DOI: 10.25349/d9fc8d
title: Data for: A morphological basis for path-dependent evolution of visual systems

description: Path dependence influences macroevolutionary predictability by
 constraining potential outcomes after stochastic evolutionary events.
 Although demonstrated in laboratory experiments, the basis of path
 dependence is difficult to demonstrate in natural systems because of a
 lack of independent replicates. Here we show two types of complex
 distributed visual systems, each recently evolved twice within chiton
 mollusks, demonstrating rapid and path-dependent evolution. The type of
 visual system a chiton lineage evolves is constrained by the number of
 openings for sensory nerves in its shell plates: lineages with more
 openings evolve visual systems with thousands of eyespots, whereas those
 with fewer evolve visual systems with hundreds of shell eyes. These
 macroevolutionary outcomes shaped by path dependence are both
 deterministic and stochastic because possibilities are restricted yet not
 entirely predictable.
description: 1.1 Taxon sampling and sequencing To
 generate sequencing data from specimens that were not preserved in ideal
 conditions and/or are more than five years post-collection (including a
 large library of previously sequenced chiton specimens from the personal
 collections of D. J. Eernisse as part of ongoing systematic revisions), we
 generated a suite of target capture loci. We designed target capture baits
 to predict exon regions following a similar approach to Hugall et al.,
 2016. We identified a set of 891 orthologs from 19 chiton transcriptomes
 and three reference molluscan genomes (Octopus bimaculoides, Lottia
 gigantea, and Crassostrea gigas) using the phylogenomics pipeline from
 Kocot et al., 2017. We identified conserved exons from the three mollusc
 genomes using EnsemblMetazoa and predicted intron-exon boundaries in the
 chiton sequences with a custom script using tBLASTx. From this, we
 compiled a set of predicted chiton exons from 355 genes that had at least
 four representative chiton sequences from our transcriptome dataset. Arbor
 Biosciences synthesized 19,980 biotinylated DNA baits that target these
 regions. The baits have a length of 120 bp and a GC content between
 35-55%. We used the MYbaits Custom DNA-Seq kit v. 3.02
 (Arbor Biosciences, Ann Arbor, Michigan, USA) according to the
 manufacturer’s instructions. We applied our target capture baits to DNA
 extracted from chiton specimens preserved in ethanol. We extracted total
 DNA from 87 specimens of preserved chi-tons with a phenol-chloroform
 extraction. We then produced Illumina sequencing libraries with unique
 barcodes using the NEBNext® Ultra™ II FS Library Prep Kit (New England
 Biolabs, Ipswich, Massachusetts, USA) with a 25-minute incubation at the
 fragmentation step. We used TapeStation (Agilent, Santa Clara, California,
 USA) automated electrophoresis to confirm that the libraries had an
 average fragment size of 250 bp with adapters and that adapter dimers were
 absent. We incubated these libraries with biotinylated DNA baits via the
 MYbaits Custom DNA-Seq kit, and we pulled down the baits that hybridized
 with the Illumina library fragments with a streptavidin-coated magnetic
 bead binding process before washing away the uncaptured library fragments.
 We amplified the captured fragments with the KAPA HiFi Library Amp Kit
 (Roche Molecular Systems, Inc., Wilmington, Massachusetts, USA). After the
 amplification, we confirmed that the libraries maintained an average
 fragment size of 250 bp and that adapter dimers were absent. We pooled
 enriched sequencing libraries and sequenced them on either an Illumina
 MiSeq (11 species) or NextSeq (68 species). We trimmed
 Illumina adapters and low-quality sequences (quality score of ¡20) using
 TrimGalore v0.4.1 (54). We used HybPiper (55) to map reads to reference
 sequences. To expand our taxon sampling further, we combined target
 capture data with chiton transcriptomes from recent phylogenies of chitons
 that included chitons with only aesthetes, chitons with eyespots, and
 chitons with shell eyes (56). We also included transcriptomes of several
 aplacophoran mollusks and a few conchiferan mollusks to root the phylogeny
 (Accession numbers in Supplementary Material, Outgroup and Available
 Sequences). To recover our target capture loci from chiton transcriptomes,
 we aligned the sequencing results from each target capture locus across
 chitons and determined the consensus DNA sequence for each. Then, we
 created a local blast database of each new chiton transcriptome and used
 each consensus DNA sequence from a target-capture locus as a query to
 locate the corresponding sequence within the new transcriptome. For each
 gene that we included, we retrieved only a single hit at E-value threshold
 1e-4, so we are confident in our gene sequence determinations.
 1.2 Phylogeny To generate a robust phylogeny
 of chitons, we compared coalescence and concatenation-based approaches
 using Maximum Likelihood and Bayesian approaches. We first aligned all
 loci with MAFFT v7.305 (58) to produce a single data matrix of 103 chitons
 (see Table S1) that could be partitioned by locus. We produced a maximum
 likelihood tree in IQ-TREE2 (57), partitioning by gene and using
 ModelFinder to determine the best model for each locus. We also calculated
 gene concordance factors in IQ-TREE2. Separately, we produced a
 maximum-likelihood phylogeny for each gene individually with IQ-TREE2,
 again using ModelFinder, and input these trees into ASTRAL-Pro (41), which
 estimates a species tree from multiple gene trees via the multispecies
 coalescence (MSC) model and a quartet-based approach.
