 Hello. Yes. I'm Natalie Whitaker. I am new to the the galaxy team. I'm the communication specialist for Johns Hopkins. But, you know, in my downtime, I am also a PhD student at Florida International University. I am actually familiar with Galaxy because I study population genetics and genomics. Right now I'm working on population genetic studies for magromythraxidemis, which is a Caribbean king crab. Those who have talked to me before know I am utterly obsessed with crabs right now and how stinking cool they are, and how important they are to be ecology and biology and overall health of marine ecosystems. So I'm super excited to share a little bit more about me and then some of the research I'm doing right now. A little bit of a disclaimer, I have did not use Galaxy in this chapter of my dissertation, however, I am incorporating it into my last two chapters. So it's not going to be totally about Galaxy but everything I'm going to talk about can be done in Galaxy. For my current research, I did have to use a different platform because a previous researcher that I needed his data from use that platform and so but now that I've used all that, I'm going to begin going back to Galaxy. But you guys will see a little bit about what I have done with Galaxy in the past and mostly about my current research. So yeah, thank you guys for coming today. My topic is called King of the quarries and I'm investigating the origins of quarry populations of the Caribbean king crab to advise coral reef recovery in the Florida Keys. A little bit of a special shout out to Dr. Mark Butler and Dr. Heather Bracken Grism and Dr. Pedro Perez, who are at Florida International University and they have been my mentors on this project. So, oh, it's not letting me switch. Okay, I guess I have to click there we go. Alright, so just a little bit of my background. I got my bachelor's of biological anthropology from the University of Central Florida back in 2019. And I always knew I wanted to pursue higher even higher education and go to graduate school but I wasn't sure what I wanted to study yet. I actually am a Johns Hopkins alumni I received my masters of environmental sciences and policy there in 2021. And basically immediately discovered that I loved the human relationship with the environment and specifically our marine systems and the ocean. So I transitioned over to Florida International University to work towards my PhD. But I am thrilled to be back with Johns Hopkins. Like I said I am the new communication specialist with Johns Hopkins so you know repping my, my old sweatshirt there from my master's time. And so yeah that's just a little bit about my background. So, I'm new to the galaxy team, but so far it's been a total blast. I've been learning a lot more about the galaxy project in general and the amazing team that's behind its function. I've relatively experienced galaxy user. So most of my experience with galaxy comes from things like sequencing assembly for genes and gene analysis. And but I do hope to become more of a regular galaxy user as I learn more as I'm learning more about the capabilities so I'll be kind of going back to galaxy I'm super excited about that because I'm very passionate about open science. I'm also familiar with using galaxy training so it's actually been a fantastic tool for me to help my undergraduates and my interns in the lab, learn about the basics behind sequencing editing gene alignment analysis, and all that fun stuff. So you guys know all about that sort of thing. So, yeah, here we go. But now that you guys know a little bit more about my background. I'm excited to talk to you guys about my research so let's talk about crabs. All right, so our story begins, close to home for me, I am in Miami right now. So just south of me is the absolutely gorgeous Florida keys and some people probably don't know that the Florida keys are actually home to one of the largest barrier reefs in the world. Depending on the research you look at were either the third or fifth largest barrier reef. So it really depends but that also comes from the fact that it's currently declining. So the Florida reefs foster a diverse and absolutely gorgeous ecosystem. But as I'm sure we are all aware coral reefs around the world are disappearing at an astonishing rate, and the beloved Florida keys are no exception to that. So actually, the Florida keys have lost 95% of their coral cover since 1975. So that's, that's pretty shocking. It's pretty disturbing. So the reefs have undergone an ecological phase shift that's caused by anthropogenic stressors like eutrophication, pollution, climate change, all that fun stuff overfishing. And as a result, there's actually been an increasing presence of macro algae that is now dominating the reef systems. So there's efforts all around the globe aimed at restoring and conserving the remaining coral reef ecosystems. But we have a kind of more novel method of coral reef recovery that might be a little crappier than you think. So meet the Caribbean king crab magma my tracks been a system is the Caribbean king crab is in the spider crab family and its populations range from Venezuela all the way to North Carolina so it is native to Florida and the Florida keys. And it's currently accepted that the distributed populations of the Caribbean king crab have low to moderate genetic diversity due to short polygic larval duration, and they're limited migration ability. So they are not like these guys, but they are not like the brightest right so they're, they're