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Daniel Toledo talks about his ongoing research on Coral Reef Tunicates

Daniel Toledo-Rodriguez, a coral reef ecologist and recipient of the 2022 Seed Fund, updates us on his research on the genetic diversity of coral reef tunicates in the Southwest part of Puerto Rico.


Tell us what you have accomplished so far since starting your Seed Fund project?

Since the beginning of the project, we’ve been able to collect a total of 28 species of various types of sea squirts (solitary, social and compound) from deep areas located on the slope of the La Parguera National Reserve (LPNR) and some of the [mangrove] keys. Of these, DNA has been successfully extracted from 20 samples and all microscope work has been completed. It is expected that I will collect more samples of different species in the upcoming months. In addition to the successful extractions, I was also able to amplify many of the samples using polymerase chain reaction (PCR) as well (which is the next step prior to genetic sequencing).


What has been the most challenging aspect you have encountered? How did you overcome it, or how are you working to overcome it?

The most difficult challenge has been field sampling because most of these organisms are cryptic and easily confused with the actual reef structure. The second challenge is the DNA extraction process itself. Nonetheless, I have overcome this challenge with patience and perseverance. We began collecting organisms in deep areas (40-45 meters) first, which is the most difficult logistically due to the safety and organization of the divers, in order to observe a variety of species that may not be common because specimens are not normally caught at this depth, as these organisms are typically sampled in docks, under ships/boats, ports, and so on (in shallow areas).

"We began collecting organisms in deep areas (40-45 m) first, which is the most difficult logistically due to the safety and organization of the divers..."

Do you have any preliminary or final results to share?


Preliminary results have yet to be generated because we are still analyzing several DNA extractions and performing PCR operations with the remaining collected samples. Admittedly, due to the "limited" number of processed species, we have not yet submitted any DNA samples for sequencing; nevertheless, we have set a personal deadline by July 2023 to submit at least 35 "different" species to the sequencing facility for initial identification. However, we can say that we have found several known and probably unknown sea squirt species!

Did you discover something new about your field of interest and/or your topic that you didn't know before?


With the opportunity to study in this research project, I've been able to gain new scuba diving techniques in order to investigate the class Ascidiacea at previously unexplored locations in Puerto Rico. I've also realized that compound ascidians aren't all that tough to deal with; my ascidian detecting skills have improved, allowing me to capture more species at each sampling site.

What are the next steps to finish up your project and when do you aim to have it completed?

On or before July 2023, all samples will be sent to a specific laboratory for DNA sequencing, with the goal of using the resulting DNA sequences to genetically identify samples to their lowest taxa (species ID) and also to generate phylogenic trees and other useful information to answer evolutionary questions pertaining to the class Ascidiacea. Furthermore, the data will be submitted to GenBank (a DNA repository of genetic files), and a peer-reviewed publication will be published at the end of the study. We estimate to complete the study within October 2023.

Noteworthy, I am preparing to attend an Ascidian Taxonomy Workshop on June 5th through June 9th, at the Soka University of America in Aliso Viejo, CA, USA, to formally learn to dissect and identify living ascidians, although I'm currently employing some known procedures from the literature for this project.

Daniel's Seed Fund project is supported by Medalla Light. Follow along here and on our social media to watch for the final updates on his project!


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