και είναι ότι είναι ο εργαλός που βλέπουμε. Παρακολουθούμε, όλοι και ευχαριστούμε πολύ για την συμμειλώνα του Σασαικού Σεμίναρ. Είναι ο Σασαικός του Σεμίναρ, και είναι ο ίδιος κοιμήρς του Σασαικού Σεμίναρς. Είμαι ο Γεωργή Καφετζής, ένας φορματοσυστικής κοιμήρς, σε το Μασσολαιρήσωλετ's λόγο. Και τώρα ήρθε σε ένας θεούής με τον Μπαδεμ. Και as your host for today, I would like once again to begin by thanking Tim Vogels για να ξεκινήσουμε να ευχαριστούμε τον Τύμ Βόγγελς και τον Πάνος Βοζέλος για να προσπαθήσουμε αυτό το very initiative στους the greener and much more accessible seminar world. Όχι όμως, αλλαόμαστε να πάμε back to the reason we all gathered here for today and introduce our guest from the University of Maryland, Prof. Karen Carleton. With undergraduate studies in chemical engineering from Yale University, she went on to University of Colorado and acquired her PhD in physical chemistry in 1987. Following postdoctoral years in Berkeley with Bradley Moore, Karen started her own lab in 1989 in Andover and physical sciences incorporated. In 1995, she then joined University of New Hampshire as a member of the Hubbard Center for Genome Studies and the Department of Zoology before moving about a decade later to University of Maryland, where she has been located ever since and nowadays holds the title of professor in the Department of Biology. With research interest revolving around photoreceptors, color and visual communication in coral reefs, it is with great pleasure that we are having current today here with us and we will be hearing about cyclids in her talk entitled Using Obscene Genes to See Through the Eyes of a Fish. So without any further ado from my side, please all welcome Prof. Carleton. Karen, this stage is officially all yours. Thanks very much, George. I really appreciate the invitation and the opportunity to speak with everybody. And I wanted to thank Worldwide Neuro for this initiative and especially the Sussex Group for keeping the vision community going during these tough times. So I really appreciate that and it's the vision community I think is a really wonderful group to work with. I've enjoyed collaborating and so a quick shout out to Team Opsen out there. So as George said, I have sort of a little bit of an unusual background and with my physical science training, I think about the visual system primarily by following the path of the photon. And so this helps break the visual system down into some key steps that we need to think about as biologists. And so we have an illuminant that's going to send photons out through the environment. They're going to reflect off some object that is then going to be detected by a receiver. And the light will go through the cornea, be focused by the lens and form an image on the retina. And in the retina, there are these photoreceptors that are going to detect that light. Now today we're going to ignore the rods for the most part and focus mostly on the cones and there are multiple types of cones that are useful for bright to daytime vision. And these cones differ because they have different ops and proteins within them that produce visual sensitivities in different parts of the spectral range. And so these different signals can go from the retina into the brain, where the organism can then make decisions about what sort of behaviors to follow, whether to avoid something or whether move towards it. And so vision is an incredibly important part of animals behavior. So vision is really fascinating because there's a very clear link between the genotype and the phenotype. So the genotype is the sequence of the ops and proteins that surround this 11-cis retinal molecule that absorbs the light. And it's the amino acids close to that retinal that shape the absorption wavelengths of that visual pigment. So the sequence or the genotype of the ops and shapes the phenotype or the visual sensitivity of the organism's eye. And this has important functional implications for that organism. Likely the visual system is under strong selection based on whether the animal is foraging or mating or trying to avoid predators. And there can even be differential selection for organisms that live in different environments or maybe are performing different visual tasks. And those that selection might vary even throughout the day, across the seasons, through an animal's lifetime, say between larvae and adults or across evolutionary millennia. So there's a lot of opportunity for the visual system to evolve and for us to understand how and why the visual system works the way it does. I'm going to talk about visual systems today and fish are a wonderful system because there's so much diversity across fishes. So fish make up half of all vertebrates and they've been around for many hundreds of millions of years. Even our model zebrafish is several hundred million years divergent from many of the fish we're going to talk about today.  And for those of you who maybe don't think you know too much about cichlids, if you've ever gone to a restaurant and ordered tilapia, you have eaten a riverine cichlid. And these cichlids come from the river Nile in Africa, so they're riverine generalists, but they're now raised around the world in aquaculture facilities and they produce about 5% of the world's aquaculture output. So tilapia are farmed everywhere in an important source of protein for the world. But in addition to the tilapia, there are something like 2000 species of cichlids that are distributed around the globe. And that includes cichlids in the new world, cichlids in Africa, Madagascar and even a few in India. But if you look at the species distributions across these four different groups, the vast majority of the diversity occurs in Africa. And so we'll spend most of our time today talking about African cichlids. Now, much of the diversity in Africa is within the Great Lakes, although there are cichlids all in the riverine systems throughout Africa. And as these lakes were forming relatively recently, several of them are in the Rift Valley that's forming as the plates of Africa are spreading apart. So as these lakes are forming, the riverine species invaded one or a few times to seed the radiations in each of these lakes. And so in these newly forming habitats, there was sort of a blank slate and cichlids rapidly diversified to fill all the available ecological niches. Now this diversity has arisen quite rapidly. Lake Victoria is less than a half a million years old and it has hundreds of species of cichlids within it.  But there are literally hundreds of species and this makes Lake Victorian cichlids some of the fastest radiating vertebrates that we know of. Lake Malawi cichlids are a close second to that. Now, this image here is a satellite picture that's color coded to show water clarity and the blue or the coloration, the smaller the attenuation coefficient, so the clearer the water. And so you can see that both Tanganika and Malawi are quite clear lakes. There's some of the clearest freshwater lakes in the world, whereas Lake Victoria is quite a bit more murky. And so this allows us to kind of do an evolutionary experiment of each of these lakes has an endemic radiation of cichlids. So we get to run the evolutionary experiment multiple times and see what happens when you take cichlids and add large bodies of water. And because these vary in their environment, we can see sort of what the impacts are of different environments. Now, in addition to habitat differences, cichlids have huge variations in behavior. Some cichlids will pair bond and in that case males and females look quite similar to each other.  In this species, these females live in these individual houses of these little shells. And then oftentimes, cichlids will be female polyandrus and that means that females control mating and they choose multiple males to breed with. And this typically occurs on a large lec. And so here is a rocky lec from Lake Malawi. And you can see several males here all holding territories, all relatively close to each other. And females will then come swim to this lec when they want to breed. They have to find the male of their species and then choose which of the males they're actually going to breed with. And so males tend to try and be quite flashy and try and entice the females to breed with them. Now you might notice there's some other fish that are quite muted in coloration and females tend to be very muted. They want to be cryptic in this environment for reasons I'll tell you in about in just a second. So in addition to mating behavior changes, cichlids also can vary in parental care. Some cichlids will actually lay eggs on a substrate, other cichlids will actually mouth brood their young.  And so because these females are sort of mobile nests carrying their eggs around with them and they do this for several weeks of time, they want to be quite cryptic so that other cichlids don't notice that they're there because those cichlids will try and ram them and have them spit out their eggs so that they can be eaten. So females tend to be quite cryptic and males want to be quite conspicuous. Now, most of the cichlids I'll talk about today, including the tilapias and the cichlids from Malawi in Victoria, are these female mouth brooders that are polyandrus feeding with breeding with multiple males. So the females will visit a male lec to choose their mates. This requires species recognition, then mate choice.  Again, males are very colorful and females are not. Now within Lake Malawi, which I'll talk about quite a bit, there are two major habitats, sand habitats and rocky habitats. And over the sand, you get different cichlid species where males will build these breeding sites or bowers, as we call them. And each species will have a different bower shape. Sometimes they're castles, sometimes they're pits with mounds around them. And again, each species has its male build a different shaped bower carrying huge volumes of sand that they have to then maintain. Over the rocks, the fish have it a little bit easier. The males just have to defend various rocky regions, cracks in some cases, caves and other cases. And they will then try and entice the females to breed. They will rise up and either use kind of vibratory motions or auditory signals or chemical signals, hoping to entice the female to go back to his breeding site where she will lay eggs, he will fertilize them and she'll pick them up in her mouth and then go off and decide whether to mate with additional males. So that's a little bit about cichlid behaviors. So again within Malawi, we have these sand and rock dwellers and from the time of the riverine colonizer, there was this first split between sand and rock that was then followed by massive diversification. Again, 2000 species that have all kinds of different body shapes and colors. And if we look at some of these species, we start to put them in piles and we start to first have piles that are genera and genera typically differ trophically based on what they feed on. So we have predatory cichlids that have these fusiform shapes with large jaws. We have snail eaters that have strong jaws for crushing snail shells. We have fish that delicately pick invertebrates out of the algae or plunge into the sand to capture invertebrates. We have fish that rip scales off of other cichlids. We have zooplanktevores up in the water column sucking in a little zooplankton. And then we have the cows of the lake scraping algae off the rocks. And all of these diverse genera arose from this omnivorous riverine ancestor. Now, if we look closely at a given genus, the thing that differs between species is male color. And so all of these cichlids here are in the same genus and they all feed generally in the same way and they have similar behaviors, but they differ by the flashy male color pattern that they use to try and attract females to breed. And so female cichlids like cichlid biologists use male color to kind of distinguish species. So going to Malawi is great for doing field work. We've been