DOI: 10.5061/dryad.9cnp5hqpk
title: Nematostella vectensis full length opsin sequences and transcriptomes

description: Opsins are the primary proteins responsible for light detection in
 animals. Cnidarians (jellyfish, sea anemones, corals) have diverse visual
 systems that have evolved in parallel with bilaterians (squid, flies,
 fish) for hundreds of millions of years. Medusozoans (e.g. jellyfish,
 hydroids) have evolved eyes multiple times, each time independently
 incorporating distinct opsin orthologs. Anthozoans (e.g. corals, sea
 anemones,) have diverse light-mediated behaviors and, despite being
 eyeless, exhibit more extensive opsin duplications than medusozoans. To
 better understand the evolution of photosensitivity in animals without
 eyes we increased anthozoan representation in the phylogeny of animal
 opsins and investigated the large but poorly characterized opsin family in
 the sea anemone Nematostella vectensis. We analyzed genomic and
 transcriptomic data from 16 species of cnidarians to generate a large
 opsin phylogeny (708 sequences) with the largest sampling of anthozoan
 sequences to date. We identified 29 opsins from N. vectensis (NvOpsins)
 with high confidence, using transcriptomic and genomic datasets. We found
 that lineage-specific opsin duplications are common across Cnidaria, with
 anthozoan lineages exhibiting among the highest numbers of opsins in
 animals. To establish the putative photosensory function of NvOpsins, we
 identified canonically conserved protein domains and amino acid sequences
 essential for opsin function in other animal species. We show high
 sequence diversity among NvOpsins at sites important for photoreception
 and transduction, suggesting potentially diverse functions. We further
 examined the spatiotemporal expression of NvOpsins and found both dynamic
 expression of opsins during embryonic development and sexually dimorphic
 opsin expression in adults. These data show that lineage-specific
 duplication and divergence have led to expansive diversity of opsins in
 eyeless cnidarians, suggesting opsins from these animals may exhibit novel
 biochemical functions. The variable expression patterns of opsins
 in N. vectensis suggest opsin gene duplications allowed for a
 radiation of unique sensory cell types with tissue- and stage-specific
 functions. This diffuse network of distinct sensory cell types could be an
 adaptive solution for varied sensory tasks experienced in distinct life
 history stages in anthozoans.
description: <strong>Opsin Identification
 and Phylogenetics (References numbered as in the
 paper)</strong> Initial <em>N. vectensis
 </em>opsin sequences were collected from three published datasets
 [5,6,47]. All potential <em>N. vectensis </em>sequences were
 aligned in Geneious Prime v.11.0.12, and identical matches and fragments
 were discarded. BLAST was used with the final set of opsins as bait to
 search against the <em>Nematostella vectensis</em>
 Embryogenesis and Regeneration Transcriptomics database (NvERTx) [67], our
 own reference transcriptome (generated for this study), and two publicly
 available chromosome level genomes: v2 genome hosted by the Stowers
 Institute [50,71], and the genome from the Darwin Tree of Life Programme
 at the Wellcome Sanger Institute (ENA submission accession: ERA9667479)
 ([51] no associated publication). Hits were added inclusively and combined
 with a modified alignment including non-redundant sequences from Vöcking
 et al. 2017, Picciani et al. 2018, and Gornik et al. 2021 opsin
 phylogenies [5,6,29]. We then added opsin sequences from new anthozoan
 transcriptome and genome data. We added opsins from 12 species with new
 transcriptomes, and 3 species from new genomic data (Additional File 3:
 Table S1). This was done using <em>N. vectensis </em>and
 <em>Acropora digitifera </em>sequences from ASOI, ASOII, and
 Cnidops groups as bait for BLAST searches. The top 100 BLAST hits were
 initially conservatively kept. All sequences were aligned in Geneious
 using MAFFT v7.450 with default settings [72]. FastTree was used in
 Geneious to check the tree, removing unannotated outgroups from the
 multiple cnidarian and <em>N. vectensis </em>BLAST searches.
