DOI: 10.5061/dryad.ncjsxkt73
title: Systematic and taxonomic revision of Emerald and Tigertail dragonflies (Anisoptera: Synthemistidae & Corduliidae)

description: Libelluloidea is the most speciose superfamily within dragonflies
 (Odonata: Anisoptera), yet intrafamilial relationships have remained
 contested for the past 150 years. Here we present a phylogenetic
 hypothesis for two families within Libelluloidea, Corduliidae (Emeralds),
 and Synthemistidae (Tigertails) based on comprehensive taxon sampling
 (Corduliidae: 141/165 species, Synthemistidae: 123/150) for which we
 generated Anchored Hybrid Enrichment (AHE) high-throughput molecular
 sequences (10 - 1054 loci). Furthermore, we combined our molecular dataset
 with 100 discrete morphological characters based on wing, body, nymphal
 and genitalic characters. Using our molecular data, and an evaluation of
 morphological characters via ancestral character state reconstruction, we
 propose a new classification for these taxa.  Here, three new
 families are erected: Lauromacromiidae fam. nov.,; Macromidiidae fam.
 nov.,; Aeschnosomatidae fam. nov.. and the status is revised for six
 families: Pseudocorduliidae stat. rev., Lohmann, 1996; Gomphomacromiidae
 stat. rev., Tillyard & Fraser, 1940; Austrocorduliidae stat. rev.,
 Lohmann, 1996; Idomacromiidae stat. rev., Tillyard & Fraser 1940;
 Idionychidae stat. rev., Tillyard & Fraser, 1940; Neophyidae stat.
 rev., Tillyard & Fraser, 1940. Furthermore, we sink the genus
 Procordulia Martin, 1907 into the genus Hemicordulia Selys, 1870. Finally,
 we recover five enigmatic taxa (Archaeophya Fraser, 1959, Libellulosoma
 Martin, 1907, Austrophya Tillyard, 1909, Apocordulia Watson ,1980, and
 Cordulisantosia Fleck and Costa, 2007) for which no molecular data was
 previously available within these new families with high support using a
 total-evidence approach. Character state reconstructions revealed
 widespread homology among traditional characters used to identify groups
 within each family. We estimate the ancestral Libelluloidea possessed a
 compact anal loop, prominent uniform labial dentition in the nymphs, and a
 reduced ovipositor. Finally, time-divergence analyses estimate
 Libelluloidea to have originated within the Late Jurassic, with subsequent
 families diversifying throughout the Cenozoic.
description: <em>Taxon Sampling</em> We
 acquired specimens of Corduliidae and Synthemistidae and our outgroups
 from both freshly caught field-collected and museum specimens. Specimens
 were hand caught using aerial nets within New South Wales and Victoria
 Australia during the summer months of January and February 2023. Specimens
 were placed in glassine envelopes and submerged in acetone for
 preservation and were transported to the American Museum of Natural
 History for molecular and morphological analysis. However, most specimens
 used in our work were sourced from natural history collections. Specimens
 were sampled from collections in the American Museum of Natural History
 (AMNH), Florida State Collection of Arthropods (FSCA), Naturalis
 Biodiversity Center, Natural History Museum (NMNH), Brigham Young
 University, Monte L. Bean Life Sciences Museum (BYU), Natural History
 Museum, London UK (NHMUK), the Australian Museum (AM), and the United
 States National Museum of Natural History (USNM). In total, we sampled 141
 of the 165 species of Corduliidae (85%) and 123 of the 150 species of
 Synthemistidae (82%). We also sampled 9 additional Libelluloidea taxa from
 other families (<strong>Libellulidae:</strong>
 <em>Pantala, Libellula, Orthetrum</em>,
 <strong>Macromiidae:</strong> <em>Macromia,
 Epophthalmia</em>), and families within Cavilabiata
 (<strong>Chlorogomphidae:</strong>
 <em>Chlorogomphus</em>,
 <strong>Cordulegastridae:</strong> <em>Cordulegaster,
 Anotogaster</em>, <strong>Neopetaliidae:
 </strong><em>Neopetalia punctata</em>). Specimen
 provenance data including locality, date, author, collector, determiner,
 and sequencing code are listed in Supplemental Table S1. 
 <em>DNA Extraction and Sequencing</em>:
 We removed the hind leg from each field-collected or museum
 acquired specimen using sterilized forceps, and extracted DNA using
 ZYMOBIOMICS DNA miniprep kits (Irvine, CA). We quantified DNA yield using
 a Qubit 4 fluorometer, and sent to RAPID Genomics (Gainesville, Florida)
 for library preparation and sequencing. Loci were amplified using modified
 Anchored Hybrid Enrichment (AHE) probes first implemented in Bybee et al.
 (2021); the modified probe set consists of 1,306 loci covering
 approximately 500 kilobases (Goodman et al. 2023). We sequenced loci for
 representatives of each genus using the full 1,306 probe set (herein
 referred to as 500kb set), while a subset of 92 loci (herein referred to
 as 20kb probe set) were sequenced for the remaining species (See
 Supplemental Table S2)(500kb: 60%, 20kb: 40% of taxa). We re-sequenced
 several monotypic genera (<strong>Synthemistidae:</strong>
 <em>Archaeophya, Austrophya, Apocordulia,
 </em><strong>Corduliidae:</strong>
 <em>Williamsonia, Cordulisantosia</em>) using the 20kb loci
 probe set due to failed assembly during the first round of 500kb
 sequencing. <em>AHE Assembly and
 Analysis</em> We trimmed adaptors from raw reads
 using <em>fastp </em>(Tang and Wong 2001),<em>
 </em>and checked for quality using <em>multiQC
 </em>(Ewels et al. 2016). We followed methods outlined in Breinholt
 et al. (2018) to assemble and assess orthology for each target capture
 locus. In brief, we assembled each locus individually using iterative
 baited assembly with <em>SPAdes </em>(Prjibelski et al. 2020)
 and a chromosome-length genome assembly of <em>Tanypteryx hageni
 </em>(Petaluridae) as reference (Tolman et al. 2023). We then
 screened each locus for orthology by first ensuring that the locus did not
 have multiple BLAST hits in the reference genome and, secondly, by
 ensuring best reciprocal hits between the reference and the query
 sequence. <em>Phylogenetic
 Analysis</em>: We generated multiple sequence
 alignments for each locus using the ‘MAFFT-linsi’ algorithm in
 <em>MAFFT v.7.475 </em>(Katoh and Standley 2013) and trimmed
 alignments using a 0.75 threshold cutoff using <em>trimAI
 v1.2</em> (Capella-Gutiérrez et al. 2009). We concatenated the
 alignment using <em>FASconCAT v1.11</em> (Kück and Meusemann
 2010), and generated an optimal partitioning scheme using relaxed
 clustering with the model fixed to GTR + G for each subset in
 <em>IQtree v2.1.</em>3 (Minh et al. 2020). We then selected
 the best nucleotide substitution model for each subset in the partitioning
 scheme using ModelFinder (Kalyaanamoorthy et al. 2017) and estimated a
 maximum likelihood tree (ML). We estimated branch support using SH-like
 approximate likelihood ratio tests (SH-aLRT) and 1,000 ultrafast bootstrap
 replicates (UFboot) in <em>IQtree v2.1.3</em> (Guindon et al.
