Screening of a deeply sequenced transcriptome using Illumina sequencing as well as the genome of the tardigrade Hypsibius exemplaris (referred to as Hypsibius dujardini in the published article) revealed a set of five opsin genes.To clarify the phylogenetic position of these genes and to elucidate the evolutionary history of opsins in Panarthropoda (Onychophora +Tardigrada+Arthropoda), we reconstructed the phylogeny of broadly sampled metazoan opsin genes using maximum likelihood and Bayesian inference methods in conjunction with carefully selected substitution models. According to our findings, the opsin repertoire of H. exemplaris comprises representatives of all three major bilaterian opsin clades, including one r-opsin, three c-opsins, and a Group 4 opsin (neuropsin/opsin-5). The identification of the tardigrade ortholog of neuropsin/opsin-5 is the first record of this opsin type in a protostome,but our screening of available metazoan genomes revealed that it is also present in other protostomes. Our opsin phylogeny further suggests that two r-opsins, including an “arthropsin”, were present in the last common ancestor of Panarthropoda. Although both r-opsin lineages were retained in Onychophora and Arthropoda, the arthropsin was lost in Tardigrada. The single (most likely visual) r-opsin found in H. exemplaris supports the hypothesis of monochromatic vision in the panarthropod ancestor, whereas two duplications of the ancestral panarthropod c-opsin have led to three c-opsins in tardigrades. Although the early-branching nodes are unstable within the metazoans, our findings suggest that the last common ancestor of Bilateria possessed six opsins: Two r-opsins, one c-opsin, and three Group 4 opsins, one of which (Go opsin) was lost in the ecdysozoan lineage.

Specimens of Hypsibius exemplaris (Gąsiorek, 2018) (Eutardigrada, Hypsibiidae) were obtained commercially from Sciento (Manchester, UK). Several hundred specimens were used to extract total RNA using TRIzol reagent (Invitrogen, Carlsbad, CA) and RNeasy MinElute Cleanup Kit (Qiagen, Hilden, Germany) according to the manufacturers’ protocols. Library preparation for double indexing (Meyer and Kircher 2010; Kircher et al. 2012), 76 cycles paired-end sequencing on an Illumina Genome Analyzer IIx (San Diego, CA), and postsequencing processing (adapter trimming, removal of reads with falsely paired indices and filtering of reads at three different levels of stringency; Filter15: reads with more than five bases below a phred quality score of 15 were removed; Filter30: reads with less than 20 bases in a row with a phred quality score of at least 30 were removed; Filter25: same as Filter30 but the threshold value was set to 25 to keep as many high-quality reads as possible) were performed as described by Hering et al. (2012). Each of the three obtained data sets (Filter15, Filter25, and Filter30) was then assembled de novo using two different software packages to assess the occurrence of opsin transcripts in a broader methodological framework: CLC Genomics Workbench v5.1 (CLC bio, Århus, Denmark), and IDBA-Tran v1.1.0 (Peng et al. 2013). The IDBA-Tran assemblies were done twice, allowing for the retention of one or up to three isoforms of a transcript, respectively, using the --max_isoforms option (for details see statistics_and_BUSCO_values.xls).

An explanation of each file is given in the README.txt file.