 Trimmed (using TrimAl) and untrimmed alignments were generated, but
 untrimmed sequences generated the best-supported phylogeny and are
 reported here. Final maximum likelihood gene trees
 were constructed using IQtree2 with the following command: iqtree2 -s
 &lt;alignment.phy&gt; -st AA -nt AUTO -v -m TEST -bb 1000 -alrt
 1000 [73] on the Minnesota Supercomputing Institute’s (MSI) High
 Performance Computing system at the University of Minnesota. The LG+G4
 model of protein evolution was auto-calculated from this command, and 1000
 ultrafast bootstrap (UFboot) replicates and SH-aLRT tests were used for
 evidence of support. Clades were considered supported only with
 &gt;80% SH-aLRT/&gt;95% UFboot support. The tree was rooted based
 on known outgroups from previous
 phylogenies.<strong> </strong> Genomic and
 opsin sequence analysis To identify distinct opsin paralogs,
 we used BLAST with transcripts as bait to identify genomic loci, resulting
 in the identification of 29 opsin genes. We manually identified
 intron/exon boundaries at all cnidopsins with introns by aligning coding
 sequences to the genome and translated these to identify their location
 and phase on the protein. We then aligned the cnidopsin amino acid
 sequences with a xenopsin and bovine c-opsin in Geneious and mapped exon
 boundaries manually onto the alignment. Opsin genomic coordinates for both
 new Nvec genomes [51,74], and previous Nvecv1 scaffold coordinates [75]
 are found in Additional File 3: Table S2. We used InterPro scan in
 Geneious to predict the seven transmembrane domains. Using bovine
 rhodopsin as a reference, we aligned our 29 opsins, <em>Drosophila
 </em>r-opsin and <em>T. cystophora </em>cnidopsin
 sequences to identify residues at canonically conserved
 positions. A few tandem loci next
 to <em>NvASOII-8a, NvASOII-8b, NvASOII-9a, NvASOII9b, NvASOII-7,
 </em>and<em> NvASOII-4</em> genes were found in the  v2
 genome [50] whose nucleotide sequences were nearly identical (&gt;97%
 similarity). We noticed adjacent non-coding intra- and intergenic regions
 were also near-identical, suggesting these may be haplotype differences
 and not real paralogs. One of these adjacent pairs also was split by a
 section of Ns in the genome (Additional File 4: Fig. S3), an indicator of
 difficulty with assembly in this region. We compared these trouble spots
 with the Wellcome-Sanger genome [51] and for each, found only one of the
 two near-identical pairs, further evidence that these copies are a result
 of assembly error, while other high-similarity but distinct loci were left
 in our final opsin count. RNA-sequencing and
 analysis Libraries were generated from two
 independent spawns each at blastula, gastrula, planula, primary polyp
 stages, two adult males and two adult females. Adult animals had been kept
 in normal laboratory conditions at 18°C with a 12-hour light/12-hour dark
 photoperiod and at least one week since they were last spawned. Live
 animals were placed directly into Trizol reagent and tissue was
 homogenized in a microcentrifuge tube with a pestle before performing RNA
 extraction.
 Total RNA was
 extracted from Trizol (Invitrogen), gDNA removed by gDNA cleanup kit
 (Qiagen), RNA was precipitated using isopropanol and then cleaned up using
 ethanol precipitation. Libraries were prepared using Kapa Stranded RNA
 Hyperprep (Illumina), quality control and size selection were performed by
 the Bauer Core at Harvard University and paired end, 150bp reads were
 sequenced on an Ilumina NovaSeq. Raw
 sequencing libraries were processed for erroneous k-mers and unfixable
 reads using rCorrector [76]. Adapter sequences were trimmed using Trim
 Galore (Babraham Bioniformatics, UK), and rRNA reads were mapped to
 the <em>N. vectensis </em>mitochondrial genome and removed
 using Bowtie2 [77]. All libraries were combined to generate a single
 <em>de novo</em> transcriptome assembly using Trinity v2.12.0
 [78]. Transcriptome quality was assessed by checking alignment statistics
 and quantifying BUSCO completeness [79]. We used EvidentialGene to combine
 gene and transcript evidence from multiple sources and reduce the number
 of genes in our transcriptome [80]. We used our own transcripts, and three
 other assemblies [50,67,81], and the coding sequences from v2 genome [50],
 as input. We used the EvidentialGene output for our final transcriptome.
 mRNA quantification was done using Kallisto for each of our 12 libraries
 using our <em>de novo </em>assembled transcriptome [82].
 Stages were compared using the time course analysis in Sleuth run in R,
 and adult libraries were additionally pairwise compared using Sleuth [83].
 The data for heatmaps were extracted from the Sleuth object using
 normalized transcripts per million (TPM) in either the timecourse analysis
 or male v. female comparison. Heatmaps were made with the heatmap command
 in gplots package [84] using R v4.2.3 run in RStudio v2022.12.0+353
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