fortunate to go for a number of field seasons. We work at the University of Malawi's field station, which has a compressor for scuba diving. I often go with my husband and some of our students and we collect and dissect lots of fish. We can go out on this boat for several weeks going up and down the lake. It's a huge lake kind of the size of Lake Michigan. And it's a really interesting and fascinating place to be. We've been fortunate to entice some visual scientists to join us. So Justin Marshall and Tom Cronin came for one field season and this is Justin kind of gathering some spectral reflectance data of substrates. And the graduate students are generally really happy to be there enjoying the field work, looking at this huge diversity. This might be, you know, 50 different species all swimming next to each other. And you literally can't decide what to look at first. So people have been studying cichlids and cichlid vision since Russ Fernald first started this work back in the late 70s. And all the cichlids that were looked at early on proved to be trichromats. So just like humans, cichlids have three different cone types. The short wavelength cone is in a, the short wavelength visual pigment is in a single cone. And the longer wavelength pigments are in these joined double cones, which we think neurally are independent of each other. These form a highly organized retinal mosaic and they are sort of evenly distributed across the retina. So we expected because they had three different cone types. And to produce three different visual pigments, you need three different ops and genes that they would indeed have three ops and genes one for each cone type. And it's the ops and surrounding that retinal that causes the option shift, shifting the absorption spectrum of the retinal out to different wavelengths that produces these different spectral sensitivities and the different types of cones. So we were surprised as we started doing more micro spectrophotometry with help from people like Ellis Low and Farron Sarosi, that we started to find multiple types of visual pigment combinations. Some species were UV blue green and green sensitive, others switched off the UV to become violet sensitive, and some cichlids even had a sort of more human like blue green and red sensitivity. So I thought this was going to be really exciting, we would go in there and sequence our three ops and genes and we find these major sequence differences and we would totally understand cyclic vision by looking at ops and gene sequence variation. And I couldn't have been more wrong. So we started sequencing ops and genes and we found that there were actually seven different cone ops and genes. And we were able to work with David Hunt and Jim Bowmaker and Phyllis Robinson. You can actually take each of these genes, express them in a protein expression system, sort of add 11 cis retinal and stir, and you can measure that we have a UV sensitive visual pigment from this gene, a violet, a blue, a blue green, two very similar green pigments and a red pigment. And these are all because each of these ops and genes produces a visual pigment in a different part of the spectrum. So, cyclic therefore have sort of a whole range of visual pigments. And it seems that the way they build their visual system is to take one of these short wavelength sensitive genes and express it in the single cones, and two of these longer wavelength genes and express them in the double cones. So, it became rather than a story about gene sequence, it was a story about gene expression changing which set of these available genes were expressed. So, I'm going to talk a little bit about the molecular mechanisms that cyclics can use to tune their visual sensitivities. Just first of all, we know that ops and sequence can have big impacts on visual sensitivity, changing individual amino acids that are close to this retinal molecule can produce measurable shifts in the visual pigment absorption. So, the absorption will peak at a different wavelength depending on the sequence. But when we went to sequence the ops and genes across cyclics, what we found was most of these ops and were the sequences were quite similar across many different species. And it was only the shortest and the longest wavelength genes that actually had significant sequence variation to produce small spectral shifts. And so this confirmed that it was not ops and sequence differences that could explain the large visual pigment differences we were observing that it had to be something else. So again, the shortest and the longest genes could vary visual pigments by a small amount, but most of the genes in the middle were pretty stable, they didn't vary much. The big way that cyclics change their visual sensitivities is by expressing instead of say an SWS1 gene and SWS2 gene. And so by changing totally the amount of which gene is expressed, they can produce big shifts in their visual sensitivities. And this then explained these large differences we had observed with microspectrophotometry. When we went to do gene expression using quantitative PCR, we found that this species was UV blue, green and green sensitive because it expressed the UV blue, green and green genes. This one switched off the UV gene and turned on the violet sensitive gene, and then this species expressed the blue, green and red genes. And I'll just say here we've got seven different cone ops and genes, but two of them are so similar that we can't genetically distinguish them always and so we often group them together here. So we have six different genes that are varying or just six different classes that are varying between species. So now if we look across 50 different species we can see, so we call these the short combination, the medium combination and the long combination, and we can compare how frequently cyclics can switch between these different combinations. And we found when we looked at a bunch of sand dwellers and a bunch of rock dwellers that all lived within the same bay, that we could identify genera, where one sister taxa was say, medium sensitive or one was long sensitive, or one was short and one was medium, or one was even short and long. And so sister taxa are differing by which of the ops and gene combinations they're expressing, and that suggests it must be a very labile process easily changed between species, which was sort of totally surprising. So cyclics have some of the most variable visual systems between species that we know of. Well, we wanted to see if this occurred in the other lakes as well and so, working in Lake Victoria with only sea housing and in Lake Tanganyika with Hans Hoffman we collected a bunch of species. And just to give you a hint, here's some of Justin's downwelling light data in Malawi to compare with only sea houses data in Lake Victoria. Lake Victoria being more murky has essentially no ultraviolet violet or even very little blue light present. And so these are very different light environments. And in agreement with that, we found that the Lake Victorian cyclics don't express all of the options. They primarily only express this long pallet combination, being longer wavelength sensitive because there's no UV or violet light to detect. That differs from again Lake Malawi where we have multiple combinations, both the long, the medium and the short. And then we found that in Lake Tanganyika, which is just as clear as Lake Malawi, we also get species that have all three expression combinations. And so both of these lakes are clear and have the species that differ in which ops and combinations they're expressing. We were then able to use this phylogeny to kind of reconstruct what we thought the ancestral taxa might have been. And so these pie charts show the probability that the ancestors of each of these groups have had one of these three different pallets. And you can see that the ancestor of all of these groups was most like the riverine species, again the tilapia, which also has the long wavelength pallet. So this suggests that both within Tanganyika and within Malawi, they have separately re-evolved this ability to have species that differ in which of the ops and genes are expressed. And so not only is it labile within lakes, but it has re-evolved in both of these lakes independently. We wondered why all of these species would have all of these genes if they weren't being expressed. And so we decided to look through development and we chose to look at this riverine outgroup species to begin with. And so the tilapia is easy to raise in the lab and it produces lots of young. And it allowed us to look at how much opsum was expressed in species or individuals as a function of their age. And so we took a couple of different brews and we would just sample a couple of individuals every couple of days. And what we found was that the very young fry expressed mostly the UV gene. That then turned off by about two months and was replaced by the violet gene, which was then turned off and replaced by the blue gene so that the adults have blue expression in their single cones. The double cones do something somewhat similar.  And the red gene really takes over just like this blue gene does in the tilapias. And we've got a little bit of green ops and expression as well. So we have a lot of variation through development with all seven of the cone ops and genes, including the two very similar ones that we have distinguished here being expressed. So we started to wonder then what was happening in the lake species if this riverine species was showing so much variation. And so I'm going to code this this way. So here again is the tilapia results for larvae, juvenile and adults in the single cones and in the double cones. And what you see when you compare what's happening in the riverine tilapia with other species, what you find is that in the lake species, they turn on the larval genes in some species and then just keep those on. Some species go partway to the juvenile stage of the ancestral type and become medium wavelength sensitive. And then some species just turn on this adult expression combination. And so we have highly modified the developmental patterning seen in the riverine outgroup in the lake species. And so this might be because the lakes are more stable environments. And they've modified sort of that developmental progression to produce the three different visual combinations that we see varying between species. We even see one species here which actually has changes in the single cones but no longer changes its double cone. So it seems that single and double cones are decoupled in this developmental programming. So as we were doing these studies we were collecting fish in the wild. And then when we're starting to bring some of them back into the lab. And we noticed that fish that in the wild had very clean ops and expression combinations. When we brought them back to the lab would start to express additional genes. We wondered were they now going from being trachromatic to pentachromatic. So were these being uniquely expressed in individual cone types or was there some sort of co expression going on. And it turned out to be the latter. And so we started to do in situ. So Brian Dalton was actually a graduate student with Tom Cronin who came and worked in the lab. So you can see the retinal mosaics that he's taken a picture of with the single cone in the middle and the double cones around. And here he's just labeling with one gene. You can see the alternate double cones that are getting labeled as you move down a row. When he labeled with two different genes you can see that some of them are always in opposite members of a double cone. But other genes are only co expressed in double cones. And so what turned out was that these genes, the extra genes that are appearing in the lab are being co expressed and are not in their own cell type. This gene expression turned out to be even more complicated because it turned out that, well, so sorry. So producing two different visual pigments in the same cell