 2010, Minh et al. 2020). Finally, to assess the degree of incomplete
 lineage sorting, we performed a coalescent-based species tree estimation
 in <em>ASTRAL</em><em>III v5.6.1 </em>and
 estimated branch support using local posterior probabilities (LPP)
 (Mirarab and Warnow 2015). We identify nodes of high support possessing ML
 and SH-aLRT values &gt; 95, and LPP values &gt;0.95 We rooted the
 tree using <em>Neopetalia punctata
 </em>(Neopetaliidae). <em>Post-Hoc
 Modifications to Phylogeny</em>: Preliminary
 analyses recovered 38 taxa of varying genera and families that clustered
 together, forming several odd clades in our phylogeny (See Supplemental
 Information). These taxa were misplacements (herein referred to as
 <em>‘uncertain status’ </em>taxa), and these have been
 observed in other, previous AHE datasets and are the result of low locus
 count, low overlap among recovered loci across taxa, contamination, or a
 combination of factors (Goodman et al. 2025, Goodman et al. 2023). When
 contamination was exhibited based on other information, those sequences
 were removed. To improve resolution in our analysis, we omitted those taxa
 for which there was little to no overlap in recovered loci as several taxa
 possessed fewer than 10 loci that were recovered among congenerics with
 high support. After omitting these taxa, our family and genus-level
 representation decreased, with four monotypic genera no longer present in
 our analyses (<em>Austrophya, Archaeophya, Apocordulia</em>,
 and <em>Cordulisantosia</em>). Preliminary phylogenies
 recovered sequences of <em>Archaeophya </em>and
 <em>Cordulisantosia </em>within <em>Neocordulia,
 </em>while sequences of <em>Apocordulia </em>recovered
 within <em>Idionyx </em>and <em>Somatochlora,
 </em>and sequences of <em>Austrophya </em>recovered
 within Libellulidae and sister to <em>Tonyosynthemis</em>; we
 also observed short fragments and unusual gene alignments. Visual
 inspection of gene trees suggested other <em>‘uncertain status’
 </em>taxa that were grouping within <em>Neocordulia
 </em>and <em>Somatochlora</em>, most likely due to
 contamination. Our final, filtered data set included 211 taxa, down from
 the 249 taxa originally sequenced (See Supplemental Table S2).
 <em>Morphological Analysis: </em>
 We collected morphological data from approximately 430 specimens
 to characterize the variability in states among individuals and across
 sexes; for all our species we examined at least two specimens unless only
 one was available (Fig. 1 – 4). When different specimens from a single
 species varied in their character state, we coded all possible states
 present in the specimens for that species. We scored 30 wing venation
 (Fig. 1), 13 external body, 13 accessory genitalic (Fig. 2), 23 nymphal
 (Fig. 3), and 11 penile characters from Ware (2008) (Fig. 4); we further
 expanded on this dataset with an additional 10 <em>de novo
 </em>penile characters described herein totaling 100 characters.
 Wing-vein terminology followed Riek and Kukalová-Peck (1984); characters
 were based on previously published characters from Needham and Broughton
 (1927), Fraser (1957), Miller (1991), Carle (1995), Needham et al. (2000),
 and Garrison et al. (2006), as well as unpublished characters from
 Tennessen and May. When we lacked specimens to examine for particular
 species, we scored adult or nymphal characters from the literature
 (Theischinger and Watson 1978, 1984, Theischinger 1999, Theischinger and
 Hawking 2000, Carvalho et al. 2004, Machado 2005a, b, Theischinger and
 Hawking 2006, Carvalho et al. 2008, Fleck 2008, Theischinger 2009, Pinto
 and Carvalho 2010, Pinto and Lamas 2010, Fleck 2012a, Fleck 2012b, Fleck
 and Neiss 2012b, a, Machado 2012, Fleck and Legrand 2013, Theischinger and
 Endersby 2014b, Fleck 2017, Neiss et al. 2018, Fleck and Juillerat 2019,
 Roberts et al. 2019, Tennessen 2019, Ehlert and Parise Pinto 2020, Fleck
 and Haber 2022, Pinto et al. 2022). We mapped characters onto our ML tree
 under parsimony using the Trace Character option in <em>Mesquite
 v3.4</em> (Maddison and Maddison 2007). We were unable to estimate
 character evolution under maximum likelihood due to the morphological
 characters possessing more than two states, prompting us to utilize
 parsimony-based character mapping only. Further, to
 assess the degree of impact that specific parts of our morphological
 dataset  had on relationships in a combined molecular and morphological
 topology, we first subdivided our morphological characters into discrete
 groups (wing venation, external body, accessory genitalic, nymphal, and
 penile), then performed total-evidence Maximum Likelihood phylogenetic
 analysis combining our molecular dataset with each individual character
 subset. We applied an MK model of discrete character evolution for our
 morphology partitions in our ML analyses otherwise using parameters as
 described above (Lewis 2001) (See supplementary Information). We compared
 topological differences for each of our trees using Robinson-Foulds (RF)
 Distance (Robinson and Foulds 1981), and branch length differences using
 Euclidian Distance (Felsenstein 1984). We then used analysis of variance
 (ANOVA) to determine statistical differences in topology and branch length
 between character subsets. In particular, to determine the degree of
 homoplasy and variation among our morphological data matrix, we compared
 the rescaled consistency index (Farris 1989) for each character using an
 ANOVA.   <em>Fossil Selection
 and Time Divergence Analysis: </em> We employed
 fossils as calibrations for divergence time estimation following the best
 practices outlined by Parham et al. (2012). However, choosing fossils for
 calibration of odonate divergence time estimation analyses can be
 challenging due to the restricted suites of characters that are present in
 most fossils. The taxonomy of odonate fossils relies predominantly on wing
 characters due to their high preservation potential, and litany of wing
 venation traits (Fraser and Tillyard 1957). However, wing venation is
 highly prone to convergence and should be used in conjunction with other
 traits if available (Fraser and Tillyard 1957, Gloyd 1959, Hennig 1981,
 Fleck et al. 2008). Amber fossils of adult and nymphal Odonata are rare in
 the fossil record (Wighton and Wilson 1986, Bechly 1996a, Karr and Clapham
 2015, Schädel and Bechly 2016, Zheng and Jarzembowski 2020, Boudet et al.
