DOI: 10.5061/dryad.bg79cnpkt
title: Data from: Untangling the ant claws: The army ant (Formicidae: Dorylinae) Labidus mars is a Neivamyrmex

description: The New World army ants (Hymenoptera: Formicidae) comprise the five genera
 of the Eciton species group, and together they are important
 keystone predators in tropical and subtropical environments. Generic
 boundaries in the group have been considered solid and stable for nearly
 100 years. Workers of the widespread and diverse genus Neivamyrmex are
 readily separable from the other four genera by lacking a subapical tooth
 on the tarsal claw, while males can be separated with genitalic
 characters. The genus Labidus is also widespread and is often abundant,
 with several species that are conspicuous surface foragers. The least
 known species of Labidus is L. mars, the workers of which have the tarsal
 tooth but otherwise share many traits with some Neivamyrmex, being
 completely eyeless and subterranean. This led us to question its generic
 placement. Here, we used ultraconserved element (UCE) phylogenomics to
 show that Labidus mars belongs to the genus Neivamyrmex. All phylogenies,
 inferred using multiple partitioning schemes and a species tree analysis,
 recovered the same topology, placing Labidus mars workers within
 Neivamyrmex. Sequenced males of L. mars were found to be within Labidus
 and thus incorrectly associated with L. mars. Based on these results and
 review of key specimens, including types, the following taxonomic changes
 are made: Neivamyrmex mars (Forel, 1912) is a new combination; Labidus
 nero (Santschi, 1930) (rev. stat.) is a male-based taxon revived from
 synonymy under L. mars; and L. denticulatus (Borgmeier, 1955) (new stat.),
 a male-based taxon and former subspecies of L. mars, is raised to species.
description: Here we have deposited contigs representing UCE loci, unfiltered
 UCE alignments, the concatenated UCE matrix, tree files, and additional
 data analysis files (partitioning schemes and log files). All new sequence
 data have been deposited in the NCBI Sequence Read Archive under
 BioProject#PRJNA1158819. The methods used to generate these data are
 described below and in the article. Molecular Taxon
 Sampling:  UCE sequence data were newly generated for
 12 specimens and combined with published data available from the Dryad
 repository referenced in Borowiec (2019) for 3 specimens (Table 1).
 <em>Leptanilloides femoralis </em>Borowiec & Longino
 2011 was used as an outgroup following Borowiec (2019).
 <em>Neivamyrmex </em>specimens were selected using the
 morphological similarities between <em>Labidus mars </em>and
 some <em>Neivamyrmex</em> species, using the key to
 <em>Neivamyrmex</em> species in Watkins (1976) and information
 from a current project on the evolution of
 <em>Neivamyrmex</em> (S. Powell & L. Barros, personal
 communication). The six <em>Neivamyrmex </em>selected
 represent 5% of the total described diversity of the genus.
 DNA Sequence Generation:  We employed the UCE
 approach to phylogenomics (Faircloth et al. 2012, Faircloth et al. 2015,
 Branstetter et al. 2017),<strong> </strong>combining target
 enrichment of UCEs with multiplexed next-generation sequencing. All
 samples were extracted, quality-checked, and sheared, following the UCE
 methodology described in Branstetter et al. (2017). For two samples, the
 sheared DNA was shipped to Rapid Genomics (RG; Gainesville, FL) for
 library preparation, UCE enrichment and sequencing. For each library, RG
 aimed to sequence two million paired-end (PE) reads of enriched library
 plus one million PE reads of unenriched library. Enrichment was performed
 using the ant-customized version (“ant-specific hym-v2”) of the
 Hymenoptera v2 bait set, which targets 2,524 UCE loci common across
 Hymenoptera (Branstetter et al. 2017). Sequencing was performed on
 Illumina NovaSeq instruments (2x150). For eight samples, the sheared DNA
 was sent to the University of Utah Genomics Core Facility for processing,
 following the same general approach as RG, with both enriched and
 unenriched libraries being sequenced separately. Finally, for two
 additional samples, library preparation and enrichment was performed
 in-house following methods outlined in Branstetter et al. (2021) and using
 an ant-bee version of the bait set (Grab et al. 2019). These samples were
 sent to Novogene (Sacramento, CA) for HiSeq X sequencing (2x150) and only
 enriched libraries were sequenced. The utility of the Hym-v2 bait set to
 resolve relationships, both deep and shallow, in ants has been
 demonstrated in several studies (Barrera et al. 2022, Branstetter et al.
