Can we control a deadly infectious amphibian disease before it is too late? Vance T. Vredenburg San Francisco State University [Margaret Mantor]: Intro: Hello, everyone We’re going to get started. I’m Margaret Mantor, I work for the CA Department of Fish and Wildlife Habitat Conservation Planning Branch. And today, we’re going to be having a lecture, it’s part of the Conservation Lecture Series If you want to see the schedule of upcoming lectures we have it up on the intranet for employees and we also have it on a schedule in the back of the room. I wanted to note on that schedule we’ve had a cancelation of our next planned lecture which is the Alameda Whip Snake and San Francisco Garter Snake; that’s going to be rescheduled, not canceled, we will let you know the date as soon as it’s rescheduled. Today, we’re excited to have Dr. Vance Vredenburg who is going to be presenting his research on yellow-legged frogs. He is an associate professor in the Department of Biology at San Francisco State University He is also a fellow at the California Academy of Sciences and a research associate at the Museum of Vertebrate Zoology at UC Berkeley He grew up in Mexico and received his Bachelor’s degree from UC Santa Barbara and his PhD from UC Berkeley. His PhD research showed that it is possible to reverse the decline of a threatened frog in the wild, and his approaches developed during that have been implemented in montane areas globally. His current research is focusing on the impacts of an emerging infectious disease in amphibians and the role amphibian skin microbiome has in that disease We’re really very pleased and happy to have him here today! [Vance Vredenburg]: Well thank you Margaret And thank all of you for being here. I’m really excited to tell you about the work I’ve been doing since about 1994/1995 on the mountain Yellow Legged Frog. Let me just say it’s been a pleasure to work on this species. It’s an amazing species, and well it’s my favorite species, what else can I say! Let’s move on So this is my amphibian slide. Amphibians are just cool. That’s one part that’s really important to understand. They’re really amazing animals; they’ve been around for hundreds of millions of years. They’re incredibly diverse We’re lucky here in CA to have a really amazing array of amphibians- the number of species here in CA, the count keeps going up. For example, just one example, the CA Slender Salamander group that some of my graduate students are working on now, 30 years ago if you came to CA there were 3 or 4 species described, now there’s somewhere around 30 species described. So we really have an amazing array of species here in CA including of course the Yellow Legged Frog down there in the bottom, but also things like the Ensatina Salamander in the top left here. And then if you start looking at other places worldwide, amphibians are really interesting because they do all these interesting behaviors. There are dozens and dozens of different types of behaviors that have been described in terms of how they breed and what they do And they’re in big, big trouble right now, more so than any other group of vertebrates And in this slide you can see that amphibians are, as a group, worse off than any of the other vertebrates including fishes, mammals, birds, and reptiles. And it’s something that has been talked about quite a lot in the press, so I’m sure all of guys have heard about this stuff. But this is a really big problem And when you look at the big picture, it’s a conundrum, really. Because if you look at what’s happened, if you take the really longest view we have, which is life on earth, and you look at amphibians, they are actual survivors They are the oldest group of vertebrates on land. They’ve been around for over 360 million years, depending on how you measure it exactly But they’ve made it through mass extinction events that lasted millions of years and wiped out enormous groups of (well, including vertebrates), enormous swaths of life on earth. And yet amphibians, all three major areas of amphibians, all three major groups, survived these mass extinctions in the past, not just one, they survived the last four mass extinctions quite well. And yet here we are today, and they seem to be really taking it on the chin. So we might ask, why are these long term survivors in such bad shape today? And I really like this figure right here from the New York Times, it came out last year, actually 2012, and I really like this visual because it shows you in red the proportion

of species that are in trouble today. So this is the decline that’s been occurring, not in geologic time, not over millions of years, but probably in the last hundred years, or even much less, probably in the past few decades So we’re very, very concerned about this And the Sierra Nevada, I’ll argue, is one of the places that has been a hot bed for our study of this problem, of this global problem. This is a photograph I’ll get back to later. This is here in California, the Sierra Nevada, in Kings Canyon National Park And this is a photograph taken by Joel Sartore (you can see in the bottom right), he’s a photographer from National Geographic and this is in a story that came out in 2008 National Geographic. But if we look at amphibian declines and the hypotheses that people have talked about, the mountain Yellow Legged Frog group is one of the most important species to understand amphibian declines worldwide. Although, habitat destruction is not a big issue in the Sierra Nevada, nor is over exploitation of these frogs. Many of the other potential factors have been studied for these frogs. So things like invasive species (which I’ll be talking about today), UV-B radiation (which I’ll mention today), climate change, infectious diseases, and a slew of other things but these are probably the most important things out there Again, just to give you the global perspective on it, on amphibian declines, this is not something we just have to worry about in our backyard; it’s something that is truly global in its implications. If you look at this map produced by the World Wildlife Fund and the Nature Conservancy, it shows you all the different biomes on earth, instead of showing things geopolitically it shows you things geo-biologically And you can see that in major areas, for example in the Andes, Central America region, parts of Japan, a huge proportion of the species in those areas are threatened with extinction Some places between 80 to 100 % of the species are in trouble. Now we’re lucky here in CA, that although we have experienced amphibian declines, we haven’t experienced extinction events of entire species. But that probably has more to do with the fact that we live in a temperate zone, where species tend to have bigger ranges than they do in the tropics This is the poster child of amphibian declines, called the Costa Rican Golden Toad. And it was last seen in 1989. This is the species people talk about when they’re talking about amphibian declines. And it’s really interesting and alarming at the same time because this species was discovered and then disappeared in a nature reserve called Monte Verde in Costa Rica. And so this species is really important because we have really good evidence that it used to be super abundant up until about 1987 and then it declined rapidly for no apparent reason and disappeared in 1989 Well, it turns out we have an example here in CA of the same sort of situation. This is a paper that wasn’t published until 1991, but it’s titled “Mass Mortality and Extinction of a High Elevation Population of Rana muscosa,” which is our Sierra Nevada Yellow Legged Frog And you can see here in the year 1979 in Figure 1 on the left, the number of live frogs plummeted in a couple of these populations in Sequoia National Park. So, fully protected habitat, just like the Golden Toad, really abundant frog, boom, dies off en masse. Now, unfortunately, this paper wasn’t published until 1991 but it happened almost, at about the same time, as the frogs started going extinct in Central America. So this species is really important for our global understanding of what happened and I’ll get back to these data So the Sierra Nevada, we’re incredibly lucky to have places like the Sierra Nevada here in CA. It’s a protected landscape, as all of you know, there are huge swaths of roadless areas. And I can tell you as a biologist going back through trying to understand what’s happened to these species that have declined in protected areas, that’s a whole different league when you have species that are declining in protected habitats, that’s something that is really alarming, and it reminds us that you can’t just put a wall around a nature reserve and assume that everything’s going to be ok. And I think that’s indicative of the time we live in today where our biosphere has been affected now at a scale, by humans, that’s never been affected by us What’s really cool, as well, about the Sierra Nevada, is that we have a really good baseline to compare our data to. This is something