 The placement of Schizochiton has been uncertain across studies
 of chitons, and our study is limited by the inclusion of only a single
 representative of the genus Schizochiton, S. incisus. Specimens of the
 only other accepted congeneric species, S. jousseaumei (Dupuis, 1917),
 were not available, and this is the only genus in the superfamily,
 Schizochitonoidea. Some morphological characters, most notably pectinated
 insertion plates and adanal gills, suggest an affinity of Schizochiton
 with Chitonidae. However, differences of opinion persist as to whether
 these traits are useful apomorphies. For example, the insertion plates of
 Schizochiton were once termed “obsoletely pectinated” (59), although they
 have strong pectination comparable to members of Chitonidae (DJE, pers.
 observation). Additionally, the caudal sinus is absent in Chitonidae but
 present in Schizochiton (65). However, caudal sinuses evolved multiple
 times across chitons, so the presence or absence of a sinus may not be a
 useful morphological character. Schizochiton has been recovered as sister
 to the remaining Chitonina, but with weak support (36). Our placement of
 Schizochiton requires that pectinated insertion plates evolved
 convergently in Schizochiton and Chitonidae. To further investigate the
 placement of Schizochiton in our phylogeny, we used an iterative pruning
 method to verify that Schizochiton was not an unstable branch (90) and
 determined that Schizochiton did not have any indicators of being a long
 branch or a highly unstable branch (Supplemental Data,
 RogueNaRok). 1.3 Ancestral state reconstruction and
 morphospace determination To determine how many times
 distributed visual systems evolved across the chiton phylogeny, we
 performed ancestral state reconstruction using RayDISC and corHMM in R,
 which takes a tree file and a corresponding character matrix for a given
 trait and estimates transition rates and ancestral states for binary or
 multistate traits (Beaulieu and O’Meara 2014). We used the consensus tree
 file from our Mr-Bayes dated phylogeny run with 15 loci from sortadate as
 our input tree, and inferred ancestral states for shell eyes, eyespots,
 and the maximum number of insertion slits on the anterior shell plate. We
 gathered information on slit number from direct observations when
 possible, then from recent species revisions, and finally from from
 volumes of Kaas’s monographs (1985-2006). This was critical to accuracy,
 as some synonymized species in Kaas have dramatically different slit
 numbers (and often different visual systems). We took the highest recorded
 slit number for the anterior shell plate to account for the impact of
 animal size on slit number, where slit number can increase across growth.
 Whenever possible, we removed anterior shell plates from the specimens
 included on the tree and counted slits manually.
description: # Data for: A morphological basis for path-dependent evolution of visual
 systems --- This supplementary data contains the raw input/output files
 for all phylogenies described in the manuscript, as well as the raw image
 files for the morphological measurements taken. ## Description of the data
 and file structure This supplementary data is divided into the following
 directories: * `SEMimages`: Raw scanning electron micrographs of all taxa
 included in the phylomorphospace. We took multiple images for each taxon.
 Within this folder, examples of the counted macraesthetes can be viewed in
 the subfolder `CountedExamples`. Numbers in image titles correspond to the
 museum catalog of the Santa Barbara Museum of Natural History. * `MLtree`:
 The input and output files that resulted in our maximum likelihood
 phylogeny of chitons via iqtree2, using Model Finder on a partitioned
 dataset. * `TimeTree`: The input and output files that resulted in our
 time-calibrated phylogeny of chitons via MrBayes. This analysis is run on
 the 15 best genes as selected via sortadate. Split deviations were
 &lt;0.025 when the chains were stopped. * `OtherScripts`: Short,
 additional scripts that we used in this analysis and that may be useful to
 others in using our data. * `ChitonLoci`: Consensus sequences for all the
 loci used in this analysis, to provide an easy way to mine new chiton
 sequences and add them to our phylogeny. Within this folder, the
 `ConsensusAAsequences` folder contains the consensus amino acid sequence
 for each bait (useful for mining loci from additional species), and the
 `EachLocus_AllChitons` folder contains a file for each locus with the
 sequences for all chitons in the analysis. * `Baits`: A fasta file
 containing the sequences of the probes designed to pull down chiton genes.
 ## Sharing/Access information NA ## Code/Software IQ-TREE 2 2.0.3
 ASTRAL-PRO 1.0 RogueNaRok Accessed May 2022 SortaDate 1.0 MrBayes 3.2.6
 Deconcat.py reads in a .txt file that corresponds with the partition file
 used with iqtree2, as well as a concatenated matrix file (.fas; typically
 the result of an alignment and subsequent concatenation).