 2023)(See table 1 in Schaedel et al. (2020))(paleodb.com), limiting the
 possibility for analyses including accessory genitalic, thoracic, penile,
 or nymphal traits. Kohli et al. (2016) published a list of vetted fossil
 calibrations for Odonata, as part of the Fossil Calibration Database
 (fossilcalibrations.org), providing recommendations for fossil selection
 in our phylogeny; these were updated and expanded upon in Kohli et al.
 (2021). We employed fossil calibrations for the crown nodes from Kohli et
 al (2021) for Cavilabiata, Chlorogomphidae + Cordulegastridae,
 Macromiidae, Corduliidae, Libellulidae, and Corduliidae + Libellulidae.
 Phylogenetic and age justifications for divergence time estimation of our
 fossils are outlined in detail in Kohli et al. (2016) and Kohli et al.
 (2021).   <em>Fossil
 Validation</em> We surveyed five additional
 putative fossils proposed to belong in Synthemistidae as calibration
 points, extending our sampling beyond Kohli et al. (2021). We used the
 five principles outlined by Parham et al. (2012) and Ksepka et al. (2015)
 as best calibration practices. In brief, the five criteria are as follows:
 1. Fossil accession number for fossil and referrals, 2. Apomorphy-based or
 phylogenetic analysis, 3. Reconciliation of morphological and molecular
 data, 4. Locality and stratigraphic data for fossil taxa 5. Radioisotopic
 age or numeric age references for fossil (See Table 1 in Goodman et al.
 (2025) for fossil calibrations). Two putative fossils
 were originally assigned to the defunct subfamily Gomphomacromiinae as
 defined by Tillyard and Fraser (1940), both being estimated as late
 Jurassic/Early Cretaceous (113 – 125mya) in origin. <em>Mesocordulia
 boreala, </em>Ren and Guo, 1996 is from Western Liaoning Province,
 China. The holotype consists of an adult female with all four wings, head,
 thorax, and abdomen, as well as the paratype consisting of a complete
 hindwing. <em>Mesocordulia boreala </em>was subsequently
 placed within the extinct family Araripelibellulidae by Bechly (1996b), in
 the subfamily Mesocorduliinae. A new specimen of <em>M. boreala
 </em>was recently described, possessing exceptional preservation of
 both the forewings and hindwings, as well as several body, head, and
 external genitalic characters (Nel et al. 2024); several new diagnostic
 characters identified by Nel prompted the elevation of Mesocorduliinae to
 family-level. A second fossil is <em>Eocordulia cretacea,
 </em>Pritykina, 1985 from Western Mongolia. The holotype consists of
 fragmentary fore and hindwings, there is a paratype of an adult female,
 and paratypes consisting of the hind wing of an adult male, and posterior
 end of the abdomen with appendages of an adult male. <em>Eocordulia
 cretacea </em>was placed within the extinct family Eocorduliidae by
 Bechly (2007), but not peer-reviewed. Although both taxa exhibit very
 similar wing venation traits to Synthemistidae, such as a compact
 (<em>Eocordulia cretacea</em>) or smoothly curving uninflated
 anal loop (<em>Mesocordulia boreala</em>), both taxa possess
 unfused sectors of the arculus, a trait present only in non-libelluloid
 Cavilabiata (Cordulegastridae + Neopetaliidae)(Nel et al. 2024). As such,
 we chose to exclude these two fossils as calibration points within our
 time-divergence analysis. Two other putative fossils
 for calibration belong to the defunct subfamily Cordulephyidae as defined
 by Lohmann (1996b). The older fossil is <em>Paleophya
 argentina,</em> Petrulevičius and Nel 2009, from northwest Argentina
 and is estimated to be<em> </em>from the early Paleogene (58.7
 - 55.8 mya). The holotype consists of a very fragmented hind wing, with
 anterior and posterior portions missing except between the arculus, anal
 loop, bridge, and the third postnodal crossvein. The second fossil,
 <em>Neophya legrandi </em>Nel and Fleck 2013, is from the late
 Eocene (33.9 – 33.8 mya) from the Isle of Wright. The holotype only
 consists of the base of the hindwing, between the first antenodal
 crossvein and the pterostigma, and above the triangle. Paratypes consist
 of the hindwing base, the costo-apical part of either the fore or
 hindwing, and the wing apex of the fore or hindwing.
 Lastly, <em>Somatochlora brisaci </em>(Nel et al.
 1996) is significantly younger, with a Miocene origin (8.7 – 5.3 Ma),
 discovered in a deposit from Southeastern France. The fossil is a
 near-complete hindwing except for a few posterior regions of the wing
 margin missing near the third and fourth medial veins, the cubital (C) and
 anal (A) veins. Although previous morphological analysis of the wings of
 <em>S. brisaci </em>failed to recover it within
 <em>Somatochlora</em> (Goodman et al. 2025), this is an
 appropriate fossil within Corduliidae. To determine the
 utility of these fossil taxa as calibration points, we scored wing-trait
 data for <em>Neophya legrandi, Paleophya argentina</em>, and
 <em>Somatochlora brisaci</em>, and performed a total evidence
 ML phylogeny consisting of our molecular + all morphological characters.
 Furthermore, we included morphological data for our ‘<em>uncertain
 status</em>’ taxa for which we had ambiguous molecular placement
 (n=40) including <em>Austrophya, Archaeophya, Apocordulia,
 </em>and <em>Cordulisantosia</em>. Finally, we scored
 the Malagasy species <em>Libellulosoma </em>to infer their
 placement within Libelluloidea. Previous research has hypothesized its
 relationship as close to the South American and Australian taxa
 <em>Aeschnosoma </em>and <em>Pentathemis</em>
 respectively (Corduliidae)(Fleck and Legrand 2013, Roberts et al.
