 Good day. I'm Max Hegblom, Editor-in-Chief of FEMS Microbiology Ecology, and it is my pleasure to welcome you to this webinar in Ecology of Soil Microorganisms. Our webinar series enabled us to highlight different topics of microbial ecology to a broad audience. Those of you who have tuned into our previous webinars knows that FEMS, the Federation of European Microbiological Societies, invests in science. We use the income from our journals to fund charitable activities and support our community. It is important to note that learned societies and their journals provide grants to scientists, organize and support conferences and summer schools, and sponsor a range of events such as this webinar series, which provides a forum for the presentation and discussion of key research enabling the flow of ideas to a worldwide audience. As an author, when you publish in FEMS journals and when as a reader you use these publications, you directly support the various activities in which FEMS invests back in science. As Editor-in-Chief, one of my rewarding tasks is to select topics and speakers for these webinars from our collection of papers. Note also that if you missed some of our earlier webinars, they are available via the FEMS OUP websites. Indeed, before we start, I wish to thank the staff of FEMS and Oxford University Press for all their work behind the scenes in making these webinars happen. Also a big change for the journal is that from January 2024, the journal will be fully open access. This means that all the archives will also be available free to read regardless of your institutional subscriptions. In addition to ensure that article processing charges do not become a barrier for authors, there are a number of discounts and waivers available, so please check the website for more detail on this. Today, our three speakers will take us on a captivating journey into the ecology of soil microorganisms and learn how soil bacteria and fungi respond to stress conditions, habitat restoration, and change in climates. Our three expert speakers will unveil some of the adaptations and roles of microorganisms in regulating soil ecosystems. First, Emily Sully from the Department of Environmental Systems Science at Eteha, Zurich, and now at the Helmholtz Center for Environmental Research in Leipzig will discuss the impact of water limitation on soil microbial communities involved in nutrient cycle. Then Nicholas Barber from the Department of Biology at San Diego State University will discuss the restoration age and reintroduced bison and how they may shape soil bacterial communities in restored tall grass prairies. And finally Anna Escalanta from Institute of Ecology in Universidad Nacional Autonomia de Mexico will present on the functional significance of microbial diversity in arid soils. After the talks, we will open the session for your questions and for discussion. So please submit your questions and comments via the Q&A link and we will get back to these at the end of the session. Also to note before we get started that if you're interested in learning more about the ecology of soil microorganisms, we have a thematic issue on this topic coming up in early 2024. So again, take a look at the journal website. But now it's my pleasure to introduce Emily Sully, our first speaker and she'll get us into nutrient cycling in soils. So Emily, welcome. Hello everybody. I will share my screen. Thank you very much, Max, for the nice introduction and also for inviting us to present our work in the FEMS Microbiology webinar today. I will talk about the impact of water limitation on soil bacterial and fungal communities involved in nutrient cycling in Scottspine mesocosms. And I would first of all like to thank also my co-authors, in particular Astrid Jaeger, who did the assessment of the microbiome for this study. So you might be wondering why we're interested in understanding the impact of water limitation on the microbiome in the soils associated specifically with Scottspine trees. The reason for this is that climate change is predicted to continuously increase the frequency and severity of drought periods, mainly due to the continuing increases in temperatures. And this is unfortunately leading to several episodes of tree mortality, which have been recorded at many sites globally. Also in Central European Alps, several in several regions, episodes of tree mortality have been recorded, where the dominant species is Scottspine, Pinocilvestris. And also reductions in tree growth and vitality have been observed in the southwest of Switzerland that you can see this area marked in red in the map. Here also from the figures, you can see that the forest is not doing well. Scottspine trees have actually been suffering from drought. And you can see from the pictures that the trees in orange are actually dying. This is not in autumn, so they're not discolouring because of autumn and seasonality, but really because they're suffering from drought and and losing vitality. The valet, the canton valet is a very bright area of Switzerland, and also in general of the European Alps, mainly because the mountains are blocking the air masses. And so this results in lower precipitation, in comparison to other areas. In this canton valet, the precipitation has not really been changing over time. However, the temperatures have been increasing, and so mainly the loss of water through evaporation from the salt has caused tree mortality episodes during the last decades. And the situation, unfortunately, seems not to be improving until the end of the next next century. Predictions of climate change are actually indicating that the temperatures will continue to increase without some efforts, mitigation efforts, by four to seven degrees. And possibly also during summer, the precipitation rates will be reduced by 40%. And this is, of course, very problematic for forest functioning, because trees also sustain ecosystem services. For instance, especially in mountainous areas, they protect against rockfall, avalanches, and soil erosion. Also, not only mature trees are dying, but also very young trees which are developing in the forest due to drought. And therefore, it is important to understand how young trees are also resistant to water limitation, to understand how forests can actually recover after extreme events of drought. Drought and water limitation doesn't only affect trees, but it also can influence the soil microbiome. In forest, organisms such as fungi, bacteria, archaea, protists actually play key functions such as decomposition, nutrient cycling, and symbiosis with trees. And an alteration of the soil microbiome due to drought might also lead to then influences, negative influences on soil health and the nutrient cycling. Water limitation does not only affect the soil microbiome directly, but it can also alter the soil microbiome through changes in physical chemical properties in soils and through alter plant parameters. For instance, due to water limitation, plants can undergo a series of physiological changes. For instance, they probably altered the amount of inputs and the chemistry of the inputs to the soil, which are very important energy source for some microbial communities, and also changes in their growth. For instance, an alteration in the root growth, usually the growth of trees is reduced due to water limitation, can also affect the amount of exudates released to the soil, which can in turn induce shifts in both in the functioning of the microbiome, but also in the community composition. So the main aim of our study was to assess the response of soil prokaryotes and fungal communities to different levels of experimental water limitation in Scottspine mesocosms. And we were also interested in understanding how changes in soil physical chemical properties and plant growth would affect the soil microbiome. We hypothesized that water limitation and alter resource abilities would induce a community succession towards an enhanced abundance of sapiotrophic tax involved in the decomposition, degradation of organic matter derived from plants, that organic matter from plants. At the same time, we expected that water limitation would decrease in biotic tax and rather increase oligotrophic bacteria and desiccation tolerant tax under water limitation. Finally, we also expected the composition of the soil fungal community to be more resistant to water limitation as compared to prokaryotic communities, mainly due to the ability of fungal communities to produce hyphal network, which allow fungal to basically scavenge larger areas of the soil for nutrients and water, of course. So to basically tackle these hypotheses, we set up a mesocosmic experiment in the greenhouse of VTH Zurich in Switzerland. We set up 18 mesocosms. You can see in quite large pots, as