Dataset: 18s DNA sequence of potential prey of siphonophores

The methods below are adapted from Damian-Serrano, Hetherington et al. (2022).

We built an 18S gene barcoding database of potential siphonophore prey items to expand on the available reference sequences in public databases. To do this, we collected 60 specimens of 30 species of zooplankton and micronekton from the California Current using a Tucker trawl. We targeted plausible prey species from motile open-ocean taxa that cohabitate with siphonophores and are underrepresented in SILVA databases (high quality ribosomal RNA databases, arb-silva.de), including fishes, crustaceans, jellyfishes, urochordates, chaetognaths, polychaetes, and mollusks. Specimens were photographed alive, then tissue was sampled and frozen, and finally the rest of the animal was fixed in formalin as a voucher to be identified and preserved at the Yale Peabody Museum of Natural History. 

DNA extraction, quality control, PCR, and amplicon cleanup was carried out in a similar fashion as the metabarcoding protocol described above (and detailed in Damian Serrano (2022)), using the PCR program with an annealing temperature of 54°C, and a single pair of primers (166F and 134R), spanning the full extent of the sequence containing all barcode regions used in the gut content metabarcoding (from V3 to V9). Purified amplicons were sent in plates with the forward and reverse primer separately for Sanger sequencing from both ends at the Yale DNA Analysis Facility. A total of 89 newly-submitted sequences were then assembled and trimmed at a 95% quality cutoff in Geneious and concatenated with the latest SILVA database (SILVA_138_SSURef_NR99 downloaded on February 23, 2021) pruned to remove non-eukaryotic sequences. 

Taxonomic identities of the reads in siphonophore gut contents were assigned using the assignment software METAXA2 (Bengtsson‐Palme 2015) with a 70% reliability cutoff, comparing the sequences against our custom-built library built using SILVA138 that includes the new prey sequences.

Siphonophore collection

In order to sample a representative set of taxa across the siphonophore phylogeny, we targeted a set of 41 species (aiming for 10 specimens per species) including cystonects, apolemiids, pyrostephids, euphysonects, and calycophorans from shallow and deep waters. Most species were sampled from the Offshore California Current Ecosystem (OCCE) except for the Portuguese man-o-war P. physalis, which was collected off Bermuda in the Sargasso Sea; Sulculeolaria chuni and some Nanomia spp. (labeled as “Atlantic”) which were collected off Rhode Island in the Block Island sound; Forskalia sp. M123-SS8 and shallow Nanomia sp. KiloMoana2018-BW7-4 which were collected off the coast of Hawaii. While all the Nanomia populations sampled in this study have been referred to as Nanomia bijuga, we suspect that there may be undescribed cryptic Nanomia species among the specimens sampled based on the disparate tentillum morphologies we observed. Therefore, we decided to have them labeled at the genus level. One Nanomia specimen (KiloMoana2018−BW7−4) was collected off the coast of Kona, Hawaii. The pleustonic (surface floating) Physalia physalis samples were collected manually using a bucket from a small boat. Species found between the 0-20m deep were collected using blue water diving techniques following the guidelines in Haddock & Heine (2005). Species from 200-4000m were collected using ROVs. All animals were collected live and brought back to the ship (or field station in Bermuda for P. Physalis) for dissection. Live colonies were photographed (sometimes recorded on video), and zooids of diagnostic value (nectophores, bracts, tentacles) were dissected, when possible, fixed in 4% formalin, and stored as vouchers at the Yale Peabody Museum of Natural History (voucher catalog numbers provided in specimen metadata S15 Table of Damian-Serrano et al., 2015).

Gut content metabarcoding

Shortly after collection of the live specimens, we dissected and pooled several gastrozooids from each colony, making sure that those with visible gut contents are included in addition to several other without conspicuous prey, and also including visible egested food pellets at the bottom of the sampling container. 

To extract DNA, we digested the samples with proteinase K at 56°C for 1-2h, and used the DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany) eluting twice at 56°C for 10min into a final volume of 100μl. For barcode amplification, we used a set of six primer pairs that amplify six barcode regions within the 18S gene (‘V3’, ‘V5-V7S’, ‘V5-V7L’, ‘V7’, ‘V7p+V8’, and ‘V9’). The primers were designed using Geneious 11.1.5 (Kearse 2012), constraining the search to short (>300 bp) amplicon products with a high chance of remaining uncleaved after digestion in the gastrozooid, flanked by priming sites conserved (to a maximum mismatch of 3bp) across metazoans. The search for conserved priming sites was conducted on an alignment of 18S genes from 975 species across all metazoan phyla downloaded from GenBank (available in github.com/dunnlab/siphweb_metabarcoding/Primer_design). The primer search was optimized to only retrieve non-degenerate primer pairs with compatible annealing temperatures and without problematic dimerization and hairpin temperatures. Primer sequences are shown in Table 1 (Damian Serrano 2022), and their properties can be found in Table T1 in the protocol (Damian Serrano 2022).

Prey reference database

In order to enhance the accuracy of the taxonomic assignments of reads, we also built an 18S gene barcoding database of potential prey items to expand on the available reference sequences in public databases. To do this, we collected 60 specimens of 30 species of zooplankton and micronekton from the OCCE using a Tucker trawl. We targeted plausible prey species from motile open-ocean taxa that cohabitate with siphonophores and are underrepresented in SILVA databases, including fishes, crustaceans, jellyfishes, urochordates, chaetognaths, polychaetes, and mollusks. Specimens were photographed alive, then tissue was sampled and frozen, and finally the rest of the animal was fixed in formalin as a voucher to be identified and preserved at the Yale Peabody Museum of Natural History. DNA extraction, quality control, PCR, and amplicon cleanup was carried out in a similar fashion as the metabarcoding protocol described above (and detailed in Damian Serrano 2022), using the PCR program with an annealing temperature of 54°C, and a single pair of primers (166F and 134R), spanning the full extent of the sequence containing all barcode regions used in the gut content metabarcoding (from V3 to V9). Purified amplicons were sent in plates with the forward and reverse primer separately for Sanger sequencing from both ends at the Yale DNA Analysis Facility. A total of 89 newly-submitted sequences were then assembled and trimmed at a 95% quality cutoff in Geneious and concatenated with the latest SILVA database (SILVA_138_SSURef_NR99 downloaded on February 23, 2021) pruned to remove non-eukaryotic sequences.