kind of they're kind of dumb little crabs, they don't go very far compared to other crustacean species, their larva when dispersed into the water column does not live very long. So they are very much segregated populations of when you look at their genetics. So, what makes these guys extra unique is that they actually are the largest in in herbivore in the Caribbean. So they love to eat the dominating macro algae that's currently plaguing the coral reefs in the Florida keys. So actually a study by Dr Mark Butler and in his lab at Florida International University's Biscayne Bay campus have actually identified that the Caribbean king crab, when it's in slightly larger populations on the coral reef actually reduces macro algae cover by 50 to 80% which in turn has shown to influence a three to five fold increase in the mass spawning of coral sperm and eggs into the water column, the dispersal of coral larvae and coral reef fish community abundance and diversity. So these findings are super cool. And they indicate strong potential for successful large scale coral reef recovery in the Florida keys through the introduction and maintenance of the Caribbean king crab populations. So while populations of the Caribbean king crab are found naturally in the Florida keys there are also populations found in isolated saltwater quarries like the ones you see here. And these quarries are distributed throughout the region so these are human made structures that are filled with saltwater are not connected to the ocean directly. And they have been invaded by the Caribbean king crab. So it is proposed that these quarries be used as sort of aquaculture reservoirs for breeding the crabs for release on the coral reefs. However, our current understanding of the population dispersal the Caribbean king crab obviously hints at that separate populations throughout the Florida keys including within these quarries could be could have low genetic diversity. So that's something we need to investigate before we do any sort of conservation or restoration utilizing the populations. So genetic diversity plays a significant role in ecological processes and thus influences ecological consequences to populations communities and entire ecosystems. And presumably as a result of low genetic variation populations with low genetic diversity have limited adaptive potential, such as like weekend response to a changing environment of environmental circumstances, which would make them less suitable for conservation efforts. Therefore, to better inform and hopefully fingers cross employee deploy these innovative conservation and restoration efforts, we wanted to understand three main objectives for the first chapter of my dissertation. Number one would be what is the origin of the quarry populations do they come from Florida. Logically, we would think yes of course they came from Florida they're this close to the ocean. However, people are known to release species into into the wild or into quarry populate quarries. And we needed to confirm that they actually came from Florida because we do know that they're genetically distinct and within their the species natural range so from North Carolina to Venezuela. Secondly, we wanted to know do the quarry populations have lower genetic diversity when compared to the Florida reef populations. That is incredibly important we need to make sure they are they still have genetic diversity, and are still hopefully connected to the reef populations for that introduction because we don't want to be mixing different genetic populations together. And is the relocation of the populations of the Caribbean king crab in from the isolated quarries back onto the native coral reefs a viable conservation and restoration effort. So these are kind of the three things that I'll be going over with you guys today. And basically outlines chapters one and a half of my dissertation so to answer these questions. We collected 260 individual crab samples. They were collected from six different reef locations, which are outlined here, along with four different quarry locations. And we used data that was previously collected and analyze from Dr. Antonio Baez, which he had 78 samples from Mexico Costa Rica and unspecified Florida locations. So together, we had over 300 different samples that were used in this analysis. So for with each of the new 260 samples as well as the 78 previously analyzed samples, the crabs DNA was extracted PCR is for run and we use Sanger sequencing to calculate haplotype and nucleotide diversity as well as to build a haplotype network that would allow us to visualize the genetic diversity amongst the species populations. So for PCR three mitochondrial genes were chosen to be amplified partial sequences from 12 s co one and 16 s were chosen to conduct population level genetic comparisons, because their evolutionary rate is faster than the nuclear genome, which makes them the ideal choice for population genetic studies such as this one. If you were to be doing a similar study to this on a population that was not a crustacean different genes would be used but these are the top three genes that are regularly used for crustacean species and population genetics. Sanger sequencing was chosen because we wanted to target a smaller genomic region in a larger number of samples. Additionally, it's fast and it's cost effective which is, you know, great for an entering PhD student, which allowed me to include more samples from more population locations in the analysis. So in