that all of you and your colleagues when you’re looking at actual geography that you know well, you can go back in time and see what used to be there. Because some forward looking biologists, Grinnell in particular, and Storer from the MBZ at Berkeley, decided it was time in 1915 to do some surveys in CA, because they thought that at the time that CA was overcrowded. And they knew that there were going to be effects on nature. And they did all these biotic surveys and now that treasure trove of information is now available in museums So I can go to this lake, for example this is Roosevelt lake, and I can go and look at frogs that were collected at this lake, and I can go back to that lake today, and say ok they collected I think it’s something like 76 Rana muscosa frogs (mountain Yellow Legged Frogs) from this lake, who’s there today? Well it turns out, there are no frogs in that lake today but hopefully that will change, and I’ll get back to that So one of the things I did during my dissertation was to just try to say, ok how bad have the declines been in the Sierra Nevada for this species of frog? And I went back to the museum records and then with a whole bunch of people, including some folks from at the time CA Fish and Game, but USGS, CA Academy of Sciences, many other people (this was not something I did on my own) went out and resurveyed areas where we knew these frogs used to be, because we have them in jars, they have locations, and we know when they collected them, how many they collected, etc., etc., and we went back and basically looked at these sites And all of the red areas are areas where the frog had gone extinct by 1997. So for the Northern Frog, and I’ll explain why I split these frogs into two different species in a moment, there was already a 92.5% decline in comparison to historic sites as of 1997 in this northern species, and in the southern species it was even higher, a 96% decline, which actually includes the southern CA populations So if you look at just the Sierra Nevada as a whole, 94% have declined and if you look just at Southern CA populations: 99% decline So it’s only a few hundred animals left in Southern CA. And in 1997 the picture wasn’t that grim in the Sierra Nevada but it’s become decidedly worse So before I go on, I need to explain something that always confuses people. And in fact, it confuses me when I go to write papers about this species. And that is that this species has now been split into two species. So the history of this frog, the mountain Yellow Legged Frog, is described by a graduate student at Berkeley in 1914, he noticed that it was very similar in coloration to some of the foothill Yellow Legged Frogs and he assumed that this frog was a subspecies of the foothill Yellow Legged Frogs, which is now called, and was at the time, Rana boylii. So Rana boylii or the foothill Yellow Legged Frogs, he called the mountain Yellow Legged Frogs subspecies of that so he called it Rana boylii sierrae in the Sierra Nevada and Rana boylii muscosa in the southern CA areas. And muscosa means lichen like, it doesn’t have anything to do with other stuff. So what happened then in the 1960s (that’s how it stayed until about the late 1960s) and then a professor by the name of Zweifel came through and looked at these frogs morphologically and realized there was a big difference between the Sierra Nevada, that although there’s a big difference between the Sierra Nevada frog and Southern CA frog, there’s also a lot of overlap. So he decided that these two subspecies were actually too similar to each other to be called different subspecies, so Rana boylii sierrae and Rana boylii muscosa, he decided were the same thing, but he also decided that they were quite different from the foothill species Rana boylii. So he split them out because “m” comes before “f,” he called them Rana muscosa, all of them together. That’s the history of how we got Rana muscosa Then I came along 30 years later and applied some genetic techniques to this and realized there were morphological changes, frog vocalizations changes, and mitochondrial differences, so genetic differences between these populations of Rana muscosa in the Sierra Nevada and in

Southern CA. And so, this is the paper that was published in the Journal of Zoology in 2007, where we simply looked at these three types of data and based on these three types of data, we separated the species into two distinct species, so they’re coming all the way back to the original descriptions in the sense that the Sierra Nevada frogs, especially from central Sierra Nevada north, tend to be lake adapted frogs with shorter legs for their body size, different vocalizations that are hard to characterize, and also quite a big break genetically, which you see in the mitochondrial DNA phylogeny on the left. So Rana sierrae broke up into three clades in the northern part of the Sierra Nevada and then three clades for Rana muscosa which is the southern species. Two of the clades are in the Southern Sierra Nevada and one clade is in Southern CA Here they are again: you can see these a little bit more closely, showing the two different groups of frogs. And one of the reasons we decided to call these different species, was that the morphological break seen on the right hand side and the genetic break seen on the left hand side coincide in space. So somewhere right in here in what’s called the monarch divide you have the two different groups of frogs coming close to each other but not actually overlapping in the same population. They might be, but we won’t know that for a long time because those populations right at the contact zone, unfortunately, they’ve gone extinct So maybe in the future with better genetic techniques using specimens, we might be able to tell a little bit more of what has happened in those areas. So you can see the clades here in colors and of course any conservation work that goes on we need to pay attention to this, because there are big differences, depending on whether you believe in mitochondrial genetic clocks or not, the difference between the two species is about 5 million years of difference with no contact based on general models of no contact for about 5 million years between the species and then within the species much shorter time frame in terms of much less difference between them. But I’m happy to tell you that you can actually take a frog and measure it, although it seems crazy and they look really similar, you can actually measure it and you can have a really good estimate of whether you’re looking at a northern Rana sierrrae or a southern Rana muscosa You can actually tell those species apart morphologically These are sort of the evolutionary significant units that we use, which in this case we were just using the mitochondrial clades, and there’s a lot more work that I think needs to be done in