Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • BCO-DMO Processing Description
• Data Files
• Supplemental Files
• Related Publications
• Parameters
• Instruments
• Project Information
• Program Information
• Funding

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. Phylogenetic trees were rooted on Cinachyrella apion (Uliczka, 1929), HM592753.1 and HM592667.1 for 28S and COI phylogenies respectively. 

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.

- Imported original file "primary.csv" into the BCO-DMO system.
- Marked "NaN" and "-" as missing data values (missing data are empty/blank in the final CSV file).
- Replaced non-standard character em dash ("−") with hyphen ("-") in Long column and all string columns.
- Converted Date format to YYYY-MM-DD.
- Renamed fields to comply with BCO-DMO naming conventions.
- Saved the final file as "986886_v1_tetractinellid_sponges_genetic.csv".
- Converted the supplemental file named "Supplemental file.docx" to a PDF and renamed to "986886_v1_supplemental_file.pdf".

NSF Award Abstract:
Coral reefs are among the most species-rich ecosystems on the planet, occupying only about 1% of the seafloor, but housing more than a quarter of known marine biodiversity. Sometimes called the rainforests of the sea, coral reefs have great intrinsic biological, cultural and economic value. Nearly a billion people across the planet rely on coral reef ecosystems as a significant source of their diet, and the annual economic benefits of coral reefs are estimated to be around $9.9 Trillion USD. Thus, the global decline of coral reefs by an estimated 30-50% since the 1980s is of considerable concern as scientists struggle to understand whether species are being lost before they are even discovered. While coral reefs are spectacularly diverse, the majority of this biodiversity actually lives hidden deep within the three-dimensional framework of the reef itself. This hidden (or cryptic) community of organisms are both dramatically understudied and fundamentally important for the persistence of coral reefs. Sponges are a dominant group among these cryptic organisms within the reef which provide food from the bottom of the food chain and help sustain coral reef biodiversity. Despite the vital ecological role of sponges on coral reefs, little is known about their diversity, abundance or species ranges across the Indo-Pacific. For example, the most striking marine biodiversity gradient on the planet is described from several of the visibly dominant groups on coral reefs, including corals and reef fishes. From the global hotspot of species richness in the Indo-Pacific Coral Triangle there is a sharp eastward decline in species numbers to more remote oceanic islands in the Central Pacific, such as the Hawaiian Archipelago. However, no survey to date has evaluated whether the diversity of poorly known cryptic coral reef species, such as sponges, show the same pattern as the visible species that dominate the surface of the reef. Summer training modules introduce at-risk Pacific Islander youth to coral reef biodiversity to recruit and train a new generation of sponge taxonomists. Identification guides are being produced to help resource managers in establishing a baseline of sponge diversity, which allows resource managers to identify and protect native species, improves detection of alien species introductions and serves as a tool for monitoring changes in the ecosystem in response to human impacts. The work is being disseminated widely through scientific literature, public and professional presentations, popular press articles, and an educational display about sponges and coral reef biology in collaboration with the Waikīkī Aquarium.

This important knowledge gap is addressed by analyzing an existing backlog of standardized sampling devices (ARMS) collected from throughout the Pacific Ocean to determine whether sponges that live largely unseen within the reef framework follow the same diversity gradient as has been previously reported for fish and corals. By integrating taxonomy with multi-locus DNA barcoding and metabarcoding, this project is documenting species richness and biodiversity patterns among the cryptic sponge community across five ecoregions spanning over 10,000 km of the tropical Pacific. These collections include many new species and are providing vouchered DNA barcodes to existing reference databases that currently include fewer than 1% of sponge species across the planet. Sponges are a rich source for pharmaceutical development, so discovery of new species also provides opportunity for exploration of natural products from both the sponges and culturable microbes associated with them. By examining sponge species occurrence and diversity along both environmental and anthropogenic gradients in each ecoregion, the data also address whether coral reef sponges can serve as indicators of human impacts. Collectively, these results are transforming our knowledge of tropical Pacific sponge biodiversity, species ranges, and providing much-needed reference barcodes to global sequence databases. By determining whether sponges show the same Indo-Pacific richness gradient as reported in fishes and corals, this project is testing how well generalizations made from the visible subset of species that live on the surface of coral reefs apply to rest of coral reef biodiversity. This study is greatly advancing our knowledge of Pacific coral reef sponges and will ultimately inform the scale over which vital ecological roles performed by this understudied taxon, such as the production of nutrients at the bottom of the food chain, are acting across the Pacific.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

Description from NSF award abstract:
The objective of this Research Coordination Network project is to develop an international network of researchers who use genetic methodologies to study the ecology and evolution of marine organisms in the Indo-Pacific to share data, ideas and methods. The tropical Indian and Pacific Oceans encompass the largest biogeographic region on the planet, the Indo-Pacific. It spans over half of the Earth's circumference and includes the exclusive economic zones of over 50 nations and territories. The Indo-Pacific is also home to our world's most diverse marine environments. The enormity and diversity of the Indo-Pacific poses tremendous logistical, political and financial obstacles to individual researchers and laboratories attempting to study the marine biology of the region. Genetic methods can provide invaluable information for our understanding of processes ranging from individual dispersal to the composition and assembly of entire marine communities.

The project will:
(1) assemble a unique, open access database of population genetic data and associated metadata that is compatible with the developing genomic and biological diversity standards for data archiving,
(2) facilitate open communication and collaboration among researchers from across the region through international workshops, virtual communication and a collaborative website,
(3) promote training in the use of genetic methodologies in ecology and evolution for researchers from developing countries through these same venues, and
(4) use the assembled database to address fundamental questions about the evolution of species and the reservoirs of genetic diversity in the Indo-Pacific.

The network will provide a model for international collaborative networks and genetic databasing in biodiversity research that extends beyond the results of this Research Coordination Network effort.