DOI: 10.5061/dryad.m905qfv6q
title: Data from: Vestigial organs alter fossil placements in an ancient group of terrestrial chelicerates

description: Vestigial organs provide a link between ancient and modern traits, and
 therefore have great potential to resolve the phylogeny of contentious
 fossils that bear features not seen in extant species. Here we show that
 extant daddy-longlegs (Arachnida, Opiliones), a group once thought to
 possess only one pair of eyes, in fact additionally retain a pair of
 vestigial median eyes and a pair of vestigial lateral eyes.
 Neuroanatomical gene expression surveys of eye-patterning transcription
 factors, opsins, and other structural proteins in the daddy-longlegs
 Phalangium opilio show that the vestigial median and lateral eyes
 innervate regions of the brain positionally homologous to the median and
 lateral eye neuropils, respectively, of chelicerate groups like spiders
 and horseshoe crabs. Gene silencing of eyes absent shows that the
 vestigial eyes are under the control of the retinal determination gene
 network. Gene silencing of dachshund disrupted the lateral eyes but not
 the median eyes, paralleling loss-of-function phenotypes in insect models.
 The existence of lateral eyes in extant daddy-longlegs bears upon the
 placement of the oldest harvestmen fossils, a putative stem-group that
 possessed both a pair of median eyes and a pair of lateral eyes.
 Phylogenetic analysis of harvestman relationships with an updated
 understanding of lateral eye incidence resolved the four-eyed fossil group
 as a member of an extant daddy-longlegs suborder, which in turn resulted
 in older estimated ages of harvestman diversification. This work
 underscores that developmental vestiges in extant taxa can influence our
 understanding of character evolution, placement of fossils, and inference
 of divergence times.
description: czi. files of confocal stacks of whole mount HCR in situ
 hybridization, multichannel. Taken on a Zeiss Confocal
 780.
description: <strong>Supporting Text. Relates to STAR
 Methods.</strong> Additional descriptions of the
 results and discussion. This supporting text is cited in the main
 manuscript as "Supporting text in [DOI].
 <strong>Data</strong><strong> S1 (available in
 Dryad). Relates to STAR Methods.</strong>
 Spreadsheets containing (1) the list of genes used in the HCR in
 situ hybridization experiments (Data S1, Table S1); (2) the list of
 species and accession numbers of arachnid terminals included in the opsin
 phylogenetic analysis with metazoan opsins (Data S1, Table S2); (3) the
 primer pairs and two sequenced plasmids with incorporated cloned fragments
 of <em>Po-eya</em> used in the RNAi experiments (Data S1,
 Table S3); (4) fossils and age estimates implemented for node dating in
 PhyloBayes, with ages are in million years (Myr) (Data S1, Table S4); (5)
 the reference sequences and probes sequences used in the HCR experiments
 (Data S1, Table S5). <strong>Data
 S2</strong> <strong>(available in Dryad). Relates to STAR
 Methods.</strong> Raw .czi files of the confocal
 scans presented in the paper. Files are named according to the figure
 numbers where they appear.
 <strong>Data</strong><strong> S3 (available in
 Dryad). Relates to STAR Methods.</strong> Zipped
 Folder containing raw data files of the phylogenetic analyses for the
 ground plan coding matrix, original matrix, and strict matrix, and dating
 analyses. The folders contain the morphological matrices (.nex files),
 alignments, molecular data analyses, and the description of morphological
 characters. Folder information is in the Readme.txt file
 enclosed. Detailed instructions for the phylogenetic
 and dating analysis are available in the STAR Methods.
description: # Vestigial organs alter fossil placements in an ancient group of
 terrestrial chelicerates Supporting Text: Additional descriptions of the
 results and discussion. This supporting text is cited in the main
 manuscript as "Supporting text in [DOI]. \**Dataset S1 (refers to
 STAR Methods): **two spreadsheets (Table S1-S4 as four sheets; and Table
 S5) containing: (1) Data S1, Table S1: the list of accession numbers of
 the genes used in the HCR in situ hybridization experiments. (2) Data S1,
 Table S2: the list of species and accession numbers of arachnid terminals
 included in the opsin phylogenetic analysis with metazoan opsins. (3) Data
 S1, Table S3: the primer pairs and two sequenced plasmids with
 incorporated cloned fragments of Po-eya used in the RNAi experiments. (4)
 Data S1, Table S4: fossils and age estimates implemented for node dating
 in PhyloBayes, with ages are in million years (Myr). (5) Data S1, Table S5
 (separate spreadsheet, containing several sheets): the reference sequences
 and probes sequences used in the HCR experiments. \**Dataset S2: **Raw
 (.czi) files of all the confocal scans presented in the paper. Files are
