13:07:00 So, yeah, I would like to start by thanking the organizers for bringing all of us together it's been a super exciting program so far, and I am just so grateful to be here with all of you in person in a long time. 13:07:16 So I am going to tell you guys today, 13:07:21 some thoughts that I've had about why bacteria might fix nitrogen. 13:07:26 So before I get into that, since I knew I wanted to give you a little bit about my sort of academic background. So I started off working in one of the most model of model organisms equal I. 13:07:40 This was during my undergrad degree with Michael wF at UBC. And so we've already heard a lot during this program about how when you grow equally on glucose, you could get this crossbreeding polymorphous that arises repeatedly. 13:07:55 So I characterize that using both essays and then built an adaptive dynamic model, demonstrating that you could get evolutionary branching in this system. 13:08:08 However, during the course of working with Michael. I also got really interested in the evolution of mutualism and did the calculations and discovered that these large and small colonies are still actually competing with one another, they're not actually 13:08:23 meatless. So during my PhD at UC Davis. I interacted with a whole bunch of amazing people. They're getting news done. My learned how to do evolutionary genomics, either testing, one of the people really pushing the envelope in terms of coexistence theory, 13:08:44 Richard McCurry working in Game Theory Judy Brownstein working on the evolution ecology of mutualisms and spouse, who's done a lot of really exciting work looking at the evolution of plant interactions with various species. 13:08:59 So I just wanted to shout out to all of the people, not in this room who thinking has contributed to my own work. And so, yeah, so I got really excited about the legumes Libya mutualism and have also, you know, because just growing things in the lab and 13:09:18 greenhouse. Still was a little bit too disconnected from the real world for me, have had the incredible opportunity to do work at a number of. 13:09:30 Yeah, model sort of field sites. So that ultra our site Pullman, the Great Lakes biology research center and site that Sharon and I have been getting going up Bodega Bay, looking at like him coexistence. 13:09:44 So, over all you working across these different scales, the questions that really drive my research program are what promotes beneficial interactions between organisms. 13:09:56 And then how does diversity arise and persist. 13:09:59 So mutualisms are kind of interesting from a theoretical perspective because diversity within them seems like somewhat of a paradox. So, under antagonistic evolution as are all of you know, and appreciate you readily generate negative frequency dependent 13:10:17 selection and get numerous deals and phenotypes that are maintained and just really fast, you know, like with the Delta variant, all these really, really, you know, fast publisher dynamics dynamics. 13:10:32 So mutualistic couple mission. 13:10:34 Yeah, yeah. Can you define antagonistic evolution, oh sure frequency dependent selection. Yeah. 13:10:41 Yeah. So antagonistic evolution is co evolution between two species where the interaction is negative for at least one of the partners. 13:10:51 So to competitors, or a hosted a pathogen say. 13:10:57 So co evolution is when you have reciprocal evolutionary change in each partner and frequency dependent selection is when the fitness of a given you know type or phenotype is a function not only if it's phenotype but of that unit types frequency within 13:11:12 the population. 13:11:16 Yeah. 13:11:23 Okay, so right so mutualistic revolution. On the other hand is predicted to generate positive frequency dependent selection, and many models show coexistence of only two levels or phenotypes if you get diversification at all. 13:11:28 Good. Yes. 13:11:38 But when we look around in nature, mutualisms are really diverse. So some people have wondered whether they're actually undergoing antagonistic coalition. 13:11:48 And so there's been a sort of a cottage industry of people sort of thinking about and looking for examples of cheating and mutualisms. And so this really goes back to, you know, this sort of anti group selection, you know, kind of mentality within the 13:12:20 And so, to kind of build consensus within the field. 13:12:24 Emily Jones and I ran a MCs Working Group back when those were I think in 2012. And so we brought together a bunch of mutualism researchers, and did a big survey of people who identified as ecologists and evolutionary biologist and have them, you know, 13:12:42 sort of fill out this online survey about how well they agreed or disagreed with these different common definitions of cheating that were in the literature. 13:12:51 And so the consensus that emerged was the cheaters must prosper. So in terms of their fitness, a cheater must have increased relative fitness while decreasing its partners relative. 13:13:04 And this is important in terms of what we think that you should do in these systems, because if cheaters can't spread, and lead to mutualism erosion, they're not actually threatening the mutualism. 13:13:16 I'm looking through the empirical data, there's actually very few examples of this. And so this kind of left us wondering, maybe it's really hard to collect these data, or maybe cheating is actually where it's sort of hard to determine. 13:13:39 So thinking about sort of going more quantitative and trying to think about, oh yeah that's a quick quick question. 13:13:48 Yeah, so if. 13:13:52 So real fitness relative to who. Yes, okay, fitness relative to the population in. 13:13:52 Yeah. 13:13:57 Yeah. So, okay, so we have two species. So we have our folklore mutual list that has different phenotypes. And then we have its partner, which is Junior types of different species. 13:14:10 And so, you know, if it was possible to interact everyone by everyone and like, compute those population means witnesses of like every single interaction. 13:14:19 So basically each of these dots on the spinners correlation diagram, would be one of those interactions. And so then you would take the mean of like everything. 13:14:30 And basically asked whether or not. 13:14:34 Yeah, whether or not there are phenotypes president of one species that have increased fitness relative to the mean of their population, but the decrease the fitness of their partners that they interact with relative to the population that make sense. 13:14:53 That make sense. Okay. 13:14:55 So, um, yeah so there's basically this cheating quadrant, down here. 13:15:02 Just a quick follow up. 13:15:11 Doesn't make that a cheater, though, right so it's only a cheater if it also increases actually reducing, not just giving a smaller benefit to the partner. 13:15:21 Right. 13:15:21 Yeah. 13:15:22 Yeah, because you could have, say, like this mutation here that has that gives less benefit than the population benefit to the partner, but it also it itself has lower. 13:15:40 Can I ask another technical question. Yeah, so when you calculate means do you weigh them by the genotype frequency in the population or is this yeah I mean so ideally you would like, in reality, no one has that kind of data, but it like ideally you would 13:15:53 right because you what you care about especially you would like to weigh them by the genotype frequency. Yep. Yeah and you could also you know if there's facial structure or non random interactions, you know you could wait things by the frequency with 13:16:06 with which they actually interact and 13:16:09 If you have that kind of data, which we don't. 13:16:17 So if the two species so in tackling by say Jackson and benefits. 13:16:22 And if the partners are relying on on you for example if you reduce the supply. How then you will, by definition right reduce the average fitness of the partner. 13:16:33 Yeah, I will get to them. Yeah, yeah, I have exactly that example. 