these; this is only based on 100 samples There are many out there and we’re hoping to do more in depth work in how different these are from each other. But you can see all of the different groups 1 through 6 there, they’ve all declined dramatically, so they’ve all suffered terribly. And I think the newer data that looks like this, I think clade 1, 3, and 6 are the worst ones in terms of conservation Alright, so let’s shift to what happened: why did they go extinct? So I’m going to start with introduced species because it’s a very simple story. It’s so simple that I can give it to kindergartners, and they actually get it and I’ve shown them data. I always tell myself that if I can convince a kindergartner, I can convince anyone. So native fish did not actually invade the higher parts of the Sierra Nevada except for a few exceptions, that’s because we had these amazing barriers to fish colonization. Of course, frogs don’t have that same barrier and the Sierra Nevada, don’t forget, is a very old mountain range somewhere between 70 and 100 million years old depending on how you measure it, so very, very old mountain range. And if you look at, here’s a GIS map and we try to sort of reconstruct the distribution patterns of native fishes in the Sierra Nevada you can see there, we’re looking North, you can see the Bay area off to the left and there’s the chain of the Sierra Nevada and you can see in the Lake Tahoe area, there’s some native fish there. All of the foothills, especially on the Western side, have native species of Salmonids and other fish as well, and of course that golden area down there is the area where the California

Golden Trout is native. But most of that area inside that red line, that red circle, which is where these frogs exist, almost all of it is fish free up until not very long ago So when I actually started doing my dissertation work, I had lots of people tell me why are you working on the effects of fish on frogs? Because there is no way the declines that started in 1978 were due to fish introductions People have been introducing fish in the Sierra Nevada for a really long time. And of course there’s already some overlap, so why would you expect fish to be a problem? I tell this story now, and people are surprised that I got this reaction. But there are people and places where you can go see overlap between this species and introduced fish. And when you see overlap, you think that they must be able to coexist. So here’s a photograph for example from the 1890s, showing mules carrying fish back up into the mountains and of course back in those days, the thought was they were actually creating a fishery and doing a really good thing. There wasn’t a really good understanding of how moving an aquatic predator that didn’t exist in the ecosystem, what it would mean to that ecosystem Of course, the frogs, I’ll try to convince you, were the ones that suffered greatly The thing that’s really interesting about fish introductions is that they didn’t happen in the same way over the whole time period So they started out on mule trains and those you can only go so far before the fish just can’t make it, or the people for that matter (They were tough back then, but not that tough!) Things changed in about the 1950s/ 1960s (I’m a little fuzzy exactly when those things change) but it’s about that era when fish hatcheries started producing so many fish that they started thinking, oh, well where else can we drop these fish so that people can go fishing? And of course when you’re dropping things out of an airplane, fish can climb those mountains really easily. And that’s exactly what happened for quite a few decades actually And here’s just an example from one of my collaborators Rolan Knapp on just how extensive the damage can be, and there have been many papers now published on this issue both in the Sierra Nevada and globally for that matter If you go back to some of these remote areas when Grinnel and Storer were doing surveys back in these really far away areas, they didn’t find any fish except in the front country, and in the back country like in Humphrey’s Basin , there were no fish and there were no native fish. But if you went out very recently and surveyed those areas, all of these lakes in yellow are lakes that have introduced fish in them. So if you’re a frog or a mayfly or some sort of species that is not evolved with fish predators, you don’t have very much habitat left over to survive in. And of course, that’s what I wanted to look at for part of my PhD work Now I’m going to tell you a little more about the characteristics of this amazing frog, and I’ll just talk about both species together for most of this talk. But they are really, really good at making a living in these high elevation environments. They lay eggs underwater, the eggs take about two weeks to hatch, those eggs when they hatch turn into tadpoles, and the tadpoles are extremely long lived and so that’s really a great adaptation for living in a relatively low energy environment. So they store up a lot of energy year after year and can be extremely abundant in lakes and then when they metamorphose, they do so with quite a lot of success because the tadpoles are really big when they start metamorphosing Adults, by the way, are very long lived, we pit-tagged, with little microchips, adults, and I’ve had 6 to 7 year old frogs recaptured And a study by Kathleen Matthews looking at bone growth, they estimated that some of the adults lived more than 10 years, which is pretty amazing So how do we test the effects of introduced fish? I didn’t want to take a frog population and put fish right on top of them, so the national park service beginning in about 1998, allowed me to go in and gillnet fish out of some of these lakes and by me I means lots of people helping me do it. This was a nice way to test this idea because the frogs don’t get caught in gillnets and the fish, we didn’t poison the fish, we just took them out mechanically It was a lot of work, but it also helped in

terms of interpretation So here’s the place where I did this work It was published in 2004. And the data I’m going to be showing you is from 1996-2003 And these are lakes in a chain here, what’s called 60 Lake Basin in Kings Canyon National Park, Fresno County. And we have these lakes draining from the big lake down through all these smaller lakes. And the experimental procedure was pretty simple. We moved fish from some of these lakes in red and then didn’t remove fish from other lakes, whether they’re frog lakes or fish lakes and then just count the number of frogs in these lakes. And one thing that’s really amazing, is that before I started these experiments, if I went into the site at 60 Lake Basin and counted the number of frogs on these shorelines, you could just walk a few meters from one of these frog lakes that had no fish to a fish lake and count the frogs (there were frogs in the fish lakes) but the numbers of frogs were just dismal. It would be like thousands of frogs in that bottom, big lake in green for example and then you’d walk downstream 10 meters sometimes and you’d find two or three frogs in a really big lake. So the expected effects were very, very strong. The sample size for this study was 5 fish removal lakes and 8 control lakes and there were two different types of control lakes, the fish lakes that we didn’t manipulate that already had the introduced fish in them and then the frog lakes. And these are all connected to each other, so we couldn’t choose randomly which lakes to do the fish removals on, we had to choose lakes that had no fish up above them and waterfalls down below them so we wouldn’t just be taking fish out and having them recolonize every summer And here’s what the data look like, it’s really straightforward and nice. If you look on the y axis, we’ve got number of frogs per 10 meters of shoreline (that’s how we count these frogs; we walk along the shorelines and the frogs hop