As an undergraduate student at New York City College of Technology in Brooklyn, New York, I conducted research on the molecular characterization of Antipatharians (black corals) under the guidance of Professor Mercer R. Brugler and Dr. Estefania Rodriguez in the Black Coral Lab at the American Museum of Natural History. This summer I was fortunate enough to participate in a NSF funded summer research exchange program in Professor McFadden’s lab at Harvey Mudd College in Claremont, California. I’ve been working closely with Dr. Andrea Quattrini and fellow student researcher Alicia Pentico (Harvey Mudd ’19) on the Ultraconserved Elements (UCEs) project in an attempt to clarify phylogenetic relationships within the class Anthozoa. For my portion of the project, I’ve been focusing on relationships among orders of Hexacorallia. The subclass consists of six orders: Actiniaria, Antipatharia, Ceriantharia, Corallimorpharia, Scleractinia and Zoantharia.
Over the last nine weeks I’ve split my time pretty evenly between working in the wet lab on library preparation and hybridization and working in the dry lab on bioinformatics. I’ve constructed three phylogenetic trees using three datasets: mitochondrial DNA, nuclear rDNA (18s and28s) and UCEs. I look forward to the next steps of this project and seeing what evolutionary relationships will be revealed once Alicia and I combine our data.
This summer the Anthozoan UCE’s project and Dr. Quattrini have given me the opportunity to take part in my first ever deep-sea research cruise! We are currently on day 7 of the 17-day OP2017 Restore Cruise in the northern Gulf of Mexico. We are targeting 4 species of coral (Hypnogorgia pendula, Paramuricea biscaya, Swiftia exserta, and Callogorgia delta) to sample across 16 sites. The cruise is a collaboration between labs at Harvey Mudd College and Lehigh University and is funded by a grant from the NOAA Restore Act. Populations of the target species were heavily impacted by the Deepwater Horizon oil spill in 2010 and by the subsequent use of chemical dispersants to clean up the oil. The overarching goal of the project is to eventually determine a model of gene flow of these 4 species in the Gulf of Mexico to guide restoration efforts for the impacted populations. The models of gene flow and larval dispersal will be created using the results of population genomics conducted by the Herrera Lab at Lehigh University and DNA barcoding efforts conducted by Dr. Quattrini in the McFadden Lab at Harvey Mudd College. The genetic results combined with larval dispersal models (A. Bracco, GA Tech) will give us an idea about patterns of connectivity between populations across the GOM. My roles on the cruise include both watch standing and sample processing. My sample processing duties involve preserving a clipping of all the coral samples we collect in 95% Ethanol for later DNA barcoding. I am not only doing this for our own barcoding purposes at Harvey Mudd, but I am also preserving mesophotic corals (H. pendula and S. exertia) in ethanol for Dr. Etnoyer’s Lab at NOAA. My watch standing duties include logging dives in the ROV van when the ROV is collecting samples and otherwise aiding anyone in anything they need done while I am on watch. While those are my official duties, they are not the only activities I have been participating in during my stay on the ship. I have been aiding other scientists on board with their projects when they need help and I have learned a lot about other ongoing research in the field of deep-water corals and their associates. Beyond this I have been spending time making lasting connections with great young minds in the field at institutions such as Lehigh, Temple, Penn State, University of Hawaii, NOAA, and USGS. I am very appreciative of this amazing opportunity that has solidified my ambitions of continuing to pursue a career in deep-sea exploration and research.
-Mike Adams (Harvey Mudd College ’18)
For the past month, I have been assembling and annotating the genome of Renilla muelleri, commonly known as a sea pansy. We isolated DNA from organisms acquired through the aquarium trade, which were then sequenced using both an Illumina Hi-Seq, and MiSeq and a Pacific Biosciences RS II. Our paired-end Illumina reads had an average length of 166 bp at 190x coverage, and our PacBio reads were at 10x coverage. I fed the Illumina reads and the PacBio subreads to MaSuRCA-3.2.2 for a hybrid de novo assembly; the resulting scaffold was run through stats.sh (bbmap) and Quast to calculate genome statistics. Both programs reported the genome to be ~185 Mb with a GC content of 36.3%. The assembly had 6,036 contigs with a N50 of 63.191 Kb. To compare different assemblies, we used SPAdes to assemble a genome with only the Illumina reads. Using the same statistics programs, the Illumina-only assembly had a similar GC content 36.9%, but a genome size of ~ 255 Mb. I attribute these differences to the difficulties that short-read-only assemblers have in resolving repeat regions. The assembler is unable to identify the length of a certain region because of numerous base pair repeats unless it has longer reads like PacBio.
