 So our last speaker is Dr. Bridget Williams. There's Bridget. So Bridget will be discussing her title is advancing plant conservation with genetic and epigenic tools, a case study of the federally threatened Kentucky Gladecrest. And Kentucky Gladecrest is a federally listed plant here in Kentucky. I worked with Bridget's colleague, Dr. Christy Edwards on the field work for this project and in their lab does a lot of collaborative projects with several of the federally listed plants in Kentucky so I'm super excited we don't have many talks on conservation genetics so I'm super excited to hear your talk Bridget and see what the results are of of a Kentucky project are so take it away Bridget. Can you hear me. Yep. Okay, sometimes that just somebody let me know if the volume goes out while I'm giving a presentation sometimes the computer that I'm on will worry about it. Okay, get to it. Okay, thanks so much for that introduction Tara said today that the projects that I'm going to be talking about or part of the work that I conducted as a PhD student at St. Louis University, where I was co-advised by Dr. Christy Edwards here at the garden and I left for a postdoc and now I've, I've been lucky enough to come back and do a postdoc with her so I'm really excited to to update you guys on the work that we've done it's a it's a pretty cool system and we found some really interesting some interesting things so. So first of all I just want to get everybody kind of thinking on on, you know, kind of frame what the talk is going to be about and so I first want to cover what the conservation genetics paradigm is. The focus is typically on small populations of organisms in our case plants, and the small populations are typically characterized by low genetic diversity. And the reason that we're interested in these small populations with low genetic diversity is because that low genetic diversity leaves them susceptible to factors that can further lower their genetic diversity. So things like population bottlenecks genetic drift and in breeding depression. And that's going to lead to the loss of adaptive genetic variation, which reduces their capacity to adapt. And so it's based on this chain of events that theory predicts that this leaves them at an increased risk of extinction. And the goal of conservation genetics then is to protect the capacity of organisms to adapt by conserving variation on which selection can act in order to drive adaptation. And something that I'm really interested in personally is our species with low genetic diversity, doomed to extinction. So now I want to introduce you to our study system. This is the genus of Levenworthia. It's in the family Brassicae, is comprised of eight species of small winter annuals, and they're adaptive academics so they're all adapted to limestone glades and like open habitats with those rocky shallow calcarous soils. And it's also a model for studying the evolution of mating system because there's a lot of diversity going on in this genus with mating systems. So we have five species that are known to be self compatible to species that present mixed mating systems meaning that they can self fertilize and cross fertilize. And then we also have one species that's been confirmed to be self incompatible. And interestingly, we also have three of these species that are self compatible that have been listed as endangered or threatened and at least a portion of their ranges. And so today we're mostly interested in Levenworthia exitua species. So this is a poorly known species, but we do know that it's distributed throughout Kentucky Tennessee Alabama and northern Georgia, and it's been further divided into three varieties. So over here on the left, this is Levenworthia exitua variety exitua which is identifiable by white petals and lamp under sepals, and it's found in Tennessee and north Georgia. Then we have Levenworthia exitua varlutia, and it's identifiable by yellow petals and it's only found in Alabama. And finally, the star of today's talk is Levenworthia exitua varlutia, which is identifiable by white petals and green sepals and it is only found in Kentucky. So this is the one that we're focused on today. So Levenworthia exitua varlutia is narrowly restricted to only two counties in Kentucky. It has a very narrow range. That range is located directly south of Louisville in an area that's undergoing a lot of residential and commercial development. Over half of remaining populations have experienced serious declines and many have been lost or extirpated, and it was recently listed as threatened under the US Endangered Species Act. But there's a lot that we don't know about this system genetically, and so it was really important to kind of get an idea of what was going on with the genetic diversity to try to estimate its adaptive capacity and to better inform conservation management decisions. And so the study designed for the conservation genetics portion of this work focused on Levenworthia exitua varlutia exitua, and then Levenworthia exitua varlutia. So I've kind of listed out here just briefly. Again, you know, exitua exitua is found in Tennessee and northern Georgia versus the very narrow ranging exitua listen a and just to Kentucky to counties in Kentucky. Exitua varlutia has been shown to be self fertilizing, but we weren't really sure exactly what the mating system of exitua variety listening to was. So we thought that it could be self fertilizing, but that was something that we, we wanted to test. And so we sampled six populations of exitua varlutia for a total of about 136 individuals, and we sampled 21 populations of exitua listening to us. And the reason for that is because we really just needed a handful of populations of exitua varlutia against which we could compare genetic diversity metrics in listening to. And then we used a panel, a final panel of 16 micro satellite markers that were selected based on whether or not they exhibited to or more genetic vary or gene variations or alleles. And we identified these as being the most variable in exitua varlutia exitua, with the idea that if there's, if these are the most variable markers, then we should see some variation. So we really tried to isolate the most genetic variation that we could in this, the system in order to try to determine how much genetic diversity, we can detect in this, this narrow ranging species. And so we were interested in, in identifying or understanding the genetic diversity and structure or the distribution of genetic diversity within and among populations of exitua varlutia. And we also wanted to know whether it was genetically distinct from exitua varlutia exitua. Because that will also affect how it's managed if it turns out that it was just a, you know, kind of disparate population or distant populations of exitua varlutia, then maybe it doesn't need as much attention to protect. And so it was, it was really important to determine both of those things. And also because we expect that exitua varlutia exitua is self fertilizing, or we know that, then