in). So imagine from here to the edge of the room here, 10 meters or so, you’d see about 5 frogs on average in a frog lake. And that’s what all these light grey lines are at the top, it’s over 900 surveys from 1996 until 2003 of those 8 control lakes, just frog lakes. And then at the bottom, there’s two different kinds of lakes, the ones that stay flat near zero and are really light grey and squares are the fish control lakes where we didn’t remove the fish and just showed that yes, sometimes there’re frogs there, but very few. And then the lakes numbered 1,2,3,4, 5 are the lakes where we actually removed the fish and those horizontal lines on the bottom starting at 1997 with number 1 are the years that we did the gill nets, that we did the gill netting of those lakes And you’ll notice these lakes, some of them we didn’t finish them in the study, like lakes 4 and 5 aren’t done yet. But the frog numbers, as soon as you start knocking the fish down, the frog numbers start increasing dramatically And what’s really cool is that we were able to repeat the study in different places, for example in Marmot Lake area, LeConte areas, where we removed fish, in this case that’s what the arrows are (the year that we removed fish from the lake) and the two axes there are either frog density or tadpole density through time. So when you remove these nonnative fish, frogs recover. And it’s a really great story. What’s really cool is that it turns out this story is an important story not only in Sierra Nevada but in the Pyrenees in Europe and in Patagonia, South America, and in Australia and in New Zealand and in Kilimanjaro. And you wouldn’t believe, I was just down in Peru last summer working on some frogs there (we were actually working on disease) but the rivers are all filled with rainbow trout from CA. This problem of introduced species is really, really big and it’s global, I mean it really is everywhere. And species that are sensitive to these fish predators are ones that we really need to pay attention to. But the good news is, in at least some cases we can reverse the effects of these introduced species. These frogs are going to need it, as you’ll see later because this isn’t the only issue affecting the frogs Another story I wanted to tell you about is one where we look at what happens when fish are introduced into these food webs, they’re not just affecting the frog, they’re affecting the entire aquatic food web. They’re really good predators, they’re super effective, and they can really change the way energy flows through these systems. And the way we’ve measured this, the way ecosystem ecologists measure this, is using stable isotopes, where you can actually track where carbon or nitrogen

comes into the system and how using stable isotope analysis, how that carbon tracks through systems. For example, if you have some algae growing in the water that’s fixing carbon, that carbon is going to have a different signature than a plant on land that’s fixing carbon because they’re using different pools of carbon, one is the atmosphere that’s very well mixed and the other carbon pool is from, literally from the pool, from water and that has a much different signature of carbon. Plants when they photosynthesize, prefer the lighter version of carbon as opposed to the stable isotope version, the heavier one, so you can see that signature. And what we were able to show in this study is that the fish are not only eating the frogs directly, they’re also competing with them indirectly. So even if you have fish in the lake next door, they’re still affecting the frogs in the next lake over because they’re changing the food web of those lakes. And these frogs, unlike some other frogs like red legged frogs, these frogs get almost 100% of their energy from the aquatic food web even though they’re up on land. So that’s something to think about when you’re out in these aquatic areas in the Sierra Nevada looking at these frogs, their food is coming, not from way up in the forest, it’s coming from the aquatic system. So this is really important to understand that So science informed conservation, you know we really do have some good successes. But of course, we’re just beginning; they’re happening not only here in CA but really all over the world. It’s pretty exciting. There’s a paper that should be coming out soon from the Pyrenees that I’ve been working with folks on there, where a very similar frog in Spain is recovering after fish removals in those areas So let’s get back to this mass mortality issue though because this is one of the things that distracted me and a lot of my peers when I was working on introduced species, and that is that those introduced species don’t describe all the declines. Now that doesn’t mean that they’re not important, that just means they don’t describe everything that’s out there That’s one of those things that’s always really hard to get across that there are multiple factors here that are involved in amphibian decline, it’s almost like the “death by 1,000 cuts” idea This is a photograph I took in one of these lakes that had had a big recovery of frogs and then something else happened. These frogs are floating upside down in the water, you can see my sharpie pen at the top. And there’s one poor frog that managed to climb up onto a rock. And I’d been to this lake, two days before this, I didn’t note of any sick frogs What is going on? So now for the remainder of my talk, I’m going to talk about the Chytrid fungus, that I’m sure many of you have heard of. The Chytrid fungus is called Batrachochytrium dendrobatidis, I’ll be calling it Bd and the disease that it causes is called Chytridiomycosis. Now here’s where the name come from just in case you’re curious. It was first described in the dendrobatid frog in the National Zoo here in the US. So it’s called the dendrobatid frog killing fungus and it’s from this group of fungi called Chytridiomycetes (they’re microscopic, so you can’t actually see them They’re super interesting because they have a flatulated stage called the zoospore that swims through the water to find its host and infect it Now we’ve made a lot of progress on what’s going on with this pathogen and I want to tell you about it because it’s really, really interesting. And this gets back to this question of globally, what’s going on? I’m going to tell you about a bunch of studies in the Sierra Nevada, but it’s happening all over the place Here’s what the life cycle looks like: these are aquatic zoospores on the bottom, they swim, they infect the skin of the frog, they reproduce asexually really rapidly and then they produce more of these zoospores, which are then shot out of the body of the host frog and then they can either reinfect that host or infect a new host And what’s really cool is that we’ve been working really closely with microbiologists to come up with methods to measure the presence and the amount of this fungus in nature. And it turns out it’s really difficult. For example, that study from Bradford that I showed you with the big declines of Rana muscosa in Sequoia in 1978 where all those frogs died off, we don’t know why they died off but they died off almost immediately and nobody was able to figure out what was going on. Well that’s because this fungus wasn’t described until 1999. So it’s relatively recently described to science So what does it do? It affects the skin, and the skin in amphibians is really important organ, it’s very active physiologically, more so than our skin really. For example, there are huge number of amphibians, salamanders in particular, that have no lungs at all because