you can see from the image here, and the soils were filled with natural forest soil from the forest that you have seen before in one of my slides affected by drought during the last decades. And also the tree seedlings that we or saplings that we planted here had an age of three years at the time of the start of the experiment. And the experiment was set up in September 2019, and then we basically watered, we irrigated the mesocosms to field capacity for four months in order for the mesocosms to basically acclimate the new conditions in the greenhouse. And after this period, we then started the irrigation treatments. We had three treatments, a controlled treatment, which was for which the mesocosms were maintained at field capacity, and then we had two water limitation treatments, an intermediate and the severe water limitation treatment, for which the amount of water was reduced by 40 and 75 per cent. After the start of the treatments, we then sampled on a seasonal basis, and we analysed all sorts of parameters for the soils and for the plants, for instance pH, nutrients in the soil, but also growth of the plants and gas exchange. And we also analysed and assessed the soil microbiome using DNA sequencing and the QPCR methods for the quantification of fungal and bacterial ribosomal markers. And I would like to mention, again, that the analysis of the microbiome was done by Astrid Jäger, who was the PhD involved with the project, and unfortunately she could not attend the seminar today, and this is why I am presenting. As you can see from this graph, we did a very good job with the treatment, so that at the beginning of the experiment, all of the treatments had basically the same gravimetric water content in the soil. And after the start of the irrigation treatments, the amount of water in the soil started to decrease for the water limitation treatments, as compared to the control, and we always had the lowest amount of water in the severe water limitation treatment. You can also see in the graph that there was an seasonal variation over time, and this is because there is a confounding effect of temperature on soil moisture when the temperatures are higher in summer. We regulated the temperatures manually in the greenhouse to basically follow a seasonal pattern. And during warmer temperatures in summer, then there's higher evaporation from the soil and also higher evapotranspiration by the trees, and this is why the amount of water in the soil also decreases. Nevertheless, the amount of water in the control treatment was higher as compared to the water limiting treatment. You can also see that it was a bit more variable. One year of water limitation, very interestingly, did not alter the relative abundance of either the prokaryotic unfungal communities in the soils over the control course of the experiment. Nevertheless, we did see a peak of abundance for both communities in summer. And this could be likely because of the temperatures might stimulate the growth of organisms which favor high temperatures, which grow better under high temperatures. But it could also be related to the fact that plants continue to sustain their growth during the growing season. And so the higher input of carbon by the plant, which continue to sustain growth, might have also acted as a good energy source for the microbial communities, especially during summer. Nevertheless, water limitation had a very strong impact on the community composition in the soils of the mesocosms. Here you can see the cap analysis for the prokaryotic community. And you can see that basically the prokaryotic community strongly diverged among treatments. This you can see from the second axis, the y-axis, but there was a very strong diversification among treatments. And this diversification actually increased along the course of the experiment. Towards the end of the experiment, you can see towards the right side of the graph. So in the second winter of the mesocosm study, also the reclassification values of the cap analysis were much higher, also indicating that basically there was a diversification over time for the prokaryotic community. Instead, the fungal community did not show a very clear pattern, neither for the treatments nor for the seasonality or for the sampling times during the different season of the experiment. And also you can see that the reclassification values were quite low, usually lower than 50%. So we cannot basically say that there was a diversification of the fungal community for the treatments. And so this basically implies also that the fungal community is less sensitive to the conditions that we had in the mesocosms in particular. It was less sensitive to water limitation as compared to prokaryotic communities. And we relate this pattern mainly to the ability of fungal to basically create this large hyphal network to basically scavenge for nutrients and water over longer distances in the soil. Similar supporting results were also obtained by the Bipartit Association network analysis that we did. Here you can see the graph of the network for the prokaryotic community. Basically, the network which I show is indicating the clustering of communities according to treatment, so irrigation treatment and sampling time. And you can see very nicely that there was a very clear separation of the treatments for the prokaryotic community. Also, you cannot see from the graph that on the right side where we had the severe water limitation treatment in brown that the community composition was mainly dominated by Actino Bacteriota. And this makes sense because this type of organisms are actually known to be very resistant to water limitation, tolerant, and also good decomposers of organic matter. So when there's maybe less easily available carbon to be used, they can also decompose more difficult and complex organic matter. On the other hand, we didn't observe the same pattern for the fungal community composition. Sorry, not composition. For the fungal community, in fact, you can see that there was no clear effect of treatment, or at least this was less evident from the Bipartit Association network. And you can also see from the colors that the community of fungi was mainly dominated by Ascomicota. So again, it seems from this analysis quite clearly that prokaryotic communities are more sensitive to water limitation as compared to fungal communities in soils. We also did an indicator species analysis. And as you can see, quite nicely from this graph, some groups of microbes increased with water limitation while others decreased. So I would like to start with a group of, with a tax which actually increased with water limitation, as these were not so many. And you can see these at the bottom of the graphs. You can see that we observed an increase in Actina Bacteriota, as mentioned earlier. We also saw a lot of the purple dots close to severe water limitation nodes. And these are again known to be desiccation tolerant and decomposers of organic matter. We also saw an increase with water limitation of Thermoplasmatota, which are organisms which are also found in extreme environments. The taxa which were mostly affected, as you can see from this graph from water limitation, where appear to be Mucormicota, which are well-known symbionts of plants and which rely on carbon inputs from the host tree. So probably under drought there was less available carbon directly from the trees. And this is why they reduced their relative abundance and their water limitation. At the same time, Nitrospirota, which is a taxa which involved in nitrogen cycling also was reduced in relative abundance. In our study, we also observed that at the genera general level, we observed that there was an increase in desiccation tolerant and subrotopic fungal organisms. And we also observed a decrease of genera involved in nitrogen fixation and symbiotic genera. So overall, we observed quite a strong change in the community composition of our soils. Also, physical chemical properties strongly played a role on altering the composition of the fungal and prokaryotic community in our study. As you can see from this graph, both prokaryotes and fungi were strongly affected by gravimetric water content, soil temperature, as we have already widely discussed during the presentation, but also in changes in total nitrogen pH and soil organic carbon concentrations. And just to give you an idea, the pH basically was higher at the beginning of the experiment and then the pH declined over time. It remained more basic for the control treatment as compared to the other treatments. And we also observed that the pH was more strongly affecting the prokaryotic community as compared to the fungal community. And we also observed clear changes or like small changes in organic carbon and total nitrogen