13:16:38 Yeah. Okay, so right so this is just to show that there can be these, you know, these genotypes that when you do these fitness calculations, you would call a cheater so like this unit type F. 13:16:53 But when you look at the fitness correlation overall it's really strongly positive. And so under fitness alignment, even though you can have some of these phenotypes that you know that we would call cheaters. 13:17:05 There are these others you know types that are basically super cooperators that have much higher fitness than the cheaters, and also increase the partner fitness so selection overall on the population move towards more holistic interactions. 13:17:19 So, the converse would be fitness conflict. So here, definite correlation is negative and so the population is going to move towards the cheating quadrant eroded. 13:17:34 Okay, so what determines fitness conflict versus alignment. 13:17:38 What are the traits that might give us a sort of biological understanding of this. And so we thought about two fundamentally different types of, you know, I work I suspect group so microbial immediate host traits, but you could, you know, think about 13:18:01 that are expressed when you have these interactions. So the first is one dimensional traits and then the second is this to a resource exchange. So with a one dimensional trait that has a multi genomic basis. 13:18:06 So there's basically a trade value so say like foreign time or root length of a plant. And if that value is determined by both the genotype of the host and the genotype of the microbe, Then you could imagine a fitness function for the host and if it was 13:18:23 function for the microbe and selection acting to change both partners within this sort of by fitness landscape. And so, what is kind of interesting to think about is that there's, you know, the sort of shape of fitness correlation depends on where you 13:18:57 are. So if you are in between the host and the microbe fitness Optima then you'll have this conflicting selection happening on the two, but on these other sides there's actually fitness alignment. 13:18:59 And so you could make predictions like you know maybe novel Association would be far from either optimal and you would want alignment, things like that. 13:19:09 And so it and assignments and, and a few other people. We recently did a little bit of population genetic evolutionary modeling on this talk about more kindness good, the blood. 13:19:22 I'm confused about precisely the question from the previous slide, which is whose trait is this, in this case, yeah, it doesn't it's both of their traits like flowering time or if understand if there's a mutation of flowering time it increases hot host 13:19:36 fitness decreases micro fitness, but it's a mutation of post in this case well so yeah so there could be mutations in the host foreign time and we know what lots of those are there can also also be mutations and microbes that influence the host firing 13:19:53 time. Yeah. 13:19:58 Yeah. So calling these just like plant traits. 13:20:03 It's really, you know, if you have say over a trade, you know maybe that trade is partly yours. 13:20:08 Okay, so getting into the resource exchange. so in this situation so we have mutual trust one that produces resource one consumed by me plus two, which produces resource to lots of diagrams, like this. 13:20:22 And so you could have a teeter. 13:20:25 Like, when you pointed out that could increase its fitness by producing less of the resource, which then would decrease the fitness of its partner, a partner was depending on that resource. 13:20:38 And so this is assuming that it's still taking the same amount of resource to. 13:20:46 Yeah. And so the. 13:20:48 Yeah. 13:20:50 But if the amount of resource to or the effect of mapping resource one to mutual lyst who's fitness is coupled to the amount that is being provided, then you could actually still have fitness alignment. 13:21:10 And so there's various mechanisms that people have proposed, and in many cases demonstrated that broadly fall under the idea of reciprocity, where if you give less resource that your partner is going to get less resource in exchange, and how fitness line, 13:21:27 this way. 13:21:30 Okay, So now getting into a biological system. 13:21:34 So, I will argue that biological nitrogen fixation is really a model system and a lot of ways for resource exchange mutualisms. 13:21:43 So, first of all there's a lot of nitrogen in the atmosphere the air that we're all breathing is 70% nitrogen, but it is as inaccessible to us and to plants. 13:21:54 As the water out there would be to this person on a sailboat. 13:22:00 And this is because the nitrogen nitrogen triple bond is extremely hard to break, so it can be broken by lightning. So very high amounts of energy. 13:22:15 Also using a lot of energy they were Bosh process, which uses really high temperatures and pressures to reduce the nitrogen gas with hydrogen to make ammonia fertilizer, leading to the Green Revolution. 13:22:25 And then there's biological nitrogen fixation which is done at ambient temperature and pressure. 13:22:31 So biological nitrogen fixation converts about 195 mega tons of this is helpful or not, of nitrogen per year, into the rest of the nitrogen cycle that we heard about parts of, but so I'm not going to get into paper Bosch converts about the same amount 13:22:52 through industrial fixation and then apply it as fertilizer, and then lightning gives an order of magnitude less. 13:23:01 So, nitrogen fertilizers have some issues that many of you are probably aware of. So they've been proposed to be where the numbers. Yeah, so that number is a factor of 1000 smaller than the amount of carbon that goes in and out of the atmosphere each 13:23:24 year. Does that mean that much of much more of the nitrogen is recycled without going into the atmosphere and back and back down again. 13:23:34 That is a question that I've never thought about before. 13:23:37 So I don't have an answer for you, but it sounds reasonable. 13:23:42 Yeah. Because there it's there. There's hundred and whatever it is gigatons yeah yes yeah I'm 13:23:55 surprised I mean the carbon nitrogen mission. 13:23:58 Yeah. 13:24:00 Yeah, so it's either recycled for now recycled laughs. 13:24:13 Yeah, yeah, yeah, no, that makes sense that's yeah 13:24:21 yeah okay so nitrogen fertilizers, how problems. It's been proposed that were beyond the nitrogen planetary boundaries. 13:24:29 And the main bad thing that they do is cause eutrophication of aquatic systems like the famous the dead zone in the Gulf of Mexico. 13:24:41 Yeah. 13:24:57 It contributes about 30% of their protein in our diet globally. 13:25:03 Through these delicious peas and lentils and there is growing interest in using biological nitrogen fixation for sustainable bio energy production. 13:25:12 So okay so this is kind of the most boring pathway there's none of this pathway branching or splitting or anything that we've been hearing about, it's really just like a one step thing where the nitrogen gas is reduced in the presence of redundant and 13:25:27 a lot of new ammonia and hydrogen gas. 13:25:31 It is done by this very elaborate enzyme system, nitrogen as that has a iron protein H, that feeds electrons to this tetra rumor. That's have tremors active site and either have a iron molybdenum co factor and I medium co factor or an iron co factor. 13:25:53 The molybdenum one is the most common and when people first discovered the iron, iron and the Canadian ones. 13:26:01 They were it was thought that these people were crazy, it was wrong. 13:26:05 But we now know that these alternative nitrogen aces do exist, the proteins overall are highly conserved, there's no other enzymes known that can do this reaction. 13:26:28 There was a report in the 90s from a German group of a super oxide dependent nitrogen a system, but I was part of an international team that showed that this does not actually exist it was due to a series of lab artifacts. 13:26:30 So the actual project enzyme is irreversibly inhibited by oxygen, which has consequences for its use in metabolism, as well as making it very challenging for the people who are trying to engineer it directly into plants nitrogen fixation evolved. 