we use that to compare, listen, eat against because we would expect similar patterns of genetic diversity, if it was also a self fertilizing system. And so I just want to jump right in and show you some of our results because I have a lot to get through in this talk. So we use those micro satellite markers to look at genetic diversity within exitua exitua and exitua sinida. And interestingly, what we found the so this, this table shows the partitioning of genetic variation among populations among individuals within populations as well as individuals so we're looking at multiple scales of genetic diversity in each of these species. And what we see here is that 20% of all of the genetic variation that was measured or detected was found among populations, whereas essentially 0% was found among individuals within populations. And so what that means is that all of the individuals of exitua varlutia sinida within a population essentially are genetically identical. And we found 80% some, the vast majority of genetic diversity that we detected in the species was found within individuals. So that means that heterozygosity they have a lot of, they have a lot of alleles with, you know, packaged within individuals but again, these individuals are identical within a population. So that's a really interesting pattern and does not reflect what we would have expected to see if this was a self fertilizing system. We also observed in the system that observed heterozygosity was greater than expected at some loci within this species, such that there was even fixed heterozygosity, which does not make sense for a self fertilizing plant. You would expect that that genetic diversity within an individual would be further and further eliminated. And instead we saw that it was, it was pretty substantial. So I think it was like eight or 10 of the micro satellite markers that we used reflected some kind of fixed heterozygosity at those loci. So that was really weird. And, and then beyond that, whenever we look at what we found an exigua of our exigua among populations this is where we see the most genetic diversity partitioned in this species at 50, roughly 55, 56% among individuals we see about 13% of variations so we do see genetic variation between individuals within a population whereas we didn't see any over in this species. And then within individuals there is about 30%. So we have like individual levels of heterozygosity are much lower than what we observed in that in the very rare and endangered species. And so this this pattern where we see most genetic diversity partitioned among populations is much more along the lines of what you expect whenever you have a self fertilizing system. And so that that was definitely a big indicator that the Leavenworthy Exigua of Arlesinida is very unlikely to be a self fertilizing plant. And so, next what we looked at this is maybe a little bit easier to see than a table is genetic structure within and among the populations of both of these species. And so what you see here this is called a structure of a biogram and what you see here is that these blue of the excuse me the colors represent genetic clusters to which each individual has been assigned so it's really hard to tell because there's such a solid block of blue here but all of these blue there are kind of individual lines you might be able to see over here and the exigua for population each one of those lines indicates a genetic individual that was included in the study. So this large blue block represents 440 individuals that we genotype in that study. They're all just so genetically identical that it's hard to tell that there are individuals and each one of these bigger blocks represents a population. And so then the red over here represents the six populations of exigua and you can see that there is some genetic diversity it's largely contained within exigua for and so the blue indicates that there is some genetic similarity to what we saw in exigual which is not entirely unexpected since this exigual of our listen Eda has been classified as a variety of little more the exigua species. And I also want to point out, we've ran. We also did an analysis of clonality. And interestingly, in exigua of our listen Eda, I, they were all assigned to a single clone there was only one genotype. So again, that's very, very strange and not at all what we would expect to see in a cell fertilizing system. And so this is this is known as a heat map. And so what we're looking at here is warm colors represent greater genetic similarity and cool colors represent less genetic similarity. And so I hope you can tell, we've kind of divided up each one of these lines indicates an individual that was genotype in the city and so we've got all of our exigual listen Eda individuals are clustered together and all of our exigua exigua individuals are clustered together. And what you basically the, the, the message or the take home from this image is that we see a lot of very strong genetic similarity again among these exigua of our listen Eda individuals where we seem a lot of the red coloration and warmer colors versus and exigua exigua. It's much predominantly cooler colors. And also you might notice there is zero genetic similarity found between the listen Eda individuals and the exigua exigua individuals as well and so that was, that was really, really fascinating to see this again and this other analysis where essentially, these listen Eda individuals have a high degree of relatedness among all 21 populations regardless of where the individuals were sampled and of course you can see some variation in there. But, but again that's the predominant feature is that these are all very, very closely related genetically. And from this work, basically, that's, that's basically what we found is that, surprisingly, individuals are essentially genetically identical and this variety of exigua. And we actually think that Levenworth exigua listen Eda could be an APA mech and for anybody who might be unfamiliar, an APA mech is the clonal propagation via seed. So a maternal plant will produce clones of itself via seed. And there are a couple of different methods for that that I don't really have time to get into but that's what we think might be going on with exigua of our listen Eda that would be one explanation for why is able to maintain high levels of fixed heterosagosity among individuals and all populations are identical. And so that that's what we think might be going on there. There are some instances of other plants in the brassa casee that are apomeptic, as well as some diploid apomix, which we think this one, we do think it is deployed, but we've only done some preliminary analyses that would give us more information on that so there's more work to be done there but right now that's that's our prediction is we think that Levenworth exigua of our listen Eda is probably apomeptic. So that kind of that brings us back to this conservation genetics paradigm, where we're concerned about small populations with low genetic diversity, and