concentration, the nitrogen rather declined during the course of the experiment, which also led to an increase in the CN ratio of the soil, which also likely played a role in shaping the microbial community composition. So this is already my conclusion slide. And I would like to summarize a bit, the results that I showed you during the presentation. We observed that water limitation basically did not affect the relative abundance of neither the prokaryotic nor the fungal community composition during the course of the experiment. We only observed a peak during the summer season. We also observed that fungal communities were less sensitive as compared to prokaryotic communities in response to changes in soil moisture contents. We also observed that the diversification of prokaryotic communities was gradual and rather was very marked towards the end. And water limitation overall promoted the prolification of microbial groups which were tolerant to environmental stress, such as suffer of trophic taxa and rather induced a decrease in those taxa which were involved with nutrient cycling and those plant symbionts. And so overall water limitation will likely have potential effects on the soil microbiome of Scottsbank forests and will likely affect the functioning of forests due to changes in the critical functions which are provided by soil microbiome communities. And with this I would like to thank all of the people which were involved in the project, the Swiss National Science Foundation for the funding of my ambition project. And I would like to thank you for your attention and again Max for inviting us to present our work. Thank you Emily. Really interesting. I already have a whole bunch of questions to get back to but we will wait till the end and continue with the session. So thank you and just a note to everybody. I think Zoom has gone into AI mode and is loving these webinars and the talks and sending applause and reactions all over the screen and they are not being turned off. Unfortunately there's some glitch here in the system. So please go to your own reactions arrow and you can hide the various reactions that that seem to be just popping up to the very nice talks that are going. So again we'll come back to the Q&A later. So thank you Emily. So our next speaker is Nicholas Barber from San Diego State University and he's working on a really interesting system on reintroduced bison to tall grass paris and how this shift is now changing soil bacterial communities and their functions. So Nicholas welcome. Thanks so much Max. Thanks for inviting me. Thanks to everyone for joining around the world today. That was really fascinating talk Emily. Thank you and I'm looking forward to hearing from Anna as well. One thing I'll start with is that when Max invited me for this webinar I was honored and excited but I wanted to make sure he knew I'm not really a microbiologist. I came to this work very much from the ecology side. I'm a community ecologist. I've worked a lot with plant and insect communities to understand the forces that shape their communities but I began collaborating with microbiologists especially Wes Swingley shown here at the bottom and this paper I'll talk about today was in collaboration with him and other members of his lab. Does Climac who was a master student and Jennifer Bell who was a postdoc and this paper also sort of grew out of COVID times when we went into lockdown in 2020. I had to cancel plans to spend the summer doing research in Germany and was stuck at home but I was sort of fortunate and privileged to have a lot of flexibility without all the challenges and responsibilities a lot of our colleagues faced so I was able to take the opportunity to learn how to work with Amplicon data and this paper grew out of that. So I'll actually start with the ecosystem to frame this study and the questions we asked to orient everyone. Here's the lower 48 states of the US I'm way over here on the west coast in San Diego and this work took place a little west of Chicago in the US state of Illinois where you see this red star and in much of this region of the central US if you go outside of cities into the surrounding countryside most of it looks like this large-scale agriculture primarily a rotation of corn or maize and soybean but if you were to take a time machine back 150 or 200 years it would it would probably look more like this most of the center of North America the Great Plains was grasslands and this includes the eastern mesic regions of grasslands called the the tall grass prairie this enormous ecosystem stretching in a broad band from what's now southern Canada to what's now Texas and eastward toward the US Canadian Great Lakes this is one estimate of its extent these maps are very little depending on the exact definition of the habitat it's a highly diverse ecosystem with high plant diversity high plant a lot of characteristic animals but like lots of grasslands around the world this ecosystem which had deep rich soils in combination with generally high rainfall especially that eastern part of the prairie made it ideal agricultural land and so by the mid 20th century much of it was converted to row crop agriculture something like 90 percent of the habitat lost most of the remaining remnant or relic prairies are tiny patches of land mostly too small to be seen on a map like this which makes it one of the most endangered habitats in North America so then by the start of the 21st century prairies have really become important targets of ecosystem restoration to try and reestablish prairies support their unique biodiversity their functions and services so former farm fields are planted with a mix of native seeds to regrow these diverse grassland plant communities but it still requires a lot of ongoing management including control of invasive weeds and importantly from my talk today it also requires the application of regular disturbances two of these common disturbances are fire and grazing prescribed fire like you see here is applied frequently anywhere from annually to every few years because in the absence of fire that that suppresses woody plants most tall grass prairies will grow up and no longer be prairies if you saw our yumpinans talk in this webinar series last year he saw an example of this this fire mimics historical fires from lightning strikes but also importantly it mimics the fires that were set as land stewardship by American Indian tribes who used fire as a cultural practice to maintain the land in which they lived the work I'm talking about today took place in a region that's the traditional land of multiple tribes including the Peoria, Potawatomi, Ocetisakawan, Myami, Kikapu, Sok, and Meskwaki people who all lived on and helped to shape these lands the other important disturbance is grazing by large mammals historically large herds of grazing bison inhabit of the great plains but were hunted nearly to extinction in the absence of grazing disturbance many restored prairies lose plant species richness over time although many species may initially establish over a decade or two a lot of them don't persist and are often out competed by large high biomass grasses but those grasses are often preferred food by bison so bison reintroduction to restored prairies may be able to maintain higher plant species richness by suppressing those grasses and by adding heterogeneity to the environment through their activities like grazing and trampling wallowing and depositing dung and urine so a number of large prairie restoration projects have reintroduced bison over the years but it's a really big undertaking because of the resources needed so a lot of effort then goes into reestablishing these prairies and a lot of ongoing maintenance and management but restoration is often focused on on plants and animals even though as we all know soil microbes are a critical part of the system especially when restoring function as part of the the overall goals that importance is underscored by how much plant biomass is is below ground in by roots in the soil that can go meters down as shown in this famous photo by Jim Richardson and yet a lot of management choices are made based on the above ground portion of communities with the assumption that a diverse plant community means diverse assemblages of other organisms so in this study we looked at soil bacteria and asked a question how do diversity and composition of these communities change following prairie restoration and potentially in response to management disturbances from prescribed fire and the presence of these reintroduced bison we worked at natusa grasslands a large ecosystem restoration project owned and operated by the nature conservancy that includes original unplowed prairie remnants and where the staff and volunteers have been carrying out restoration