13:26:48 A long time ago, and is very widely distributed across both pretty bacteria across bacteria sort of all bacteria, many different. 13:26:59 Yeah, bacteria and all kinds of habitats, as well as, as archaea. 13:27:04 It's, there's some contention in the literature about whether the iron, iron or the iron molybdenum of all at first, but it's very clear that there's been significant horizontal gene transfer of this through leaves bull protein. 13:27:20 So with regard to plants. There are two basic categories of plants, it is a trip interactions. There's the associative interactions, which occur in the riser sphere. 13:27:36 Primarily, there are some reports of energetic interactions, like in sugarcane. And so these rises sphere associative nitrogen fixers tend to be diverse and consume carbon exited from plant roots. 13:27:50 There's a variety of different oxygen environments in these kinds of systems in the symbiotic system, there's a much stronger regulation, the bacteria are housed inside the specialized structured called nodules, and the plant modulates the oxygen environment, 13:28:09 you think like hemoglobin, which is what also gives the impossible burger it's just so nitrogen fixation seems like it should be costly cooperation, right, because the bacteria is doing very energetically expensive process. 13:28:28 The plant is speeding carbon. 13:28:30 And so there's been a lot of work. 13:28:34 Both theoretical as well as empirical demonstrating that there are these mechanisms. So things like partner choice, where plants will preferentially associate with more beneficial strains of razor Bo. 13:28:51 There's many examples of this in the literature. There's also a strong evidence for sanctions and reciprocity, where plants will allocate differentially to nodules that more or less beneficial strains. 13:29:01 And so these are sort of positive on the idea that for this to happen you need to have selection on the symbiotic to fix nitrogen, even at the expense of its own potential fitness. 13:29:14 Um, but I think that there are also cost free reasons for nitrogen fixation. So the first very simple one is that microbes fix nitrogen, because they need nitrogen. 13:29:26 And then plants just kind of benefit as bystanders microbes could be fixing benefits that are fixing nitrogen because plants are giving them extra carbon and sort of creating this environment to favor fixation sort of passively. 13:29:43 And then the most devious one is that vision fixation isn't actually part of a like fair exchange between the plant and the microbe, but the plant is really just kind of using the microbe and gene that happened to be present in the microbe as an organ 13:29:59 L, and is basically forcing it to fix nitrogen, which could be happening again, not. 13:30:05 So just to illustrate this sort of passive potential mechanism so if we imagine a soil with a plant roof growing in it, soils are typically carbon limited and they're readily available carbon. 13:30:20 And we know that plants are typically nitrogen limited and exude carbon. So, in the rise of spirit zone around the root, all of the nitrogen is going to be taken up by the plant. 13:30:31 And there's going to be high levels of carbon. So this is exactly the environment that would favor a free living nitrogen fixer, because there's lots of carbon but not very much nitrogen. 13:30:41 So if you want to grow you just mentioned and grow. 13:30:45 Yeah, so this sort of system has been called screening by rk and others were instead of sort of operating directly on your partner, you're changing the environment conditions to think of our partners that are beneficial for you. 13:31:01 So, yeah, so this would kind of set up a situation where you could have passive trade and byproduct benefits but the plant. 13:31:09 Okay, so I'm going to hopefully show you guys. 13:31:14 Data from two different systems. So one associative system. The switchgrass system in the field. And so this is a relatively short bit of ecology, mostly because it's interesting and cool. 13:31:29 And the second is in the Lake Erie PA system and the growth chamber. And this is where I'll get into that economic modeling of benefits. 13:31:43 Okay, so right so the switchgrass project is part of a big team that I became part of when I was at Michigan State University. 13:31:54 And so we are piggybacking our project off of this observation from the teal PRC that as switchgrass establishes, it becomes less responsive to nitrogen fertilization. 13:32:06 So that suggests that is accessing some other pool of nitrogen and the hypothesis is that this pool of nitrogen is nitrogen fixation. 13:32:16 Um, and so just to kind of orient you so these are plots, sort of mid season switchgrass by the end of the season it's above everybody's head. you can't really tell there's people there, the plots are all set up as blood clots that there's a fertilized 13:32:38 and unfertilized half, and then replicates in the field. So we went out and sorry I didn't really understand the graph on the right, maybe yes yeah okay so we have the switchgrass yield. 13:32:48 And so this is for one Junior type of switchgrass that's been planted and given different amounts of fertilizer. And so in 2009. There's a response to nitrogen, up to about a little over 100 kilograms per Hector. 13:33:07 But then, in 2010, it only responds to nitrogen up to about 50. And then, in 2011, these are the same plant because it's a perennial. But why am I supposed to ignore the fact that the yield is going up so much, and only look at the inflection point, because 13:33:21 it's the fertilizer response. Yeah, so as switchgrass establishes so because it's a perennial. They have really massive systems but it takes time for them to develop those root system. 13:33:33 And so their final biomass will will increase select their, you know their biomass at harvest will increase as the standard establishes. 13:33:42 Does that make sense. 13:33:44 And so this is just because they've been in the ground for longer and so they've had more time to store carbon below ground for the next season's growth. 13:33:52 And those are weird. 13:33:56 No, I understand I just. 13:33:59 Okay, maybe maybe won't be. 13:34:01 Yeah. Yeah, it's really not important. 13:34:09 Yeah, yeah. Know what yeah what's what's important is that is that this convince do to give us billions of dollars to do this like microbial ecology project. 13:34:23 Yeah, that was not a joke. 13:34:26 Um, anyways. Okay, so we went out. 13:34:29 So one of the sites happens to be right beside KPS. 13:34:36 And the piano projects there Evans lab is based there so it was very easy for us to go out and we decided to sample every two weeks, and we actually did this for three years but the first year we did acetylene reduction and then realized partway through 13:34:51 that the soils were generating ethylene and so, so we are ignoring all of the Southern production assets so this is 15 and two gas operation. 13:35:09 Data from these little Minnesota quarters that we take into the lab and we put them in little vials and give them sort of optimal conditions for nitrogen fixation. 13:35:20 And, yeah, and so this is what our data looks like. 13:35:25 So the first year of data has these really interesting patterns, where if we compare the unfertilized plots. 13:35:28 They got four carbon sources, they got good soil moisture. 13:35:35 In this light gray line, and the fertilized half of the same plots in this dark gray line, we can see that early in the season, the unfertilized plot of higher fixation rates. 13:35:47 As expected, because we're not giving them, fertilizers, and then it kind of tapers off during the growing season. And then, when plants, start to shift material below ground in the fall. 13:36:00 And so nothing starts there's this really interesting spike were like, Wow, this is amazing. 