 I also am super excited to be in this session and I'm not in a very enviable spot. I'm in between two of the luminaries of fungal ecology these days between Peter and Kavir, so I'll do my best with my time to share with you some of my enthusiasm about endophytes and why I think they're a great fit for a session that's centering on interactions and communities. I am coming to you from Tucson, Arizona, my living room to be specific, and in doing so I'm coming from the traditional lands of the Tahuna'od and the Pasco Yaqui peoples here in Arizona. So what I'd like to do today is actually start with thinking about the phyloplane, right? So the phyloplane that is the leaf surface that we see all around us is often acknowledged as the largest biotic habitat for microbes on the planet. These leaf surfaces are what we see when we walk into a forest like this tropical forest. However, if we were to do a deep dive into any of the leaves around us we would see an immense internal surface area that is the surface of each of these cells and the spaces between them that together represents an area far larger than the phyloplane itself. That is an area that is encompassed by a tremendous number of microbes, including fungal endophytes. This is a reality that we see when we dive into a seed as well. Again, many cell surfaces that together create what we refer to as the endosphere or the apoplast, and it's in this environment that we find the vast majority of fungal endophytes in a habitat that is orders of magnitude larger than what we can see when we walk into a forest and look only at leaf surfaces. The study of endophytes has a really nice history going back well over 100 years with a special focus early on on grass-associated endophytes and a widespread appreciation that when you take a healthy piece of leaf and you surface sterilize it and you place it onto a growth medium, out will grow and endophyte in many cases. And so for a long time, endophytes in most plants outside of the grass system where they sicken herbivorous and have other impactful ways to interact with humanity. The endophytes that we see in most other plants haven't gotten a lot of attention, but I think they're pretty charismatic. This is a photo of a few of the endophytes that I studied back in my dissertation days. And as someone coming to mycology with an appreciation for organismal biology and a desire to learn about these things, they spoke to me and I hope that you see their beauty as I do. So what do we know right now about fungal endophytes? Well, first of all, we know that in general endophytes are phylogenetically diverse. The vast majority are filamentous ascomaicota and we see them occurring frequently, especially in the sardaria mycetes and dothidia mycetes. Also, we're appreciating them ever more in the leotium mycetes, not only in certain conifers in the pineaceae, but in, for example, high montane habitats in the tropics. Eurotium mycetes, which we encounter in a variety of settings, including, for example, tropical soils where they infect seeds or in aquatic habitats. And Pzizomycetes, which are holding some really interesting surprises, a greater prevalence of endophytism perhaps than was previously recognized. Because these are a common feature across these classes that together comprise the vast majority of the known diversity of ascomaicota outside the lichen-forming fungi. We consider them to be quite foundational symbiotes when it comes to plants. If we go back to the origins of the Pzizomycotina, recent estimates placed that on the order of 500 to 600 million years ago, such that the capacity to make a living, living in close association with the photosynthetic partner likely preceded the great diversification of land plants. Today we know that endophytes are likely hyperdiverse, kind of an unusual term, but one that we can maybe define as representing hundreds of thousands of species. And we know that they're underrepresented in culture collections, in part because of their lifestyle of simply living inside healthy tissue and not necessarily fruiting during the time in which that tissue is alive and looking good. Through the past 20 years, I've been very fortunate to work with a wonderful group of collaborators, including Francois Lizzoni, Jenna Uren, Chuzo Oeta, and our Panama team, especially Jim Dallin, Karolina Sarmanto, and Camila Salamea, as well as many others to collect endophytes at a nearly global scale. Basically going into environments and asking in the common plants lichens in those leaves and in the seeds that we find, what are the fungi that are present? And how can we start to understand something about how their communities are assembled and what they do? One of the nice things is that with more than 200,000 strains now at the University of Arizona, the vast majority of which are barcoded for ITS, we're able to start the long process of describing those that are gracious enough to fruit and culture. That's a lovely subset of beautiful fungi, and it's been a real pleasure to be exploring them, for example, these beautiful species of coniokita. So let's go back then to the endosphere or the apoplast. So this area inside leaves and seeds and other structures is really interesting from an ecological perspective because it represents a continuum with the outside environment. We see this in mature leaves of adult trees, and we see it even in the earliest stages of plant establishment. Here we have an