since the mid 1980s each year they plant new sites usually former ag fields and they slowly expanding the reserve and the methods they use are really helpful for scientists because they use very consistent methods to seed and manage these sites with regular weed control and prescribed fire all on very similar soil textures so near the end of 2014 then they introduced a herd of bison into a large fenced unit covering about half of the preserve and within it were a range of remnant and restored prairie sites which the bison had free access to so we took advantage of this to choose a set of focal restorations that varied in age planted from 1987 to 2013 and they varied in bison access the blue sites here show where bison have access and yellow they don't and we also selected two large remnants shown in red and two nearby agricultural fields the green striped areas and these are going to rotate it between corn and soy all the prairie sites but not the farm fields get prescribed fire the schedule varies a little but on average it's about every second to year we began sampling these sites in the fall of 2013 and then spring summer and fall sampling from 2014 through 2018 taking surface soil that we pooled together from each site so we had a chrono sequence of sites from newly planted to over 25 years old at the start and we repeatedly sampled these sites so we could see how they changed individually over time and in burn versus unburned years also the bison were introduced after we completed our 2014 sampling so we have pre and post bison samples for those units from these soil samples we measured basic abiotic variables and then we targeted 16s ribosomal RNA genes using a chime 2 and data 2 pipelines to process the sequences and assign taxonomy in this study we collapsed tax at a genus level to calculate community metrics for each sample I'll show results today for alpha diversity and for community composition based on beta diversity so looking at our results let me first orient you to these next few figures starting with soil characteristics this is showing carbon nitrogen ratio of soil in restorations only just in the replanted prairies plotted here against the age of those restored prairies and all the figures I'm about to show there was a significant quadratic relationship with age so here in the years following replanting these former ag fields gained soil carbon and probably lose some nitrogen that's remaining from a legacy of fertilization and the ratio is also slightly higher with bison present those are all like I said restorations looking at overall mean values and comparing them to ag fields and to remnants um you can see carbon nitrogen ratio seems a little higher in remnants the yellow point here but the effect wasn't significant there's a similar overall pattern for soil pH in this case ag fields have a higher soil pH than the either the other query site types so then do these differences in soil conditions with age and bison translate to differences in prokaryotic diversity kind of again this hump shape pattern for both richness and Shannon diversity suggesting soil diversity increases after restoration to a point and then declines in the oldest plantings sort of like the the plant richness decline that we see in older communities here however the that apparent impact of bison on soil abiotic conditions doesn't translate to differences in microbial diversity only age effects were present and then if we look at site type on the right side of each figure there's also lower richness and lower diversity in remnants so that decline in older restorations is actually making them more similar to these low diversity remnant soils now this is all alpha diversity what about composition one issue is that the sites with bison access include both the youngest and the oldest restorations at the sites and these represent two very different site histories then in the case of older sites they're well established restorations decades old to which a new disturbance was added when bison were brought in and for young restorations these sites went through some of their earliest development with bison present those years shortly after planting when we see a lot of plant community composition changes as these long-lived perennial plants establish and spread and from earlier a week we suspected that soil microbial communities are also going through a lot of turnover in these early years but we didn't know whether that turnover would change with this new eating stomping rolling pooping disturbance out on the prairie here's an ordination from principal coordinates analysis of restoration samples so no remnants or ag fields here from just this 2013-2014 samples pre-bison each circle is a sample and if you can see the number in each circle it's the age of the restoration in years so here these are site sampled before bison were introduced just noting where they would be occurring eventually in light and dark green and samples in the sites that would remain bison free and gold at the start without bison young sites one to three years since planting in the in the light green here we're definitely distinct which which confirms some of our earlier work while all the older sites five or more years older are clustered together so the question that is what happens as these young sites get older with bison impacts present what's going to happen to them we can hypothesize different outcomes in the following years one possibility is that succession over time is a really dominant factor our previous work before bison showed up showed that age is a strong gradient so maybe over time the younger bison sites will just converge with the other site types and there's a typical prairie soil microbiome composition or maybe bison are really big drivers acting as a filter for which microbes thrive or don't and the the older and younger bison sites will converge so there ends up being distinct bison and non-bison soil communities or maybe both age and bison are drivers as I suggested earlier and bison could shift the trajectory of successional change resulting in bison sites being distinct and the compositions between younger and older bison sites again still being distinct so going back to the the pre-bison conditions what happened in the following years this is the first growing season after bison were introduced in 2015 then 2016 2017 and then in 2018 when bison had been present for three or four growing seasons we do start to perhaps see three different compositions like that third hypothesis but as additional support for that third hypothesis bear with me as I jump back to the pre-bison communities here the young sites on the right are one to three years old while just slightly older restoration older restorations marked with red stars are five six seven years old but compositionally similar to much older 10 to 20 year old sites and yet by 2018 when those young bison sites on the right and light green here are themselves five six seven years old they're remaining distinct suggesting that bison impacts it really might be playing a role in changing the succession and the outcomes of these restoration activities so we also looked at which taxa might be driving these changes we see the work I'll talk about here was was led by Wes Swingley using a machine learning approach to identify taxa with higher average information gain for random forest decision trees either across years or following bison introduction and then we could example examine the relative abundances of those taxa in relation to the three site groupings I'll just point out a few interesting highlights first a few taxa were more abundant in these younger bison sites than at the old sites here's two examples an alpha proteobacterium and a beta proteobacterium this one on the on the left here ellen 6067 is particularly interesting it's a member of the mattress of monodacy it's been documented in other tall grass soils as well as increasing in abundance along ph gradients and we did see increasing ph over time in those young bison sites but like many mattress of monodacy there also are many of them are ammonia oxidizers that respond positively to fertilization and this alpha proteobacterium in the micro pepsaceae is also abundant with fertilizers but there's no longer any fertilization in these sites except for the possibility that bison dung and urine deposition could be in the right form and maybe a sufficient amount to subsidize these microbes there were also taxa that were generally lower that were significantly lower in there we have in relative abundance in the young bison sites here's a three genera following that pattern all in the actinobacteria these genera these genera have all been found in other grasslands too and found to be rare in cultivated soils or under heavy grazing