13:36:06 Let's see if it happens again in 2018 and it didn't it all. 13:36:12 Basically, yeah, basically nothing really happened in 2018, other than the rates just being high. 13:36:20 And, yeah, based on funding and then covered. We haven't repeated this detailed time series since then. 13:36:30 So, okay, so we went and looked at the new hm pecan communities of these back. 13:36:43 Yeah, so this is the this is the free live in nitrogen fixation rate. So it's the amount of of into that's being that's being like harvested by the soil community. 13:36:56 Right. 13:36:58 Yeah so so we take these little soil plugs and put them in vials and fight the atmospheres with 15 and two gas, and then add more carbon sources and bring us almost a 60% water holding capacity, and then incubate them for about a week so there could be 13:37:17 some, some turnover. 13:37:19 Yeah, some of our rates were really low, when we were doing the pilot and so we think impatient for a little bit longer. And then at the end of the incubation you dry the soil grind it up and then do you 15. 13:37:34 So, regular 1400 analysis. 13:37:40 Yeah. Yeah, and the gases all like acid washed and. 13:37:43 Yeah. 13:37:48 So, yeah, okay, so I shouldn't fixation happens varies by year, sometimes it varies within your but not always. And inconsistently with fertilization. 13:37:58 So we wanted to know who might be doing this nitrogen fixation. 13:38:01 So we did eight o'clock on sequencing, and we found that there were quite diverse and variable communities, but there wasn't really strong clustering. 13:38:13 There, you know, was some variation explain some very small amount of variation explained by data here replicate and whether or not it was fertilized but yeah not super clear patterns. 13:38:29 But clear that there's a lot of different things there, and that they're kind of wiggling around, we did do an indicator species analysis and identified a number of bacteria that are. 13:38:44 Yes. Sorry, just to understand the based on your answer to bankers question. 13:38:49 So you said you add carbon to this treatments. 13:38:55 What, why is that, mostly because adding carbon is standard for when people do nitrogen fixation. 13:39:05 And do you know why people do that. 13:39:07 Um, I mean I think it just it stimulates the committee's so that there's enough activity, sir. Yeah because typically so we've done a little bit of meta genetic sequencing of these and like the total abundance of like nitrogen fixers and pieces very low. 13:39:39 So in a way with me just to clarify what what you want to know is the potential for exact nitrogen fixation yeah naturally what's happened. Oh yeah, yeah, yeah, yeah there's, there's really yeah i mean if anyone has ideas of how to measure actual fixation 13:39:41 in the field. that would be amazing. But I, yeah. 13:39:45 Yeah, there are not related to that. Yeah. 13:39:52 In principle, we can get and we're doing some experiments yeah jars that I won't talk about it. But yeah, yeah, we can do that too. 13:40:02 Yeah. Okay, so these feel communities. 13:40:05 So, yeah, these are basically the taxes that are the most strongly associated with the rates of reliving nitrogen fixation. 13:40:17 So you can see if your favorite taxa is on this list. 13:40:21 Um, we also did random forest regression to see whether any of our copious amounts of metadata could explain variation in the rates of free living nitrogen fixation, and we found that soil moisture has the strongest predictive value. 13:40:37 And, you know, the correlation looks pretty messy, but it does come out as being a relatively good explainer. 13:40:46 So just to sum up this part. So the switchgrass prizes fear has diverse Day is a terrific communities. 13:40:55 There are multiple taxes that correlate with nitrogen fixation rates, you know, so it's not like there's one magical nitrogen fixer that's going in and fixing lots of nitrogen. 13:41:06 Yeah, we do have a culture collection in my lab of fixers that were doing genome sequencing and planting calculations with. And we also found that soil moisture is a big driver and this has been found before the switchgrass system, and is thought to be 13:41:20 due to modulating oxygen levels rises here. Just a quick question is, Are there many different types of plants they can have associative or it's a very special to switch, oh no water. 13:41:33 Yeah. Yeah. So, yeah, basically anywhere that you go in the world and tuna fish they go insane like there's always nitrogen fixers there, we just what we don't understand is, you know, what makes them active ones. 13:41:46 Yeah, yeah, yeah but they're, you know like, there's episodic nitrogen fixers and the tropics. 13:41:52 One sure the soul of this one has some fixers. 13:41:57 Yeah, then 13:42:04 these are unwell plan to. 13:42:04 These are perennials opinion so yeah and you know if there is any effect of previous condition so like the previous here. 13:42:11 Rainfall see so most most reason. So what's the effect of the historic variables on nitrogen fixation. Yeah. 13:42:20 Yeah. So Sarah Rowley has done some work in a slightly more controlled conditions. 13:42:30 And yeah has done some of that kind of modeling and her data suggests that it's rainfall in the lat like within. I think like three or four days, that's the, the absolute best predictor. 13:42:44 Rather than going like further back, but yeah. 13:42:49 Yeah, and we have definitely not done all of the time series analyses that we can do. 13:42:54 So if anyone has Yeah, interesting, talking to me about. 13:42:59 Yeah, so sorry if this is too similar to when you things question. So the question, going into this is that we, we don't actually know who nitrogen is in plants or is it that we know which species these genes are in but we don't know Institute who's actually 13:43:13 doing exactly, yeah. Yeah, okay. Yep. 13:43:16 So does this kind of analysis Do you feel like we were closer to this now or because of the issues about nitrogen fixing potential versus nitrogen fixing rate it's still unclear. 13:43:27 Yeah, so the the thing that we're doing now with the sample so we took just normal cores and then we also took fours and flashbulbs them, that we're working on doing the RNA extractions. 13:43:40 And so looking at, which packs are actually expressing nitrogen news is going to get us more of the way to answer. 13:43:49 Just a quick question. Yeah, so people don't like in situ measurement and see whether, especially does more associative naturally fixing activity near the roots versus some, some somewhere like even farther away, so I'm just curious like whether maybe 13:44:14 they're doing this with other microbes are they doing, are they doing exchange with that the parents have people done that spiritual 13:44:14 Have people done that spiritual waited, people are really there are some studies like that coming out. So, and in fact, even within you know like even close to our route, there's yeah what people call like hotspots on cold spots. 13:44:32 Because, you know, if you have if you think about a route that's growing. 13:44:36 It's really variable where the compounds are actually being exuded from so most explanation is happening at the root tips, and then it like functions. 13:44:46 So, yeah, so the the nutrient environment like varies a lot even like within a single. 13:44:53 Yeah. And, yeah, I'm not sure if that completely answered your question, but yes there's lots of stories, and a lot of interest in spatial variation. 13:45:05 Okay so that is all I will say about especially with nitrogen fixation. So switching over to the legumes area somebody who says, My first true scientific love. 13:45:18 So, This involves a highly profile etic clade of bacteria. 13:45:31 But the classic ones are all within the author pretty of bacteria. And until the early 2000s. We thought that our eyes were office until some people in France, decided to do a modulation asset because all they were getting on their plates, when they played 13:45:41 it out nodules were contaminants, and lo and behold, they discovered that Berkeley area are able to modulate and fix nitrogen association with a lot of tropical legumes, and they do this using nitrogen as genes that are more closely related to other beta. 13:46:03 Pretty good bacteria, but they're not factor gene which are essential for symbiosis came from the alpha particle back. Yeah. 13:46:12 So that is pretty cool. So rise up and live in the soil. 13:46:17 They're all around us, they in fact legume roots, through root hairs. So the root hair first forms the shepherd's hook that kind of grabs wraps around the bacteria, and then they sort of coordinate Lee create this infection thread that the bacteria enters 13:46:37 the root and the root grows. These nodule structures that are integrated with a vascular system. 