electron micrograph. This was an image taken by Yiling Huang, who was a graduate student with me, and what we see is a spore that is germinated on a leaf surface. It's sent out a hypha, and that hypha is entering into the leaf through the stomata lipidine. And so in this situation, with the vast majority of fungal endophytes having some elements of horizontal transmission, we know that the spores have to be dispersed, the spores have to survive, they have to germinate successfully, and then the foraging hypha needs to find its way into the plant tissue. Now once inside, in the vast majority of plants, we can see the hypha proliferating between cells. So this is another image from Yiling's work. This is coming from Juniper, and what you can see are the fungal hyphae growing between cells here. So they are truly in this apoclastic space, and that's not unique, of course, to plant leaves. We see that here. This is again an electron micrograph. In this case of a tropical seed, this was taken by Camila Salamea, and the seed has been buried in tropical forest soil for a period of time and brought back out. And you can see the covering of fungal hyphae adhering closely to that seed. And as the seed ages or is acted upon by forces like temperature and water, you get the rupture of the colossal plug here at the base of the seed and the entry then of fungal hyphae. So in those early stages, then, we have that continuum from, in this case, growing from soil into seeds and proliferating inside that seed tissue. Now a similar situation occurs with lycanthalae. And in this case, endophyte-like fungi associate preferentially with photobiont cells. So here we're looking at a cross-section of a lycanthalus with our photobiont. And if we micro-disect that photobiont layer out, we get a large number of fungi, very similar phylogenetically and functionally, to fungal endophytes of plants. And Janna Uren did some really nice work on this that helped us understand that even in the same habitat, the endophytes that are occurring inside lycans can be quite distinct from those occurring in angiosperms, in conifers, or ferns, and actually have a very high community similarity to those occurring in nonvascular plants like mosses. And we can see this similarity happening even in complex environments like this, where we have a lycan growing with a variety of mosses and with local angiosperms, we still get this strong difference. So there's something about growing in a nonvascular host that unites those groups of fungi. So another interesting element that's come out of surveys of endophytes is that endophytes appear to be distinct from other fungal communities, and they differ in composition among biomes. So this is exciting because as we move around the world, we can find more and more endophyte species. But it's also interesting to think about the implications for essentially localized co-evolutionary processes. This has been studied, perhaps most thoroughly, by the Uren lab and with the work that I'd like to reference coming out in 2019, focusing on endophytes of boreal systems. And in that study, one aspect of what the group did was to compare sequence data from culture-free and culture-based surveys of endophytes all around the boreal forest built and compare the degree to which those OTUs were represented also in soil fungal data sets using the Tettersue et al. 2014 data set. And the extra is only about 1.5% of the fungi that were present in these data sets were in common, and that was 10 times fewer than expected by chance alone. So the take-home is that by the time we're looking at endophytes that are present in a breadth of ground tissues of plants, they're representing quite a different community than one might find with similar methods in surveying only soil. One way to think about that is that they're potentially enriched for by the conditions within plants, and we'll talk about that in just a moment. Another thing that Janna and the team did was to compare sequence data with similar endophyte surveys that were done in the temperate zone and other biomes. And in that work, Janna pointed out that about 90% of the culture endophytes from the boreal system were uniquely boreal compared to known databases, as were about 97% that were observed by culture-free methods. So this suggests that there is a strong turnover as one moves from major biome to major biome, and that we might expect a collection of relatively distinct boreal endophytes, temperate endophytes, and so on. It's interesting that as we move, for example, from the temperate zone to the tropics, we see similar disparities in those communities. And then finally, we see even if we focus on a single genus of hosts that is widespread, we see high turnover among biomes, even in that one case. So not looking at a global perspective, but only at hosts that represent the same genus. And we can see that illustrated here. This is information for endophytes from cladonia, and we can see temperate forests being quite separate from boreal forest and arctic endophytes in the tundra. So this sort of pattern then is exciting. It suggests a tremendous number of endophytes out there. But when we say that endophytes are unique, sometimes we want to put a little asterisk there. And just one example came out of the beautiful work led by some members in the Zoom room, looking at morels and identifying new morel species in North America, including a mortella kaivensis, which is an