essentially is like finger painting. It allows you to mix and match visual pigments to produce a pigment that's intermediate. And this is a way then by tuning the ratio of these two genes to infinitely tune the visual pigments that are present in the retina. And so cyclics now have sort of an infinitely tunable set of visual pigment sensitivities. Well, this co expression that we are observing turned out to be quite a bit more complicated because it turned out it also varied spatially across the retina. And so for this set of genes here, this gene was present in every single double cone across the retina. And that's shown in the regions where it's blue. So where there's pure RH2A expression, that's in blue. But there's co expression of this LWS that increases as you go dorsal ventrally. And so where it turns red, that means 100% of the cones are co expressing both genes. So we have some dorsal ventral variation with this pair of genes. When we looked at these two genes, which are co expressed, they vary temporal nasally with pure expression of this blue gene in the nasal region and the temporal region and then co expression in the nasal region. And then for the single cones, we had this region right here in the middle, which we call the Aryan centralis, it has high ganglion cell densities. And so in the center here, it's pure UV expression, but in the periphery, we have pure co expression. And so these different variations suggested that there's sort of a region in the middle here, this area centralis where co expression is quite low, but in different parts of the periphery we're getting different amounts of co expression. So crazy so much variation every time we started to look at a different factor we found more variation. Well, this co expression turned out to be responsive to light, and it turned out to show some environmental plasticity. And so Pratima in the lab was able to take some wild caught fish that we had shipped, and we started sampling the first day they arrived from the airport, and then she sampled them several days later, having them live in the fluorescent lighting of our lab. To compliment those experiments Miranda took some fish that were raised in the lab and moved them either to bright lights, or to more solar like illumination which you get out of these metal halides. And she also sampled over relatively short time scales. And what we found in both of these cases was there was a lot of plasticity and response when we changed the fish from a wildlife to a lab environment. So in the wild the fish have pure expression, and when they start being raised in the lab, even after three days they're co expressing these additional genes in these cells. Similarly fish that were raised in the lab have co expression but when we move them into a more solar like environment with a lot more UV content. Now they go back to being more pure expression. And this happens over just a few days. And so this gene expression shift is very rapid. And amazingly so and might help cyclids adapt to environmental shifts as they're moving about in their local environment. So we have then a whole bunch of different mechanisms by which cyclids can tune their visual systems. So we have shifts in gene expression, major shifts turning genes on and off, and then subtler shifts that are due to co expression that are in response to changes in the environmental lighting. Well, we wondered then were any of these this variation functional, did it. Sorry, matter at all I'm going to leave the head. And so, move back. So we have these many different mechanisms that cyclids are using. They change sequence they change gene expression. I'm running out of time so I'm not going to show you that they change in lens transmission, they change in chroma for expression, and they even have some gene losses, particularly in the central and south American species. And in doing sort of a survey across a whole bunch of species. We found that many fish now use these same mechanisms for tuning their visual systems. And so, cyclids are just the tip of the iceberg, as it were, with fishes ability to tune their visual systems to adapt over both evolutionary and then shorter term time scales to adapt to different light environments and likely also ecological foraging and and behavioral traits. So I encourage any of you who are interested in different groups to keep looking because I think we'll continue to find a lot of variation across different groups of fishes. So again, we wondered whether any of this variation really might matter. Would this is maybe this variation is just random noise, or maybe there's just a lot of individual variation. And so I'm going to tell you three quick examples of why we think this variation is correlated and important for both behavior and ecology. So I'm going to start with an example of coding sequence variation, which I probably convinced you was not important but which actually turns out to be important in some instances. So, working with only Seahouse and again on the Lake Victorian cyclids, we found that there were two species that varied in the sequence of their ops and genes having three amino acids that differ. And these cause functional shifts in the visual pigment sensitivities, both based on MSP done in Jim Bowmaker's lab and doing protein expression that work that was done in the Japanese lab of Norio Cata. So these functional differences might be important. Again, remember that the Victorian cyclids only ever expressed the blue, green and red opsons and so this red opson might have some impact. And so only was able to go to the field and measure the light environment at the surface one meter down and two meters down. And what you can see is that the lights environment shifts quite rapidly with depth in murky Lake Victoria. Well correlated with that they found that the species that had the longer wavelength LWS version occurred at depth and fish that had the shorter wavelength LWS version occurred shallowly. Coincident with that, they also found that the fish that lived shallow were blue in color and the fish that lived deeper were red in color. And so this suggested that this environmental gradient produced a visual gradient and evolution of the visual system, which then fed back on female mate preference, such that male colors evolved to produce red males at depth and blue males in the shallows. And these two groups are actually different sister taxa their species and so vision and visual sensitivity differences have actually driven speciation in this case. So just a small difference in sequence tuning can actually have a big impact. We haven't found anything that spectacular in Lake Malawi, but there we focused more on differences in gene expression. And so one example of the variation that we found in Malawi is that we wanted to explore how gene expression might relate to foraging types. And so we took 50 different species that come from different foraging guilds, and we broke them up into whether they were breeding in the water column on algae or down in the mud or feeding on other fish. And we then compared those groups as to how much of the UV ops and they expressed. What we found was that fish that were up in the water column had higher UV ops and expression fish that were down and feeding in the mud or feeding on other fish had lower UV ops and expression, suggesting that UV vision is important for foraging, and that shouldn't surprise anybody. Even these algal feeders often will opportunistically feed on zooplankton when they are available. So because UV vision is so important for contrast and detecting these little zooplankton, it makes sense that these fish would maintain their UV sensitivity. The last example I want to talk about for functionality has to do with thinking about the co-expression. Again, this co-expression is fairly complicated because again we have one pair of genes that increase in co-expression dorsal ventrally and another pair that increases co-expression from temporal to the nasal region. And we think that these gene expression differences are the result of the fact that different parts of the eye are looking at different backgrounds when they view their world. And so because the ventral retina is looking up against the downwelling light, that downwelling light is quite broad and it might be advantageous to actually co-express genes because they can better collect the light, the broader light. And this variation might also be beneficial for looking at the space light. And so you can see now in black here's the downwelling light and in black here is the space welling light and you can see the co-expressing gene is broader and actually will detect more of the background light, giving the fish an ability to better detect contrast. And we did some calculations to kind of confirm that there was an enhanced ability to detect contrast with this co-expressing combination. Now, not only as the co-expression spatially variable, but it's not totally random which genes are being co-expressed. So if we look at how the genes are laid out in the genome, these three genes are next to each other, these three genes are next to each other and this gene is on its own chromosome. And the genes that are co-expressed are actually fairly far apart. So these two genes are co-expressed, these are co-expressed and these are co-expressed. Sorry. And so it's the spectral nearest neighbors that are being co-expressed, suggesting that has evolved to produce the visual pigments that would best tune the collected light when they're co-expressed in the same cell. So I hope that gives you some confidence that what we're seeing is not totally random and that it is perhaps beneficial for the fish. Well, at this point, we had done as much as we could to try and explore the variation in ecology, and we still couldn't explain a lot of the variation that we're seeing. And I didn't know at that point if we were just not very good ecologists or whether there were some ecological events that maybe happened some long time ago that we no longer could observe. And so we decided at this point to kind of switch gears and start to try and focus on genetically how these visual systems were shifting from one to the next. And again, remember that they're modifying the developmental program of the ancestor, which goes from short to medium to long as they grow from larvae to adults. So cichlids are amazing for another reason and that is they can actually be crossed between genera. So we started making crosses between different genera that had different expression combinations. And that allowed us to use classical genetics to try and identify the genomic regions that were important for turning on these different ops and genes. And so here's a cross that we made between this species that expresses the blue and red gene, and this species which does not. And what you can do is you can genotype a whole bunch of markers. So each of these little black tick marks is a different marker on each of the chromosomes, so chromosome one, two, three and four. And at each of these markers on the different chromosomes we could test whether those markers were or were not correlated with ops and expression in the F2 of this cross. And so using those F2 it kind of mixes up the whole genome and allows you to determine whether individual parts of the genome are important for a particular trait in this case that trait being ops and expression. Now the opsons are on this chromosome here and this chromosome here. And we found some pretty significant factors that were important say for expressing the blue ops in here, and for expressing the red ops in here. And so we wanted to see whether we could actually figure out what genes were doing this and genetically what was the mechanism for impacting this. And because these genes are far away from the opsons, the genetic change is not some cis regulatory change of the ops and promoter it has to be some kind of gene that's going to act on the opsons. So probably it's going to be some transcription