13:46:46 And you can see the pink like hemoglobin, and so throughout this process the bacteria are encased in plant cell membranes, the action happens once they differentiate into these Bacteroidetes structures, larger and have more gene copies, and they up regulate 13:47:02 a massive portion of their genome and end up fixing nitrogen. So yeah so important facts and thinking about the evolution so the transmission is all horizontal. 13:47:14 Unless, like that what I was mentioning, you know, a farmer like what's the seed, with the right idea that doesn't happen in nature, the natural is essentially global. 13:47:24 So, yeah, there's a lot of, like thinking that oh maybe like you to revise maybe I can get into a mixed module that happens rarely, rarely from the data that we have. 13:47:35 Okay, so, assume. Yeah. 13:47:38 Hi. 13:47:40 Yeah, for for the rise opium when when during the mode of a fixing nitrogen inside the room. 13:47:49 Are they growing themselves are they are they just sitting the electromagnetic cells just, just a Tony. 13:47:56 Tony woven nitrogen to ammonia. 13:47:58 Yeah, so they're not actively dividing. So they do, they do end over duplication so they get really big, but they're not actively dividing and they're constrained inside of these plants on the brand via zones. 13:48:11 Yeah. 13:48:13 Very interesting and. So, but then that's the stage where, what the show is about the plants and feeding successfully and so forth to these bacteria. Right. 13:48:36 Bacteria right. And so I guess the public will not be much need if they're not growing the database, they're using these capital, just as a energy source to fix nitrogen, or other repaired, people looking to, um, yeah so yeah so one of the things that 13:48:48 they do with carbon and you can kind of see these panels. So, so most Rabia will accumulate probably had Roxy Peter eight. 13:48:59 that has lots of carbon in it. 13:49:02 That has been proposed as a potential sort of heating mechanism. So if they just like hoard all the carbon as PHP and then after they, you know, after the plant decays and they get back into the soil then they have this sort of store of carbon that they 13:49:16 could future fitness, but that will not that will not that they wouldn't. Well, there's not much room to go into the space and saturated, you can't add most of humor. 13:49:27 When body science was supposed to be. 13:49:32 Yes, I agree. 13:49:34 Okay so, so. 13:49:36 Okay so so so so okay it's not clear where the carbon from from the host is going to 13:49:48 nitrogen fixation itself very expensive energy wise. 13:49:53 Yeah, I mean it takes 16 ATP. Next one. 13:49:59 if it's if it's not growing Oh, let me ask another way that people replicated the situation of a nitrogen fixation say in vitro in maybe in a spill spill device or something so that they cannot grow and it just sort of sitting there, fixing nitrogen and 13:50:16 not a were of experiments with it but there are a lot of free living nitrogen fixers that often, but they're there. That's different. Yeah, yeah. 13:50:28 Okay. Yeah. Um, yeah I mean it is possible so like, yeah, and I'll show some of the much more complex metabolism from the pools group where you know so what they do is basically harvest back droids and then they can get, I don't. 13:50:46 Yeah, they can measure things about the membranes transporters and things. 13:50:53 Yeah, but in terms of having them, like actively fixing nitrogen in vitro I'm not aware of the can do that. 13:51:01 Or another way is the rate of natural fixation of Purcell the biomass No, this is the only one fixing nitrogen anyone can do a cell count to two to see it yeah yeah yeah there's estimate things estimator. 13:51:19 Yeah, 13:51:19 yeah, yeah it's it's really variable, and it also depends on the type of model. 13:51:25 Yeah. 13:51:28 Yeah. Yeah, kind of related question. 13:51:30 Should I think of these and the symbiotic nitrogen fixers as somewhat analogous to captured cyanobacteria or plastics, do we see a genome reduction, a substantial reduction. 13:51:43 Yeah. 13:51:43 Yeah, so there's only two daughter cells how, what do we know about that analogy. Yeah, so there's not. So, we actually have pretty big genomes they usually like seven ish Meg seven to eight. 13:51:58 Yeah, they use. 13:52:01 Yeah, they use a, they people have done gene expression, studies. And so they they definitely turn on different genes when they're in the soil, or broth versus in symbiosis so so they have this pretty big like suite of genes that we think that they're 13:52:17 probably not using when they're not in the host. Yeah, but there's no genome reduction, which is interesting, probably because you know they do have to go and live in the soil. 13:52:28 Okay, everybody else. Yeah, there's also kind of related to that. 13:52:34 There's a lot of right Sofia including some very closely related to the symbiotic recipe that don't contain the symbiosis genes. So they'll lose. So the symbol is the sort of core symbiosis genes are on transmissible islands point by certain sequences, 13:53:02 or cases on plasmids. And so, so they can be lost, these genes and Joel sacks Riverside spent his postdoc, trying to isolate non symbiotic rosacea from rises fears and there. 13:53:11 Yeah, I mean they're, they're pretty, pretty common in you know rises fear and microbiome studies, they're relatively common and the fights. 13:53:20 So like these guys IBM is like one of the most common rabid dog so screwed and to fight, but it doesn't have it doesn't populate or fix nitrogen, it's just, they're living in the plant. 13:53:33 Yeah, so it's it's more, you know, so I think of it more as the genes for nitrogen fixation, or maybe the organ Holland the bacteria is just kind of this carrier of the system that the plan is how I think about it, 13:53:51 Okay, but it's Yeah, it's kind of different. But if I, if I'm not mistaken, there's no example you had known of a captured Nigerian fixer obtaining the food status of an Oregon now, so to speak, is completely it's a replication is fully coordinated and. 13:54:12 Not that I'm aware. So I'm wondering why do you think yeah why not. Yeah, it seems like it would be a good idea. Yeah, yeah, I find it interesting that it's inside of bacteria were captured and became organ house with nitrogen fixers, we're not. 13:54:26 Yeah, no, yeah I mean there are lichens that have a nitrogen fixing symbiotic as well but yeah that's probably the closest example that I'm aware of. 13:54:40 Thanks. 13:54:42 Yeah. 13:54:45 They really captured though. 13:54:49 You know what the size of those sounds are. 13:54:54 Okay. 13:55:01 Oh, you should get a mic. 13:55:07 So sorry. 13:55:16 As Ola the Waterford has some cyanobacteria in those amounts that show that people are making that same proposal that it's on its way to organize status with reduced, you know, highly controlled metabolism by the Zola Fern, and this is my fun favorite 13:55:28 thing to research besides my own research so we can talk more. 13:55:34 Yeah, it's the only example of vertical transmission, that I know of, yeah. 13:55:39 Quick question so the annual the rhizome via that are associated with annual plants have no choice but to go back to the soil at the end of the growing season yeah plants and so, but yeah for any oh yeah system. 