 2017, Branstetter & Longino 2019, Pierce et al. 2017, Ward
 & Branstetter 2017, Williams et al. 2022). All
 new sequence data have been deposited in the NCBI Sequence Read Archive
 under BioProject#PRJNA1158819. UCE Matrix
 Assembly:  Sequences from RG and UU were demultiplexed
 by the providers and reads from Novogene were manually demultiplexed using
 BBTools (Bushnell 2014). For the RG and UU samples, we combined the
 enriched reads with the unenriched reads before read trimming and further
 processing. For all newly sequenced samples, the sequence data were
 cleaned, assembled, and aligned using the Phyluce package
 1.7<strong> </strong>(Faircloth 2016). Within the Phyluce
 environment, we used Illumiprocessor (Faircloth 2013) and Trimmomatic
 (Bolger et al. 2014) for quality trimming raw reads, SPAdes v3.14.1
 (Bankevich et al. 2012) for de novo assembly of reads into contigs, and
 LASTZ v1.02 (Harris 2007) for identifying UCE contigs from all contigs.
 All optional Phyluce settings were left at default values for these steps,
 as these settings have been found to work well in similar datasets. For
 the bait sequences file needed to identify and extract UCE contigs, we
 used the ant-specific hym-v2 bait file. To calculate assembly statistics,
 including sequencing coverage, we used scripts from the Phyluce package.
 For the published data from Borowiec (2019), UCE contigs were downloaded
 from Dryad (https://doi.org/10.5061/dryad.bj83h5d). After extracting UCE contigs, we aligned each UCE locus using a stand-alone version of the program MAFFT v7.130b (Katoh & Standley 2013) and the L-INS-i algorithm. We then used a Phyluce script to trim flanking regions and poorly aligned internal regions using the program Gblocks (Talavera & Castresana 2007). The program was run with reduced stringency parameters (b1:0.5, b2:0.5, b3:12, b4:7). We then used another Phyluce script to filter the initial set of alignments so that each alignment was required to include data for 80% of taxa. Additional matrices with 70, 90, and 100% taxon completeness were used for data exploration, but the 80% matrix was selected for most analyses as it reduced missing data without resulting in significant loss of loci. To calculate summary statistics for the final data matrix, we used a script from the Phyluce package (<em>phyluce_align_get_align_summary_data</em>). UCE Phylogenomic Analysis: We performed several analyses on the 80% concatenated supermatrix using the maximum likelihood-based program IQ-TREE v2 (Minh et al. 2020). An unpartitioned search was conducted using the GTR+G model of sequence evolution as an initial check of tree topology. Following this result, we partitioned the tree by UCE locus and conducted separate analyses using IQ-TREE v2, first merging loci using  “-m TESTMERGEONLY” and “--merge rclusterf -rclusterf 10”, then conducting a second search with the resulting partitioning scheme using model selection from ModelFinder (Kalyaanamoorthy et al. 2017). The supermatrix was also partitioned using Sliding-Window Site Characteristics based on entropy (SWSC-EN; Tagliacollo & Lanfear 2018), which breaks UCE loci into three regions, corresponding to the right flank, core, and left flank. The theoretical underpinning of the approach comes from the observation that UCE core regions are conserved, while the flanking regions become increasingly more variable (Faircloth et al. 2012). After running SWSC-EN, the resulting data subsets were merged using “-m TESTMERGEONLY” and “--merge rclusterf -rclusterf 10”, and phylogenetic relationships were inferred from the resulting partitioning scheme using model selection from ModelFinder in IQ-TREE v2. For all IQ-TREE runs on the concatenated matrix, we performed 1,000 replicates of the ultrafast bootstrap approximation (UFB; Hoang et al. 2018) and 1,000 replicates of the branch-based, Shimodaira-Hasegawa-like approximate likelihood ratio test (SH-aLRT; Guindon et al. 2010). To address possible topological conflict between UCE loci due to incomplete lineage sorting, a coalescence-based phylogeny was reconstructed using ASTRAL-III v5.7.8 (Zhang et al. 2018). We first created a set of gene trees for the set of 2,030 UCE loci using IQ-TREE v2. For each gene tree analysis we performed model selection with ModelFinder (option “-m MFP”), using the AICc model selection criterion, and conducted 1,000 UFB replicates. Once the gene trees were generated, we followed the recommendation of Zhang et al. (2018) and used Newick Utilities (Junier & Zdobnov 2010) to collapse branches with ≤ 30% UFB support. Using the modified gene trees, we performed a standard ASTRAL analysis, leaving all terminals as separate entities, and assessing support as local posterior probabilities.