factor. So we narrowed down this region here by adding lots more markers and adding more individuals, and this was work done by Jane and the lab. And we were able to narrow it down to about a megabase region, but it's still contained 180 genes. And we started then to use transcriptomics and some other methods to try and identify which gene in this region was going to be a transcription factor important for opsies. The idea is that there will be some gene in this group where when it's up regulated turns on the ops and and when it's down regulated down regulates the ops. So it should be correlated with ops and expression across many different species. And what we found was a gene called retinal homeobox one, which was indeed highly correlated with the blue ops. And when Jane started sequencing this gene, she found that some species had an intact regulatory region upstream of where the gene was, and others had a big deletion there, about a 400 base pair deletion. So we then decided to look across the diversity of the African species, the lake Malawi species that I've shown you a couple of times now. And across both the sand and the rock dwellers, we found that there were species that had this region intact and others that had it deleted. And when that region is intact, you can see if an individual has either both its copies, good or even just one of its copies having that intact region. The blue ops and is highly expressed, but when that region is deleted, shown by these white boxes, the blue ops and is quite weak. And so across the whole Malawi flock, we can sort of explain a large amount of the blue ops and expression by the genotype or the presence and absence of this regulatory region upstream of this gene. And so we can actually explain about 60% of the variation with this individual locus, which genetically is a huge effect. Well, we went on to try and explain and find the genes that were in some of these other regions. So, then was a postdoc in the lab and he was able to show that tbx2 tbox2 was a transcription factor important for turning on the red gene, and actually turning off some of the green genes. And he did this work in collaboration with Laura in a Roops lab at the NIH. And then Pratima was able to show that MITF is the gene that explains this contribution to the blue ops and expression. And again, it works in combination with this RX1 gene to control blue ops. Now you'll notice MITF has a smaller effect than this gene, but we wanted to test whether these genes actually do indeed have the effects that we think they do. And so we've started to work now with Scott Junty, who recently came to the University of Maryland and has the capabilities to make these CRISPR-Cas9 mutants. And his lab was making a mutant of the MITF gene and it happened to be the exact one that we were interested in. So we've been raising families to try and test the effect of having the coding sequence of this gene mutated. And what we find is that if we have homozygous mutants, heterozygous mutants and homozygous wild type, they do actually differ by the amount of blue ops that is expressed. And so by actually knocking out the MITF coding sequence, we actually get more blue ops and expression than we see in the wild type. Now the effect is a little bit weak, and so we're trying to repeat this in some more families. But we think that this supports the idea that MITF is controlling blue ops and it gives us hope that we can knock out other genes to try and explore their effect on how ops and are expressed. Now, for all of these locus or loci that we've discovered now, we started looking to see why it was that these genes had an impact. And we found that each of these three genes actually had some large, either insertions or deletions. And so for this RX1 gene, we found again, some of the species have a deletion, but actually some of the species actually have an insertion. Similarly for the TBX2 gene, the causative effect is this deleted region, but we found that some species have this insertion. And for the MITF region, the effect is actually caused by an insertion in this locus. Now these insertions are not just random sequence, it turns out that they are transposable elements. And we think that then what's actually happening in these cycloads is you get a transposable element pasting itself in, and then it goes to cut itself out and it creates a gap. And so by having these cut and paste transposable elements, it's creating regulatory variation that is explaining ops and spectral tuning. So here's a little cartoon that shows this network, this genetic network. And we've got our RX1 and MITF gene that are both acting on the blue opson. We have the TBX2 gene that's acting on the red opson. And also we think on a regulatory region for the RH2 opsons, the green opsons. And so we think what's been happening over relatively recent times is that there's transposable elements kind of hopping around the genome that are cutting, being pasted in and then cut out. And that then creates these mutations that cause this whole network to be modified. And so this seems to be something that might happen and explain sort of the rapid diversification that we're seeing. So before I end, I want to convince you that we're not just genetic nerds that spend all our time in the lab. We also like to think, we do go to the field, but we also like to think about behavior. And so Danny is a student who recently graduated and he really wanted to think about cycloid behavior. I kept telling him I don't do cycloid behavior, but he forged ahead anyway. And he was able to train cycloids to recognize blue targets and to tap them and to get rewarded for tapping them. And he then started asking these fish, can you tell the blue target apart from say gray targets of different brightness or even other colors. And so here are some of his experiments to show you kind of how the fish behave. Again, the fish will tap the target and then get rewarded. You can make it a little bit harder. Sometimes the fish has to think about it then. And he came and asked the fish to look at and choose between different colors. And so this was a way that Danny was able to prove that cycloids do indeed have color vision. They aren't just using all these visual pigments for brightness, they actually can use them for color. And working with Karen Cheney and Justin Marshall, he was able to actually quantify cycloids' ability to discriminate, say blues from reds going through purple. And he could figure out when cycloids got confused and use that to quantify how good their color vision was. And it turns out cyclic color vision is about eight times worse than human color vision, but still quite sufficient for discriminating the colors that are so important for their many activities in the wild. So with that, I can perhaps answer some of these or discuss some of these unanswered questions that I think would be very interesting to ponder more about some of the things we don't know about cycloids. There's still so much to learn. I want to end by just thanking all of the people who did the work. Several of the folks working on genetics. Miranda and Melissa are now trying to make some more crisper knockouts. We've had some great bioinformatic help. Zika is a new student working on some more behavior in the lab. Again, a huge shout out to the wonderful vision community who've been greatly helpful in our thinking about the cyclic system, but have also involved me in thinking about coral reef and even deep sea fishes as well. And so thanks to all of those folks and also thanks very much for all the folks who have funded our efforts. And so thanks for listening. Thank you very much Karen for this fascinating talk and thank you for stopping screen sharing already. I have a couple of questions of mine but at this point I would like to remind to the audience that they can go ahead and post their questions in the chat or join us in the very Zoom room that we are currently sitting in and I have provided the link in the YouTube chat. So the first question is from Tom Badden as cyclics very obscene expression etc do spectrally dependent behaviors and up shifting accordingly. I think it was the last word. What what came before accordingly. Sifting. Oh sifting. But I do not know what he means by up maybe he will join us soon and he will clarify that. He says end up and up accordingly. So, I mean there, the opposite expression variation does correlate to some degree with behavioral differences. And some of that has to do with foraging but there's many other behaviors that all of the cyclics do. So, you know, I can explain the fact that they've got UV sensitive cones or not, but all of the other variation, I have no idea why it's occurring. It's not correlated with anything that we've come up with it's not correlated with cyclic color it's not correlated. I mean there's some correlation with light environment. Beyond that, there's really, you know, in Malawi, all of those types occur in Tanganika all of those types occur. Why, I don't know. Yeah, it's really interesting. What one question I have is, so when you take them from the wild and you put them in different light conditions in the lab. Do you observe that this change is also age dependent, in a sense that does plasticity go away. The older the animal is. So I should say, so the species that have the short wavelength combination, they remain plastic through their whole life. We can move adults back and forth and they're still plastic. They will be more plastic if they've gone through different environments through development, but they still remain plastic. However, the tilapias. Well, we don't actually know about tilapia plasticity early in development. I should ask Miranda that question, but the adults do not seem to be plastic. And so it seems that some species are more plastic and others and the ones that kind of retain the larval combination of option expression are the most plastic, which may make sense because if plasticity is something that arises early in development, if you keep those early genes on, you're going to retain that plasticity. Yeah, that is right. I see where you're taking this. As there are not any questions right now in the chat. I would like to remind people, I mean, Tom is already here. I would like to remind people that they can be joining us in the zoom room for a postdoc informal get together. The live transmission may be in 10 minutes from now. I would like to remind to the audience that this was the last but certainly not least talk of the season of the Sussex Vision Seminar Series and we will be continuing beginning of September with a talk from John Dowling. And yeah, like Tom if you want to continue with what you were saying earlier. But if not, I would like to ask a really naive question like do we know anything about bipolar cells and RGCs in secrets apart from like gradients of topographic distributions and so on. No, nothing. Okay, very interesting. Yeah, so I would love to know right what's happening developmentally as these options are shifting on and off. Presumably, well, Russ from Fernald had shown that the cones, they're not dying so the cones are remaining stable, presumably the rest of the neural pathway is also stable but I have no data to support that. And you did mention that when we have like these changes, they are also retina region dependent right, or did I get this wrong. Yes, you're correct. So we can. If we change the room lights right the the ventral region of the retina will change. We can actually we did one experiment Brian did this, where you can actually use fluorescent lighting from below. And then we could induce a small change in the dorsal retina as well so it really depends on the light which direction it's going and where it's falling in the retina. Very, very interesting. Yeah, so maybe I can just chime in. So, yeah, that was really interesting. So I just sort of follow up for my question. I mean, I think you're sort of answering it in. I guess the answer is we don't know