this study the program genius was used to evaluate and edit the resulting sequences to create alignments and to concatenate the three resulting sequences for each of the almost 300 samples that over 300 samples that we have. They can easily be done in galaxy as well. So using the concatenated alignment DNA SP was used to calculate the number of haplotypes as well as polymorphic sites per location. And in addition, the DNA SP was also used to calculate genetic diversity using the metric haplotype and nucleotide diversity also per location. Finally, the software pop art was used to estimate a concatenated haplotype network using the TCS statistical analysis. All right, so here we can see the most recent haplotype network for the Caribbean king crab for people who have not, you know, looked at a haplotype network before or maybe unfamiliar with what it is. The haplotype network visualizes relationships among DNA sequences within a population or an entire species. So, just to kind of tell you a little bit about how to read a haplotype network, the size of the circle correlates with the number of samples that have that one specific capital type. The color represents a different population location, and each tick on a branch represents how many mutational steps occurred between haplotypes. In this case, we can see that there are 66 haplotypes among the tested populations and that quarry populations are most closely related to Florida populations therefore it is most probable that the Caribbean king crab populations that initially invaded the quarries did originate from Florida. So this could have happened in a number of ways either they were released there by fishermen, or you know just general citizens, or maybe hurricanes with flooding they washed into the quarries. But it's nice to confirm that they did in fact come from Florida. Also, there is a clear distinction between populations from Mexico, Costa Rica and Florida, which does, you know, put out some concerns there about the genetic connectivity. However, we can see that all the populations do still share one main ancestral haplotype. So this is actually called what this result for a haplotype network shows it's a star shaped, meaning they are all still closely related genetically, even though there is some genetic variation amongst the farther sites in the populations that we studied. And we do also see unfortunately that the isolated quarry populations have developed unique haplotypes that are not found in the reefs. So this raises some, some concerns about their use in aquaculture for release on the coral reefs. But you know, we'll get into what exactly that means in a little bit. It's still exciting, I promise don't worry. Additionally, we calculated the haplotype diversity for each of the populations and then found the average for the reefs in the quarry separately haplotype diversity represents the probability that to randomly sampled alleles would be different. So when considering the relocation of the Caribbean king crab populations that are in the quarries back onto the native reefs for coral reef conservation and restoration purposes, ideally we would like to see relatively equal haplotype diverse haplotype diversity between the reefs and the quarries. But as you can see the quarries do have lower genetic diversity than the reef populations, which is, you know, that's not great. But, and then if we, we also calculated nucleotide diversity for each of the populations and found the average for the reefs in the quarry separately again. And then nucleotide diversity is the average pair wise difference between all pairs of individuals in a sample so it's a little bit more robust than just haplotype diversity. And once again, we do see that the quarry populations do have lower genetic diversity when compared to the reef populations. When we considered these results alongside our haplotype diversity calculations and our haplotype network. It becomes clear that the quarry populations do have lower overall genetic diversity than the tested reef populations. So that all said and done, but what does it mean. So if we go back to the quarries that were sampled for this study. These results show strong evidence of genetic consequences caused by the founder effect. So essentially, these quarries were invaded by the Caribbean king crab at an unknown time and from a previously unknown location. And since these populations are now isolated, unique haplotypes have been established and, and exchanged that were not that are not found on the reef populations. So while these genetic consequences don't seem, you know, drastic they do raise questions and concerns about the genetic health of the populations in the quarries, and the potential implications that could arise from their use in relocation to Florida's use for restoration purposes. The founder effect is a extremely powerful force it even drives an entire speciation events, but all hope may not be lost, which is fantastic news for me and all of us. So, in the, in the case of the quarries, we do see genetic evidence of continued connectivity to Florida reef populations. So we don't know how this is happening. I'm interested in kind of figuring out how maybe doing more analysis to see which haplotypes came first, doing some branch diversity analysis to try to see more. But there is, you know, three haplotypes that remain connected to the Florida Florida reefs. So again, maybe these quarries are flooding. They're connecting to each other. They're connecting back