 This, this particular symposium is really exciting for me. And I wish I could be there to see all the familiar faces and colleagues. But it is really exciting to get to share the science so thank you for this opportunity. So, my talk is about hunting for dark matter fungi, and it's going to have a tree of life talk. And let me sort of explain why I chose that title used to sort of like metaphor of a tree in the dark, and that is our fungal tree of life. We, we generally know its structure we know it's big, but there's a lot that we don't know about it. We sample extant taxa and we, and we observe their characteristics but we know that that's just a small proportion of the total diversity. And, you know, we want to use this tree to do many things in biology, but there are dark areas of the tree where we don't have samples. And when we, you know, try to infer a phylogeny and trace some kind of character on it. We're subject to what we know, and what we don't know has an influence on the result that we, that we get. For example, this is a reconstruction of say the, the flagellation or the presence of a flagellum and fungi. And this is maybe an earlier view. What we want to do is to know the tree better and to understand that it's all all the various regions where we, we didn't have data before the so called dark branches of the tree. And when we do that, and when we look at the character states of those, the total tree we can see how having better sampling completely changes our understanding of the evolution of a character so like for example if it was a flagellum. So, you know, this, this term dark matter fungi is used to describe the fact that there's this huge gap between what we know and what we predict in the dark matter is the stuff that we predict on the basis of estimates of total diversity. And those estimates have been a major rallying cry for mycology in the last two decades. And we know that there's a great diversity out there that we've yet to really describe well. And what we have described is just you know maybe 10% or even less of the total diversity. And we're told so well, I want to make a point that even of the stuff that we have described. We don't have DNA data for it so in some sense it's hard to compare with the, the rest of the predicted diversity, but you can go out and do a study like this really important study by teachers to at all, where they did this global sampling and cataloged everything they found and found you know about 45,000 species, and only about 10% of them fit in with this described taxa. So, here we're tapping in and saying that there is this dark matter of taxa that are we know exists because they basically appear when we do a DNA sequencing study but we don't actually know what those species are. And, you know, and if we try to use approaches like classical methods of culturing, they, they only reveal very small proportion of the total diversity, but those studies are really fundamental, and we'll talk about one reason why or one example and which this was the case. So our lab is is trying to tackle this dark matter problem by one having a well constructed phylogeny that serves as a is sort of a roadmap or a backbone for for putting this novel diversity into context phylogenetic context. We are also using the using these approaches of like meta bar coding to try to explore areas of the tree which we think are under sampled and are likely to contain this, these dark matter taxa. And we're specifically interested in going after taxa that we, we know have been described so that we only, you know that there's only two thirds of the tax that have ever been sequenced. I'm sorry if only one third of the tax have ever been sequenced we know there's a lot of stuff out there that we that have been observed, and we want to try to track those taxa down and sequence them and then try to compare them with the, the tax that have been described on the base of DNA sequence alone so we want to bridge basically microscopy and DNA and and then once we have sequence we want to to to predict what those organisms that we're detecting it can actually do so we're working on on approaches to take genome sequences and then say something about the ecological function or cell biology of this organisms. So, I'm going to first talk about our tree of life work and make the point that a lot of what we think of in terms of fungi when we when we discuss fungi is focused on the, the dye carrier, and there's a good reason for it because these are 98% of the described species. But it has a particular life cycle that's attached to it in the in the name dye carrier comes from this heterocariotic stage but the the general idea and this is something we teach in in basic biology and introductory genetics is that fun I have a very different life cycle than other kingdoms like animals and plants where the animals have a diploid dominated life cycle and plants have alternation of generations and fungi are supposed to have mostly haploid mitosis and in very little diploid mitosis but the point is that this is really based on observations and by carrier, and you can even, you know, listening to Anna talk you can see we really don't even have a great understanding of, of life cycles of AMF fungi these ubiquitous fungi. And but then within fungi, there's all this diversity of lineages that have been poorly studied and especially in terms of genetics. Okay, so our, our, our understanding of fungi is very tilted towards dye carrier. And our lab is been really interested in exploring many questions using so sport fungi and so sport fungi are just basically the fungi that reproduce with a modal spore. And they do things like attack pollen and attack algae. And local zoo sport fungus, sometimes they're called kittrids