Hello readers! I am in my final week at OIMB (this is my second-to-last blog post), and while time certainly has flown by, I am amazed by all that has been accomplished during these past eight weeks. Despite all that I have learned during my internship, in many ways I feel that I am leaving OIMB with a greater certainty than before that I know very little about science; this is both a humbling and exciting realization, and it drives me to further pursue the fascinating subjects that I have been exposed to during my time here.
As is often the case with research, nearly everything for my project came together very quickly at the last minute. On Thursday, I evaluated the quality of my newly-arrived sequences (most of which were high quality—yay!), trimmed the nucleotide ends of the sequences, and created consensus nucleotide sequences by pair-wise aligning the forward and reverse sequences for each gene. I then entered the gene nucleotide sequences into GenBank (an open access resource that matches sample sequences with over 150 million known nucleotide sequences) so as to contextualize my specimen sequences with established sequences. As expected, my sequences for the undescribed species did not fully match with any GenBank sequences; however, all of my sequences did most closely match with nucleotide sequences for the genes of fellow genus Micrura species, indicating that these specimens do indeed belong to genus Micrura. On Friday, Svetlana and I created phylogenetic trees for both the CO1 and 16S genes using a program called Geneious.
After devoting weeks to re-extracting DNA, amplifying genes, troubleshooting amplification, then purifying and quantifying gene samples for sequencing, it was rewarding to finally see the fruit of my labor—these phylogenetic trees! As you can see from the distinct groupings (or clades), both trees provide evidence for the presence of five cryptic species, exceeding our preliminary expectations of three. Both trees group specimens BdT007.1 and BdT007.2 together as members of the same species, with BdT007.5 as a separate (but closely-related) species. Likewise, both trees group together BdT007.3 and BdT007.4 together as members of the same species, with BdT007.6 as a separate (but closely-related species). We also see that the closest specimens matched via GenBank to these species (Micrura 132432 and Micrura 133724) are closely-related to BdT007.1, BdT007.2 and BdT007.5 but in fact belong to a fifth separate species. We contextualized these relationships by adding an outgroup species (Micrura 132529) to the phylogenetic tree.
This findings illustrate the necessity of utilizing DNA barcoding when assessing species. Note how similar in appearance the yellow-hued Micrura 132432 and Micrura 133724 specimens are, but how orange-hued specimen BdT007.1 and red-hued BdT007.2 have noticeably different appearances from one another. Both pairs belong to the same respective species—this demonstrates that while morphological differences often serve as an indicator of the presence of multiple species, we see that this is not always the case.
I am excited to share these findings with you, and am excited to share further findings as I delve more into the analysis of my research results. Comment below if you have questions, and look for larvae pictures next week!
“Instructions for living a life./ Pay attention./ Be astonished./ Tell about it.” (Mary Oliver)