they need those muscles to shoot tongues out of their mouth and so they breathe right through their skin. These frogs, Yellow Legged Frogs, breathe through their skin as well as through lungs, they also have lungs, but the point is that their skin is really important for osmoregulation, breathing, other things. And what happens when they get infected by this fungus is that a complete dysfunction occurs with the epidermis So here’s the picture of the Yellow Legged Frog on the cover of Nature from a couple of years ago and I know it’s cut off on the side, but I wanted you to see the title on the outside of the cover of Nature, which is “fear of fungi,” and the subtitle here is “emerging pathogens threaten natural ecosystems and food security.” So it’s not just an amphibian decline thing It turns out that fungal diseases are on the rise in humans, animals, and plant systems, and they’re really scary, and this seems to be something that’s happening very recently And the role of science in studying our frogs, our species of amphibians, here in CA is very important, that we’re able to do this. We can’t do these kinds of studies that we do with amphibians on humans, for example. We can’t experimentally infect a human, but you can do it to a few frogs. And so doing, in a controlled environment, you can learn a lot about what’s going on with this pathogen, why does it not fit the evolutionary history of all pathogens. This fungus that I’m talking about, Bd makes the bubonic plague look like a drop in the bucket. It really does. The bubonic plague killed off a third of the human population and one of the biggest outbreaks in human history, a third of the population in Europe in the Middle Ages, that’s a lot of people, that’s nothing compared to what’s happening to amphibians. We’re talking about 500 plus species infected, probably 200-300 species driven to near extinction by one fungus It’s not just happening in frogs though, here’s a figure from bat conservation international that shows the spread of white nose syndrome, which is a fungus that’s infecting bat populations in the US. It started in that little black dot in eastern new York, and has spread mostly towards the west over the last few years And in many cases, the entire colony, millions of bats are driven all the way to extinction So what’s the connection, is there any connection? Well, I think the important thing for us to understand is what’s the underlying biology behind all of this? Because the underlying biology is what links all of us together If we can understand the worst outbreak of a pathogen in vertebrates in recorded history, which is what Bd is, we’re going to be in a better position to protect ourselves and the species we really care about in terms of food and other things in the future if we understand the underlying mechanisms that could have led to this out break. And the Sierra Nevada system and the Yellow Legged Frog system has been really important for that Now we get back, here’s another picture of 60 Lake Basin. What I’m going to show you is repeated visual population counts (over 900) from this time frame 1996-2008 and then I’m going to show you data from skin swabs which is (I don’t know if I have the picture in there) but one of the ways we can measure this fungus is by swabbing the frog with what looks like a cue tip, letting the frog go, and taking that back to the lab and extract the DNA from that and look for particular combinations of DNA that only match Bd or the chytrid fungus. Then we can also compare it to known standards of the Bd, so known quantities of DNA from that fungus and then we can measure how infected those animals are. So is it there and if it is, how much of there is it on the individual frogs. And that’s what I’m going to be showing you from 60 Lake Basin. This is a study again based on the same place where we did the fish removals that were published in 2004 and here we have the map of the basin showing you those same lakes that are draining towards the north All of the green lakes are lakes that have frog populations in them, the green lakes with black outlines are ones where reproducing populations of these frogs existed back then And then the yellow ones are ones that become infected with the fungus, and then the black ones are ones where the adults all get killed off. Grey and white ones are ones where we have no data or where there are fish present

So this is what the basin looked like after all the fish were removed from those five lakes, but there are still plenty of lakes that contain fish in the basin. Frog populations rebounded, it was the biggest meta population of frogs anywhere in the Sierra Nevada. And for some reason it showed up in these lakes in yellow, which turns out where some of the most remote lakes within the basin- so we’re not really sure how it got there. But what I’m going to show you now is from 2004 to 2008 and I’m going to show you the spread of this fungus over a relatively small area, it’s only a few kilometers wide and long So about 2005 it had spread, it had already caused the extinction of a few of these populations in the top of the screen and it had spread to almost half the basin. 2006, 2007, 2008, so you can see (let me just back up and show that again) the progression of the fungus That’s interesting, it’s an aquatic fungus and yet it’s spreading upstream, so that’s a really important finding. The second important finding is that it’s happening, it took four years for it to move over this tiny landscape, so that also tells us something pretty interesting about what’s going on. Just so you know, there is a silver lining, there’re still frogs left in that basin and I’ll get to that later But here’s the interesting thing: if you look at the 60 Lake Basin data that I just showed you, which is in Sequoia National Park, (sorry, Kings Canyon National Park), in this lower part of the graph, in this area what we found is that Bd has been spreading in these recent years leading to literally hundreds of local extinctions. There were about 600 populations of Yellow Legged Frogs in that watershed area you see cut out which is part of Kings Canyon National Park. In 1997 there were about 600, now we’re down to about a dozen. All of those extinctions are due to this fungus, not to fish introductions or other factors. In all of those cases when the populations disappeared, it was right after the fungus got there. In direct contrast to that, if you go to Yosemite National Park and you look at the Yellow Legged Frogs that live there, everyone was already infected by the time we started our study and no populations (I don’t think anyway) had gone extinct recently (so in the last five years or so). So we have a situation where you have persistence of the host and the pathogen in the Yosemite area and you have rapid population decline or local extinction and epidemics or epizootics of the frog pathogen system in the southern part of the Sierra Nevada. So what we think is really different between these areas is the timing, but we’ll get to that So I need to back up one second and tell you just a little bit about Bd worldwide because as I told you it has infected 500 species But it turns out there are really different outcomes depending on what species you’re talking about. If we’re talking about our favorite Rana catesbeiana at the top (or American Bullfrog), those guys are carriers. They get infected but they rarely die from the disease Same goes for Xenopus laevis, the African Clawed Frog. The closely related species of Xenopus are highly susceptible and other species of frogs, many of them, are highly susceptible So what gives, why are these so different between these areas? So what we discovered in that 60 Lake Basin study that was published in 2010 in PNAS, that one where I showed you the map and the Bd spreading throughout that system, was that we discovered a mortality threshold for these populations. And that was really quite different between, that showed that the dynamics of the infection was very different in Sequoia/Kings Canyon than they were in Yosemite. In Sequoia/Kings Canyon we had a true epizootic, where the prevalence of infection was rising rapidly after they got infected and the infection intensity on the individuals became very high before the population started collapsing In contrast, if you went to areas in Yosemite and looked at the infection dynamics there, they’re very stable. So although they have a high proportion of individuals in the population infected, their infection intensities, or the load of infection on animals, was 2 to 3, sometimes 4 orders of magnitude lower than what you find in these disease outbreak areas And in a study we just published in 2011, we looked at different species and found that this rule sort of applied to different species as well. The species that are surviving like that first one in panel A, this is days on the x axis, infection intensity on the y axis, where 10^4 was the zone where above that, right around there is where animals start succumbing to the disease. So you have three