produces like a sporangium and the sporangium has these filamentous rhizoid that are involved in anchoring and nutrient absorption. And these are, these are the zoas bork fungi and they are that zoas bores is sort of an, an old homology with the animal flagellum and sperm cells. And so that's a trait that existed in the early branches of the fungi and was lost as we go deeper into into time in the fungal tree. And we talk about our phylogenomic reconstruction based on the kittrids and it's really, you know, it really owes itself to the people pictured here. Joyce Longcore and Martha Powell and Pete Letcher and here's Raven Simmons, Joyce's PhD student who's now postdoc in the lab who, who did a lot of this work and. So the point is that these, these great biologists have have devoted their lives to understanding the diversity of the kittrids and they developed these massive culture collections. And that's something that I've been fortunate to become the steward of and we have this emerging collection called the collection of zoas bork you fungi at the University of Michigan. And just to say I wanted to give credit to to these wonderful biologists before talking about what the data show. So we have all these cultures and we're generating a phylogeny from them. You know, it's not a ton of points I'm going to belabor here but we, we sequenced about 70 kittrids, and we predict their, we add them to the existing data we then search for homologous genes, and then we filter them and then we have you know a couple of simple approaches that are pretty standard now for phylogenomic methods one is that you take all the data and you concatenate it together, or you estimate the individual gene phylogenies separately and then you try to fit them into this best fitting species tree. At the end we had about 487 proteins that we felt were pretty reliable. And this is what the data look like. So obviously this is pretty hard to read in it, and a lot of it is some interesting detail in terms of the kitchen taxonomy. The main points are that the trees relatively well supported the the star or the diamonds here are nodes where there's a difference between the concatenated tree and the coalescence tree. And, but what we, what we recover us is seven distinct phylozoasporic fungi. So we have rosello mycota which go with micro spritia aphelidae mycota, then branched to catrida mycota, neocalamastica mycota and monobloffardo mycota man this is a really mouthful. But then there's and then there's two additional phyla here. And I'm going to kind of gloss over these characters and focus really on on the one which has to do with life cycles. And this is where we got the data from so when you do, when we do this genome sequencing, if we use short read approaches so like Illumina genome sequencing. We have a very high coverage and that coverage and it's pretty accurate to, we can use that coverage to look at heterozygosity and Anna talked about this in her last talk, but what the, the two things we can look for our coverage of particular words so in here we have a plot of the coverage which is also depth of 23 base pair words. And here's sort of our modal coverage here and around 700x and where the word is interrupted by a polymorphism, it divides our coverage in half. It will split this, those that particular word into two equal words, equally abundant words. And when we have high coverage we can observe that the frequencies of these two alleles like this a and this T and this in this particular example are right around centered at 50%. So, when we look at and see that our leal frequencies look like this we have very high confidence that this is a bona fide heterozygosity. So we're taking all this data together and we tried to reconstruct the ancestral states of ploidy in the early branches of the fungi. And what we observe is that also in this, in this tree this is like the outgroup here, the blue circles are diploids that we recovered and the reds are haploids that we recovered. And what you can see is that blue is spread throughout the phylogeny. And there's some clades that are almost always haploid and this would be like Spicella mycetes and mucormicoda always came out as haploid. But most of these zoosporic lineages have a large proportion that are diploid. And just down here are the likelihood estimates for each of the two states at some critical nodes. So, this sort of the thing that this has told us is that the fungi for a large portion of their evolution were either diploid or could could be diploid in the dominant part of their life cycle and I say this the dominant part of their life cycle is because these are mostly cultures right so where they're growing in this mitotic state and they turn out to be diploid. Okay, so now I'm going to go to the second thing that we're doing which is to, you know, add into the tree in places where we know that there should be these interesting environmental DNA based dark matter so things that parts of the tree that have very high diversity, very little voucher material. To do this, this is my cartoon of the current gold rush we're in is you take, you know, your sample and you put it into the sequencer and outcomes is really interesting by our diversity, and it's really, it's really fun stuff. But, you know, how much longer can this go on how much, how much more will we be gaining in discovering in terms of novel diversity. So, at least in my area of the of my college yet. This was a major discovery, which was that right at the base of the tree, right at the base of the fungi. There's a group that we call either crypto my coda rosella my coda that has a ton of diversity, but based almost entirely on environmental DNA sequences. So here's the paper that was now it's 10 years ago, where