how that leads to the increased risk of extinction. And so whenever we assess genetic diversity in species that are typically characterized by low genetic diversity. It's almost like we're overlooking where we're kind of knowingly overlooking potential alternative forms of biodiversity, because if you are measuring that you expect to be low already. There's, there's a chance that that may not be you may not be maximizing what your diversity that you're capturing and what you're able to protect. And so it brings me back to this question that I have which is our species with low genetic diversity to extinction. Because many species with limited or fiction and diversity display variation and traits and some are very successful in a range of environmental conditions. And base of plant species are a really good example of this because many display low genetic diversity. Many are clonal or capable of self realization. They often respond favorably to disturbance and they can display extensive trait variation. And I point this out because it indicates that there are non genetic forms of variation that can promote the persistence and survival of these genetically depopulated taxa. As we've seen invasive species real they're invasive because they are excellent at invading and taking over. And yet they share a lot of the genetic characteristics of some of these rare endangered species so you know what what's the difference there. There's no doubt this non genetic variation. One form of non genetic variation is found in the epigenome and this is a regulatory system of the genome that can alter gene expression without changing DNA sequence. And so one form of epigenomic variation that I'll just touch on today is DNA methylation. And what this is this is just the addition of a methyl group it's a chemical modification to the cytosine nucleotide of DNA. And this is completely natural it's found in every branch of life although it is not necessarily found in every single organism and every branch of life. But it's one of the most well studied forms of epigenomic variation and so that was one reason we decided to focus on this it's a really practical form of variation across the genome in order to measure. And so what do we know about DNA methylation plants. Well we know that DNA methylation can be heritable environmentally sensitive, it can be linked to phenotypes and trait variation, and it can also be independent from genotype. We also know that it can play a role in shaping and generating genetic variation and so it's a really, it's a really kind of complex but elegant system that we're just beginning to understand how it might contribute to additional forms of variation in living organisms. And so this brings us back to this conservation genomics paradigm again and whether or not species of genetic diversity are doomed to extinction. And so we think that the conservation genomics paradigm needs an update where we look at also conservation epigenomics. And this is looking at epigenetic and epigenomic diversity as a form of non heritable genetic or heritable non genetic variation environmental memory and phenotypic plasticity. That could actually act as a substrate for natural selection and produce adaptive responses and alleviate stress in these genetically depopulated systems to actually increase their chance of survival. And so the goals, then, after that became in this potentially epimetic system to look at epigenetic and phenotypic variation. And so we did this because we want to understand how do phenotypic traits and patterns of epigenomic diversity vary among populations in the species and do patterns of epigenomic variation correspond to patterns of phenotypic variation. As well, what are the implications of these studies for the conservation of the species. So this study I have here that it included four species three cell fertilizing our punitive epimect. I'm only I only have time to just present a little bit of the results for our rare endangered species so that's what I'm going to focus on but this is the overall study we have a sample seed from the field and grew them in a greenhouse under two different conditions reported a bunch of traits and conducted quantitative genetics, as well as whole genome by cell by sequencing which allowed me to measure DNA methylation. And so, let's just, let's look at some results that get at both of these questions here. So the patterns of epigenomic diversity. This is looking at DNA methylation differences genome wide in the rare endangered species. Exigua varlas in the ADA, and each of the populations, there are three here are indicated by the shape of the symbol, and then the colors indicate different maternal lines. What we see immediately is that individuals predominantly group by population so we have all of our little circles or McNeely Park individuals down here. Sportsman's Club individuals all the squares are here, and then the Pine Creek Barrens individuals are over here at the triangles, but they also seem to cluster by maternal line within population which is fascinating because again these are all genetically identical. You can see that there's this blue ellipse here is really big and whenever I dug into that it looks like the all of this variation is largely driven by the single individual here. I think there's something interesting going on there. But again, I'll have to save that for another time. And so we also like the patterns of phenotypic diversity. And what we found is, despite these individuals being genetically identical, we actually found significant variation in traits by population. And that was also very curious because it doesn't, you know, you wouldn't expect a genetic diversity is the basis of trait diversity, then why are genetically individual genetically different individual genetically identical individuals showing different patterns of trait variation. The possible conservation implications for this are based on genetic data to protect and collect seeds from the five populations that exhibit small mutations and at least one population that has dominant genotype to capture all geographically representative genetic diversity. But in considering the epigenetic phenotypic data, we would think that non genetic variation could be an important source of biodiversity in the species. And targeting this diversity conservation can actually expand the diversity being protected within it, which would boost its adaptive capacity and potentially promote its persistence. There are some things that we still need to work out we need to understand whether this epigenomic variation is heritable is DNA methylation the basis of that trait variation, and what environmental factors might this variation respond to. So a big picture wrapping up here, limited genetic diversity does not equal a lack of biodiversity epigenetic and phenotypic variation could be especially important and genetically to popper species as measuring genetic diversity alone in species is likely to underestimate specific biodiversity overall. Thank you.