different species in the bottom right, A, B, and C (these are actually salamanders from Central America), and these guys, some get infected and stay infected for 40 plus days, but their infections (those little lines you see squiggling at the bottom) never get very high. By contrast, B and C in those panels the infected animals rise sharply, their infection intensities rise sharply over just a matter of a few weeks, and then they die. And on the bottom of B and C, you can see a black line with dots that stay down low, those are control animals that were not infected and survived. So it’s not an animal housing issue, it’s something where it really does look like we’ve hit upon something And if you look at other people have published on the same issue and embarrassingly they called it the “Vredenburg’s 10,000 Zoospore Rule,” (they should have just called it the 10,000 Zoospore Rule). But what they found, they found a similar thing in their system, where animals below this mortality threshold survived, animals above it seemed to die off So we think we finally got some predictability in terms of what’s going on with these fungal infections. So now, I would propose that if you brought me a swab from frogs where you are working, I can analyze those swabs and I can give you an idea of whether animals in those populations are truly threatened by the fungus or not Of course, not all species are the same. So here’s a species of frog that I wasn’t supposed to talk about today, but I couldn’t help it this is the Pseudacris regilla ,which may or may not be split into several different species depending on who you talk to. But they’re a wide ranging frog, the chorus frog or tree frog. These guys co-occur with Rana muscosa and Rana sierrae and they can sustain very high levels of infection. So if you look at the same map in 60 Lake Basin, this is a paper my grad student published a couple of years ago, you can see on the left hand side, all those little extra notations are places where the Pseudacris frog lives in 60 Lake Basin. And then on the right by 2010, you can see that all the black arrows are places where Rana muscosa disappeared because of Bd. But the blue lakes and the yellow lakes, are lakes where the Pseudacris frog survived, in fact they actually expanded their distribution in the basin. In fact, we propose in this paper that they’re sort of the Typhoid Mary of the system. They can sustain high levels of infection and they can move. (They’re much more terrestrial). So they could, we don’t know for sure, but we propose that they could have been the way that the disease spread across the basin This is just to back up that statement a little bit, if you look at trials we did in the lab, we had a whole bunch of animals, 35 animals, sorry 39 animals, of this species of frog, the tree frog, in the lab and we looked at what happened with the infections of those animals. So if they’re uninfected, the top panel of four frogs, a couple of them started out infected, but they stayed uninfected and they survived in captivity just fine. And all these other guys down below were animals that were infected sometimes at very high levels so approaching that 10^4 infection status, and in fact they weren’t completely immune to the pathogen. There was one animal which had the highest infection load, I believe it was panel number 31, that had the highest sustained infection load of any of those animals did succumb and die of Chytridial mycosis But most of these animals survived just fine and didn’t even lose weight by the end of the experiment. The way that we think they did it, when we looked at those animals, it looked like they had the pathogen on their skin was organized quite differently than it was on an animal that suffers from the disease. So these guys had very concentrated but very high levels of infection in little areas of their skin but they also had large parts of their skin that appeared to be uninfected or almost completely uninfected, so they still had a big functioning part of their skin That’s how we think they did it So where did this fungus come from? How do we manage it? What do we do about it? One of the first things we have to do is understand where it came from. I can tell you that in 60 Lake Basin, it wasn’t there until 2004 And then when it arrived, there were drastic consequences. However in other parts of the world, if we start looking back to museum records for example, we see that the oldest records of Bd in the world come from a study from Brazil, so Brazil has the oldest ones, South Africa has the next oldest in the 1930s,

and in both of those areas we have situations where species that live in those areas seem to survive with the infections. We don’t yet have the careful studies that we need to look at the infection dynamics of those, but we do know that there aren’t major population declines in the species that are infected In California, up until very recently, we believed that this fungus emerged in about the 1970s or 80s when the first declines happened in frogs, but it looks like it happened a little bit earlier. It got here in the early 1960s. And in fact, we published a paper recently where we looked at introduced bullfrogs and we found it all the way back in 1920. So you can see where I’m going with this It looks like introduced species, again getting back to that theme, could be responsible, it could be one of the pathways that this pathogen could get into new host populations, in particular naïve host populations that don’t have an evolutionary history with that pathogen. Here’s one example, this is an African Clawed Frog, they were used for human pregnancies up until the 1970s, in fact my mother told me she did a frog test and it came back positive Frogs and humans, you know, we share the same hormone systems, there’s so many similarities If you inject urine from a pregnant mother (human) to a female frog, that frog will ovulate and within 9-12 hours, you’ll have your answer, positive or negative. Thank goodness we have a better way of measuring pregnancy these days. But at one point that’s how it was done, and these African Clawed Frogs had been in science as a really important species to study since the days of Darwin. And because they’re easy to keep in captivity they were shipped all over the place, many places like hospitals had these, and probably, once they had better ways to do things, they didn’t want to kill them, so they just let them go. And now we have feral populations of African Clawed Frogs all over, and thank goodness we now have laws in place that don’t allow that sort of thing to happen anymore They’re not the only culprits, Rana catesbeiana, the American Bullfrog turns out is really tasty, very easy to grow in farm situations Look at this farm. Can you believe this? This is 10s of 1,000s of American Bullfrogs. This is in a farm in Yumen, in China. These frogs are raised in farms all over the world in places where labor is cheaper. But it turns out, I don’t know why, but for some reason they like raising these frogs. Either they taste really good or they’re really easy to grow or both. But that is also a really big problem, because