 2019). All divergence time analyses were conducted on
 the nucleotide dataset in MCMCtree as implemented in the software package
 <em>PAML</em> <em>v.4.7a</em> Yang (2007) using an
 ultrametric (equal branch lengths) version of our ML tree as input. We
 used our full unpartitioned dataset due to computational limits since our
 dataset consists of over 1000 loci and 90 partitions were suggested by
 PartitionFinder. Fossil calibrations were set using uniform prior
 distributions with hard upper and lower bounds. Our root maximum age was
 set at 158.1 million years, based on the earliest fossil within
 Cavilabiata (<em>Juralibellula ningchengensis</em>) (Huang and
 Nel 2007). We set default parameters for defining prior distribution and
 used the General Time Reversible (GTR) nucleotide substitution model for
 calculating the hessian matrix for our dataset. For each scenario, we
 performed two independent MCMC runs with 500,000 iterations, sampling
 every 100 trees with a 2000 tree burn-in, and checked for convergence
 using <em>Tracer</em> <em>v. 1.6 </em>(Drummond
 and Rambaut 2007). Finally, we examined the prior distributions of each
 run to ensure reasonable fossil choices and placement on the tree (Warnock
 et al. 2012). Divergence time estimates, distributions, and outputs are
 provided in our Supplemental Information.
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description: # Systematic and Taxonomic Revision of Emerald and Tigertail Dragonflies
 (Anisoptera: Synthemistidae & Corduliidae) --- We have submitted:
 Specimen provenance data including locality, date, author, collector,
 determiner, sequencing code, and if scored for morphology of species used
 in phylogeny (**Supplemental_Table_S1.csv**) Specimen loci count,
 sequencing dataset (500kb or 20kb), and if removed in downstream post-hoc
 analyses of species used in phylogeny (**Supplemental_Table_S2.csv**) Raw
 AHE reads of Corduliidae and Synthemistidae species + outgroups from our
 AHE pipeline. We followed methods outlined in Breinholt et al. (2018) to
 assemble and assign orthology to each target capture locus
 (**1_Assemblies**). Supplemental files, figures, and job files of
 phylogenetic analyses conducted within our manuscript
 (**2_Phylogenetic_Analysis**) Output files from different morphological
 scoring analyses in order infer the degree of influence morphological
 characters possess in topological and branch length differences in our
 molecular phylogeny, using taxa retained after ad-hoc pruning
 (**3_Morphological_Analysis**) Output files from our total-evidence
 (Morphology + sequences) phylogenetic analysis to infer the placement of
 species which we failed to amplify sequences for
 (**4_Total_Evidence_Analysis**) Output from our time-divergence MCMCtree
 analysis (**5_Time_Divergence_Analysis**) --- ## Data Formats Excel/csv
 files: * **.csv**: Utilized for organizing metadata of specimens used in
 phylogenetic sequencing, including taxonomy, author, locality, longitude,
 latitude, institution, and collection (**Supplemental_Table_S1.csv**) Text
 Files: * **.txt**: Contains output from parameter settings/output files
 from our phylogenetic analyses and divergence analyses (**mcmc.txt**)
 Python/Shell/perl Scripts: * **.py/.sh/.pl**: Python or bash scripts for
 running phylogenetic analyses (See Breinholt et al. 2018)
 (**FASconCAT_v1.11.pl**) Cluster Job Files: * **.job**: SLURM job script
 submitted to the cluster, contains parameters of various phylogenetic (ML)
 analyses (**combined_analysis.job**) Phylogenetic Files: * **.tre**:
 Output phylogenetic trees generated from IQ-tree, can be viewed using
 Figtree
 ([http://tree.bio.ed.ac.uk/software/figtree/](http://tree.bio.ed.ac.uk/software/figtree/)) (*Astral_tree.tre*) * **.newick**: Output phylogenetic trees generated from IQ-tree can be viewed using Figtree ([http://tree.bio.ed.ac.uk/software/figtree/](http://tree.bio.ed.ac.uk/software/figtree/)) (**ML_Tree_Renamed.newick**) * **.phy**: Input alignment file in phylip format of AHE sequences used for sequences, can be opened using Aliview ([https://ormbunkar.se/aliview/](https://ormbunkar.se/aliview/)), or Mesquite ([https://www.mesquiteproject.org/](https://www.mesquiteproject.org/)) (**dna.phy**, **wings.phy**) * **.fasta**: Input unaligned Raw AHE reads of Corduliidae and Synthemistidae species + outgroups, can be opened using Aliview ([https://ormbunkar.se/aliview/](https://ormbunkar.se/aliview/)), or Mesquite ([https://www.mesquiteproject.org/](https://www.mesquiteproject.org/)) (**GEODE_17697_Corduliidae_Antipodochlora_braueri_targets_ORTHO.fasta**) Time-Divergence Files: * **.ctl**: Control files which define parameters for creating time-divergence analyses using the MCMCtree package in PAML (**mcmctree.ctl**) * **.BV**: Pre-computed branch lengths and their associated variances for the specified tree topology in our time-divergence analysis (**out.BV**) Additional Files: * **.out**: Standard output file generated by jobs run on a SLURM-managed cluster. Provides inputted parameters for phylogenetic analyses and produced output files (**slurm-3619918.out**) * **.nex**: Nexus format data generated to store both molecular and morphological-data in text format (**partition.nex**). * **.bionj/.gz/.iqtree/.log/.mldist/.parstree/.ufboot/.ckp/.mcmc/.parts/.p/.t/.probs/.tstat/.vstat/.txt/emf**: various output files generated from Maximum Likelihood (IQ-tree) phylogenetic analyses --- ## Recommended Software for Data Analysis In order to run/reproduce analyses from **1_Assemblies**, requires the use of bioconda ([https://bioconda.github.io/](https://bioconda.github.io/)) with the packages MAFFT v.7.475, trimAI v1.2, and FASconCAT v1.11 In order to run/reproduce analyses from **2_Phylogenetic_Analysis** requires the use of iqtree2 ([http://www.iqtree.org/](http://www.iqtree.org/)) In order to run/reproduce analyses from **3_Morphological_Analysis** and **4_Total_Evidence_Analysis** requires the use of iqtree2 ([http://www.iqtree.org/](http://www.iqtree.org/)), Mesquite v.3.81 ([https://www.mesquiteproject.org/](https://www.mesquiteproject.org/)), R and RStudio ([https://posit.co/download/rstudio-desktop/](https://posit.co/download/rstudio-desktop/)) In order to run/reproduce analyses from **5_Time_Divergence_Analysis** requires the PAML command line package ([http://abacus.gene.ucl.ac.uk/software/paml.html](http://abacus.gene.ucl.ac.uk/software/paml.html)), using the MCMCtree command --- ## Usage, Compatibility, and Accessibility This dataset encompasses a wide breadth of file formats, due to the span of analyses conducted within this manuscript, ranging from phylogenetic, morphological, and divergence. The majority of these files can be viewed using a standard text editing software such as BBedit ([https://www.barebones.com/products/bbedit/index.html](https://www.barebones.com/products/bbedit/index.html)), as