out to the reefs, even whatever is happening. It's fantastic news when considering the proposal, the proposed coral reef conservation restoration efforts, because we can see that some of the haplotypes are already shared naturally between populations. And then furthermore, going back to our haplotype and nucleotide diversity analysis, we can see that a few of the quarries, most notably kujo key east and big cockpit key may have that lower genetic diversity, but the calculations are comparable to a few of the reef populations. So this does give us even more evidence to believe that it is possible to use the quarry populations and coral reef conservation and restoration. So let's recap. We wanted to know where the quarry populations of the Caribbean king crab originated from and we found that they most likely originate from Florida. We wanted to calculate the genetic diversity of the reef and quarry populations and found that the quarry populations have lower genetic diversity than the reef populations, which is most likely a consequence of the founder effect. And finally, we wanted to know if relocating the quarry populations the Caribbean king crabs onto Florida's coral reefs is feasible in terms of conservation and restoration and we determined that this conservation reference conservation restoration effort is still viable. However, further research is needed to better understand the genetic diversity and the genetic connectivity of the populations. So you know, as all science goes we now have more questions than we do answers. So where do we go from here now that we have identified these potentials can concerns but have also identified that coral reef recovery is still plausible. Additional genetic studies are needed. So this is where I'm going to be incorporating galaxy back into my research. So from here we have already begun expanding our genetic studies using DD rad sequencing and modeling gene flow to further enhance our knowledge of the genetic diversity and the connectivity of the Caribbean king crab. So by using DD rad sequencing we will be able to identify specific locations of the genome that have strong selection and identify any environmental variables that may be impacting the genetic diversity and connectivity of the Caribbean king crab. Additionally, we have plans to collaborate with a fellow lab at Florida International University to sequence the entire genome of the Caribbean king crab. This is extremely exciting news, I know I'm kind of a nerd. But by creating an entire genome we're going to have the ability to conduct a whole slew of re sequencing analyses with a reference genome to refer to. And this will truly expand and strengthen our results. I'm very excited about this. So it's even going to I'm even going to be able to use this full genome to redo the analysis from the first chapter of my dissertation which we just kind of went over to kind of confirm and enhance those results as well. So one of the main, one of the main techniques that I'm hoping to use once we have a reference genome is called high C. High C was actually talked a lot about in the last galaxy community call so hopefully you remember a little bit about that and how important it is to genomic studies and populations and species. So high C will allow me to perform comprehensive genome research, such as identifying interactions and variances in specific parts of the genome. So by taking these new strides in our population genetic studies, we hope to develop a vast understanding of the Caribbean king crabs overall genetic diversity and connectivity in order to best inform and hopefully employ these novel conservation efforts at the Florida keys, desperately need. So while this story isn't over yet we remain optimistic that the king of the quarries may just be the key to saving Florida's coral reefs. So thank you guys so much for your attention. I'm happy to happy to answer any questions that you have. Sorry, my presentation was kind of short, but I'm always happy to talk about crabs with you guys. Awesome. Thanks so much Natalie for this presentation and he's really informative. I'm going to open up open it up now does anybody have any questions. I guess I'm. You mentioned it, but I'm not. I don't think I connected all the dots. Why do we need the equal levels of genetic diversity or population diversity to sort of release and back like or is there a threshold right I mean by those numbers were within 20% like what what like if they're not the same is 20% acceptable like you know maybe. Yeah, so that's a big question and it's something I'm researching right now so this has not been done with crustacean species before which is why we're kind of moving on to larger genetic studies with DD Rad and full genome sequencing. So it has been done successfully with, with, with populations that show that sort of differentiation between the genetic diversity with haplotype and nucleotide diversity, but those were in species like bats and butterflies and things like that so there are crabs. And the coral reefs are already so sensitive we need to confirm that, okay we see the lower genetic diversity we don't know what the threshold is. So we need to get as much information under our belts as possible in order to inform these conservation and restoration efforts. It's a huge deal right like the populations in the quarries. Yes, our inbred, but that's to be expected right they're not connected to the ocean, but how inbred is to inbred to be released back on on the reefs. So, as, as