if you remember these guys carry the pathogen but don’t succumb to it There’s another pathway where they could be spreading frogs into the wild Here’s a photograph of the San Francisco Bay area, and just as an example, and I’m sure there are more around here locally. But we have African Clawed Frog in Golden Gate Park (I think until recently) and American Bullfrogs right next to campus at SF state at Lake Merced And these are just a couple of examples. By the way, they were infected with Bd in those areas So those two species aren’t the only ones There’s the amphibian trade that happens, people love to have pet frogs and those pet frogs get shipped all over the world and many get released when parents get sick of taking care of their kids animals. (As a parent I can say that!) So these are some of the pathways, not all, that we think could have led to the extreme introduction of this pathogen globally, and we’re calling it pathogen pollution is one way to think about it. And it doesn’t have to happen just once, it can happen, it may be happening, sort of multiple events and multiple pathways. If you look at other species of frogs, sorry, if you look at the museum records of frogs in California, there was a big die off of Yosemite toads in the Sierra Nevada that happened in 1977/78, and one of my students has gone back to museum records and looked for the arrival of the fungus and shown that the first positives were in 1967, two animals in 1967 showed up positive, and then by the 1980s and 90s she was able to find a higher prevalence of infection in these animals. Sam McNally, another one of my students, I know those numbers are really tiny, but hopefully you can see at the right hand side of that graph as we go from the timeline, from 1900 to 2000, there’s a bunch of positives showing up beginning at about the 1970s (there’s a few positives that show up a little earlier than that) but the real changes in prevalence, big time prevalence changes, happened in about the 1970s, which is just before those declines

happen If you don’t believe those, here’s another example in California Slender Salamanders where if you look at decadal data there (and these sample sizes are quite big), we’re finding no positives until the late 1960s (a few), and then the number of positives increase through 2000. Luckily for us, species like this, there are lots of samples in museums that were needed to be collected to identify all these cryptic species, but in the meantime, now we have bodies that we can go back and swab and look for the fungus and it’s worked out quite well. One of the things that’s interesting to point out here too, is the percent of infected animals is quite low in this species, it never gets above about 15%. So you might wonder why, well they have a really different lifestyle And if anybody has questions, I’d be happy to talk about that So we’ve used this technique going back though museum records to show that the lot of the declines that happen in Central America happened before 1999 when the fungus was described There were mysterious declines, including the decline of that Golden Toad. Well, we went back and showed that at these mountain tops in Mexico and a couple of volcanoes around Mexico City and Chiapas and down into Guatemala and Central America, that in all of these sites, the fungus shows up about a year before researchers note that they can’t find any more animals for their studies. They had no idea that this fungus was happening and it’s a really clear pattern that we’re seeing across Central America South America also had major declines. This is a map that was published in a 2008 paper, where someone went through museum collector’s notes and noted all the times and places where the collector noted, “Gosh, I can’t find any more of my study animals.” The earliest two in South America were in 1977 in Venezuela and 1980 in Ecuador. And then what Karen did, is she basically took all those dots and years and just connected the dots. And said, well, if it was a pathogen (and a fungal pathogen), then it probably spread in these directions In Ecuador it spread south into Peru and north into Columbia. And in Venezuela it spread southwest down into Columbia and that way And what we’ve done is we’ve gone back through museum collections (here’s where all the data are) and over 8,000 specimens. And this is what the data look like, so the prevalence is on the y axis, the date on the x axis, and these are two different genera of frogs Atelopus and Telmatobius which are two of the most highly affected species, for example the Atelopus frog, there’s about 100 species, 93 are extinct in the last few decades and we think because of Bd, that’s the one that the Costa Rican Golden Toad is from. Telmatobius, same story, about 25 species and at least 80-90% have disappeared very, very recently And if you look at when the fungus showed up, it lines up very, very nicely with those outbreaks in South America. And in this case the prevalence in those samples goes way, way up So what I’m proposing is that we need to do the kind of science that includes not only lab work where we’re doing things like susceptibility trials but also fieldwork where ground truth our results to try to see, well if this species is susceptible in the lab, what are we seeing in nature? Is that population declining or not? We’ve done some of these studies in Peru, we’ve done them in the Philippines, and we’ve done them here in California So these are some of the species we did in Peru, I’m going to skip over this section This is the last little bit. I’m going to talk maybe five minutes or so. And I just want to say, what can we do about this infection? First of all, can we control it? We don’t know how to control it, in the sense that we can’t just go out and spray fungicides and stop the spread of the fungus because we don’t, honestly, it was described in 1999, we still don’t know exactly how it’s spreading from place to place, so if you don’t know that, then you can’t control it. In fact there’s been one study in Spain, in Majorca where someone tried going into these little ponds, catching all the frogs and using a fungicide to treat the area and rid the whole population of the fungus, but then of course the next year the fungus was back again. It’s probably because they didn’t catch every frog or maybe the fungus has a different stage that can survive and sort of pops up when it needs to. One of the interesting things that we found with these populations of frogs that differed between the populations in the Sierra

Nevada and populations in Yosemite and in the Southern parts in the Sequoia Canyon area was that they had different skin microbes, the populations of microbes on their skin were quite different. And that’s what I’m going to tell you about here. Here’s a frog sitting in a little vat of bacteria And the idea comes from this figure here, where you have a salamander that has these interactions, interesting interactions going on between its skin microbiota on the bottom right, or bacteria, which in this case are symbiotic and sort of a positive influence on these guys (they’re growing on the salamander, but not hurting the salamander). And then when this fungus, here’s our Bd on the bottom left, when it arrives it’s competing with that bacteria on some level for the space on that frog. The bacteria actually produce antifungal compounds, there’s a couple of them there including Violacein, which medical companies actually have been really crazy about finding out what they do because they want to treat fungal infections in humans and in so doing we’re learning more about what’s going on with amphibians, which I think is quite interesting So it turns out some of these microbes appear to protect their host species against fungal infections and this is an experiment that we did with Rana muscosa in the lab a few years ago, it was published in 2009, and the title is “Skin microbes on frogs prevent