phylogenomics Resolves Evolutionary Relationships among Ants, Bees, and Wasps. Curr. Biol. 23:2058– 2062. * AMAR: Morales-Briones D.F., Kadereit G., Tefarikis D.T., Moore M.J., Smith S.A., Brockington S.F., Timoneda A., Yim W.C., Cushman J.C., Yang Y. 2021. Disentangling Sources of Gene Tree Discordance in Phylogenomic Data Sets: Testing Ancient Hybridizations in Amaranthaceae s.l. Syst. Biol. 70:219–235. ## Code/Software The novel program CAnDI (Conflict And Duplication Identifier) is available at [https://github.com/HollyMaeRobertson/CAnDI](https://github.com/HollyMaeRobertson/CAnDI) For gene expression data analysis, Welch’s t-tests or ANOVA followed by Tukey’s HSD post-hoc tests were conducted in R [[https://www.r-project.org,](https://www.r-project.org/) (R Core Team 2017)] Gene Ontology analyses were performed with PANTHER16.0.