you like going back here, you can see no name key right down here is out of the question we can we're not even looking at no name key anymore for this possibility because actually until we got more samples no name key had zero nucleotide diversity which was very concerning, but even though we're looking at like big copper key and kujuki east and and kujuki west. Those are relatively more equal to the reef population so we're kind of concentrating our efforts on to those right now. And what's actually super neat about kujuki east and west is they are literally spitting distance from each other. So we suspect that they're actually exchanging them amongst themselves as well. I have like an exact answer for you, I, I, we don't know how different is too different, which is why we're trying to get a better understanding. We also are going to try to identify if the other than inbred and being inbred, we're trying to identify if the reasons that the quarries are different from the reef populations is the environmental influences. So I'm actually going to be doing seascape genomics with my DD rad analysis, and I'm going to be projecting the genetic connectivity between the populations along with environmental variables like salinity pH temperature all that fun stuff to see if any environmental changes are even you know anthropogenic cause is influencing the genetic changes in the, in the quarries and so if they're suitable to be implanted back out onto the reefs. So yeah that is the question of my basically entire dissertation, and I just don't have an answer yet I'm sorry. How long do they live. The Caribbean king crabs, not very long but they grow very quickly so to we estimate two to three years for an individual as an individual's lifespan here. They are fairly regularly eaten by by by predators, but they grow very quickly so they start out obviously small little tiny baby crabs and within six months they're already bigger than golf balls so that's kind of what we're hoping to be able to implant is that is that size of the species. But yes it's, it's sad that they don't live very long because I mean we're also we're raising them in our lab we have an aquaculture reservoir in our lab where we're raising them to study them more. And it's good for the reefs though right because when we're with that we can easier control the population we know the larva is not going to go anywhere we're no they're not going to migrate very far if at all. And we know that they're going to die within two to three years which makes the populations easy for us to control and manipulate from, you know, conservation perspective. Sorry to dominate the one more thing. What's their radius, you said they kind of stay local but do you know what like in terms of the miles, how far did they move. So, not very far at all. We used to have an estimate of not even miles I mean we're talking a couple hundred yards from the coral reef, if that was a center point. However, after Hurricane Ian, we went back to the reefs, and some of the populations we had been studying are actually gone. So we're trying to determine okay did they leave, or were they brought somewhere else as a result of the storm. So we had an answer for that but now we're questioning it again. Now we need to kind of confirm. Sorry, this is my first like big presentation I've done for my dissertation so I know it's not the greatest I'm still working out all the kinks. How big is the genome is that something that we might potentially feed to the GP. So the genome, we don't know for sure yet because we haven't sequenced the whole one yet however spider crabs in general are between two to three billion base pairs long. They're pretty good size so we're estimating because with the sky about three billion base pairs. And what about sex determination is that how does this work. I've only done sex determination like with the actuals without with the species like individuals in my hand. X, Y, or yes. Yeah, they're X, Y. And we don't kill these guys right so I'm clipping little little crab keel a little toenails to do all this so we have a lot of information about each of our individuals size. If they were a grab it and all that fun stuff so there's a lot that can be done, but I'm super excited about the full genome. I probably were saying this but what's the heterosagosity that you estimate. Sorry, what's the heterosagosity that you. I don't know yet. Yeah, I'm sorry. That seems to be my answer for everything we're not we're not sure yet. We have so much more work to do. What was your workflow like. I think that was the initial part of the study. I think you were mentioning that you didn't initially use galaxy because most of the data were not. There was kind of a different way that you were accessing it so. And then in your kind of future plans to use galaxy with the whole genome sequencing and and that what is that going to look like. So, oh, I'm so excited about it so I am going to now that I have access to his entire data collection, Dr Antonio's. Now that I have access his I have it all saved on my my school computer and with all my hundreds of individuals as well. I'm going to be using galaxy for the DD rad portioning of my sequencing for my analysis to start so we're going to be uploading all the data I'm going to be kind of filtering out identifying areas of the genome that are under song strong selection. And we're still working on funding for the whole genome part it's it's getting wrapped up right now so we kind of move forward with