but let me see if it really is so in fish or let's say in lower vertebrates whatever that means. So this this idea that keeps popping up that certain behaviors are linked to certain cone systems at the circuit level right so I think an extreme example would be maybe the UV dependence of break up trade take out the UV cones that goes. Right. So you could argue that, therefore those cones are responsible for that behavior and if then the UV cones were to shift spectrally or otherwise, the entire behavioral program would shift automatically. So, in taking that then in the reverse, maybe using these fairly stereotype behaviors, you could get at the circuits that might be existing in the fish. Right. So, so for example, the fish after source of writing reflex right which happens to be spectrally approximately like the red cones at least in supernates. Right. So, if you now have a sicklets that is not expressing the red option is those are writing behavior like what spectral sensitivity with that then follow with it. It doesn't go away. Does it use a different cone because that would then need a different circuit. Yeah. So I'll tell you a different behavior that we did a little bit of work on we've done some op the motor response work. And for that work we took fish and we split them between the lab fluorescent lighting and these fluorescent metal halide lights. So we had fish with more LWS expression and fish with less LWS expression. And we were able to show that the optomotor sensitivity tracked the amount of LWS expression. And so, and that has to be an only one cone type. So, it didn't matter what how much green ops and was expressed it wasn't sensitive to the single cone options. It only depends on how much of the LWS expression, how sensitive they were in the in to that optomotor so. But the cell types are still there. And so it seems to me that it can't be just one ops and gene that determines a particular behavior it has to be something wired to a cone type, and because we can put multiple options in those cones. So I'm not doing both sides. I realized of the same answer. It seems like it has to be something that's not spectrally dependent only. Yeah. Now I can see what you mean. I mean, this would be a very unhappy fish, but if you take out the red ops and stick UV option and then see if they suddenly track UV stripes but not red ones that might give you a way too difficult an experiment to actually do it but. Well, I think that, you know Fabio Cortesi and Justin Marshall are making knockouts in, you know, anemone fish, and I think are intending to do some of those kinds of experiments. So I've been trying to knock out the UV gene but have not yet succeeded and so perhaps we'll be able to do those experiments at some point. So I will step right in, even though I have waved my moderator rights at this point and I would like to communicate to you a question from Cendric van der Berg that appeared in the chat right now. What do these environmentally induced changes correlate to in terms of delta s indetection or discrimination behavior, both luminance and color, are they greater than one. So I'm going to answer it a slightly different way. When you co express options in a double cone cell that will increase contrast by two to 5%. So if Justin is listening, he will say that's a pretty small impact, but as an evolutionary biologist, I would say a two to 5% increasing contrast is a huge effect. It might allow you to detect a predator, you know, one foot closer or one foot further away than you might have and that would maybe make the difference between whether you're dinner or not. So even small contrast changes could have a big impact. Some of the some of the effects on color can be three, four and five JND. So they're not insignificant impacts. One really nice atom you want to go ahead. I can but you had a question. Really nice one like how deep are these legs like what depth are we actually talking about, even for the deep inhabitants. So the lakes are 700 meters deep. However, being near the equator, they actually go and not sick below 200 meters because the water doesn't turn over. So, so the fish only live in the top 200 meters. Because when we are there and we're scuba diving and there's no anechoic chain or no, you know, pressure chamber anywhere close. So we have not really sampled deeper although there are sicklets that live quite deep and presumably have different visual systems I think there's a group from Hull in that has done some work on the deeper species and they've done some transcriptomics on that on those fish. Thank you very much. I just wanted to say hi real quick. I really enjoyed your talk. Thanks a lot for coming. It's really good to see where the sick would work scone to I haven't had an update in a while now. Great and you're in the UK now. No, I'm back in Sweden again. Okay. Okay, lots of cool stuff going on there. Oh yeah. Only for only for me right now everyone else is on vacation. I'm the only one working. You're the only one without the summer house. So maybe next year I'll have one. I haven't integrated enough yet. So you think you're there for a while then. Yeah I think so I have funding for five years and my wife actually has a job she really likes this time so we're probably going to hang out for a little while.


So the other day I was having an interesting conversation with a friend of mine who's a dating coach for men basically helped shy guys build the confidence to meet women and how to connect with them both in an online and offline way And what was interesting is he shared something with me that said that women have more options than men in the dating realm And I go well tell me more about that because from a dating coach for women They seem to think it's the other way around so What he intimated was that women often times since men typically pursue women They tend to have more options because men are doing the initiating more often than men But what he also shared with me was something kind of interesting I thought I'd lean into today because I think oftentimes there are women that pass up really good men And let me just say this as dysfunctional as the dating realm is and it really is dysfunctional There are a lot of good men out there. Just some of them are bad daters So what he shared with me is 80 percent of women Only pursue or are interested in 10 to 20 percent of the men online That's probably even less than that. So I thought that's kind of interesting I said, well, how does that benefit women? And he says it doesn't he says women are passing up a lot of really good men Because those particular men that they might consider Don't don't seem to tickle that woman's fancy if you will Now I think some of the challenges is as we age for those of us in midlife I think it's I really oftentimes believe that those folks in midlife have the eyes of a 20-year-old When they're in the dating marketplace Not forgetting that they're in their 40s 50s and 60s and don't look like they were in their 20s And I think men and women do this combined But I also believe that there's something else that happens that many men and women do because of the Disfunctionality and because so many human beings do little or no personal development work to heal their childhood wounds and traumas Oftentimes women choose men as well as men choose women that are not emotionally healthy In fact in some of these cases these people are rather toxic for them Which I'll lean into for a second because I think it's important to recognize if you're if you seem to be dating the same type of man over and over again It might possibly be because you're experiencing what's called as love attachment love attachment If you're not familiar with the book by a mere Levine and Rachel Heller Here's a copy of the book I recommend attached by the way all the books I recommend is in the link below in the description I Recommend this book because maybe you're choosing somebody who is not right for you based on your attachment style Now in addition you might also be choosing men based on what's known as the amago and by the way since many of you Have asked me to spell it. It's I am a GO and I highly recommend reading the book getting the love you want by Harvelle Hendricks and Helen Hunt By the way again the links below Why I recommend reading this book is you might find that you're choosing men That are similar to one or both to your parents oftentimes. It's usually your father It's either you're choosing someone like your father who might have been emotionally unavailable or might even been abusive or You choose someone completely opposite of your father So you're either Rejecting a behavior or you're accepting a behavior that might not actually be healthy for you So you might be choosing the wrong men Because of this childhood wound inside of you This is why I wrote my book what the heck is self-love anyway a journey of personal development self-open spiritual work Self-love the book why because I believe that when we actually shore up our emotional Fortitude if you will We can actually make better choices in the dating marketplace because I shared with you a moment ago That there are some people are quite toxic and if you're not familiar with a chart that I created a while back It's called inner personalities and behaviors. This is new. You haven't really seen this in a while By the way, this is not an effect It's an opinion, but I believe that 20% of the population has talked that can feel like toxic behavior to us And then while I say 20% have genuine caring behavior The vast majority of human beings are in this confused behavior confused And what I mean to say is and I understand why it's very frustrating for you in the dating marketplace Because a lot of men are confused at midlife What do they keep confused about they really don't know what they want and This is why it's interesting to me. I've seen a proliferation lately I don't know if it has to do with the fact that I am Well, I don't even consider myself in the long-distance relationship before because I only have to drive to the airport to pick up my girlfriend Actually, I'm traveling there to meet Visit her but we spend so much time together We spend three or four weeks of time together each time we're together It's not a traditional long-distance relationship. And yet why I brought this up is I'm noticing a Proliferation of women who are having long-distance connections and what I mean to say is long-distance communication Sometimes for months sometimes for years with men thinking that they're actually in a relationship But it's just a cyber relationship. I Think the reason why I brought this up is my girlfriend turned me on to a show called 98 90-day fiance Which to me is garbage drama I'll just be candid with you, but it was kind of funny to watch human beings in trainwrecks I'm sorry that sounds very crass to say that but it is kind of interesting how really ridiculously dysfunctional human beings are Myself included I've done absolutely loot stupid things in my dating life Based on a not understanding human behavior and not understanding my own behavior This is why I'm such a big proponent of self-reflection in relationship All right now some of you might be going well Jonathan What does this have to do with the five ways to tell a guy is you know? is worth keeping Well, I think it's important to recognize that maybe the type of man you're looking the maybe the type of men You choose are not the healthiest for you because of love attachment style because of the amago So first recognize that And then maybe operate from a different premise You know, it's interesting in my private coaching program By the way, there's a link below to schedule a discovery call with me if you'd like to find out more about it What I help women do is get crystal clear on who they are and what they want in a relationship And it fascinates me how many women go through my program and they're literally at the end going I can't believe I didn't know what I want. I thought