13:55:52 Yeah, the novels are perennial to. 13:55:56 So, so what I was asking is actually quite interesting in that because perhaps those environments that are associated with perennial plants don't have that are aren't relying on that separate topic lifestyle during the time of the year when plants aren't. 13:56:12 So I guess the follow to that, do you do know of any, maybe comparative genomics where you could look at rise OBO that are associated with annual versus perennial plants, that would be very cool. 13:56:25 So, one issue with perennial plants. Yeah, so the models are perennial, but as the root systems, grow, there would be no way, you know, like they, they still need a new a fresh rise OBO if they want to make a nodule on their growing, given. 13:56:46 Yeah, is the only problem. 13:56:49 Um, yeah I'm not aware of competitor my studies but that would be really. 13:56:54 Yeah, especially in places where there's like multiple annual. 13:56:58 Yeah. 13:57:02 What is the what is the characterization of the chemical environment set of these naturals Is it like anoxic neutral pH is it. Oh, um, I don't know about the pH, the oxygen levels are micro Arabic So around like 1% ish oxygen. 13:57:20 Yeah. And so the pink pigment is the legacy weapon that modulating the amount of oxygen. 13:57:26 Then there's there's additional for Denison has done some work on. There's some kind of, I want to say it's super in but don't quote me on that around the outside that also controls oxygen diffusion rates. 13:57:41 Yeah, but the auction environment is pretty clearly under the control of the plant. 13:57:47 Yeah. 13:57:49 Okay. So, is there a fitness conflict, so from everything that I've told you probably you'll be like well obviously not. 13:57:58 But I wanted to quantify this, so I compiled all of the examples that I could find in the literature where people go out and isolate a bunch of Arabia and inoculated on their favorite plant and measure performance and measure some aspect of somebody on 13:58:13 performance, so I could pull out lots of these fitness correlations and found to not my surprise, but many other people surprise that these are overwhelmingly positive. 13:58:27 Basically, yeah, for these different metrics of symbiotic fitness, which are imperfect, But still, what people have been able to measure on a wide scale. 13:58:36 So my conclusion from this work is that he's not fixing race OBO that everyone had been calling tutors are actually defective rather than being defectors. 13:58:47 Yes, quick comment. Yeah, because it's just intrinsically stripped specially structure environment. So in fact you will expect that so the, the real experiment which I presume you cannot do right now well mixing environment. 13:59:01 Whether you will record the investors, the system itself is actually very clever remix specialist. Yeah, Yeah, no, it's definitely yes. 13:59:10 Yeah, 13:59:13 yeah, yeah and you know you'd also want to turn off things like, you know, like most of the upstream signaling and you would want to turn off sanctions and you know there's all of these physiological mechanisms that we know are operating in the system. 13:59:36 Yeah, yeah. So the infection threads. Yeah, Dan kh from Sharon long club has done absolutely beautiful work on using personally labeled cells, and so he's shown so even if you have, like, if you're very lucky, you get like two distinct cells that found 13:59:55 the infection thread, because the infection thread is only usually about four cells across that are dividing end to end, you see the stochastic resolution of the infection thread into one type of the other side, by the time that actually. 14:00:09 Yeah, so there's like a really strong bottleneck. 14:00:16 okay. So, if there's not lots of cheating, we still know that there's a ton of variation in this mutualism so how are we going to understand that. 14:00:22 Yeah, that's 14:00:28 So with my former PhD student my very first PhD student, Colleen file, and then graduate student of Dr soccer Hill Theresa Clark, and our collaborator Emily Grumman. 14:00:42 We decided to try to take a quantitative look at mutualism by doing kind of a simple as possible thing and just manipulating nutrients. 14:00:51 Right. So previously I've been like, Oh, we need to like use all of these meetings and look at the creation, yada yada. 14:01:00 But, yeah, we decided to just manipulate nutrients and basically see what happens in our system and see if we can explain quantitatively. 14:01:11 The amount of variation. So, the sort of idea is that a plant can either infested carbon in growing roots that then go into the environment and take up nitrogen for its growth, or it can invest in its modules and feed wise idea to fix nitrogen for it. 14:01:30 And so the first experiment that Colleen did was to very both nitrogen and carbon. 14:01:37 So she, she varied carbon by changing the light levels. 14:01:42 Yes, sir. Can you say that again I didn't understand. I didn't catch the two scenarios. So you're saying invest in roots or yeah it's basically so they can either take up nitrogen through their roots and the environment or they can invest in nodules and 14:01:56 get nitrogen. 14:01:57 Yeah. 14:02:00 Yeah, so we buried the nitrogen environment, and then had low light and highlight, so at low light. 14:02:08 We're sort of using that as a proxy for carbon so it's just a proxy it's not perfect. 14:02:13 And the first thing that we observed was that the amount of allocation to natural biomass compared to the sheet weight decreases as you increase the amount of nitrogen. 14:02:27 This is a milligrams per liter. 14:02:29 And we didn't go above at because they just didn't make any modules. 14:02:35 So plants allocate less than nodules when they have lots of nitrogen and in fact, this is well known. 14:02:42 There's pathways that are well understood about how this works. 14:02:46 I'm more interesting, was our observation that at low nitrogen one plants are investing substantially nodules. 14:02:54 More interesting was our observation that at low nitrogen when plants are investing substantially in modules. They invest more when there's high amounts of carbon in the environment compared to when there's low amounts of carbon. So we interpreted this as 14:03:04 plants, paying more for nitrogen. When carbon history to them. So that's kind of makes sense. 14:03:10 So we wanted to get quantitative with this. So we use this economic model to basically will predict when trade is beneficial for both partners. 14:03:21 So this is basically just using the bags loft the minimum, where the growth of the plant, and the growth of the rice OBO is determined by whichever resources limiting to them. 14:03:33 And so we know for plants, they are limited by nitrogen. So we have our yield of biomass, which is multiplied by the amount of nitrogen is able to get for itself. 14:03:48 And then, the amount of nitrogen, that is able to treat for with its rise. 14:03:59 And then similarly the growth of the right so via is the yield spectrum about a carbon equation. So the cabinet could get by itself, which is nothing that's trapped model. 14:04:03 So it's basically dependent on the carbon, the plant will give it. 14:04:10 Okay. And so this kind of scenario sets up a situation that's called comparative advantage, where both parties can gain from trade. 14:04:20 And so basically if this exchange rate teeth is in between these two values then both partners will benefit from trading their extra resource, with their partner. 14:04:35 And so this is a win win deal. Everybody wins. And in fact, if you are a plant that has extra carbon, not giving that carpenters idea is actually costly because you don't need the carbon, nitrogen from your race. 14:04:51 So you could get the plant side of this model but I'm so curious about the microbe side so at the beginning of the taco to totally agree with you but now since we've been thinking about these. 14:05:01 Yeah, yeah. What are they nodules like should I be thinking the bacteria need to get in a nodule at some point in order to stay in their population or Yeah Where does that back to your growth term kind of come from. 14:05:13 Yeah, so this is all in terms of biomass, which we had many debates about when we were starting this project. 14:05:21 Yeah, we thought about maybe like fighting them out and trying to connect it to population dynamics. 