Dataset: Morphological and genetic data of tetractinellid sponges from Kāne'ohe Bay, Hawai'i based on specimens collected between 2016 and 2023

Sponge Collection:
Sponges were photographed in situ and collected from Autonomous Reef Monitoring Structures (ARMS) inside mesocosms at the Hawai'i Institute of Marine Biology (HIMB) on Moku o Lo'e (Coconut Island), O'ahu, from ARMS that had been soaking for six years deployed on a natural reef environment adjacent to Moku o Lo'e, and from the surface of outflow pipes on Moku o Lo'e (Coconut Island), O'ahu. Field observations and measurements of morphology, color, consistency, surface, and oscules for each specimen were recorded. Samples were preserved in 95% ethanol and when enough material was available, were also fixed in 4% paraformaldehyde (PFA) for 24 hours and then transferred to 70% ethanol. Specimens were deposited in the Florida Museum of Natural History (catalog number beginning with acronym UF) in Florida, USA, and the Bernice Pauahi Bishop Museum (catalog number beginning with acronym BPBM) in O'ahu, USA. Samples from Kāne'ohe Bay were collected under special activities collection permits SAP2018-03 and SAP2019-06 (covering the period of January 13, 2017, through April 10, 2019) as well as HIMB collection permits SAP2022-22 and SAP2023-31. Samples from 2016 were collected from mesocosms where no permit was required.

DNA extraction, sequencing, and assembly:
Subsamples of sponge tissue (30 milligrams (mg)) were removed from each specimen and were preserved in 95% ethanol and processed for DNA extractions. Methods for DNA extractions, polymerase chain reactions (PCR), and Sanger sequencing are found in Vicente et al. (2022) with minor modifications as follows: Amplification of the 28S rRNA was possible using the 28S 63MODF (5'-ACC CGC TGA AYT TAA GCA TAT HAN TMA-3') forward with the 1072RV (5'-GCT ATC CTG AGG GAA ACT TCG G-3') (Medina et al., 2001) reverse primer, or the 28S C1' ASTR FWD (5'-ACC CGC TGA ACT TAA GCA T-3') (Cárdenas et al., 2009) combined with the 28S 1072RV reverse primer. COI amplification was possible using the COI dgLCO1490 (5'-GGT CAA CAA ATC ATA AAG AYA TYG G-3') and COI dgHCO2198 (5'-TAA ACT TCA GGG TGA CCA AAR AAY CA-3') (Meyer, Geller & Paulay, 2005). The PCR program consisted of an initial denaturation at 95 degrees Celsius (°C) for 3 minutes, followed by 34 cycles of denaturation for 30 seconds at 95°C, annealing at 45°C for 45 seconds, and extension for 1 minute at 72°C. A final extension hold at 72°C for 5 minutes finished the reaction. Forward and reverse reads from the Sanger sequences were assembled, trimmed, and edited by eye using Geneious R6 (Kearse et al., 2012). Sequences were checked for contamination using the BLAST (Altschul et al., 1990) function in GenBank and results that showed >85% sequence identity to a sponge were used for the alignment.

Phylogenetic analysis:
The Geneious alignment function (Geneious R10) with default parameters was used for aligning 28S rRNA and COI sequences. Alignments consisted of 676 bp of the 28S and 589 bp for the COI in both separate and concatenated alignments, which resulted in 1196 bp of the 28S+COI gene. RaxML (Stamatakis, 2006) included in Geneious was used for maximum likelihood (ML) analysis with the GTR+GAMMA model of nucleotide substations, 100 starting maximum parsimony trees, and 2,000 bootstrap replicates. Resulting bootstrap values of >50 from the ML posterior probabilities were shown on the tree (Fig. 15). Phylogenetic trees were rooted on Cinachyrella apion (Uliczka, 1929), HM592753.1 and HM592667.1 for 28S and COI phylogenies respectively. All accession numbers pertaining to each new species and each Geodia papyracea replicate are available in Table S1.

Morphological analysis:
Sponge pieces containing both ectosomal and choanosomal tissue fixed in either 4% PFA or 95% ethanol were transferred to 70% ethanol. Sponge pieces were dehydrated in alcohol using a series of 35%, 50%, 70%, and 100% before being embedded in paraffin. Sections (100-300 micrometers (μm) thick) were cut perpendicular to the surface of the sponge through the ectosome and choanosome with a microtome. Small pieces were also boiled in nitric acid for 1-2 minutes or until the solution turned clear and all organic matter dissolved. Spicules were let to settle (~15 minutes), and the acid supernatant was discarded. Spicules were then suspended with distilled water and decanted three times to remove the acid, before adding 95% ethanol for long-term storage. Spicules were suspended by shaking, and a few drops from the solution were observed under light microscopy, photographed, and measured using ImageJ (Abràmoff, Magalhães & Ram, 2004) relative to a stage micrometer. A minimum of 30 megascleres and 10-15 microscleres were measured per species (unless noted otherwise) for lengths and widths (expressed as minimum–mean–maximum, length x width in μm). A few drops of the spicule suspension were added to a stub, air-dried, and imaged under a Hitachi S-4800 FESEM Scanning Electron Microscope (SEM) at the Biological Electron Microscope Facility at the University of Hawaiʻi at Mānoa.

Comparative material summarization:
Sponges that shared morphological characters (i.e. spicule composition) in the genera Stelletta, Asteropus, and Stryphnus were included in Table S3 which lists species closely related to the new species described here. All previously described Tetractinellida species in Hawaiʻi were included for comparison in Table S2. Species locations were described following the Marine Ecoregions in Box 1 of Spalding et al. (2007). Species from temperate and cold climates (e.g. Arctic, Southern Ocean, Temperate Northern Atlantic (Black Sea), etc.) were not used as comparative material.