I then used the draft hybrid assembly for annotation with Augustus, a gene prediction software. I conducted two annotations with Augustus, one using training data from Nematostella vectensis (provided by Joe Ryan) and the other with Mnemiopsis leidyi; both used RNASeq data of Renilla provided by J. Ryan. Based on previous research, the number of genes in Renilla is anywhere from 15,000 to 25,000. With the Nematostella training set, 20,464 genes were predicted, and with Mnemiopsis 8,588 genes were predicted. The Mnemiopsis training set was able to predict exons and introns, but the Nematostella training set seemed to have predicted each exon as a gene. Because of this, the Mnemiopsis annotation had less than half of the predicted genes Nematostella annotation had. I tested these training sets out, but I realize that Nematostella and Mnemiopsisa are >500 MY divergent from Renilla. In the future, I will use a training set specific to Renilla generated from RNA-seq data. I am also planning on assembling a final genome with environmental contaminants (e.g., bacteria, viruses) removed and then running a final annotation. Stay posted for the draft genome and annotation files!
Justin Jiang, Walnut High School ‘19
For the past couple of weeks in the McFadden lab, I have been working on DNA extractions and gene amplifications for 89 samples of Xeniidae sent to us from the Queensland Museum in Australia. The samples were collected at three sites on the Great Barrier Reef, as well as from Western Australia. Xeniidae corals are a family of soft corals that reproduce at an extremely high rate, and are known to overpopulate areas where coral bleaching has affected other corals, leaving little room for any repopulation. However, corals of the Xeniidae family cannot be reliably identified to species using morphology, thus, in the McFadden lab I have been working on amplifying DNA barcodes that can be utilized to help distinguish between species with more certainty. Using Polymerase Chain Reaction (PCR), I have been amplifying three specific genes. The genes include two mitochondrial genes---COI and mtMutS, and a nuclear ribosomal gene--- 28S. The amplified genes have been sequenced, and I am currently cleaning and combining all three genes to assemble an extended barcode for each sample. I will then be able to compare the barcodes from different samples and group the specimens according to threshold levels in their genetic makeup at the three genes. The program I will be using, MOTHUR, groups samples together into molecular operational taxonomic units (MOTUs) if they have less than a 0.3% average genetic distance. From this, we will be able to get an idea of the species diversity of Xeniidae off Australia. In the following weeks, I will be working on editing the gene sequences as well as troubleshooting samples in which gene amplification was unsuccessful.
Rei Imada, Claremont McKenna College, ‘20
In the McFadden Lab, we have been studying coral phylogenetics, hoping to examine how past events have affected the evolutionary relationships between corals and their relatives, so we can better understand the effects of climate change. Over the past 5 weeks, we have started building phylogenetic trees from a) mitochondrial DNA b) Ultraconserved Elements (UCEs) and c) nuclear rDNA for different coral samples. The nuclear rDNA and mitochondrial DNA from which those trees are being developed are byproducts of the UCE assembly data. With some UCE test run data from 16 octocorals, I have extracted primarily complete mitochondrial genomes. These genomes were annotated using MITOS. When the annotated results were not complete, and genes were missing or fragmented, I returned to the original genomes to find and extract the genes. These genes were then aligned using MAFFT and concatenated. Phylogenies are going to be created from these alignments using RAxML. For the UCEs, I have prepped another 72 samples for sequencing using Kapa HyperPrep kits.
We will keep you updated!
Alicia Pentico (Harvey Mudd ’19)
Cathy and Andrea recently participated in the MASS program through Scripps Academy (http://www.scrippscollege.edu/academy/math-and-science-scholars). This program is designed to connect high school girls with faculty at the Claremont Colleges. Faculty work closely with students on a research project, with a final culmination in a research presentation.
Students travelled from high schools in Los Angeles to Claremont on four Saturdays during the fall. They were trained in DNA extraction, PCR amplification, Gel Electrophoresis, DNA alignment and Phylogenetic Analysis. The students also learned a bit about deep-sea corals! It was a fun experience, and we look forward to participating in the program again next year~
Two of our undergraduate summer researchers presented their research at the annual Summer Research Poster Celebration at Harvey Mudd College this month. Thanks again to Aaron Friend (senior), who spent the summer at the AMNH, and MIke Adams (junior) who spent the summer at HMC working on species delimitation of corals.
We have successfully designed 16,449 probes to target 1,795 loci across the anthozoa. We used several existing genomic and transcriptomic resources as well as our newly assembled Renilla muelleri genome (hybrid assembly). Brant Faircloth's amazing package Phyluce was used primarily to design these probes. Probes are being synthesized by MycroArray and we plan to test them on a subset of taxa before finalizing the probe set.. We will make the probe sequences publicly available.
And, we thank all of our collaborators who have provided essential data for use in probe design.