 named according to the figure numbers where they appear. For instance:
 Figure1c_HCR15_tube4_Ptep_rh1_647_rh2_488_peropsin_546_embryo1.czi This is
 the complete Z-stack of the image used in Figure 1C. HCR15_tube4 indicates
 the original naming of the experiment (not reported in the paper). Ptep is
 the code for Parasteatoda tepidariorum (species code). Next, the gene and
 channel used to image are indicated. Ex: gene rh1, channel 647. We note
 that the naming reflects the original file name used by the authors. For
 orientation, please use the reference to the figures in the paper and
 information reported in the paper. In case of any doubt, please the
 corresponding author. I hope this will be a useful resource. Other
 relevant names: Popi: Phalangium opilio Cscu: Centruroides sculpturatus
 Ipus: Iporangaia pustulosa **Dataset S3:** Zipped Folder containing raw
 data files of the phylogenetic analyses for the groundplan coding matrix,
 original matrix, and strict matrix, and dating analyses. The folders
 contain the morphological matrices (.nex files), alignments, molecular
 data analyses, and the description of morphological characters. ├──
 Maximum likelihood: Contains all the data for the total evidence analyses
 │   ├── 1_Datasets for IQTREE: Contains the molecular matrix and the
 morphological matrices for each coding scheme. │   │   ├──
 Molecules78.phy: Molecular matrix │   │   ├──
 Morphology2023_groundplan.nex: Groundplan coding │   │   ├──
 Morphology2023_groundplan_CyphoEyes.nex: Groundplan coding with
 Cyphophthalmi median eyes coded as "Absent" │   │   ├──
 Morphology2023_original.nex: Original coding │   │   ├──
 Morphology2023_strict.nex: Strict coding │   │   └──
 Morphology2023_strict_CyphoEyes.nex: Strict coding with Cyphophthalmi
 median eyes coded as "Absent" │   │   └──
 Morphology2023_strict_polystate.nex: New coding for the lateral eye
 reduction added, see Supplementary Results and Discussion. │   ├──
 2_Command files for total evidence ML searches with IQTREE │   │   ├──
 TE.Opis2023_groundplan.txt │   │   ├──
 TE.Opis2023_groundplan_polystate.txt: polystate analysis refers to the
 matrix with the new lateral eye reduction character, see Supplementary
 Results and Discussion. │   │   ├── TE.Opis2023_original.txt │   │   └──
 TE.Opis2023_strict.txt │   │   └── TE.Opis2023_strict_polystate.txt:
 polystate analysis refers to the matrix with the new lateral eye reduction
 character, see Supplementary Results and Discussion. │   └── 3_Tree files
 from IQTREE │   ├── Molecules only tree │   │   ├── Opi78genes.log │   │  
 └── Opi78genes.tree │   ├── Morphology only tree │   │   ├──
 GroundplanMorphology.tree │   │   ├── OriginalMorphology.tree │   │   └──
 StrictMorphology.tree │   └── Total evidence trees │   ├──
 Groundplan.bionj │   ├── Groundplan.ckp.gz │   ├── Groundplan.contree │  
 ├── Groundplan.iqtree │   ├── Groundplan.log │   ├── Groundplan.mldist │  
 ├── Groundplan.parstree │   ├── Groundplan.splits.nex │   ├──
 Groundplan.tree │   ├── Groundplan.tree.pdf │   ├──
 Groundplan_CyphoEyes.bionj │   ├── Groundplan_CyphoEyes.ckp.gz │   ├──
 Groundplan_CyphoEyes.contree │   ├── Groundplan_CyphoEyes.iqtree │   ├──
 Groundplan_CyphoEyes.log │   ├── Groundplan_CyphoEyes.mldist │   ├──
 Groundplan_CyphoEyes.parstree │   ├── Groundplan_CyphoEyes.splits.nex │  
 ├── Groundplan_CyphoEyes.tree │   ├── Groundplan_polystate.bionj │   ├──
 Groundplan_polystate.ckp.gz │   ├── Groundplan_polystate.contree │   ├──
 Groundplan_polystate.iqtree │   ├── Groundplan_polystate.log │   ├──
 Groundplan_polystate.mldist │   ├── Groundplan_polystate.parstree │   ├──
 Groundplan_polystate.splits.nex │   ├── Groundplan_polystate.tree │   ├──
 Original.tree.pdf │   ├── Strict.bionj │   ├── Strict.ckp.gz │   ├──
 Strict.contree │   ├── Strict.iqtree │   ├── Strict.log │   ├──
 Strict.mldist │   ├── Strict.parstree │   ├── Strict.splits.nex │   ├──
 Strict.tree │   ├── Strict.tree.pdf │   ├── Strict_CyphoEyes.bionj │   ├──
 Strict_CyphoEyes.ckp.gz │   ├── Strict_CyphoEyes.contree │   ├──
 Strict_CyphoEyes.iqtree │   ├── Strict_CyphoEyes.log │   ├──
 Strict_CyphoEyes.mldist │   ├── Strict_CyphoEyes.parstree │   ├──
 Strict_CyphoEyes.splits.nex │   └── Strict_CyphoEyes.tree │   ├──
 Strict_polystate.bionj │   ├── Strict_polystate.ckp.gz │   ├──
 Strict_polystate.contree │   ├── Strict_polystate.iqtree │   ├──
 Strict_polystate.log │   ├── Strict_polystate.mldist │   ├──
 Strict_polystate.parstree │   ├── Strict_polystate.splits.nex │   └──
 Strict_polystate.tree └── Node dating with PhyloBayes: Contains all the
 data for the Dating analyses ├── 1_Dataset │   └── Molecules78.phy ├──
 2_Calibration data │   ├── calib_new.txt │   └── calib_original.txt ├──
 3_Outgroup │   └── Ar.outgroup.txt ├── 4_Constraint tree │   └──
 Opi78.tree └── 5_Results ├── Dated.New_calibrations.tre └──
 Dated.Original_calibrations.tre