I knew what I want I thought I knew what I want. I thought I knew what I want But by going through the exercises I've helped create or the exercise I have created It has helped them get clarity on who they are and what they really want It fascinates me how often we get to even by the third session and women say to me. Oh my god Jonathan I've been doing it wrong Fact one of the exercises called everything you've learned about relationships is wrong and Why I'm saying this is you really have to maybe throw out the old script To be able to see the guys who they're worth keeping to throw out the old narrative and Maybe look beyond the surface because a lot of great men exist. I am witnessing I just got a call the other day from a client who'd worked with me just three months ago And she was just telling me how she met this fantastic guy and she can tell the difference I I get these calls all the time Jonathan. I met a great guy Jonathan I met a great guy and they know the difference And maybe if you're not ready for coaching, I'm gonna recommend a book for you Barbara DeAngelis wrote an amazing book Called are you the one for me? I highly recommend reading this so you can understand How to make better choices in your life and while it's not personalized the way I help you really based on your personality It's really sifting through the muck Out there because it's gonna require that listen these days. We're meeting total strangers. We know nothing of about bubba bubba about them This is why one of the aspects of my coaching program is called radical honesty Prequalifying your prospect developing the questions you need to ask to determine if they're really a right fit for you I'll be candid with you. I did this with my girlfriend. I grilled her on our first phone call I had my 15 questions that I wanted answered before I was gonna invest time Especially since it was going to be a long-distance dynamic Don't waste listen. I know everybody tells you don't interrogate people don't interview people.  They may not be the right guys anyway Because if they're not genuinely serious about a relationship, then maybe you're investing in the wrong person to begin with So let's talk about what you need to be paying attention to most importantly to know if this guy is genuinely a keeper Okay, so I've got my trusty notes. I'm gonna share this with you all bump bump bump All right number one His actions consistently match his words his actions consistently matches words You know and I say consistently because look at I get it You might be talking to someone you've been talking for a couple weeks on the phone And all of a sudden he goes MIA for 24 hours. Okay.  I mean when I was growing up. We didn't have cell phones We we bear we didn't even have answering phones if you missed a phone call you had no idea if someone called But nowadays we have this instant gratification Expectation that they're constantly in contact with us, but let me come back to actions matching words Is he consistent? Not necessarily accurate every single time, but is there consistently that is certainly Something to appreciate and value in a human being when their actions consistently match their words number two They have Victor Victor consciousness and not victim consciousness. I mean I got to tell you something here in the United States We are suckling on the nipple of victim consciousness We literally blame everybody else for our lot in life and I got to tell you Listen, I'm gonna share something with you that I witness over and over and over again with women only because I'm a man who's dated women and that is Most of the time when after a relationship ends they throw their previous partner under the bus and blame that partner for their dysfunctional relationship and to me that's victim consciousness if you're Complaining about your ex-partner then you're operating as a victim and that is a turnoff By the way, men do this and women do this as well. This is why I tell you we have an incredibly dysfunctional Society here. I'm saying by the way. You've seen my emotional maturity relationship skills chart. This is not a fact That's an opinion But roughly 20% of the population has clinical issues and while I'm being generous with saying 20% are healthy Most everybody is dysfunctional because they're operating from victim consciousness And this is why I'm such a big proponent of reading the book the Hoffman process the Hoffman process This is a deep dive to healing childhood wounds and traumas as well as adult traumas So you're not operating from a place of victim consciousness and thankfully, you know I look at my circle of male friends and they're all in great relationships. I will tell you in the last Five no seven years. I know at least a dozen guys who I think almost all of them met exclusively on an online dating site Half of them have gotten married and the balance of them are in relationships with people that they're living together And these are really good quality men. I want you to know ladies There isn't it is there is an abundance of good men out there Oftentimes you're rejecting them and by the way men reject women for stupid idiotic things too Okay, I'm not discounting that but a lot of times you're rejecting good men and you're choosing men that are in Victor victim Consciousness if someone is often complaining about their lot in life their health their professional capacity or their past relationships That's not a person that operates from Victor consciousness Number three and this is critically important And that is what I call fighting fair fighting fair every relationship needs two people that understand active listening and Validating what their partner says even when there's a disagreement So what I'm here to say is fighting fair is listening to your partner's point of view or the person's point of view Accepting that person's point of view is being true for them At least acknowledging it and then doing the same and yet we're we're we oftentimes in Disagreements, it's I'm right and you're wrong and they're saying I'm right. You're wrong. No, I'm right You're wrong. Do you know it's fascinating? Do you know most couples therapists are? Keenly aware that when two cup when a couple comes into their session, they know exactly what's gonna happen this The person's gonna point the finger at the other person By the way, if you really want to be a good partner then operate from a premise of active listening and Validating the other person when you're communicating with them, you know, it's funny Women often say to me Jonathan the most important thing a relationship is communication communication communication. Well, I get it But many of you just vomit your feelings think you're good at communicating and many of you are not This is why I want to recommend a book. I haven't shared in a while This is a great book. You should all be reading how to be an adult in a relationship Because many of you are not operating as adults and then you wonder why you're having a frustrating experience in your dating realm So learning how to fight fair is just one of the many principles that both men and women should be learning to actually enhance Their relationships with another person Now number four This is hard for men, but genuine empathy This is harder for men because we were taught to stuff our feelings, but genuine empathy I will tell you there are a lot of good men out there that are incredibly empathetic and empathy isn't just I can feel your feelings. That's just one facet of it more importantly I care about your feelings I care about your best interest as well as caring about one's own Because the end of day we have to put the oxygen mask on ourselves first to be a good partner with someone So if you're with someone who genuinely cares about your feelings your best interest That's a great starting point to build on a relationship and lastly and most importantly is Transparency and what I mean to say is if it's material to the relationship If it's material to the relationship then be transparent about it and choose people are transparent I'll share with you something personally that really impressed me about my girlfriend There's a picture of her and I on her second date if you will at a wedding She shared something very vulnerable with me But more importantly she was sharing something very transparent and while it's private to her So it's I'm not feeling I don't feel comfortable enough to share it with you what I appreciate mode because I happen to be about a past relationship What I appreciate most is she shared it Because it had relevance to possibly our relationship if it's material to a relationship then it's important to be Transparent with one another This is why I'm such a big proponent of everybody reading the book the four agreements the four agreements This is like a Bible to being a good healthy human being to show up in Relationship whether it's both people by the way, maybe ladies before you have sex with a guy you make him read this book Okay, you both read it together before the penis gets inside the vagina always talk about eight dates But maybe this is the book you should be reading because this is a blueprint For operating as a better human being as a more conscious Loving caring human being because when we operate from a place of transparency in relationship And let me just say this There are great guys out there. There really are I know you you're swimming in a sea of a lot of Dysfunctionality partially because you're no but you're no you're no picnic either Okay And yet it does require doing a little bit more work Than the average person because it's a lot harder to find those diamonds in the rough And yet I want you know they exist I do want you know they exist and if you need support again schedule a discovery call with me because maybe I can help you with this All right, the five ways to know he's keeper actions matches words He has victor consciousness not victim consciousness. He knows how to fight fair He has a level of empathy and he has transparency In your relationship and if you meet one of these guys You're on the great foundation for having a man who's potentially a keeper and I get it's frustrating out there Requires to be a little bit more of a detective. You have to be colombo these days But let me tell you Have your spy glass out because you can find these men because many of them are existing and I invite you to start with This prayer god universe spirit I invite a great man into my life. It is reigning great men Men whom I have chemistry with Men who are communication styles are mutual with one another and we can laugh and play all the time We communicate like good friends or even act like brother and sister at times To find a person who's compatible with my lifestyle and we can blend our lifestyles together and we share the same values in life And we find out we do the same things like forks forks down and toilet paper out And lastly we build the deep roots of trust that allows us to support and nurture a beautiful healthy relationship god universe spirit I invite that into my life Are you with me if you are say yes jonathan? I want that in my life if this resonates with you Please post a comment. Let me know. This is sinking in post a comment below if you're on the live stream posted in the chat box All right, those of you know that my live streams. This is the time for q&a um and During the q&a if you have a question for me post the word question and write the question thereafter or purchase a super sticker super chat All the money's from the super sticker super chat goes to a scholarship fund in the name my son connor asley That's a picture of him right there. That's him with one of his best friends This is a dear friend of his who actually tattooed connor's name About a month after a month the tattooed connor's name on his shoulder a month after he passed away So again, all those monies do go to charities or to groups like the hoffman process insight seminars and hope to Helps defray the cost