they also seem to prefer nutrient poor soils which might be what these soils look like after that legacy of fertilization goes away so some general conclusions from our results and what they tell us perhaps about grassland restoration as our restorations get older their soil bacterial diversity becomes more similar to the to the lower diversity in remnants bison don't have consistent effects on alpha diversity but they do seem to influence composition and prescribed fire had very little effect maybe because all these queries receive regular fire and rarely go more than two or three years without being burned one implication for restoration practice is that reserves that include sites with a variety of conditions different ages differences in bison presence or absence across those ages will likely lead to high beta diversity within the whole reserve because bison can drive that environmental heterogeneity which might maybe support greater overall diversity at the full reserve scale and enhance function and stability at that full ecosystem level but a couple other questions came up from this work first we use 16s sequences looking just at taxonomic composition we can ask do these taxonomic differences actually translate into differences in functional potential of these microbes and we're only looking at prokaryotes what about fungi which obviously and we saw in emily's talk are a big part of soil communities we've actually been able to address some of that in some additional work from our 2017 samples we selected the spring summer and fall samples from six restorations and then the samples in spring summer fall from the two remnants and the two ag fields and then thanks to a community sequencing program grant from the joint genome institute we're able to get 30 soil metagenomes using whole genome shotgun sequencing from these we got both taxonomic classifications and functional potential using the keg orthology database and again using both including both prokaryotes and fungi and happy to say the results of this study were just published a few weeks ago in fems microbiology ecology the qr code here can link you to the paper I want to acknowledge the lead author of this paper Kayla Mason on this paper was based on her master's thesis here at San Diego state I won't take too much time but just as a quick couple quick highlights to for some of the relevant findings in this new paper first these ordinations here are depictions of taxonomic composition at the top panel and functional composition based on keg ortholog counts in the bottom panel blue dots are restorations green are remnants and brown are the ag fields it's clear the taxonomic distinctiveness between the three site types is also reflected in their functional potential so that we might expect these different communities to support different soil functions too as one example of that when we when we look at genes encoding enzymes associated with cellulose degradation we see they're significantly higher in restored prairies and particularly are more abundant just after prescribed fire similar to what's been seen in some forest systems after fire takes place now there's a lot more in this paper I encourage you to check it out if you have time and finally I want to emphasize how much support we've had in this work especially from the staff and crew at natusa including dr elizabeth bach who is the staff ecologist and an incredibly knowledgeable soil scientist we've had lots of helpers in the field to collect soil over the years and the lab work this paper really benefited from helpful feedback from colleagues as well as editors and reviewers it funds and our financial support over the years came from several funding sources so I'm looking forward now to hearing Anna's talk as well and to our panel discussion afterwards thank you very much thank you nicolas really really interesting and we'll follow up there's already a bunch of questions waiting in the q&a so let's move forward to Anna escalante from mexico city and again looking at arid soils and what is the functional significance and microbial diversity what's happening with of course now climate change more and more reduction in in precipitation as well so Anna thank you thank you max and thank you for the invitation and I'm really honored to be here today and thank you to the all the attendees that are listening well I will just start I will talk to you about this publication from this year this is the full title functional significance of microbial diversity in arid soils biological soil crust and nitrogen fixation as a model system I want to highlight the list of authors of this paper particularly Alberto Barron Sandoval the first author and Teresa Perez Carvajal which both were master students when they started collaborating or leading this work and I'm happy to say that now Alberto is a PhD student in user Irvine and my colleagues authors of the papers also Jennifer Martini from UC Irvine, Stephen Bollock at CCCA here in Mexico and Alfonso Leija and Georgina Hernandez at UNAM as well so the the plan for this presentation today is to go through these topics or points of the paper that I want to highlight first I want to talk about a little bit about the diversity and function relationship in microbes particularly in prokaryotes and some key concepts that led to the the study first I'm sure that many of you are aware that microbial diversity is responsible for most or all biogeochemical transformations in all ecosystems but still we are debating how impactful community composition really is in ecosystem functions why well because of functional redundancy which means that different phylogenetic groups conduct similar functions in ecosystems these have at least this implication that when different communities are placed under the same environmental conditions we or many of us have assumed that these communities make function equally but we have also reflected on that the degree of to which functions and compositional changes are related in these communities may depend on the function in particular that the function of interest which might be carried out by a broad or narrow range of core co-occurring taxa for instance natural infixation is a narrow function for which not so many phylogenetic groups are capable of fixing nitrogen in contrast the composers or carbon mineralization groups are a part of a growth functional functional group also thinking about these concepts and functional significance of diversity we we post in this study that the likelihood of detecting the relationship between composition and functional processes increases when looking at functionally narrow processes such as nitrogen fixation and among highly specialized communities on this on distinct environments meaning that communities that have been exposed to contrasting conditions for a very long time like evolutionary times so with these concepts in mind we looked for a model system where we can test or evaluate this relationship between diversity and function and our model system is the arid soils and microbial communities that live in biocrosses so arid and semi-arid ecosystems are very abundant in their planet and will become more and more abundant with climate change they represent 45 percent of the planet's terrestrial surface and their biocross which are microbial communities cover 70 percent of the interplant spaces which is a lot and not only they cover a lot of space but they're the the major or main nitrogen and carbon fixers in these ecosystems and they fix 30 percent of the global biologically fixed nitrogen so it's considerable so the impact of microbial diversity in these soils the impact the functional impact might be might be quite considerable so these are the biocrosses this is like a cartoon of a biocross in some sort of sequential or succession sequence going from cyanobacteria dominated cross which is which are very thin layers dominated by cyanobacteria but there can be also moses lichens and they can evolve or success into very complex structures so this can be a succession or also there are biocrosses in different arid and semi-arid soils that they're characterized by being only cyanobacteria dominated and they never reach these these big structures on your right hand side so we were looking for cyanobacteria dominated cross because cyanobacteria main nitrogen fixers and we were looking for nitrogen fixation as a function of interest also we were looking for relatively simple in terms of composition a microbial community so we can really characterize composition and relate that to the function of interest so this is the one of the sites that we sampled and I am putting this picture on just to show you how thin these these crossed are and also to show you these tiny filaments of cyanobacteria to which soil