well as tree view software Figtree ([https://github.com/rambaut/figtree/releases](https://github.com/rambaut/figtree/releases)) and all programs, code, and data are publicly available, and free to use. Researchers are encouraged use original data formats, repeat analyses using our data for reliability and accuracy, and to email the corresponding author for any additional questions or confusions. We are committed to providing support, clarity, and guidance to facilitate the effective use of these data, with the hopeful prospect of instilling curiosity and future analyses with not only our data, but with phylogenomic datasets. --- ## Version changes **11-jun-2011:** Added additional locality information to **Supplemental_Table_S1.csv** after review of manuscript by co-authors, and renamed the family-level underscores of the *Specimen* column from **Supplemental_Table_S2.csv** to reflect the new taxonomy from the manuscript --- ### Descriptions #### **Supplemental\_Table\_S1.csv** * *Sequencing Code*: Sequencing code used for specimen upon extraction of DNA from the hind leg and subsequent submission to RapidGenomics. NOTE, lines with NA represent unsequenced specimens, but still scored for morphology * *Family*: Taxonomic rank of specimen at the family level * *Genus*: Taxonomic rank of specimen at the genus level * *Species*: Taxonomic rank of specimen at the species level (species epithet) * *Author*: Author who first described the species * *Country*: Country where specimen was captured, NA indicates that information was not present on label * *State/Province/Region*: State/Province/Region where specimen was captured, NA indicates that information was not present on label * *County/Parish/Prefecture*: County/Parish/Prefecture where specimen was captured, NA indicates that information was not present on label * *Locality*: Descriptive locality where specimen was captured, NA indicates that information was not present on label * *Latitude*: Latitude coordinate where specimen was captured, NA indicates that information was not present on label * *Longitude*: Longitude coordinate where specimen was captured, NA indicates that information was not present on label * *Altitude*: Altitude where specimen was captured, NA indicates that information was not present on label * *Sex*: Sex of specimen (Male/Female), NA indicates that information was not present on label * *Day*: Calendar day of specimen capture (1 - 31), NA indicates that information was not present on label * *Month*: Calendar month of specimen capture (Specimen roman numeral notation, I - XII), NA indicates that information was not present on label * *Year*: Calendar year of specimen origin (1939 - 2018), NA indicates that information was not present on label * *Collector*: Collector of specimen, NA indicates that information was not present on label * *Det*: Determiner of species identification, NA indicates that information was not present on label * *Institution*: Institution where specimen is housed (FSCA, BYU, RMNH), NA indicates that information was not present on label * *Collection*: Collection of specimen (If researcher donated specimens to museum, that specimen is part of that collection, D.R. Paulson Collection), NA indicates that information was not present on label * *Processed by*: Processor of specimen into database of museum, NA indicates that information was not present on label * *Type*: Specimen type status (Holotype, Paratype, Lectotype etc.), NA indicates that information was not present on label * *Notes*: Additional notes on label for specimen which does not fit into aforementioned categories, NA indicates that information was not present on label * *Scored for Morphology*: If the specimen was scored using our 100 discrete morphological matrix (See **3_Morphological_Analysis** and **4_Total_Evidence_Analysis**) #### **Supplemental\_Table\_S2.csv** * *Specimen*: Sequencing code, family, genus, and species of specimen used for extraction of dna from hind leg and subsequent submission to RapidGenomics * *Loci Count*: Number of successful loci amplified and sequenced from that specimen * *AHE_Dataset*: Probe set used to amplify loci for specimen (500kb = 1000+ loci, while 20kb = 92 loci probe set) #### **1\_Assemblies**: #### File Naming Convention Each file name follows a structured format that encodes metadata about the sample and data type: #### Components * **GEODE_####**: A unique AHE (Anchored Hybrid Enrichment) project identifier assigned to the specimen. The number (e.g., `17697`) corresponds to the sample ID used in sequencing and tracking. * : The taxonomic family of the specimen (e.g., `Corduliidae`). * : The genus of the specimen (e.g., `Antipodochlora`). * : The species epithet of the specimen (e.g., `braueri`). * : Describes the sequence content: * `targets_ORTHO`: Raw AHE reads targeting the 500kb probe region only. * `targetsFULL_ORTHO`: Raw AHE reads including both the 500kb probe region and the surrounding flanking regions. * (For the 20kb dataset, the same naming convention applies, and details can be cross-referenced in **Supplemental_Table_S2.csv**.) #### Examples * **GEODE_17697_Corduliidae_Antipodochlora_braueri_targets_ORTHO.fasta**: Raw AHE reads of 500kb loci probe set of the species Antipodochlora braueri. * **GEODE_17697_Corduliidae_Antipodochlora_braueri_targetsFULL_ORTHO.fasta**: Raw AHE reads of 500kb loci probe + flanking region set of the species Antipodochlora braueri. NOTE: remaining taxa follow same file formatting and naming, both for 500kb and 20kb dataset (see **Supplemental_Table_S2.csv**) #### **2\_Phylogenetic\_Analysis** **Unaltered_iqtree**: Here you will find all the maximum likelihood (IQTREE) phylogenetic analyses conducted after removing sequences of 'uncertain placement'. Consider these our phylogeny of all taxa prior to ad-hoc modifications * **Astral_tree.tre**: The species tree inferred using ASTRAL, which estimates a consensus tree from multiple gene trees while accounting for incomplete lineage sorting. * **concord.cf.branch**: Contains branch-specific concordance factors, showing how much of the data (e.g., gene trees or sites) supports each internal branch. * **concord.cf.stat**: Summary statistics of concordance factors, including mean, variance, and distribution across branches. * **concord.cf.tree**: A phylogenetic tree in Newick format annotated with concordance factor values for each branch. * **concord.cf.tree.nex**: Same as **concord.cf.tree**, but in NEXUS format for visualization in software like FigTree or PAUP*. * **concord.log**: Log file from the concordance factor analysis process; records steps taken and any errors or warnings. * **extract_partition_file_from_fcc.py**: A Python script used to extract partitioning information from FASconCAT or similar concatenated datasets. * **FASconCAT_v1.11.pl**: A Perl