they showed some of this diversity and all of this bit here in this in this area is environmental DNA sequences and then somewhere near towards the root of this clade is rosella my coda rosella mycis which is a endo parasite of water molds. Okay. And so, here's an example where a large amount of fungal dark matter resides. We are trying to understand like what this stuff does and in the approach that we're trying to use is to sample from different places and then to look particularly at at the type of sample where we're, we're, we're recovering or recovering and then to relate those samples to each other and habitat is of course, the very crude way of looking at them but when you do this by habitat you can see that samples taken from soil are distinctive to some degree of his samples from fresh water. And we believe that probably most of these crypto my coda rosella my coda are our parasites and so it makes perfect sense that you know soil would be distinct from from water. Another very interesting group that is also considered a dark matter clade is RKO rhizomycetes and this is the group that Anna Rosling described. Now also 10 years ago, and you can see that that. One of one of the things is that there's just a ton of diversity in this group, and all of this is is known, almost exclusively from the central DNA sequencing, but there are these serendipitous cultures that can be made. And these are really important so it sort of goes to, as a point to emphasize that not only do we need to continue to culture, we need to find better ways of so that we can can have material for which we can start to to look at and to generate phenotypes. Here's a picture of this RKO rhizomycetes inoculated onto a pine seedling seems to like growing on the seed seedling roots but it doesn't really colonize inside the plant. It's still actually a pretty big mystery what this fungus is doing in nature. So, here's two pretty, pretty large groups full of dark matter fungi. Try to answer to a little bit this question of how many more lineages could there be out there. Returning to this teeter sue study. This was done using ribosomal ITS marker. 6% of the, the taxa could not be assigned to file them. So that's pretty exciting and could potentially mean that that these taxa are novel file or at least really novel branches on the fungal tree. So, what teeter sue did was to take, so he just had this like small bits of ITS to region, and then decided to, and so you can't make a tree you can't put the stuff in a tree when you only have ITS to. So, the approach to use was to sort of use that the sequence in ITS to to PCR out to the flanks so a primer inside a known sequence and then out to general primers and track down these, these placed sequences. And, and this is the tree that resulted from this and the things in red are from that study, and you can see they placed in all sorts of parts of the tree. There's some clay in glomeromycota. There's a ton of stuff that placed in the Rosella mycota. And there's even a clay that's related to our care rise of my seats. But what didn't really happen was the discovery of many new phyla. There is this one really basal clay GSO GSO one at the base but other than that there weren't probably any phylon level diversity that was uncovered in this. Okay, so the the next part I'm going to talk about has to do with our approaches to link the historically described taxa to to the the environmental DNA. So what we really want to do is to be able to visualize taxa in in nature and to without and and skipping the culturing step go straight to sequencing. And then having our eyes on that on that cell. We now can start to say something about that organism. So, here's a single cell sequencing workflow but the bottom line is we take the environmental sample and then we have to sort out single cells. And there's fancy ways and then ways that also involve like looking at what you're trying to to sample first like laser capture micro dissection, but you need to get a single cell in a tube. And then from that single cell extract the DNA from it, and then goes this whole genome amplification step, and then from there once this works, you can get it go into the genome analysis, but one of the things our lab has been really focused on is is going just simply to, to this step here where we're just trying to get a barcode, but we don't want just it s so we were using this long art RNA PCR protocol developed by Kurt Spocker, and it involves amplifying most of the SSU and LSU, and then we run this on Oxford nanopore and then we can place it into the phylogeny. And one of the projects we've, we've focused on is the is trying to go in and sequence the predatory zygomycetes the predatory zoopigales, which are taxa that have not been amenable to culturing. And this was worked on by William Davis and Kevin Ames, where they discovered the taxa and and then got them into tubes and then sequence them. And there's some really interesting fungi here like zoophagus is the trap rotifers and then you can see that the nice hyphae growing inside the rotifer. Here's an amoeba that's being attacked by Kokolonima. And this is an example of you know how you can make a tree now that you have this this data from the single cells and start to identify and put names on some of these like environmental DNA. And these were all amoeba parasites. So this, this becomes useful now that we have matches to some of these environmental sequences we can imagine what they're doing. Here's an example where we sampled at Northern Michigan and Smith's Fenn as a place that's historically been sampled for by Kittred biologists. And we kind of attacked this place with both single cell sequencing and as well as as meta bar coding. And one of these samples, William