14:05:26 We haven't done any of that. So it's, yeah so it's it's very much like a bio mouse like not fitness, right, like so don't think about the growth as the fitness of the partner, it's like literally just like the Resource Economics of web versus natural 14:05:43 biomass we do know that natural biomass. In many systems is highly correlated with heart with bacterial population size, like about. 14:05:54 So we feel like it's not an unreasonable proxy but it's definitely not 14:06:04 is similar to Terry's question then of if they're all sort of in maintenance mode like where does the law of the minimum, come from for the bacteria. I get it for the plants because they're dividing and they need to, you know, have enough nitrogen, carbon 14:06:18 Yes, One thing that I forgot to mention, very important. Excellent question. So, in a particular class of legumes, that include meta Congo, which is what we're using. 14:06:36 I'm the nodules grow forever. They have a persistent meristem and they just going. 14:06:41 So they just keep growing sales keep getting affected by by Right. 14:06:45 Yeah. 14:06:48 Yeah, so the five, so the natural biomass does continually increase in the system. 14:06:54 Yeah. 14:06:55 Yeah, I haven't thought carefully about how we would apply this model to other types of models that grow to a fixed size and then stop, but it would be different for sure. 14:07:09 Yeah, yeah, Yeah. Does that answer your question. Yeah. 14:07:17 Okay. 14:07:18 Okay, yeah, I'm getting I'm getting close to the end. So, I'm getting close to like the cool part. 14:07:24 And as you can tell my excitement level. 14:07:28 Um, okay, so we wanted to go for it. I have a question. Yes. 14:07:34 Wait waited so long so much patients. 14:07:37 So, this is Adam Smith, and if I read Adam Smith. 14:07:42 He says, actually, if I have monopolies or mercantilist trade. 14:07:46 Then this all breaks down. And I would argue nodules look much more like mercantilism then they look like free exchange which is coming back to the same thing Ben and Terry. 14:08:00 Again, yeah. 14:08:00 Yeah, you've ever made economics point of view, I'm not sure. 14:08:03 Yeah. 14:08:05 Yeah. Yep. 14:08:07 Yeah. Wait for the punch line. 14:08:10 Okay, so first we have to like collect a lot of data. So, Theresa and Colleen through all these plants. 14:08:16 So the legal system is really cool because you can grow them and these little containers with individual watering contractions so we can manipulate the bacterial environment and the nutrient environment for them in this nice bully randomized design. 14:08:33 So we measured all of the bio masses. 14:08:37 We measured photosynthetic rates. We did a little bit of nitrogen fixation and on the side. 14:08:43 We ground up everything to get carbon nitrogen tissue levels, and basically measured and fit this model. So, just to show you that the experiment kind of worked the way that we thought it would. 14:08:56 So in the absence of Rabia plants respond to nitrogen. Surprise surprise. 14:09:05 Rise area are quite beneficial at low nitrogen, don't really provided much extra benefit of high nitrogen. 14:09:10 Okay, so when you are building these models, there is this very important parameter called the exchange rate. so this is key. So this is basically the price of carbon, Nitrogen or whatever, the ratio of that. 14:09:30 And so to come up with that you have to make some additional assumptions, reading the bottle. And so, lik, and then Emily Gorman used this Nash marketing solution where you maximize the joint game. 14:09:46 So the way you do that is you take the growth rate of the plant in the presence of trade, minus, its growth in the absence of trade So how much does the plant benefit from trade and you multiply that by how much the benefits paid any maximize that, so 14:10:02 we did that, and the model did not fit our data at all. 14:10:07 So we thought of lots of reasons why it might not work. 14:10:12 We added a variety of assumptions about like natural respiration and, like, you know, all of these different things. Nothing worked until we came across a paper that talked about the asymmetric bargaining. 14:10:27 And so in the asymmetric marketing solution. 14:10:30 It's just like an ash marketing solution, but you introduce this a symmetry parameter beta, that basically tells you how much power, each partner has over the bargaining over the exchange rate. 14:10:45 And so when we did that, we still don't have a perfect agreement with our empirically estimated trade ratios, but it's a lot better than the symmetric bargaining models, which says a symmetric bargaining models predict that the plant should pay, way more 14:11:01 carbon molecule of nitrogen at low nitrogen low soil nitrogen levels, then they actually do. And so it's kind of interesting to me that this trade ratio that we estimated empirically doesn't actually depend on the external so nitrogen level at all. 14:11:21 Yeah, which is surprised I knew that people have ideas about why this is true, I would love to hear them. 14:11:28 Yeah, what is the value of beta you get and did you allow it to their a four six at once and for all. Yeah. So it varies. 14:11:37 Um, and so at low nitrogen beta value is estimated to be about point nine, so the plant is almost in complete control over determining the exchange rate at 80, milligrams per leader you asymmetry exponent is point six. 14:11:52 So it's much closer to the fair trade of panic. 14:11:57 Yes, Yes, and I want to hear everybody's thoughts about 14:12:17 Yes. 14:12:18 Yeah. 14:12:23 Yeah. So, yeah, I don't have an intuitive, if you guys are waiting for the intuitive interpretation as to why the planet should have more power at low nitrogen I don't have one, because it certainly seems like, but I'm kind of just have a question Did 14:12:38 you ever calculate the, what in economics to call the last two cities, we did that, because I feel like what you're getting wrong as the last two cities of carbon and nitrogen that one is like a luxury good and one is like a, like a basically a free producing 14:12:52 thing, and there's models that basically think about that as the last two cities and get back stuff like Terry's models. Oh, yeah. 14:12:59 Anyway, we could yeah buddy but yeah we didn't actually last two cities look wrong to me. Okay, both of them. 14:13:05 Okay. 14:13:07 I would love to hear more 14:13:10 afterward. 14:13:12 Okay. 14:13:14 Um, Yeah. Okay, so the plant has more bargaining power than the rise of you have a counter intuitively, it has even more at muscle nitrogen, unless we did our calculations wrong and there's really something else going on, which we don't know. 14:13:30 So, okay, so it grand ambition, would be to try to determine what these negotiation roles are in terms of actual metabolism. So using some of these metabolic models prioritize them with your data, there's like a gazillion legumes and rice Apia that have 14:13:47 been sequenced and lots of, lots of data out there, because we know that in reality the interaction is much more complicated than just carbon, nitrogen, here's a couple of models metabolic models. 14:14:01 Yeah so section eight can go in, but then the plant also fees are Janine in some systems, and then that gets cycled out alanine. 14:14:11 So there yeah there's like more complicated coupling down here, so there's definitely more complexity that I'm sure other people in this room would understand much better than I currently do metabolism is not my strong suit, but it's very interesting. 14:14:30 Okay, so instead of leaving you with conclusions I'm going to leave you with questions, because I'm sure that you guys all have thoughts on some of these things. 14:14:42 So the first one relating back to the first part of my talk is whether tutors are actually rare, or are we just got it finding them or maybe they're only sheet or sometimes. 14:14:54 Yeah, it's a little bit puzzling to me why there's not more evidence for these things. Why is there diversity and mutualism, I feel like I still don't have a good understanding of this. 14:15:06 There is this interesting phenomenon, and I may have a bonus slide if I wanted to see it about frequency dependent modulation. 