particles are attached because of the polymers that cyanobacteria secret so these are biocrossed the cyanobacteria dominated ones and I want to tell you a little bit more about how the relationship between biocross the environment and function biocross are metabolically active only when they're wet they they're different in composition when you look at the hot and cold deserts the hot deserts receive rain during summer and the cold deserts receive rain during winter so that means that when biocross are metabolically active they experience different temperatures so rainy seasons temperatures are likely to be an important factor in biocrossed functioning so then we we thought about our the approach to evaluate this relationship and we went for a reciprocal transplant of biocrossed experiments both in the fields and in laboratory settings the rationale is based on a paper that was also published in famous microbiology ecology a while ago by Reid and martini and the rationale of this reciprocal transplants and the scenarios that we were like expecting or the possible outcomes are in these graphs we we see the different communities the dark and the light community mark here as dots and lines and different environments and at the same time the measure of a functional parameter let's say nitrogen fixation so the first scenario is that origin or composition of the communities is the main driver of the of the variation in the functional parameter which means that independently of the environment where the communities are placed are placed that the functional parameter doesn't change and it's specific to the community a second scenario is where the environment plays the main role in in the functional parameter response and lastly is the both the community or the origin of the community and the environment both played a role in the response in terms of function of the communities so we went to a sample the this cross into contrasting environments both are semi-arid ecosystems one is in this part northern part of mexico in the central northern part of mexico is cuatro cien negas in the state of coagula this is a hot desert and the other desert is a cold desert is in baja in the western north northwestern side of of mexico so they're their samples we brought them to the lab and we incubated them in different in contrasting temperatures this is just to show you the contrasting temperatures where the where the different deserts receive precipitation in the top side you can see a highlight in red the rainy season and the line the red line indicates the temperature corresponding to the different seasons and you can see that the me median temperature in the rainy season in cuatro cien negas in the cold desert is around 30 degrees and in contrast for the cold desert the median temperature in the in the rainy season is around 15 degrees Celsius so where which were our hypotheses and expectations well uh given that nitrogen fixation which is our function of interest is a taxonom is a is a narrow taxonomic function and also the potential of the biocross specialization to the specific hot and cold desert we we think that communities will not be functionally redundant and that we could actually see that but additionally we were expecting that nitrogen fixation would be higher when the crusts experience temperatures similar to their native environment due to this specialization or legacy effects also we were expecting that taxonomic and functional gene composition of the crust will respond to non-native environment in an origin-dependent way the legacy effect and finally that gene richness would decrease when crusts were exposed to non-native environmental conditions due to this also specialization and legacy effect what what was our experimental design to put these hypotheses and expectation to test well we went to the deserts we took the samples in quadrats on the on the b figure is the Baja desert and on the c figure is the 400 negas desert so it's cold and hot uh hot desert then we took the samples with these uh lead little white devices which are like a small soil uh cores or cakes of soils and we transplanted them from Baja to uh Coahuila and back um so we planted 16 cores of each of the of the of the samples and also we did control cores like ultra like self-transplanting to to control for for the manipulation of the samples uh and then or we also brought the cores into the lab and incubated them in 15 in 15 degrees and in 30 degrees as the median temperatures for the cotton and cold deserts for the transplants we maintain the transplants in the reciprocal sites for four months during the rainy season summer or winter as so the samples could experience the temperature and the and the ambient metabolically active so we could see some sort of composition changes and also functional changes and then we pulled all the the samples back into the lab to do measurements and for the lab setting we follow up the the responses and the changes in composition for 180 days we sampled at different times at 10 days and then at 90 days and at 180 days so it was t the t0 to 1 t2 1 t3 and for both experiments or for both experimental approaches we characterize community composition for the field experiment we did 16 s r na gene amplicons and metagenomes and for the laboratory setting we conducted a cheerful piece of the nif h gene and for the measurement of functioning or the functional response we performed an acetylene reduction acid for nitrogen fixation potentially so the results of these experiments we presented them in three categories one was the functional responses of the crust the other the compositional responses of the crust and then the richness responses of the crusts for the first part the functional responses I will try to convince you that we demonstrate that given the nitrogen fixation and taxonomic narrowness and potential crust specialization communities were not functional redundant and in addition nitrogen fixation was higher when the crust experienced temperature similar to native environment and these you can see in the following results and graphs here we see the the graph that corresponds to that hypothesis on the paper of read and martini where we compare the functional parameter in this case the the rate or the the effectiveness of the acetylene reduction assay and the response in that in that sense for both the cold desert and the hot desert in the reciprocally transplanted site for each one the hot desert is in dark and the cold desert is in light gray so we see that for well I have to say the here we lost a lot of the samples in the transplanting so we cannot have this part of the graph we could not do that but still we could conduct some statistics and we what we can see is the potential of nitrogen fixation dependent on both the origin or composition and site or environment and for both main effects so is the third the scenario of those hypotheses for the laboratory setting and after a period of acclimation communities showed higher but functional potential when placed on their similar temperatures to their place of origin this is particularly clear for the final time point where we measure this and so you can see for the hot desert which is this the the dark dots and lines it is they perform better in their native temperature compared to the cold desert which is the the light rate and the light and the light rate perform better in their native temperature which is 15 degrees celsius for the second part of the results in terms of compositional responses we see that both taxonomic and functional gene composition responded to non-native environments in an origin-dependent way or the legacy effects and here I am showing you the results from the reciprocal transplant experiment in the left hand side you can see in circles the hot desert samples the original samples are in dark and the post-transplant samples are in light gray for the cold desert the the dark and light is the same the dark is original and the and the gray is the post-transplant experiment but this in this case there are the triangles so you what you can see is that despite the time for of the transplant composition do not converge between hot and cold deserts they they're they're they're still more similar among them than between them and well that's that so they preserve or they're they're restricted by their original composition they do not converge and you can see the same thing in another in another way in the right hand side graph which is a clustering you see on the on the top part the the clustering of the otus and in the and in the vertical part you see the samples there are color coded for a cold desert in blue the dark is the original samples and the light is the post-transplant experiment the same thing for the red the red is for the cold