The Crinoid Project:Genetic bar-coding of a cryptic species?

By: Olivia Storrs

What is a Crinoid?• Marine animals inphylum Echinodermata

• Common as fossils – typically stalked

• Davidaster rubiginosa, Davidaster discoidea –Comatulidae – extant stalklesscrinoids

• These species may be found on shallow reefs

Objective

• To test, through genetic bar-coding, the hypothesis that a cryptic species of crinoid exists within Davidaster.

• Why is this important? – Identifying full genetic diversity allows better understanding of fragile tropical reef ecosystems.

Davidaster discoidea

Davidaster rubiginosa

???? Cryptic intermediate ????

Methods• 1: Obtain sample• 2: Extract DNA• 3: Clean/Prep DNA• 4: Perform PCR reactions• 5: Run gels• 6: If PRC successful, prep for sequencer• 7: Complete PCR reaction• 8: Sequence• 9: Create new primers with sequence • 10: Analysis

Collecting Samples

Sample acquisition led us to San Salvador, Bahamas where we could find two of the three crinoids in question right off the coast.

Lab Work

This particular crinoid (D. discoidea) was extracted from a reef, brought to the research facility for observation in a water table, and inspected under a microscope. The next day it was returned to its reef with no harm done.

D. discoideaPartial arms were collected

D. discoidea oral disc under a microscope with food transporting pinnules

Once all samples were collected, the extraction process commenced.

Beginning Extractions

Labeling and creating individual samples

Labeled samples

Extraction –cleaning and buffering

Back at Home – Cincinnati Museum Center Molecular Genetics Lab

Immediately upon return from the Bahamas, Polymerase Chain Reactions (PCR) began. After several unsuccessful attempts to amplify the DNA, it was decided that another round of extractions should be done in the comfort of our own lab.

Polymerase Chain Reaction- Amplifying DNA

• Literature search to find Primers

• Record list of Primers for purchase

• Order

• Wait/Receive

• Set up PCR

Performing PCR

• Requires exact measurements of

– Water

– Taq (thermostableT. aquaticus

DNA polymerase)

– Forward and reverse primers

– DNA

A Graph Illustrating PCR

www.mun.ca

After PCR, one must check to see if amplification was successful by

inserting PCR’d sample and loading dye into a Gel

Electrophoresis apparatus.

Gel is ‘run’ and then placed over ultraviolet light. This is an example of

what you should see.

Sequencing

• Used PRC’d sample for sequencing prep

• Ran a different program in PCR machine to complete sequencing reaction

• Once sequenced, collected sample sequence was used to create new primers for increased precision in amplification results

• Amplified DNA with crinoid-specific primers

The catch…

•Preliminary sequences are not adequate –sampled tissues did not yield full genetic code

Originally, to spare crinoids, we only sampled their arms (able to regenerate). Unfortunately, DNA amplification has led to mixed results. There appears to be little tissue in crinoid arms and we may need to collect larger crinoid samples for better results.

What’s Next?

• Obtain better samples (Jamaican expedition planned)

• Repeat amplification process

• Sequence and read genetic code

• Compare with knownDavidastercrinoids

• Test hypothesis of cryptic species

Thank You!

I would like to give a big thank you to the Cincinnati Museum Center, The National Geographic Society for travel funds, Professor David Meyer, his wife

Kani, Dr. Herman Mays, and my father.