My name is Craig Dawes and I am an NSF-REU Scholar at the American Museum of Natural History, in New York City, working under the supervision of Dr. Estefania Rodriguez (Associate Curator of Marine Invertebrates) and Mercer R. Brugler (Assistant Professor at NYC College of Technology [CUNY]). I am also a full-time student in the Biomedical Informatics program at NYC College of Technology in Brooklyn, NY. I have been working in Dr. Brugler’s deep-sea molecular lab since January 2015, initially as a part of the Emerging Scholars program and then as an LSAMP (Louis Stokes Alliance for Minority Participation) Scholar. Based on my experience in the lab, Dr. Brugler recently placed me in a mentoring role; i.e., I am teaching new students how to extract and quantify DNA, set up PCR, visualize PCR on an agarose gel, set up a cycle sequencing reaction, and obtain DNA sequence data using a traditional ABI-3730xL Sanger sequencer. I am originally from Jamaica and moved to NYC about six years ago to pursue a degree in Nursing. After taking a Biology course with Dr. Brugler I was inspired to explore research as a career option.
My NSF-REU summer internship includes three projects:
1. I participated in a NOAA-funded ocean-going research expedition during Summer 2015 to the Flower Garden Banks National Marine Sanctuary in the Gulf of Mexico to collect mesophotic black corals. Mesophotic corals are defined as those organisms living in the middle of the photic zone, i.e. areas of low light penetration. We collected a total of 25 black corals representing three families and six genera across a depth range of 64 - 157 meters. Using three mitochondrial intergenic regions and three nuclear genes, I am obtaining a molecular barcode for these corals, in an effort to elucidate any undescribed species and/or extend the range of known species. We also surveyed banks within the sanctuary for Acanthopathes thyoides and Elatopathes abietina.
2. Based on morphology, Acanthopathes and Elatopathes are currently classified in the same family; however, they do not group together in a molecular phylogeny. These species are considered ‘wandering taxa’ as they change position depending on the gene (mitochondrial v. nuclear) or algorithm (Parsimony v. Likelihood v. Bayesian) used to build the phylogeny. We successfully collected two A. cf. thyoides and six E. cf. abietina. Elucidating 1) intraspecific variability within A. thyoides and E. abietina or 2) closely related cryptic species could potentially stabilize their phylogenetic position.
3. We recently obtained tissue samples from ten black corals that were collected during the 2015 Hohonu Moana Expedition (aboard the NOAA ship Okeanos Explorer) that explored deep waters surrounding the Hawaiian Archipelago. Dr. Dennis Opresko (Smithsonian NMNH), the world’s foremost expert on black coral taxonomy and systematics, noted that several individuals might be new to science based on a rough morphological examination. Thus, I am also barcoding these samples using mitochondrial and nuclear DNA in hopes of elucidating potentially new species.
Other projects - Molecular characterization of Deep-Sea “Sea Anemones” from the Arctic Ocean
We also obtained three specimens from Beaufort Sea, outlying the Arctic Ocean, at a depth of 1000m. Two of these specimens were tentatively identified as Kadosactis rosea, Allantactis parasitica and an unknown species presumed to be a member of the order Actiniaria. We amplified three mitochondrial, genetic markers to confirm the morphological identification of the first two specimens and reveal the identity of the unknown specimen. Our DNA analysis of the unknown suggests that we may have found a representative of a new genus. Currently I am analyzing the morphology of the animal via histological and microscopic examination. Future work will place these three specimens in a phylogenetic context.
For the past several weeks we have been busy preparing deep-sea octocorals for multi-locus DNA Barcoding from our recent RV Celtic Explorer voyage to Whittard Canyon. Initial identification of samples, based on morphology, indicates that the octocorals contributing to the diversity within Whittard Canyon are: Isididae, Plexauridae, Primnoidae, Acanthogorgiidae, Alcyoniidae, Chrysogorgiidae, Paragorgiidae, Clavulariidae, and Pennatulacea. So far, we have extracted high quality DNA from each sample and have amplified both the mtMutS and COI markers. We have recently begun amplifying a third marker, nuclear 28s rDNA. Additionally, we are amplifying the IGR4 region of the Isididae samples in order to further delineate taxonomic relationships. Once amplified, our samples will be sent off for sequencing; these sequences will then be edited and aligned in order to construct preliminary phylogenetic trees. Our ultimate goal with these markers is to better understand the species richness of Octocorallia in Whittard Canyon. Furthermore, we will be calculating genetic distances for between our samples and compare these to previously studied specimens from around the world in order to aid in our species identification and estimates of species richness. We hope to have our analyses completed in the coming few weeks!
Thomas Byrne, Pomona College '18
UCE Project Team
All things Anthozoa, Evolution and Ecology
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