DD rad, but once I have a full genome the full genome will be uploaded to galaxy and that will be the reference genome. I think I put a little something here. Whoops, yeah here right here. So it's going to be we're going to have our set of reads and our genome, the reference genome and I'm going to be mapping it all and I'm going to be hopefully identifying areas where environmental stressors have influenced the genetic diversity of the of the Caribbean and then I'm not sure I have to kind of decide it really depends on what kind of results we get because to be honest, what we found with this first chapter. We were not expecting to see so many different haplotypes in the quarries so now I have left the end of my next chapter open for what kind of analyses I'm going to be doing so if anybody has any suggestions. Or anything like that I'm absolutely always open to knowing what you guys are going to do, which you guys think I should do when it comes to especially since I haven't done full whole genome analysis at all yet I've only been working with with individual genes or small collections of genes. So I'm not going to be doing another haplotype network but I am going to be doing seascape genomics and so I'm going to be also using GIS to map the species and into the future. And I'm, I'm just, I'm excited to contribute with galaxy and learn it more. So, I'm super excited. You mentioned that there are similar studies with bats and butterflies in terms of understanding the diversity and whether like reintroducing them into a different area would be successful. Did they do like similar studies and analyses that you kind of leveraged for this analysis. Yes. So those were, they were similar studies in that we, they took genetic information from the different populations, however it was a little different in that the reason they were aiming for reintroduction was for habitat fragmentation right so the, like the, like for the butterflies and the later patches were too far apart so they were trying to figure out if they could kind of move them all together so that they can expand out again. So yeah I did model a little bit off of, off of them. I also modeled it after Dr Pedro Perez's PhD work where he did work in Brazil with a crab species there that was separated by in a current in the water and found that they were actually very very different from each other. So I modeled it a little bit off of each right because crustacea is is so picky when it comes to picking genes and things like that. So I use his genes and and all of that from his study also Antonio study for like my primers and all that and then I use because it hasn't been really hasn't been done with crabs or actually I don't even think it's, I haven't seen much about it being done with marine species at all. So, I mean, not even like you know the megafauna the charismatic megafauna that everybody love the whales and the dolphins and things like that so I kind of took a little bit of everything this is, this is relatively unique. I mean, he followed closely by NSF and Noah and Sea Grant and all those, all those fun people so I'm trying really hard to make it worth it. That's really cool. Thanks. Yeah. Any other questions from folks. Once, once I've gotten through, I'm almost done with my DD Rad right so I got maybe like another month, month and a half of actual wet lab work for that. I will know much more about how I'm using galaxy in it, but we kind of need to see what exactly our results are going to turn out like or if we're going to get another big surprise like we did with Sanger. So, so hopefully I'll have some more answers and not too long. That was a random fun question. If you could as the user from user perspective if you could add one tool or feature for your research anyway to galaxy. What would it be. Oh my goodness, I should have anticipated that question from you Michelle. I'm not sure yet. Can I get back to you once I've I've done DD Rad for it. Yeah, I was just thinking you know in the course of working with it. You know, maybe you had something that was like a, just an interesting learning that you had. I can tell you that my undergrads are very appreciative for all the training tools. I mean it has been an absolute blessing in our lab to have it there where they can because some of the students that you know we bring in have, have barely finished you know genetics one on one. So it's been absolutely wonderful for them to actually get hands on experience on a platform that has so much information so many researchers, resources for them. So I mean I can I can actually I can ask you know some of the interns they they just graduated so I'll give them, you know the weekend to celebrate, but you know, I can definitely ask them to see if they had any, you know, thoughts about what what could be improved improved or anything but yeah I don't I don't have anything at the moment. But maybe they do but I don't know they all love it so final questions for Natalie. If not, and thank you so much for this presentation I learned a lot about crabs and the Florida keys. And really looking forward to this future work that you're going to be doing with Galaxy so obviously you know the right people to get connected to so yeah thanks again. Let me know if anybody has any ideas. Thanks everybody for signing on our next community call is going to be on May 25. Yes, same time on Thursday, and we'll be hearing a presentation on TPV from new one. Thanks everybody for joining and we'll see you in a couple weeks.