desert so you can see a clear separation in composition in terms of otus between hot and cold deserts and the composition is actually mixed between original and post-transplant for the cold desert and not so so mixed for the hot desert same thing but maybe not so clear I want to just highlight that these results are for 16s and this is for the nif-htrflp so it's a little less resolution maybe and for the lab experiment you we could not see that clear cut difference in time two and in time one and time two but eventually at 180 days composition again returns to the or to the origin in a way no the hot desert is more similar to the hot desert and the cold desert is more similar in composition to the cold desert so by the end of the experiment origin and temperature accounted for almost 30 percent of the variation in composition and finally for richness responses in the frost we found that in fact taxonomic and functional gene richness decreased when biocross were supposed to non-native environmental condition and and we see this particularly or we could see this for the for the field experiment just very quickly to wrap this up on the on the left is the 16s gene richness and on the right are different nitrogen cycling pathways a richness so in both cases we compare the original and the post-transplant samples and you can see that actually in in most case when for 16s the the diversity declines when when the samples are transplanted to the reciprocal transplant experiment as we expected from the legacy effect idea and this is also true not for all for but for many of the nitrogen cycling pathways so for our conclusions of these results I would like I hope you are convinced and if not I will be happy to answer any questions hot and cold desert biocross are not functionally redundant they function differently they have different composition natural infixation potential results strongly suggest that ecological specialization may account for this non-functional redundancy in response to environmental changes understood as these contrasting environments and put to test with the reciprocal transplants both in the field and in the lab and both compositional and functional responses show evidence of these legacy effects or the site origin effect so once again I want to acknowledge our the students involved in this project Alberto and Teresa our colleagues Jennifer Stephen and Georgina another technical support that that helped very much this is Alberto this is Teresa and the funding for this project is this with that I will end and happy to join the conversation thank you okay thank you very much and yeah thank you also Emily Nicholas and we'll continue here with a little bit of questions we have a bit of time still before we need to to note the while I'm sorting through the q&a a quick question for you Anna I'm thinking of with the transplantation experiments if you would disrupt the soil how long does it take for a new biocrust to form so what's the time and that of course would also indicate I mean how long after transplantation would you maybe see a shift in the community that got transplanted well I think I have two parts of a response to that question if the crust is like destroyed it can take a hundred years to to be restored because the the growth of the microbes is very very slow particularly to reconstruct the network that that cyanobacteria the mesh that they form it takes a long time but we did not destroy the structure of the crust we actually did this core sampling to preserve the structure and then we put that very carefully in in a like a dialysis bag so we got like deposited that in the in the soil like like a tiny little cake so we were really careful with that so yeah so it's more of how that transplanted community is going to survive under now very contrasting conditions sorry I did I didn't get the first part of the question in the sense it's really how the transplanted community that is still as an intact biocrust with the species interactions in there's going to survive now under very different contrasting conditions I think they will survive I don't know well it will be interesting to go and keep sampling what's going on but unfortunately since they're like exposed to the natural environment they're probably just like with their their cows and rabbits and animals that go around so maybe if you like cage cage it you can actually follow up more we did not do that it will be interesting to be like how much time does it take to like be part of the reciprocal biocrust in the soil interesting so this is a question from Emily regarding the actinomyces that you're seeing and again they're of course known to be quite sensitive to low pH and you often see the shift of a proteobacteria actinobacteria diminishing the acidobacterioda coming up as that pH goes down so this is a question of how you fungi then also prefer the low pH condition so how do you still have so many actinomyces are they just different species that really prefer low pH or what do you know about them that's an interesting question for me to think about yeah I haven't really thought about it much so far but we basically found yeah basically an increase in with water limitation and the pH slightly declined as well under water limitation I don't know if that could have maybe stimulated also the growth of the actinobacteria under water limitation but nevertheless water limitation alone so changes in the moisture were still more relevant for the community as compared to the pH changes the second nevertheless environmental factor which was mainly affecting the community was indeed pH and actually thinking of your conclusions and you were just mentioning not major shifts in the relative abundance of bacteria versus fungi what about absolute do you have way of knowing and again this is of course also this question of changes in just sort of organic matter or nitrogen or so on how much of that could be biomass decline as well I mean you have absolute numbers of actually bacterial and fungal biomass um yeah we have actual numbers um we always compared uh so the the ratio basically didn't really change with water limitation that's what you're asking yeah with limitation experiment yeah we didn't change we didn't observe this okay and I have a question for Nicholas again in terms of shifts in or potential shifts in microbial community and also grazing do you see a shift in the plant community that could then be the driver of the associated microbiota yes definitely and there's been a lot of work in these in these prayer restorations by others and us on on how the plant community shifts with age like I said we tend to see a decline in richness and more dominance by grasses particularly warm season C4 grasses at the expense of of forbs some of our early work advice in just the first few years seems like they're that that intended effect of suppressing grasses and grass competition allowing other forbs to spread seems to be happening and particularly legumes may be responding positively some of our new data suggests that so there's certainly the shift in the plant community going on um which we expect them to be part of the the probably part of the reason we're seeing these effects in in in microbial changes too if that's resulting in changes in you know the makeup of root exit dates that are going in or the makeup of the of the litter that ultimately is is on the ground um a lot of that litter will sit there for a whole year or even in the burden years then it's you know transformed into ash that has a lot of inputs for microbes in the soil so there is a lot of turnover happening both across time along that chrono sequence but we are starting to see shifts in the plant community because of of bison impacts yes wondering also tourism in shift with responses of plants to grazing where they might produce more phenolic compounds or so on and of course now the litter quality changes as a consequence of grazing is that anything that you looked at we haven't looked at that that's certainly something i've been thinking about too of um wanting to be able to look at at a finer scale at those um yeah at how those changes and the plants might then be changing inputs that affect other microbes too certainly a lot of these um in the grasses a lot of these grasses obviously evolved with grazers um you know they're they they can respond very positively post grazing and regrow but yeah we expect there would be changes and um i'm really curious about about the root exitate changes too okay and let's see i'm looking at this this is one question on what are the implications of changes in soil microbial community structure under water stress for ecosystem resilience and actually i'd like to expand that to the all three of you in terms of okay yes we can see shifts or so on