14:15:15 But I will say no more about why is there diversity in Houston by on specialization. So within my clover system, we've measured specialization between a different species and somebody on that they all occur with, and can interact with an only one of the 14:15:34 species is actually specialized on its face Libya everybody else is a various strength of generalist right so why 14:15:45 can we actually predict and understand nitrogen fixation rates by microbes, you know, in the lab would be a great start. But then, ultimately in the real world. 14:15:57 In terms of doing things like improving agriculture. 14:16:01 And then what is the role of the hundreds of third party like that are also hang out in these systems. 14:16:07 So with that, I would like to acknowledge various sources of funding collaborators near and far. My current lab, and then various people that I've worked with over the years. 14:16:19 I think there might be time for more 14:16:30 time for multiple questions if people have more. 14:16:34 I was so worried about getting through all of us. 14:16:37 Yeah, I. 14:16:39 So it was a Christian hearing that you said that populations within nodules are colonial. Yes, and so does one observe predominantly so so one would expect the different articles that are different little populate exactly right. 14:16:51 And so, yeah do you observe competition between naturals that like is a plant selecting for better nodules and others or yeah so there's been a really nice work showing this phenomenon that's been termed sanctions, where if you have nodules on an individual 14:17:08 plant and some of them have really beneficial strains of Arabia and others have less beneficial. 14:17:13 This is not exactly the answer to your question but it's the data that I'm the most familiar with that speaks to it. So the plan allocates more to the more beneficial nodules. 14:17:24 So, I'm just does happen. 14:17:28 Yeah, both in an absolute sense, and the relative sense. 14:17:32 So yes. 14:17:38 Yeah, so I guess, can you go back one slide to your questions yes I'm trying to understand the wire cheaters actually rare and maybe simultaneously Why is their diversity and mutualism yeah things. 14:17:49 So I guess I'm trying to think about what would happen if these things were had the opposite conclusion like how would cheaters actually take over in such a population like what would be the mechanism, but it's it seems to me that that you know if they 14:18:04 cheat in the nodules that doesn't really help them spread. So, should we be surprised that they're rare given that and that, you know, maybe the diversity comes from the fact that they always have a life stage that they have to be free living and competing 14:18:20 on other resources. 14:18:23 And that could explain both through diversity and lack of Cheaters at the same time. 14:18:26 Is that is that a reasonable way to look at it or miss. Yeah. 14:18:34 Yeah. So in terms of I mean we know very little about what they're doing in their free living stage. 14:18:40 Yeah, Joel sacks did some experimental evolution, with symbiotic strains of idea where he passage them through media and the absence of symbiosis. 14:18:52 And do they see very much K, which doesn't necessarily mean that there's, there's not differences inflection like a, like a robust around. 14:19:07 Yeah. 14:19:07 So I guess I have a question about the numbers. 14:19:10 What is the average amount of time like in generations, what fraction of the generations of these guys have spent in an agile environment. Oh, I wish I knew the answer to that question. 14:19:30 Yeah, and no i excursions into nodules mostly free living bacteria or yeah like a key part of their overall replication. Yeah, um, I have no idea. I can tell you I've like done the calculations for medical nodules and there's about 10, doubling. 14:19:39 But, Yeah, the soil, I have no idea. 14:19:44 Yeah, maybe Jay knows how to answer that question 14:19:51 on the experiment. 14:20:08 Yes, to ask, in an individual Nigel. Yep. Guys who can't fix nitrogen have an advantage. Yeah, they don't ya gotta play can function within modules. Yeah, there's actually yeah there's some beautiful work in soybeans, so you have to inoculate a really 14:20:17 really high densities to get any of these doubling doubling infected modules. And so within 30 modules, you get these like sectors basically were different parts of the module will be colonized by the fixing versus the non fixing strain the fixing string 14:20:41 sectors, the plant up regulates the like bourbon and then the non fixing sectors. It doesn't, so it can tell the difference. And then a couple of groups have shown increases mortality. 14:20:48 More reasonably additional methods. 14:20:52 Yeah, so the the non fixing one, even within a nodule that has fixing right Oh yeah, there's still increased mortality of the non fixings. 14:21:04 Yeah. Yeah, but they do tend to be indistinct plant souls. 14:21:10 Yeah, so I can't think of. So if there was like one plant cell that had some of it some by zones with a fixer and some of it sells with a non fixer. 14:21:22 I'm not aware of people having observed that. 14:21:28 Yeah. 14:21:28 Yeah. What was the life cycle of the right, Sylvia in the nodule that the nodules don't. They are not active very long Isn't that right, that they become, it depends on the type of nodule. 14:21:39 So in meta Cabo and clovers, the marrow stem of the module is persistent and so you get the sometimes it'll branch and you get these sort of like finger like module structures. 14:21:51 So there's basically there's a dividing meristem. That's the youngest planet cells, and then those are being actively infected by the infections rather than have the fixation zone and then some massive zones developments modules in filebeat modules are 14:22:06 like Lotus Japonica, the back roads. 14:22:12 Yeah, the meristem like stops growing. 14:22:16 Got a fixed all is the reason I'm asking you, how do they get out of the good bacteria, do they have to wait until the root decays or how do they spread. 14:22:30 Yeah, so, so they basically have to wait until either the plant decays or in the field what I've noticed is that there's really really high levels of module or debris, especially for plants that have large modules. 14:22:43 And so there's a number of sacks. The particular field site that I'm working on for a long time. It's a weevil that lays eggs at the base of the plant and then the larvae hatch and they climb down and they match up everything delicious inside of a module 14:23:00 and then take off. 14:23:03 Yeah, when I was a grad student, I attempted to culture Rabia from them and and failed. 14:23:11 So I don't know. Yeah, yeah so like you know so we bowls might be are like always. 14:23:24 I don't know. Yeah, yeah so like you know so we both might be are like always. Yeah. Um, is there any competitive work about these three forms of nitrogen as that you described earlier, like activity or province in the. 14:23:34 Yeah, There is a lot of work on them. 14:23:38 Okay. 14:23:41 So, 14:23:53 Okay, so alternative no drugs. 14:24:10 Okay, so. 14:24:13 Okay, so the first thing that's really interesting about the alternative nitrogen is is is that bacteria that have an alternative nitrogen is always have the molybdenum my projects as well. 14:24:27 And so they basically use the alternative nitrogen uses like a backup nitrogen. 14:24:32 So when molybdenum becomes limiting, and this has been observed in those that have active and Lindy I has. 14:24:41 I believe all three, if I'm remembering correctly. And, yeah, and so you can, you know, start it from boom boom boom and it switches on. I think the iron one first. 14:24:51 They yeah the motor one is the most efficient in terms of ATP the other ones of us more. And they also have slightly different sensitivities. 14:25:03 Yeah, 14:25:07 yeah, yeah, they all. Oh yeah. The other thing so they only very in terms of the structure of the attack armor. they all use the same page.