interesting one that was detected as a fuller endophyte through database information, and obviously lives its full morel life outside of the endophytic arrangement. So this suggests some interesting biology and the potential for plant tissue like in tissue to basically be a reservoir of hidden diversity that connects and informs other fungal ecology questions. Okay. So for a long time, early on, especially in plant pathology, there was the idea that the apoclast, the space on the surface in between cells and plant tissue, wasn't that exciting. It was mostly just carbon leakage. And that's about all. But now we know that the apoclast of most plants contains really diverse and rich nutrients, as well as diverse and dynamic interactions that we know are influenced by fungal plant communication. So I've just grabbed a cartoon here that illustrates an example where we have an endophyte in this case coming in through is tomorrow for and growing between cells like we saw in the photos at the start. And the accumulation of evidence is that in this able plastic space, endophytes can modulate plant hormone production and signaling. They can secrete cell wall degrading enzymes and trigger basal immunity. They can release effectors or not either invoking or avoiding plant responses. They can trigger jasmonic acid or salicylic acid pathways, which influence plant interactions with other organisms. In some cases, they can induce lignification or callus deposition, thickening the plant cell walls, making the plant more robust. And then finally, they can produce secondary metabolites with diverse impacts. So I wanted to take a moment and think about those secondary metabolites and just highlight some of the metabolic diversity that one can find associated even with a single strain of a relatively well-studied genus like xylaria. So this work is highlighting some of the sesquiterpines that are associated with this one strain of xylaria when it's grown on a single medium under controlled lab conditions, leaving aside how the expression of these and other metabolites might be influenced by the substrates present in a given host or environmental conditions. So what's interesting is that when we start to look at secondary metabolite production, we can see that not only the production, but also the impact varies at a landscape scale. So for this, I'll just take us on a short trip to the beautiful cloud forests of Central America. And here, I wanted to highlight the work of Sir Higginbotham, who looked at phylogenetically paired endophytes that one could find occurring in cloud forest and occurring in lowland human forest. These were phylogenetically paired to the points of being in the same genre, sometimes even the same species occurring at low elevations and high elevations. And what's interesting is that in vitro, when the secondary metabolite activity of the swengi was evaluated, what Sarah found was that the percent inhibition, that is the activity of secondary metabolites with a cytotoxic effect, was much higher for the endophytes from cloud forest and much lower for endophytes from the lowland human forest. And this was a bit of a surprise. We actually went in thinking that those lowland forests would be where we see the majority of the most potent secondary metabolites. But as we thought about it, we realized that the ever wet often clouded conditions with high diversity of tropical highland or cloud forests actually creates an environment of very high degree of competition among co-occurring endophytes. They flourish with relatively moist conditions, places with relatively thick long-lived leaves, and so on. So we think that what we're seeing is actually an ecological pattern translating to what we can observe in vitro. So with that in mind, we've just talked about how endophytes may be signaling to their host plants and interacting with them. We can see signatures of that with the secondary metabolites. But what's interesting is that even though the interactions between plants and endophytes are happening in this internal space, they can have a subtle to very visible context-dependent and interesting phenotypes. So one new one that we're excited about is basically the ability to remote sense endophytes in leaves. So I mentioned that some endophytes will induce lignification. This creates a sufficiently different signature of leaf structure that we can detect it by measuring leaf reflectance. So in certain wavelengths, we can see distinctive signatures of endophytes being present in a leaf. We also know that endophytes can be negative for their host in some cases. So where I work in the tropics is a seasonally dry forest. It has a prolonged drought during the dry season and plants are water stressed. An interesting aspect of endophytes in that environment is that they can increase minimum leaf conductance. That is the amount of water that is lost from a leaf when the stomata are maximally closed. So it raises an interesting question of potential costs and benefits being balanced in a variety of environments. And in the lowland forest where we work, this detriment we think is broadly balanced by the ability of endophytes to protect their host plants against disease. And so this is an example from Theobroma cacao. This was a project led by Luis Mejia with Alan Harry at the Smithsonian Tropical Research Institute. And because endophytes are horizontally transmitted, we can grow seedlings under controlled conditions and