description: # Data from: Untangling the ant claws: The army ant (Formicidae:
 Dorylinae) *Labidus mars* is a *Neivamyrmex*
 [https://10.5061/dryad.bg79cnpkt](https://10.5061/dryad.bg79cnpkt) ####
 Sample names DNA extraction codes, original sample names, final sample
 names, and NCBI accession numbers for all samples used in this study. File
 name: sample-names.xlsx #### UCE contigs Aligned UCE contigs for all
 samples used in this study. Names have been updated to the final published
 sample names. File name: uce-contigs.zip File list:
 *Cheliomyrmex_megalonyx_M146.contigs.fasta
 Eciton_lucanoides_EX3147.contigs.fasta Labidus_mars_EX3594.contigs.fasta
 Labidus_denticulatus_EX3300.contigs.fasta
 Labidus_coecus_EX3411.contigs.fasta Labidus_praedator_EX3412.contigs.fasta
 Labidus_spininodis_M172.contigs.fasta
 Leptanilloides_femoralis_M188.contigs.fasta
 Neivamyrmex_bruchi_EX3152.contigs.fasta
 Neivamyrmex_clavifemur_EX3302.contigs.fasta
 Neivamyrmex_mars_EX3593.contigs.fasta
 Neivamyrmex_nigrescens_D3085.contigs.fasta
 Neivamyrmex_pilosus_D3087.contigs.fasta
 Neivamyrmex_punctaticeps_EX3414.contigs.fasta
 Neivamyrmex_spoliator_EX3324.contigs.fasta* #### UCE alignments Raw UCE
 alignment FASTA files. 2030 UCE loci aligned using mafft-linsi, but not
 not yet trimmed or filtered. Names have been updated to the final
 published sample names. File name: uce-alignments.zip #### DNA matrices
 DNA matrix and partition files used in phylogenetic analyses. The matrix
 contains 2030 aligned, filtered (matrix generated with Phyluce for 80%
 completeness), and trimmed (using GBlocks) UCE loci, includes by-locus
 character partitions. Names have been updated to final published names.
 File name: dna_matrices.zip File list:
 *concat-align-nexus-gblocks-names-mars-80.nex bylocus_partitions.nex
 swsc_partitions.nex* #### Tree and log files Tree files with boostrap
 scores/local posterior probabilities and associated analysis log files.
 Sample names in trees, but not log files, have been updated to final
 published sample names. The first part of each file name indicates the
 figure number used in the publication, with most numbers (s1, s2, s3)
 corresponding to supplemental figure numbers. *fig1-swsc.log* and
 *fig1-swsc.tre*: UCE matrix partitioned using entropy-based Sliding-Window
 Site Characteristics (SWSC-EN); *s1-BS30.tre*, *s1-astral.log*, and
 *s1-astral.tre*: gene trees with branches with ≤30% bootstrap support
 collapsed, ASTRAL species tree with local posterior probabilities;
 *s2-bylocus.log*, *s2-bylocus.tre*: tree from UCE matrix partitioned by
 UCE locus; *s3-unpartitioned-gtrg.log*, *s3-unpartitioned-gtrg.tre*: tree
 from unpartitioned UCE matrix. File name: tree-log-files.zip File list:
 *fig1-swsc.log fig1-swsc.tre s1-BS30.tre s1-astral.log s1-astral.tre
 s2-bylocus.log s2-bylocus.tre s3-unpartitioned-gtrg.log
 s3-unpartitioned-gtrg.tre*