script used to concatenate multiple gene alignments into a supermatrix and generate partition files for phylogenetic analysis. * **FcC_info.xls**: Excel spreadsheet generated by FASconCAT containing summary statistics about the concatenated alignments, such as the number of characters per gene, missing data percentages, and taxon coverage. * **FcC_smatrix.fas**: FASTA-formatted supermatrix of concatenated gene alignments produced by FASconCAT. Unaltered version of **FcC_smatrix_N.fas**. * **FcC_smatrix_N.fas**: The concatenated supermatrix alignment in FASTA format, created by FASconCAT, used for tree inference or model selection, with dashes (-) replaced with N * **iqtree_model_selection_and_tree_search.job**: A job script (likely for SLURM or another scheduler) to run IQ-TREE for model selection and tree inference. * **iqtree2**: The IQ-TREE 2 binary executable or a symbolic link to it. This is the program used for phylogenetic inference under maximum likelihood. * **loci_count.txt**: Plain text file listing the number of loci recovered per taxon, useful for assessing completeness and data coverage across samples. * **odo_concat.best_model.nex**: Contains the best-fitting substitution model selected for the concatenated dataset, formatted for NEXUS compatibility. * **odo_concat.best_scheme**: The partitioning scheme inferred to best fit the concatenated dataset, listing genes or regions and their respective models. * **odo_concat.best_scheme.nex**: NEXUS format version of the best partitioning scheme, usable by software requiring this format. * **odo_concat.bionj**: A neighbor-joining tree used as the starting tree for maximum likelihood inference in IQ-TREE. * **odo_concat.ckp.gz**: A compressed checkpoint file used to resume interrupted IQ-TREE runs without starting from scratch. * **odo_concat.contree**: The consensus tree from a bootstrap or posterior distribution, summarizing common features across replicates. * **odo_concat.iqtree**: Summary output file containing the results of the tree inference, including model selection, likelihood scores, and support values. * **odo_concat.log**: Log file from IQ-TREE, detailing the process of model selection and tree inference, including run time, warnings, and performance stats. * **odo_concat.mldist**: A matrix of pairwise maximum likelihood distances between all taxa in the alignment. * **odo_concat.model.gz**: Compressed file containing the model settings and substitution matrices used for the analysis. * **odo_concat.splits.nex**: A splits graph in NEXUS format showing alternative topologies or conflicting phylogenetic signals. * **odo_concat.treefile**: The final maximum likelihood tree produced by IQ-TREE, including branch lengths and support values. * **odo_concat.ufboot**: Contains ultrafast bootstrap replicate trees, used to assess support for branches in the ML tree. * **odo_gene.best_scheme**: The best partitioning scheme inferred for the per-gene dataset. * **odo_gene.best_scheme.nex**: The same per-gene partition scheme formatted in NEXUS for compatibility with other programs. * **odo_gene.ckp.gz**: Compressed checkpoint file for the gene-partitioned analysis, allowing restart from the last saved point. * **odo_gene.log**: Log file of the IQ-TREE run on the gene-partitioned dataset, recording model fitting and tree inference steps. * **odo_gene.model.gz**: Compressed model information file for the gene-partitioned dataset. * **odo_gene.parstree**: A tree inferred using parsimony, typically used as an alternative starting tree or for comparison with the ML tree. * **odo_gene.treefile**: Maximum likelihood tree for the gene-partitioned dataset, with branch support values. * **odo_partition.best_model.nex**: The best substitution models selected for each partition in the partitioned dataset, formatted for NEXUS. * **odo_partition.best_scheme**: The inferred optimal partitioning scheme, listing regions and their corresponding models. * **odo_partition.best_scheme.nex**: NEXUS format version of the optimal partitioning scheme for compatibility with other tools. * **odo_partition.ckp.gz**: Checkpoint file from the partitioned analysis, useful for resuming IQ-TREE if the run was stopped. * **odo_partition.iqtree**: Main summary file for the partitioned IQ-TREE run, detailing selected models, tree stats, and branch supports. * **odo_partition.log**: Log output from the partitioned tree inference, including warnings, progress, and resource usage. * **odo_partition.model.gz**: Gzipped file storing model configurations used for each partition during the analysis. * **odo_partition.treefile**: Maximum likelihood tree for the partitioned dataset, typically considered the final phylogeny result. * **partition_def.txt**: The partition definition file, listing the alignment regions and their associated genes or data blocks. * **slurm-4301735.out**: Standard output file from a SLURM job submission. Contains run-time messages, errors, or print statements from the job script. * **taxa_list.txt**: A list of taxon names included in the analysis, typically used for filtering, labeling, or cross-referencing in phylogenetic workflows. **Pruned_iqtree**: Here you will find all the maximum likelihood (IQTREE) phylogenetic analyses conducted after removing sequences of 'uncertain placement' (i.e. taxa which recovered to incorrect places within our **Unaltered_iqtree**), NOTE: all file outputs, names, and files are the same as **Unaltered_iqtree** #### **3\_Morphological\_Analysis** **Distance_Metrics.R**: Code used to calculate differences in tree topology (RF distance) and branch length (Euclidian Distance) of our pruned iqtree (See **odo_concat.contree** from **Pruned_iqtree**) to that of our total-evidence trees calculated with different subsets of morphological data **Morphology_Scores_Sequenced_Taxa.nex**: Species scored from our pruned phylogeny, using our full 100 discrete character matrix consisting of characters from wings, external body, external genitalic, penile, and nymphal. Nexus file contains information and character states of each morphological character **ML_Tree_Renamed.newick**: Maximum-likelihood (ML) tree generated using our pruned iqtree (211 taxa) in newick format. Note **ML_Tree_Renamed.newick** is the same as **odo_concat.contree** from **Pruned_iqtree**, however names have been changed to remove sequencing code (i.e. GEODEXXXX, or RMNH.XXXX), family name (i.e. Synthemistidae) **1_Wings_Scorings**: Output from Total-Evidence (Morphology + Sequences) Phylogenetic analysis using our concatenated sequence matrix of our pruned species + subset of morphological matrix of just wing characters * **odo_concat.best_scheme**: The best-fit partitioning scheme selected for the concatenated dataset, listing data blocks and the corresponding models applied to each. * **combined_analysis.job**: A job script (likely for SLURM or similar