DOI: 10.5061/dryad.mcvdnck6b
title: Fossil-calibrated inference of divergence times among the Volvocine algae enables reconstruction of the steps that led to differentiated multicellularity

description: Throughout its nearly four-billion-year history, life has undergone
 evolutionary transitions in which simpler subunits have become integrated
 to form a more complex whole. Many of these transitions opened the door to
 innovations that resulted in increased biodiversity and/or organismal
 efficiency. The evolution of multicellularity from unicellular forms
 represents one such transition, one that paved the way for cellular
 differentiation, including differentiation of male and female gametes. A
 useful model for studying the evolution of multicellularity and cellular
 differentiation is the volvocine algae, a clade of freshwater green algae
 whose members range from unicellular to colonial, from undifferentiated to
 completely differentiated, and whose gamete types can be isogamous,
 anisogamous, or oogamous. To better understand how multicellularity,
 differentiation, and gametes evolved in this group, we used comparative
 genomics and fossil data to establish a geologically calibrated roadmap of
 when these innovations occurred. Our results, presented as ancestral-state
 reconstructions, show that multicellularity arose independently twice in
 this clade. Our chronograms indicate multicellularity evolved during the
 Carboniferous-Triassic periods in Goniaceae + Volvocaceae, and possibly as
 early as the Cretaceous in Tetrabaenaceae. Using divergence time estimates
 we inferred when, and in what order, specific developmental changes
 occurred that led to differentiated multicellularity and oogamy. We find
 that in the volvocine algae the temporal sequence of developmental changes
 leading to differentiated multicellularity is much as proposed by David
 Kirk, and that multicellularity is correlated with the acquisition of
 anisogamy and oogamy. Lastly, morphological, molecular, and divergence
 time data suggest the possibility of cryptic species in Tetrabaenaceae.
description: # Fossil-calibrated inference of divergence times among the Volvocine
 algae enables reconstruction of the steps that led to differentiated
 multicellularity This README is for: "Fossil-calibrated molecular
 clock enables reconstruction of steps leading to differentiated
 multicellularity in the Volvocine algae" by CR Lindsey, AH Knoll, MD
 Herron, and F Rosenzweig. DOI
 = [https://doi.org/10.1186/s12915-024-01878-1](https://doi.org/10.1186/s12915-024-01878-1) The contents of this file include the following: 1. **phytools_r_scripts.zip:** R scripts used to run simmap analyses in Phytools Specifically, these are the R scripts used to generate simmap analyses for Kirk's 12 developmental traits, certain sexual traits, and misc. traits in the Volvocine algae. 2. **MBASR_run_script_template:** MBASR template script used to run all the ancestral state reconstructions in MBASR 3. **gene_alns_263.zip)**: All 263 alignment files used in our maximum likelihood and Mr. Bayes phylogenetic analyses . 4. **phylobayes_&lt;8/16&gt;gene_input_files.zip:** 8-gene and 16-gene alignment files used in our Phylobayes molecular clock analyses. Fossil calibration include the specific node calibrations for our molecular clock analyses. Each zip file contains the following: **archaeplastida_fossil_calibrations_mins_and_ranges.txt**: fossil calibration files **ds&lt;1/5&gt;*gshop_trimalv7v3_042723*&lt;8/16&gt;genes_**: a gene alignment file  **nwk_trimalv7v3_042723.tre**: newick tree formatted file **outgroup.txt**: outgroup taxa file 5. **IQtree_files.zip:** Concatenated 263-gene alignment files used in our IQtree and Mr. Bayes phylogenetic analyses. Contains the concatenated gene alignment **IQtree_aln_file.fa** and character partition files **IQtree_charpartition.nex**. MrBayes input is **mrbayes_file_263genes.nex** 6. **astral_263genetrees_file.tre:** ASTRAL 263-gene tree file . This is file has all of our single-gene phylogenies concatenated into one file. All single-gene trees (263 total) have been individually uploaded in the following ZIP file: **single_gene_trees_trimalv7v3_042723.zip**. #### Usage Notes A) .tre files may be viewed but not limited to the following software: Figtree or TreeGraph2. B) alns = alignments C) FASTA (.fa) and NEXUS (.nex) files may be viewed using any text editor such as Sublime Text. D) MBASR template script is a text file used with the program MBASR ([https://www.biorxiv.org/content/10.1101/2021.01.10.426107v1.full.pdf)](https://www.biorxiv.org/content/10.1101/2021.01.10.426107v1.full.pdf\)) E) "CB" and "ML" acronyms are used in filenames to specify which phylogenetic analysis the tree branching order belongs. "CB" and "ML" correspond to coalescent-based and maximum-likelihood, respectively. Additional files uploaded to Zenodo: 7\. **phylobayes_fossil_calibrations_and_chronograms.zip:** Phylobayes outputs of the following items for each clock model from 16-gene analyses: chronograms, dates, and labels 8\. **command_line_arguments.pdf:** Command line PDF with software repo links.