Davis identified this Kittred here is on this green alga does medium, and we were able to match it closely with this pack bio sequence from just from extracted and sequence from from the water there. And this groups within synchytrium which is an interesting important result because synchytrium is mostly known as as vascular plant parasites. Yeah, this is just, you know, in the last example but we're, we're also working together with Mike McKay on diatom blooms in Lake Erie and these diatom blooms seem to occur under the ice in the middle of the lake. And of course where there's diatoms, there's going to be Kittred's, and we're finding a great diversity of Kittred's that are associated with these winter diatom blooms and this is work done by kensiki seito and Rayburn Simmons, and they're, they're now able to identify this major group of diatom parasites that are important in Lake Erie and also bring some of these unnamed dark matter sequences to the, to be able to say these are probably also diatom parasites. Okay, so just a little bit of time left. So we can observe those, those taxa and now we can start to, we can see those cells, the kensikas finding those cells in, in the environment and then go in straight to DNA sequence and then putting it in phylogeny. We would also like to go further and, and do genome, genome sequencing so that we can start to actually say something a little bit more about the function. So here's a cute diagram that Doina, I think it was Doina made for her recent paper on the pipeline for single cell genome sequencing. And, you know, so you've had this pool of diverse cells and eventually you want to use various approaches to get down to a single cell. And, and then you've got that and now your sequence is genome. What can you say about it? Well, one of the issues you have is that you really don't have the whole genome, because it's single cell. And, and so you need to understand that the absence of, of a gene is not really telling you anything because it could just be a technical reason why it's not there so you have to work with what you do have. One of the things we can do is we could estimate ploidy. We could look to see if the, if it makes a flagellum because that's a lot of genes associated with making flagellum, mitochondrion, ditto. Then we can look and see what nutrient transporters it has, what could it take up, what could it excrete. And then we can look at known cell wall genes, genes for high full growth, carbon utilization, and then we can also look for endosymbionts, or also effectors. So the endosymbionts thing is relatively interesting and easy if they're present in your single cell, it might stick out really, really nicely or really obviously. And single cell sequencing is known to be subject to a lot of contamination. But in this particular case we have this nematode predator stylopege, which consistently had the presence of this bacterium. And we became really convinced that this bacteria was actually inside the cells because the, the endosymbiont of the stylopege is closely related to these more Tirella endosymbionts. And I think Greg Benito is going to talk about tomorrow. So you can find this really interesting endosymbionts. That's, you know, a case where we knew the target we put that spore in the tube. And there's also cases and this is work done by Stephen Art and Doina Chivano at the JGI, where we go back to Smith's Fen, for example, and we sample random cells by flow cytometry. One of the things to notice this is like genome completeness. And, you know, some of these cells these ones in blue are the ones I particularly want you to look at have pretty low completeness sometimes or mediocre completeness. So these presented challenge but also opportunity to sort of peer into what these dark matter fungi are doing. So one of these cells BAO use you see is this here in the cryptomycota. We don't have much data for it. We have a very low number of genes but just using this approach and this isn't completely cooked down but we could say like okay it seems to have a mitochondrion but lack of flagellum. We couldn't detect chitin synthase, but there's only so many genes and genome for that and we're really incomplete here. And then our other approach where we find the thing and we put it in the tube like this so Peggy. We can say a little bit more because we know you know the host is an amoeba that the actual reactions and sequencing works better because we have many cells here. So, wrap up with the take home points. The dark matter fungi are really wherever you look, and there's plenty of surprises yet to be had but we probably have have sampled the majority of the lineages that are out there. Although there is a caveat that you know if we change approaches we might start seeing different things I not just doing it sequencing. I want to discuss these other major points I see that I'm out of time. I wanted to put up this quick note that we are trying to use this reverse ecology approach to to to really understand fungi better and to just to start to develop the methods and I'm looking for someone to join the group. I'm interested in a postdoc fellow who will be interested in working on amphibian symbiosis and model specifically model development with this dwarf African frog. There are so many people to thank for the contributions to this, these projects I discussed, particularly the folks in the lab you've been a really stimulating group to work with real honored to have the chance to work with you. And the folks at the top here were really instrumental to the work I talked about today. Also thanks to the joint genome Institute for their collaborations with the single cell genomics and thanks. I think I'm out of time. Look forward to discussing further with you.