HPC job schedulers) that executes total-evidence analysis, such as model selection and tree inference using IQ-TREE. * **dna.phy**: A PHYLIP-formatted DNA alignment file used as input for phylogenetic analyses. This file contains aligned sequences of all taxa. * **iqtree2**: The IQ-TREE 2 executable or symbolic link to it. Used for performing model selection and maximum likelihood phylogenetic inference. * **odo_concat.best_model.nex**: A NEXUS-format file listing the best-fitting substitution models selected for each partition in the concatenated dataset. * **odo_concat.best_scheme.nex**: NEXUS version of the partitioning scheme, including data blocks and associated substitution models, formatted for compatibility with downstream programs. * **odo_concat.bionj**: A BioNJ tree, which is a distance-based neighbor-joining tree used as a starting topology for the maximum likelihood search in IQ-TREE. * **odo_concat.ckp.gz**: A compressed checkpoint file generated during the IQ-TREE run. Allows resuming the analysis from the last saved state if interrupted. * **odo_concat.contree**: The consensus tree resulting from the bootstrap analysis. It summarizes the bootstrap replicates to show the most supported branches. * **odo_concat.iqtree**: The main IQ-TREE output file. It includes detailed results such as model selection, log-likelihood scores, branch supports, and partition statistics. * **odo_concat.log**: Log file that records all steps of the IQ-TREE analysis. Useful for tracking progress, runtime, model decisions, and troubleshooting. * **odo_concat.mldist**: Pairwise maximum likelihood distance matrix among all taxa, based on the input alignment and selected model(s). * **odo_concat.model.gz**: A compressed file storing the substitution models and parameters used for each partition during the analysis. * **odo_concat.parstree**: A parsimony-based tree generated as an alternative starting point or for comparison with the ML tree. * **odo_concat.splits.nex**: A splits graph in NEXUS format showing alternative groupings or conflicting signals among taxa; useful for visualizing phylogenetic uncertainty. * **odo_concat.treefile**: The final maximum likelihood tree inferred by IQ-TREE, containing branch lengths and support values. This is the primary output tree. * **odo_concat.ufboot**: Ultrafast bootstrap replicate trees. This file is used to calculate branch support values for the consensus tree. * **partition.nex**: A NEXUS-format partition file defining the start and end positions of each data block for use in partitioned analysis. NOTE: **wings.phy** is added as a partition, using MK+ASC model. * **slurm-3991242.out**: Output file from a SLURM job submission. Includes standard output from the job script, such as command progress, warnings, or errors. * **wings.phy**: A PHYLIP-format alignment file containing only wing-related morphological scores. Used in a targeted phylogenetic analysis. **2_External_Body_Scorings**: Output from Total-Evidence (Morphology + Sequences) Phylogenetic analysis using our concatenated sequence matrix of our pruned species + subset of morphological matrix of just external-body characters. NOTE: all file outputs are the same as **1_Wings_Scorings** * **External_body.phy**: A PHYLIP-format alignment file containing only external body morphological scores. Used in a targeted phylogenetic analysis. * **partition.nex**: A NEXUS-format partition file defining the start and end positions of each data block for use in partitioned analysis. NOTE: **External_body.phy** is added as a partition, using MK+ASC model. **3_External_Genitalic_Scorings**: Output from Total-Evidence (Morphology + Sequences) Phylogenetic analysis using our concatenated sequence matrix of our pruned species + subset of morphological matrix of just external-genitalic characters. NOTE: all file outputs are the same as **1_Wings_Scorings** * **External_genitalic.phy**: A PHYLIP-format alignment file containing only external genitalic-related morphological scores. Used in a targeted phylogenetic analysis. * **partition.nex**: A NEXUS-format partition file defining the start and end positions of each data block for use in partitioned analysis. NOTE: **External_genitalic.phy** is added as a partition, using MK+ASC model. **4_Larval_Scorings**: Output from Total-Evidence (Morphology + Sequences) Phylogenetic analysis using our concatenated sequence matrix of our pruned species + subset of morphological matrix of just nymphal characters. NOTE: all file outputs are the same as **1_Wings_Scorings** * **larval.phy**: A PHYLIP-format alignment file containing only larval-related morphological scores. Used in a targeted phylogenetic analysis. * **partition.nex**: A NEXUS-format partition file defining the start and end positions of each data block for use in partitioned analysis. NOTE: **larval.phy** is added as a partition, using MK+ASC model. **5_Genitalic_Scorings**: Output from Total-Evidence (Morphology + Sequences) Phylogenetic analysis using our concatenated sequence matrix of our pruned species + subset of morphological matrix of just penile characters. NOTE: all file outputs are the same as **1_Wings_Scorings** * **larval.phy**: A PHYLIP-format alignment file containing only penile-related morphological scores. Used in a targeted phylogenetic analysis. * **partition.nex**: A NEXUS-format partition file defining the start and end positions of each data block for use in partitioned analysis. NOTE: **genitalic.phy** is added as a partition, using MK+ASC model. **All_Scores_Analysis**: Output from Total-Evidence (Morphology + Sequences) Phylogenetic analysis using our concatenated sequence matrix of our pruned species + all characters from morphological matrix. NOTE: all file outputs are the same as **1_Wings_Scorings** * **MORPH.phy**: A PHYLIP-format alignment file containing all morphological scores (100). Used in a targeted phylogenetic analysis. * **partition.nex**: A NEXUS-format partition file defining the start and end positions of each data block for use in partitioned analysis. NOTE: **MORPH.phy** is added as a partition, using MK+ASC model. #### **4\_Total\_Evidence\_Analysis** **Morphology_Matrix_All_Taxa.nex**: All species scored (including taxa of 'uncertain placement'), using our full 100 discrete character matrix consisting of characters from wings, external body, external genitalic, penile, and nymphal. Nexus file contains information and character states of each morphological character. Also includes our Total Evidence Phylogeny and pruned ML phylogeny **Total_Evidence_renamed.newick**: Total-evidence phylogenetic analysis including taxa of 'uncertain placement' but scored for morphology. Note **Total_Evidence_renamed.newick** is the same as **odo_concat.contree** from **Total_Analysis_2**, as this tree possessed the highest likelihood (i.e. lowest log likelihood