of personal development for those who need it All right, let's see you and again. There's a little dollar sign sticker right there that you can Purchase a super sticker. In fact, I want to acknowledge We had one come in right now barbara. Thank you so much for that two dollar super sticker I really appreciate it. All right. Let's see what we have here in our q&a board Let's keep just keep swimming Uh raven said a rea says I found myself one of the good ones the more I do the work the more emotional connection I create my relationship It makes them feel safe and seen and valued and keeps pouring love and caring into me way to go Raise a pop. Thank you so much for sharing that everyone needs to hear there. It is raining great man All right, here we go holly. Oh, we got let's see we'll start with carolina's question What are things women do in the early stages if dating are red flags or turnoff for men? Great question I think one of the the top things women do Is they complain about? Their ex partners or ex-boyfriends. I think when you complain about that That's just by the way if history repeats themselves. Okay. I'll give you an example I once met a woman. Uh, this is some years back five or six years ago I remember on her first date. I said, you know, what happened in your marriage and she goes I married a narcissist. I'm like, okay I said what happened in your previous boyfriend that you just ended a few months ago. She goes. Oh, he was a narcissist I said any other relationship? She said, yeah, and I go. What happened those guys and they go Narcissist narcissist narcissist narcissist. I'm like, okay. Well, I'm not a narcissist You know, I'll be her hero. You know, I'll show up as her hero And I found out after a few weeks. She was rather boring to be with. She wasn't very much fun She was kind of lax a dayacle and I ended the relationship And do you know what she posted on facebook the next day? Another narcissist dot dot dot I'm like, how the fuck did I get labeled that because I said I wasn't interested in her So when you complain about past partners, it's a it's it's by the way men do it and women do it I think it's the biggest turnoff because it demonstrates victim consciousness Only those savior enable types might like those women, but those become toxic relationships later on down the road anyway So I'm not a big fan of that All right Kimberly, I'll answer your question about chicago women soon enough, but let's answer holly's question Question I recently reconnected with a man I dated nearly 30 years ago any thoughts on rekindling romance from youth Um You know what I don't know what what I mean The the good news is hopefully you have a foundation of past experiences, but just remember Who someone was 30 years ago may not be who they are right now So rather than rekindling the romance, I would do some interrogation Determine if they're good fit for you that would be my invitation for you So, um, maybe read this book I'm just thinking of this out loud if the Buddha dated if the Buddha dated. I love this book It throws out the gender rhetoric of how romance should be kindled and how to connect at a heart-centered level That might be something you might want to try holly Okay, thank you so much. I appreciate that question All right, let's keep swimming Uh Barbara says it's pizza girl here. Do you think when things are going well?  Uh really quickly, uh, if it's okay, barber I share you're part of our group called midlife love mastery in the description below is a way to connect Within my group It's a very low price group to be able to have direct access to me on a regular basis And based on the questions you ask in the group and we have a great facebook group. I shoot personalized videos just for you Here's the thing about guys Look if we get spooked for a variety of different reasons.  She said one thing and all of a sudden it triggered me and it spooked me And I went silent for about 24 48 hours and then I called off meeting with her.  I barely know this person Folks, let me give you a simple recipe that you should be using in your dating practice. I call it the three two one three No more than three email exchanges if you're meeting someone online That should turn into no more than one or two telephone calls. That should turn into One date within a three week period of time three two three one wait three two one three, excuse me Listen, it's important not to dilly dally communicating with people in the online realm In my particular case while we met a while back online. I really and we communicated once or twice I wasn't pursuing this person and she wasn't pursuing me It only happened that when we got to meet when there was an opportunity for a meet That's when the relationship began Here's the thing many of you are dragging out communication It's important to meet people right away because listen, we don't have time to fuck around And listen someone gets scared or spooked you want to find out that sooner rather than later So initiate meetings sooner rather than later And if you have distance involved then make sure you have FaceTime communication with one another But i'm not a big proponent of long distance Communication if there isn't a plan on meeting right away Because guess what happens you can spend weeks communicating with someone and all it takes is one thing to spook them And by the way once they're spooked It's like a horse sometimes they're never you're never able to ride it again So I don't know what more to share with you in this particular case I'm just saying it happens and sometimes it can be a simple trivial thing that spooks a guy And it may cause him to go radio silent or completely disappear because when you're total strangers with one another when you don't Physically do social activities hobbies mutual interest spending time with family and friends It's very difficult to actually build the deep roots of trust needed to actually support a relationship So that's my two cents on that one. So thank you so much All right Speaking of long distance. What are you some what are some things that would make your make you pursue a long distance relationship? Listen, I was very crystal clear in my situation I would not pursue something if there wasn't a game plan to shorten the distance To two people actually living in the same area and that's what we've done in my particular case um listen Long distance flirting is a lot of cyber communication And it's not real until you actually can do By the way, I'm going to sound like a broken record Social activities hobbies mutual interest spending time with family and friends traveling together teamwork building skills both in your personal and professional life intimacy both physical and emotional intimacy Emotional intimacy over the keyboard and text messaging is not building the deep roots of trust needed You have to do s*** together Long distance are very problematic. I mean, they're incredibly rare to work out You need a a strong connection right from the gecko a strong connection You need to be absolutely intentional But more importantly, you need to be with someone who is an emotional grown-up Who's probably does introspective work And lastly, there has to be a plan for things there has to be a plan of shortening the distance Otherwise You're setting yourself for a long drawn-out Drama series. Oh my god.    I laugh at myself all the time at my Disfunctionality at least my past behavior I think because I've read a lot of these books. I've short up my own Capacity to be in a healthy happy relationship but I witness Absolute crazy behavior Because I think a lot of people are addicted to the drama of it I'm sorry. I'm going off on a tangent Uh, I don't I don't know if I helped. Well, yeah, I helped you answer that question. Those four things are critical winning All right. Thank you so much Michelle says you can't say the wrong thing to the right person. I love your quote. It's not my quote I I that's not and by the way, I say if it's sincere and from the heart You can't say the wrong thing to the right person if it's sincere and from the heart All right. All right kimberley your turn Question. What do you think of us chicago women? Do you agree? We are top notch Folks kimberley knows. Um, my sweetheart is actually lived in chicago or lives in chicago By the way, she's originally from another country a south american country. She actually grew up in los angeles So um, and she lived a big chunk of big chunk of her life in chicago as well as a couple other cities. Um You know what? I think there are beautiful human beings every part of the planet Recently met a really sweet lovely woman A family member of my girlfriends who's from columbia Just a sweet kind soul and she met her husband while he was traveling This is 20 years ago on a motorcycle trip through columbia Folks, there are beautiful human beings everywhere.  You know what? My dearest closest beautiful human beings on my planet live right around the corner from me I've met beautiful human beings in dallas. I've met beautiful human beings in fargo north dakota I've met them in other countries beautiful human beings live everywhere. They are not isolated to one city I I know that You know to some degree I do believe growing up in a harsher climate might make people more resilient But also I know those people who live in harsher climates hate it So they might be you know, by the way people live in seattle are the highest suicide rate because of the They uh, the uh, what's the word i'm looking for? Ah, it'll come to me later. Anyway, you know what i'm talking about folks. There are beautiful human beings everywhere Including chicago. I'm very blessed to have met her Um while I was there visiting. All right kimberley. Thank you so much All right I also want to thank uh, cecilia for the nine 99 cent super sticker and the dollar 99 super sticker. Thank you so much Cecilia writes question. I vomited So he said only write to me three times a week. How long should I wait for him to contact me or make him Are are you boyfriend and girlfriend? Are you just meeting online? What are the particulars of this? You know folks? I can't stand this gameplay. How long should I wait to do something?  Do you want to go on a date? Do you want it to know me? Do you want to explore a relationship? folks I think it's time to start asking the men. Do you want to explore a relationship with me?  That's not my area of expertise. I don't know Text him right this minute say let's get on the phone and talk Do we want to explore a relationship together?  I guess we're riled up. All right. Thanks for allowing me to vent All right Brenda writes Question what type of things would be relevant to a relationship? Um besides honesty integrity and respect. Um, how about Mute shared shared uh values. How about blendable lifestyles? How about emotional maturity? How about chemistry? Those are all really good things to have in a relationship Thank you Brenda for your question. Lisa writes Kimberly. Of course us Chicago girls are the best All right. Dale says personal question. Have we yet? I don't understand what you wrote so, uh Let's go swim in Roller girl says hi to everybody to seal you with her super sticker. Thank you so much All right Oh, now here we go per Dale says personal question. Have we yet achieved a reliable testosterone level? I don't know what that means I I don't understand the question Uh, okay question. Why do women get so hung up on figuring out the guy's mind instead of us just pursuing what we want Okay Lisa great question. Folks, you know why I became a dating relationship coach for women Men okay women are fascinated on why men do this why men pull away and why men goes why men disappear And women will pay a fortune for trying to figure out the why with men men are very simple in this category They really have only two questions How to pick up chicks and how to pick up hot chicks. They want to learn the confidence to meet women That's all they focus on you women tried to decide dissect everything But guess what you need to because if you don't understand the mechanics to a healthy happy relationship He's most likely winging it.  