the question of redundancy so even if all the species are different do they still do the same thing so does it matter and so how would you put all of this really into this context of how resilient is the community to whether that is water stress or grazing stress or then this aridity and changes in temperature so maybe Emily you can go first yeah that's a really good question um basically we have thought about it a lot and it's interesting that basically in our mesocosm study this is similarity between of the community continued to occur so the basically the the community continued to diversify under the water treatments over time and um this basically means um yeah the community is able to sustain water limitation because we still continue to find microbes in it nevertheless the functioning probably changes i mean we didn't study functioning directly in the specific study but of course uh we saw also shift in specific communities of bacteria and so what is expected is that potentially the the functions the critical functions provided by the microbiome in the source will be altered and this will then create problems for ecosystems unless there are then changes for example in terms of the climate that there will be fewer droughts if we really do some mitigation efforts yeah so that was my answer for now yeah Nicholas why don't you continue um you know one one thing i've been curious about is if in years where we may find uh faced drought stress in this environment if it changes uh grazing patterns that because these animals have access to a pretty large area you know hundreds of hectares in size they have a lot of choice of where they particularly graze and if they seek out lower line areas that maybe aren't facing as much drought stress as upland areas where the and these sandy soils can become really dry during drought years that could sort of amplify those potential grazing effects in very specific areas and um and you know at sort of an additional level that heterogeneity by heterogeneity by changing grazing pressure at a really fine scale and maybe again drive even further these differences that we're seeing of potential grazing impacts on the on the soil microbial community at the same time the site we're working at you know the benefit of all being a relatively local compact site is that you know the the sites are at most a couple kilometers apart so they generally face the same um weather every uh during throughout the year so um it limits our ability to look at a given year of say you know our wet and dry sites because they all tend to be wet in the same year or they all tend to be dry in the same year okay uh i may have a can i ask a couple of questions um sure yeah i this is i guess it will be both for emily and nicolas um what do you think about how long the like follow-up experiments should be should be followed because for instance uh i followed my experiment for for months our 180 days and emily did the mesocosms for it was a year or something like that two years uh and well nicolas has a follow-up for many years so i guess it depends on the questions of course uh do you ever feel that you get to have a final answer of like now we we know that these communities restored or it just adapted or when do you stop and when do you actually feel comfortable when saying well now just like a philosophical question i think but it's something that i always think about what do you think emily go ahead you answer first yeah so actually the mesocosm study that i presented today was the results of one year we continued also the mesocosm study for a longer time the results were similar in the sense that the community continued to diversify over time and at the end we decided to end the experiment with with leaving the trees die and checking out what the community changes in the soils would be and of course it was a mesocosm experiment with small trees it would have been really great to keep it for a longer time uh and maybe let the trees grow a bit more than we probably would have had to transplant them to bigger pots and i think um using like one basically can have different types of experiments in combination i think that's probably going to be the best way of understanding mechanisms like combining small scale experiments such as the mesocosm experiment but also the mesocosm in the field sorry the field experiments and studies what nick has done and transplantation also is definitely a great tool for certain ecosystems for most ecosystems i would say so i think it's really important to stress um this that we need more experiment in the field but also more longer termo observations and the combination of everything together plus modeling will really be beneficial to get a better understanding but i think it's difficult to really talk about the best scales i would say um depends on the research questions that one needs to answer yeah and i would echo that i was struck to by our our three talks and this this mix of experimental approach field approaches and combinations and the different perspectives we get in terms of how long to follow it's other is a question that was posted about asking the long term changes and i i go back and forth over what long term means in in my study system that on the one hand we're looking at sites that are three plus decades old which seems very long term but these are ecosystems that are our restored sites have probably been a similar habitat for hundreds of not thousands of years and we still see even our oldest restorations that are 30 plus years old things like root density in the soil is still less than what we see in old very old unplowed original remnant sites and so although there's you know there's certainly a rapid turnover in the first few years that we see both in the plant community and in microbial community um that levels off and maybe you're not getting as much rapid change after a decade or two um there's still ongoing changes and and i don't i don't know when the end point is uh to see that um um we also often see sort of in the early years a rapid decline in in nitrogen as sort of that legacy of fertilization i mentioned goes away and then over the long term um total soil nitrogen may slowly increase again as as organic matter is is in the sort more in the soil and that root density tends to build too so there are just long term changes that may take many decades if not centuries to really um to play out no agree so i think i want to wrap up with one final question here from the q and a and this is about viruses so given that soil viruses are known to be highly sensitive to environmental condition variations are you considering investigating whether they could account for some of the shifts in microbial community uh i mean affecting both bacteria and fungi and i know i mean this is going forward because none of you had looked at the viral communities but they are microbes too and of course major influencers any thoughts well yes uh which i mean is not that i'm going to do it but it will be great to collaborate with someone that has experience on viruses that are yes there are microbes but they're like their behavior and all the techniques to study them are like very different and they're definitely drivers of both ecology and evolution so they they're part of the show and should be included that's my comment yeah i'd also love to look at this i'm in a department with a lot of virologists who bring this up a lot and my answer is often i i don't know that's a great idea we should look and do it i think that could be a really um just another additional ecological interaction we haven't considered yet and what might be helping to shape these uh the composition of these communities and of course i think also plant viruses that then interact with the uh prairie plant so yeah absolutely emily any added thoughts i totally agree with all of the comments made yes definitely important topic to to consider and this of course might be a topic for our next webinar so i would take a look at uh at papers and we're probably going to continue in the spring again with with a new topic so please keep it be posted but no i think we need to wrap up so uh thank you emily nicholas anite was wonderful to hear from you and hear more about the studies that you had published on and again thank you everyone in the audience from really what i saw over the world and again if you missed parts of this the webinar will be available on the uh under sites both vfm's and oup probably in a week or two and of course there you can also catch any of the previous webinars if you missed them so again there's information and of course you have links to the paper so you can read more so so again uh thank you everyone and uh i will see you again at our next webinar thank you all thank you thank you thank you