then introduce endophytes, in this case multiple endophyte species, to reconstitute what a natural community would look like. We then can introduce a pathogen, in this case chocler. What we see are the leaves that have only the pathogen have vastly more leaf damage than leaves that have endophytes present. What we can't see is that actually the leaves with endophytes were twice as likely to survive even before they got to this point. So we see this localized benefit, often on a per leaf basis, not a fully systemic basis. And it raises so many questions about what those interactions might be like inside leaves and how that can help us understand the rules of community assembly. One last thing that we'll note is that sometimes we can capture endophytes from leaves and apply them to other plant tissues and see beneficial impacts. And that's what we're seeing here, a case where a foliar endophyte has been applied to seeds of wheat in the top and they've all germinated and they're growing and they don't necessarily look like field grown because they've been in a petri dish but they are happy in comparison with the endophyte untreated seeds down at the bottom. So this opens up on the one hand the exciting possibility of translatable phenotypic modulation. That is you capture endophytes from one tissue or one plant, you apply them to another plant and you get a phenotype that may be useful for humans. And that has prompted some of our recent investigation looking at endophytes of wild relatives of crop plants. So this is a selenium, I'm growing in this lush field, this is here in Arizona, this is agriculture in Arizona, lots of dry space with nutrient poor soil. Here we have this wild relative of crop plants flourishing. The question is what endophytes does it have and how those contribute. So that's an area of inquiry that we're pretty excited about doing that kind of translational work. However, there are some challenges and that is that we know that not all plants will accept the same given endophyte and we see that in part through two different lenses. So the first lens is that the interactions that we observe can be driven by additional hidden components of the symbionts and that is the endohyphol bacteria that live inside the endophytes themselves. So here this is an image by Joe Spraker, we're looking at the outside of a fungal hypha now for our endophyte and in a moment we'll zoom in and on the inside we can see those beautiful bacteria flourishing. So these are endohyphol bacteria that are important in a variety of ways. When we've done removal experiments we've been able to see that they enhance growth of individual endophytes on the majority of carbon substrates that we look at. And this includes sugars that are found in seed coats, regulators of stominal closure, and drivers of seed germination and plant growth. So this is just to remind me to say that we can see it in vitro and we can see it with seed viability here with tetrazolium stain and it's something that we're now exploring under field conditions. We also know that there are some interesting additional impacts of these endohyphol bacteria. These data come from Michelle Hoffman's work back in the day and what we're looking at is the production of indel3-acetic acid an important phytohormone over time for the same strain of fungus that simply has its bacterium presence or has had that bacterium removed. Now we don't know a priori whether or not we're seeing differences in production. Those are somewhat different. We also know though that we see modulation of cellulose activity, we also see thermal tolerance. And so the presence of these additional microbes can influence how a given fungus will exist in the landscape. And in one area that this is especially exciting for me is I'm thinking about the saprotroph to endophyte sort of continuum that we see in xylaria and related fungi. So if it's the case that micro microbe associations, which are facultative, they're very flexible, different fungi can pick up the same bacteria in the Ascomycota that we survey and we'll hear more from Greg Benito's team a little bit later about other fungi. This could play a really important role in defining cellulose activity along with the intrinsic effectors that are part of the communication between particular plants and fungi. So as a result it's not surprising that when we go out in the environment we see communities of endophytes varying among hosts in most of the environments where we look. This figure summarizes a lot of work that again led by Janet Uren and here you can see our surveys that occurred around the boreal forest belt in places ranging from Sweden to Central Russia to Eastern Russia to Alaska and across North America. Here you can see the different lineages of hosts and the take home message is that when we go into an environment and sample thoroughly we will see distinctive endophyte communities associated with different hosts and different host lineages. When we're doing this kind of work it's observational but we can capture this perhaps even more powerfully by moving from the boreal environment like we see here to the tropical environment where we've set up experiments under the leadership of Camilo Salomea and Carolina Sarmiento to look at what fungi are actually getting into seeds in such a important moment of seed dispersal and establishment that is the key to fitness for tropical trees. So to do this what we've done is across BCI