I appreciate it All right Lisa says yes, you're right jonathan often I can tell from a phone call that a guy is wrong for me exactly get on the phone stop texting each other Okay kim writes question I've been seeing someone and after two months Then he wanted to keep up to keep his options open Then came back because he made a mistake He freaked out because he said he's invested in me and and support me Why you know the tricky part About choosing a partner in midlife is when we're in our 20s and 30s We oftentimes jump into relationship because of programming The programming I grew up with as a tail end baby boomer was Go to college meet a girl get or go to college get a job meet a girl get married buy a house start a family So I follow that programming at midlife People don't know how to partner at midlife Most folks don't know how to partner at midlife. So they don't know what they're looking for This is why I often say men are winging it because they don't know what they're looking for And what happens is as soon as there's something that spooks them they run away And then they do what's called the rubber band effect and within usually 90 days they come back Because they realize that they had something good there. So here's my advice to you I had a friend tell me this this might be a meme or a quote I'll forgive you, but the price just got a lot higher And what I mean to say is now it's time to have radically honest conversations About what's what are we doing here? And have a game plan Listen, you guys know i'm in a long distance relationship After we realized we wanted to be in a relationship with each other We began creating a plan of how shortening the distance is beneficial for the health of our relationship We had a plan and we talked about the plan consistently So if he wants back, then what's his plan to actually take this level this relationship to another level? By the way, this doesn't relate to this question, but i'm reading a new book Called sacred relationships And i'm i'm just starting i'm only on the first chapter. So I can't necessarily guarantee and recommend this But why am I reading it? because look Most humans settle for mediocre relationships I want my audience To settle on extraordinary and I don't mean settle. I mean choose extraordinary But if you want to be in an in an extraordinary relationship, then it requires becoming an extraordinary human being by loving on yourself This is why I continually recommend my book over and over and over and over and over and over again By the way link below because I want to invite you to become an extraordinary human being This is why i'm going to recommend this book that you check out called daring greatly by bernay brown Daring greatly. I want you to start becoming an extraordinary human being And then I want to recommend joe dispensas book breaking the habit of being yourself So you don't choose dysfunctional relationships over and over and over again break the habit of your cycles So you can actually step into your power step into your sovereignty Step step into your self-worth self-esteem self-confidence self-reliance Because nobody else is going to do it for you. We are suckling on the nipple of I need you to love me So I can feel good about myself. I need you to love me so I can feel good about myself We are suckling on that nipple so many human beings. It's the expectation that our partner feeds Our emotional well-being and it requires filling your own love cup up first Is this sinking in is this resonating? Please let me know give me an amen or give me a thumbs up By the way, if this has provided value so far, please hit that like button. Please share this video Please subscribe to my channel If this is making a difference for you.  All right, let's check out these last few questions Don't give me that look over there Ah jen thank you so much for the super stickers five dollars And I want to thank Trina for the nine dollar super sticker. I really appreciate it All right, let's keep swimming just keep swimming Kim says I told him I didn't know how I feel when I really know how I do feel Why is it? I feel a little scared when everyone else on is on his side is on his side and wants Me to give him another chance Because he he he abandoned you He ended the relationship. Of course, you're gonna feel scared. That's why if he wants back in The price has to be higher have a game plan have him come up with his game plan of what he's gonna do to create trust Folks, if you're not building trust in a relationship, um, I'm gonna recommend let me check check out this one book really quickly um Not this particular one. Um, I was just thinking of a book to build trust I don't have one handy, but what your relationship needs is a level of trust.  I want you back. What's he going to do to demonstrate that? That's the question I invite you to ask yourself because it's going to be unique to you But thank you so much for that question. I really appreciate it All right um Let's see wait Hi, john attention doesn't mean wait attention doesn't mean intention gene gene four Oh, I gotta leave that up Attention doesn't mean intentionality intentionality is the game plan By the way, read the book eight dates by doctors john and julie gotman. That's a game plan Attention doesn't mean intention. I love that big. Oh, I gotta give you a big gigantic charthamere for that all right um By the way, I the only out of courtesy to her. I don't want to share her name publicly yet, but thank you so much She's my sweetheart beloved for now Unless I get her permission all right Let's see lulus asked a question Lulu I did not see your question posted again All right We gene says would you see this as a super red flag that a man has been married four times in the past? My girlfriend just nodded yes Uh, I only give two people three chances. No, I'm just kidding. Um, you know what? All right, here's a question folks, how many boyfriends have you had?   Is it 14? What is that number?  It didn't work out. Is that a red flag? I don't know. You know what? I think a lot of is it possibly a red flag? Probably but red flag means ask better questions those better questions is find and by the way if he blames If he blames each one of his exes That's the red flag. I would walk away from that one if he doesn't take ownership for what went wrong. I'd walk away All right, let's keep swimming. Thank you Trina for the nine dollar super sticker or $10 super sticker. I really appreciate it all right Lulu if you have a question you need to write the actual question there after her I don't see it or purchase a super sticker super chat will be easier for me to find Amber says question man is very critical of me cooking my parenting the way I talk always trying to fix me He is he was hiding things. I found out set boundaries. He hung up on me. How do I balance not? perfect or let go um You know, we each have to figure out how we resolve our attachments Buddha says the number the all suffering comes from attachment to an outcome So the question I ask for you is how are you going to overcome your attachment to another human being now? Listen, I don't mean such look at I lost a child those of you know right there connor I'm never that attachment is never going to go away Okay, when you lose someone like that, but when someone breaks up to you with you An ends a relationship after to being critical of for you Why why do they hold space in you? So how are you going to figure how are you going to overcome that attachment? It's by learning to love yourself By doing things that nurture your soul and take he is renting space in your mind that he doesn't deserve there Why are you giving him up? Why are you giving him a lease on space in your brain? Why is that happening? He's not paying you the rent, but you're giving the rent for free I invite you to love on yourself and when you can actually genuinely love on yourself God universe spirit grant me the power to love on myself to do the things that nurture my soul By doing those things whether it's traveling whether it's spending time with family and friends Whether it's starting a new activity or hobby Grant me the space to lean into my sovereignty my self-worth and my self-esteem God universe spirit. I invite that in my life All right amber. I hope that helped Barbara says I bought the book reading it. Wow. That was quick Weegean says I love joe dispensa. Dr. Joe have read his book many times. Yes. That's a great book All right, ah, here's a question from lulu question He said we didn't date He felt he knows me long ago and stopped looking for women since we met don't know what he meant folks I know it's sometimes hard to decipher what men say so ask point blank What do you want and how are you going to make it happen folks?  Okay, and that might Temporarily satisfy you I only want to talk to those women who want a juicy delicious healthy happy relationship Where you're doing shared activities hobbies mutual interest spending time with family and friends traveling together teamwork building skills And you're both your personal and professional life Intimacy both physical and emotional intimacy where you're doing it on a regular basis stop spending your time here And start getting intentional Read this book if the Buddha dated read the books. I recommend I know you don't think i'm answering your question But what i'm here to say is if your relationship is on is on the phone Then it's not a real relationship according to the world according to jonathan. Okay You are more than welcome to approach those relationships, but homie don't play that game and i'm not going to encourage that Meet go out on dates spend time together see if it's worth Investing in each other and then go from there Sorry if I was harsh lulu Roller girl says amen All right. Oh, let's see what we have. By the way folks. We're getting close to wrapping up I know we have a lot of questions Um Okay, this will be our last one for the evening jonathan I've done a lot of inner work over the past few years and i'm in a great space now But I seem to be unintentionally attracting married men. I don't want That I don't want what's up with that That's an interesting question My suspicion is you might be attracting married men because you're still Healing and you might be rejecting good men ladies Many of you reject really great guys now that I know they might be holding up fish or they might have the beer god They may not be your type But oftentimes you're rejecting great guys because they don't fit your physical attributes of what you're looking for And yet these are the guys that's actions match their words They have victor consciousness. They know how to fight fair. They have a level of empathy and they have a level of transparency I can't tell you why you're attracting that only to say Is maybe you should ask the universe for something different than that Um, that's my invitation for you there All right, folks Did I provide value here tonight? Please let me know if I provided value If I'm making a difference in your life, please let me know if you have something to say post a comment below or Because we're at the end of our broadcast And I'm going to wrap up this video as I always do first off giving myself a big gigantic chanthumbarug of self-love I'm going to reach into the camera and give you a hug of love if that's okay I'm going to ask you to turn to someone a pet a teddy bear pillow and give it or them a hug of love Because hugs are a great source of love and let's face it. We could all use more love in our lives I want to thank who I want to thank sofia and jane and uh Cecilia and cc and kerry And holly and wejin and lisa and spirit dancer and jennifer and gale And I can't even pronounce your name is taylor and valerie roller girl spirit dancer. I think I said that christine Everyone, thank you so much. Hope you have a wonderful fantastic day. Be well