morbidity and mortality caused by a lethal skin fungus,” and what we did is we had seen in the field (and I’m leaving out a whole bunch of details because I don’t want to keep you here until tomorrow), but we had seen that those populations in those two areas in the Sierra Nevada, even of the same species had really different outcomes with the fungus and they also had different characteristics in terms of the fungal species that were on them. And one of the big differences was this particular species of fungus called Janthinobacterium lividum, which was in the last slide, that bacteria produces antifungals, and protects some species of amphibians from this fungal infection. So we did a lab experiment, which was vital for us to understand the mechanism behind this where we had three groups of Yellow Legged Frogs. We collected adult frogs in the wild, brought them in, and then we either added the bacteria first, which is the left hand panel J. lividum, and then infected them with the fungus or we just infected them with the fungus or we just inoculated them with the bacteria and looked at the differential success of the animals. And just so you know, in the ones that were infected with the fungus, just like the wild populations, all the animals died in about a month and a half, the ones with just J.lividum did just fine, in fact they gained the most weight, and the ones that were inoculated first with the bacteria and then infected with the fungus survived, and they survived quite well, in fact the effects of the infection never quite happened, so they never exhibited any signs of disease So the big question right now that conservation biologists are having all around the world, is can we use this idea to protect species in the wild that are being affected by these major outbreaks of fungus. In 2010, we got permission from the National Park Service to go out and try this in a new outbreak site, this is Dusy Basin in Kings Canyon National Park, and I like showing this picture because I’m the only one working, I’m the one there working, everybody else is just sitting around, no, just kidding. We had lots of people working on this project, and what we did is went in and tried to reproduce that experiment in the field. So we had a really nice experiment set up where we had multiple populations, some were going to get the bacteria, some were not, but by the time we got there, there was only one population left. So here’s what we did: we found on some of those animals (a very small proportion, one), we found the right kind of bacteria (this is before the fungus hit), we took that bacteria J. livitum, took it back to SF state, grew it up into big vats of purple, stinky stuff, and then hiked it back up into the Sierra Nevada. So we took the bacteria from one frog in that population and we spread it to other frogs and we didn’t just spread it, we took the frogs and soaked them in the stuff and gave them this big bioaugmentation of the stinky bacteria, with the idea that we wanted to sort of recreate work we’d done in the lab We microchipped all the frogs, and then of course we had to have some poor frogs we didn’t give the bacteria to, so we had control frogs in the same population. And this is what it looked like when we were actually soaking them. We soaked them on two different occasions and two different days for just a matter of hours And this is what the data look like: I’m showing you 2010 and 2011 and I’ll explain why I’m not showing you more in a moment, but we have

these two groups, the treatment group that got the bacteria, the control group that didn’t, that first line of data from 17 July 2010 is showing you the average infection intensity of those animals, most of the animals are uninfected, some of them are infected, there’s no real difference in the two groups because we haven’t actually randomly selected the groups yet, they’re all just microchipped We cultured the bacteria for 10 days from those guys, we had one success, and then we came back 10 days later and spread it to the frogs on the top on the screen and then we came back every couple of weeks the whole summer. And this is what it looked like for the first summer. If we start on the bottom panel, the control group that didn’t get the bacteria, they looked very similar to what we saw in 60 Lake Basin and many other places where the animals got infected, their infection intensities rose over 4 orders of magnitude and they disappeared, that’s why the samples by September 10 and 16 they’re all gone, we weren’t able to capture any frogs, well we captured one frog on 16 September, it was the last one we saw. On the top panel on the other hand, of the animals that we marked and gave bacteria to, although they all got infected, their infections rose over a couple orders of magnitude, they didn’t get up to that 10^4 zone, at least not very many of them, and they tended to survive, not all of them, by the way, about 50, actually about 45 % of them survived throughout the first summer. The dotted line is the winter, we came back the next year in 2011 and were able to recapture animals, only the animals that had gotten the bacteria, the animals that didn’t get bacteria, we didn’t find any in 2011. This is just done with adults. And I want to show you a couple of things, just to remind you that conservation with these frogs and probably many species that we are trying to work with out there, is not a simple thing to do, conservation’s not simple. You know, we figure out fish are a problem; we remove the fish, and then what happens; now the fungus is here. Well, look at what happens to this study lake in 2010 and then 2011 (sorry, it’s a little off the screen), but this is the same day July 16, 2010, July 14, 2011, a bit snowier. The poor frogs hardly got out that summer and then we came back on July 17, 2012 and look at that little, tiny pond There’s a big, beautiful lake behind it, but unfortunately it’s filled with introduced fish. So this is one of those lakes that’s on the list to have the introduced fish removed, but the frogs couldn’t use it. And what happened was by 2012 when we came back, all the surviving frogs had been frozen solid and were dead at the bottom of the pond. So we were finally able to at least do something with the adults to change the dynamics of infection, but it wasn’t a permanent fix because we were down to one population in substandard habitat, unfortunately this year’s going to be even worse up there. So what I just want to remind everyone here is that we need to really think dynamically about and creatively about the different types of factors that are affecting these species. These frogs, the Yellow Legged Frogs, both species, are good survivors, they’ve been there for millions of years. If we give them the habitat and we give them the right situations, they’re going to be able to survive, there are still some populations out there, and those populations for example, in 60 Lake Basin, they’re gone from Dusy now, but in 60 Lake Basin, give me some hope that there is a pathway to survival both in the face of introduced predators and in the face of climate change and in the face of disease and other factors. Pathogens don’t normally drive their host to extinction, it’s not a good evolutionary strategy, so we don’t see it very often. So what we need to do is give these species, in my mind, the room to survive the issues that are occurring, not only locally, but globally With that, I’ll end and give you a chance to answer some questions. I really want to thank my funding source which has mostly been the National Science Foundation, but also the Forest Service has funded several of my projects, as well as USGS and the National Park Service. I’ve been working on these frogs for a long time and I’m really invested in trying to keep these guys around, so if any of you have questions today and

in the future, please contact me. Thank you very much [Question and Answer Portion]