we've had five common gardens we've worked with 18 species of tropical trees we have placed seeds into these little mesh bags and buried them for up to 36 months in these gardens and what we see and as the strong emergent pattern is captured by this sort of variance decomposition in common garden with distantly related pioneer trees after about a year in forest soil we see a strong signature of plant species that is the communities differ one plant species to the next despite being in a common garden environment and that explains much more of the variation in those fungal communities than does burial duration garden location or even whether the seed is viable or not. So that's for distantly related angiosperms what happens if we look at closely related ones after 36 months in forest soil once again a strong signature of plant species. So it tells us that there is some filtering that's occurring to select or allow to flourish certain endophytes in certain posts. So where I'll end up then is just stepping back a little bit and saying when we're looking within a site we can often see that signature of a host effect variation in end effect communities among hosts. If we go to a landscape scale what we can start to do is integrate questions about for example how does environment or climate fit into that picture and here I just want to highlight some work that we did recently going across the Isthmus of Panama and working from the seasonally dry forests on the Pacific side to the ever wet forests on the Caribbean side and these data come from leaves of angiosperms and our Illumina data amplicon sequencing and what we can see here is a strong signature of temperature seasonality on species richness of endophytes that is in the least seasonal forests. We see the highest richness in the most seasonal forests okay we're able to detect a lower species richness and these results are sort of striking because our sites are less than 50 kilometers from each other yet they have a strongly different climate. Here we're looking at the community assembly and in this case with our non-metric multi-dimensional scaling we can see the structure again among these different sites that are different only by a few kilometers with a strong signature of temperature seasonality mean annual temperature and mean annual precipitation and then finally it's very interesting that host range also seems to vary as a function of temperature seasonality and we see the evidence here so it suggests that with insights we've got a powerful signature of bacterial plant fungal interaction and fungal plant interaction at a broader scale we see the role of climate and an even broader scale moving out of the isthmus of Panama we can detect in this case with our map showing mean annual temperature a strong signature of climate that we think underlies the original latitudinal gradient of endophyte diversity that was proposed back in 2007 so our stars here are showing some of the places we've been fortunate to survey over the years so that raises the question how do endophyte communities change over time as the climate is shifting and here I just want to highlight some work that we're excited about we're able to use Illumina approaches to look at endophyte communities in preserved plant specimens in herbaria and so this work originally was led by Barnabas Daru with Don Feister and PhD student Liz Bowman and now we're going to extend our sampling to the Arctic basically looking at communities that are present in herbarium specimens collected over the past 100 to 200 years and going back and collecting afresh from the plants and lichens that occur in those areas today so we hope that with transects we'll be able to look at climate change over time so just to wrap up then a lot of what I've talked about has been the ecological perspective but one of the exciting frontiers is to start to integrate endophytes into the fungal tree of life and in doing so we are working on how to deal with lots of ITS sequences so I'm very grateful through NSF dimensions and genealogy of life funding and the leadership of Ignacio Carbon and especially Francois Luzoni for the development of some new tools that allow us to with a degree of certainty place endophytes into deep phylogenetic contexts and what is the upshot of what we've seen so far the first is that we see repeated pack processes of local diversification so it's not just different sites have different endophytes but there's been local diversification of endophytes in many of the sites where we work we see that here in the Dothidium I seeds and in the Lucio my seeds and so this means that a plant in a given area is influenced by who it is the bio climatic zone in which it occurs and by the availability of the endophytes that are there so it's this that then leads us to think about the global scale of interactions and viewing that as a mosaic that is influenced by bacterial fungal fungal plant plant environment and ecology evolution interactions so that's where we are now and thinking about endophytes and I just want to thank you so much for your attention I want to thank Don and Alina for hosting this symposium and again Don thank you for all that you've done for me and so many of us in my ecology I want to thank my many collaborators and students and then finally thank the funding agencies that have supported the work on endophytes over the past couple decades so thank you so much and I'll pass it off to Kibir