score) **ML_Tree_Renamed.newick**: Maximum-likelihood (ML) tree generated using our pruned iqtree (211 taxa) in newick format. Note **ML_Tree_Renamed.newick** is the same as **odo_concat.contree** from **Pruned_iqtree**, however names have been changed to remove sequencing code (i.e. GEODEXXXX, or RMNH.XXXX), family name (i.e. Synthemistidae) **Consistency_Indices.csv**: A CSV document containing the consistency index, retention index, and rescaled consistency index (CI * RI) of each morphological character using our pruned ML tree * *Character*: Sequential Character Number (i.e. 1, 2, 3) * *Category*: Morphological category based on subset (i.e. Wings, External Body) * *Consistency Index*: Consistency index value for that character * *Retention Index*: Retention index value for that character * *Rescaled Consistency Index*: Rescaled Consisteny Index index value for that character **Rescaled Consistency Index**: R code used to calculate Analysis of Variance (ANOVA) to determine if particular subsets possess statistical significance in taxonomic importance **Total_Analysis_1**: First iteration (run) of Total-evidence phylogenetic analysis including taxa of 'uncertain placement' but scored for morphology. NOTE: all file outputs are the same as **1_Wings_Scorings** * **MORPH.phy**: A PHYLIP-format alignment file containing all morphological scores (100) of both pruned and 'uncertain placement' taxa Used in a targeted phylogenetic analysis. * **partition.nex**: A NEXUS-format partition file defining the start and end positions of each data block for use in partitioned analysis. NOTE: **MORPH.phy** is added as a partition, using MK+ASC model. **Total_Analysis_2**: Second iteration (run) of Total-evidence phylogenetic analysis including taxa of 'uncertain placement' but scored for morphology. NOTE: all file outputs are the same as **Total_Analysis_1**: **Total_Analysis_3**: Third iteration (run) of Total-evidence phylogenetic analysis including taxa of 'uncertain placement' but scored for morphology. NOTE: all file outputs are the same as **Total_Analysis_1**: **Total_Analysis_4**: Fourth iteration (run) of Total-evidence phylogenetic analysis including taxa of 'uncertain placement' but scored for morphology. NOTE: all file outputs are the same as **Total_Analysis_1**: **Total_Analysis_5**: Fifth iteration (run) of Total-evidence phylogenetic analysis including taxa of 'uncertain placement' but scored for morphology. NOTE: all file outputs are the same as **Total_Analysis_1**: #### **5\Time\Divergence\_Analysis** * **2base.t**: Time unit conversion file used in MCMCTree, typically converting between substitution units and real-time units (e.g., million years). * **FcC_smatrix_N.phy**: Input alignment file of AHE sequences used for ML tree (**FcC_smatrix_N.fas** in **Pruned_iqtree**) * **lnf**: Log file recording the log-likelihood values during MCMC sampling. Used to assess convergence and stability of the run. * **mcmctree.ctl**: Control file specifying settings for the MCMCTree run, including priors, clock model, and input/output file names. * **mcmctree.job**: Job script (likely for SLURM or a similar scheduler) used to submit the MCMCTree analysis to a computing cluster. * **No_Fossil.newick**: Input tree not including Somatochlora brisaci, Neophya legrandi, or Paleophya argentina as fossil calibration point, tree is ultrametric where nodes are labeled with fossil ages (Used **ML_Tree_Renamed.newick**) * **out**: The main MCMCTree output file. Contains the posterior means, credibility intervals, and other results for divergence times. * **out.BV**: Branch variance file, containing variances of divergence times along each branch. Useful for assessing uncertainty. * **rst**: A file containing ancestral reconstructions or summary statistics depending on the model settings. May include estimated substitution rates or states. * **rst1**: A first version of the * **rst** file. MCMCTree may output multiple such files during different steps of the process. * **rst2**: A second version of the * **rst** file. Contains additional or alternative reconstructions, depending on the analysis mode. * **rub**: Temporary or intermediate file created during the MCMC run. Often used internally by MCMCTree. * **SeedUsed**: Records the random seed used in the MCMC analysis for reproducibility. * **slurm-4413588.out**: SLURM job output file containing standard output from the MCMCTree job, such as progress updates and error messages. * **tmp0001.ctl**: A temporary control file automatically generated during the MCMCTree run, often for internal checkpointing. * **tmp0001.out**: Output file corresponding to the temporary control file. May include logs or results from a checkpointed phase. * **tmp0001.trees**: Sampled trees from the MCMC posterior, useful for summarizing tree topology and divergence times. * **tmp0001.txt**: Text-formatted version of summary results for easy viewing, often includes posterior means and credibility intervals for node ages. **approx01**: First Run of from MCMCtree analysis * **No_Fossil_divergence.tre**: Dated tree output, with estimated divergence times for nodes, posterior means or medians of node ages, with branch lengths scaled according to these times * **No_Fossil.newick**: Input tree not including Somatochlora brisaci, Neophya legrandi, or Paleophya argentina as fossil calibration point, tree is ultrametric where nodes are labeled with fossil ages (Used **ML_Tree_Renamed.newick**) * **FcC_smatrix_N.phy**: Input alignment file of AHE sequences used for ML tree (**FcC_smatrix_N.fas** in **Pruned_iqtree**) * **in.BV**: Pre-computed branch lengths and their associated variances for the specified tree topology. This information is derived from the maximum likelihood estimation of branch lengths and rate parameters based on the sequence data * **mcmc.txt**: Raw MCMC samples from the posterior distribution * **mcmctree.ctl**: Control file for the MCMCTree analysis, containing the tree topology or starting tree, MCMC parameters (e.g., burn-in, chain length, sample frequency), and priors for divergence times, clock models, and rates * **mcmctree.job**: SLURM job script submitted to the cluster, Contains: Job configuration (e.g., wall time, memory, CPUs), and the command to run MCMCTree. * **out**: main output log summarizing the MCMC run, includes details on priors, posterior estimates, acceptance rates, and summary statistics (means, medians, 95% HPDs for parameters like node ages) * **SeedUsed**: A log of the random seed used for the analysis * **slurm-4413775.out**: Standard output logs from SLURM, the job scheduler, contains messages from the MCMCTree run including command-line arguments, progress information (e.g., burn-in completion, sampling progress), and potential errors or warnings **approx02**: Second Run of from MCMCtree analysis. NOTE, same output files as **approx01**