Ep 125: A quiet place (with Dale Broder and Robin Tinghitella)

Ep 125: A quiet place (with Dale Broder and Robin Tinghitella)
Big Biology
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How do natural selection and sexual selection interact? What can observing adaptive radiation in the wild teach us about evolution?

On this episode, we talk with Dale Broder, an Assistant Professor at American University, and Robin Tinghitella, an Associate Professor at the University of Denver. They study the Pacific field cricket, a fascinating organism that exemplifies both adaptive radiation and the interaction of natural selection and sexual selection. This species uses song for mate attraction, but the song also attracts Ormia ochracea, a fly that parasitizes the cricket. The fly recently invaded areas where the cricket is common and in response, some crickets evolved different songs and even silence. We talk with Robin and Dale about how their research team characterizes these cricket songs and the different wing morphologies that produce the songs. We also discuss the role of female preference (or lack thereof) in the evolution of different mating songs and how remarkably rapidly this song evolution occurred.

This episode was supported by a grant to Dale and Robin from the National Science Foundation.

Cover art: Keating Shahmehri. 

  • Marty Martin  0:05  

    So Cam, what's new in Norway?


    Cameron Ghalambor  0:07  

    Well I'm not sure if it qualifies as major news, but I was just reading about the great moose controversy between Norway and Canada.


    Marty Martin  0:14  

    The great moose controversy, do tell?


    Cameron Ghalambor  0:17  

    Well, between 2015 and 2019 a fierce battle waged between the Canadian town of Moose Jaw and the Norwegian town of Stor-Elvdal over whose moose statue was the biggest.


    Marty Martin  0:30  

    You know, that doesn't seem like a very controversial issue, especially between two countries like Canada and Norway. And couldn't they just measure the height?


    Cameron Ghalambor  0:39  

    Well, they did, but it came down to the antlers. Here's the story. For 31 years, Mac the Moose in Moose Jaw, held the title as the world's tallest moose statue, coming in at 9.8 meters, or just over 32 feet. But in 2015 Storelgen, which translates into the big moose, was unveiled at 10.3 meters, or about 33.7 feet.


    Marty Martin  1:05  

    Okay, that's a big moose, but what makes the antlers controversial?


    Cameron Ghalambor  1:09  

    Well as a point of national pride, the Canadians responded to Norway's act of aggression by raising money to give their moose a new set of antlers, which they installed in 2019 giving Mac a new height of 10.34 meters, making him just slightly taller than Storelgen. So Norway considered building an even bigger statue, but ultimately conceded the prestigious title of tallest moose to Canada.


    Marty Martin  1:39  

    Oh, it's so nice to see a peaceful resolution to geopolitical strife,


    Cameron Ghalambor  1:44  

    If only it could always be so easy and amicable.


    Marty Martin  1:47  

    You know, moose antlers are also a good introduction into the show today. They evolved mostly because they provide a mating advantage, what we often refer to as a sexually selected trait.


    Cameron Ghalambor  1:57  

    In some cases, sexually selected traits are elaborate morphological structures which are used in fights between males for access to females.


    Marty Martin  2:06  

    But in other cases, like the extended colorful tail of peacocks, traits are just used to attract females.


    Cameron Ghalambor  2:11  

    One of the major assumptions about sexually selected traits is that they are costly and therefore also subject to natural selection.


    Marty Martin  2:18  

    So bigger antlers or longer tails might provide a mating advantage, but if those traits get too exaggerated, moose and peacocks are going to have a lot of trouble getting around.


    Cameron Ghalambor  2:28  

    Vocal signals may not be as eye-catching as moose antlers or peacock tails, but they illustrate that ongoing tension between natural and sexual selection.


    Marty Martin  2:38  

    Many listeners might be familiar with the classic work of Mike Ryan and his collaborators on the vocal signals of the Tungara frog.


    Cameron Ghalambor  2:44  

    Male túngara frogs, like many frogs, produce mating calls and females exhibit preference for certain features of these calls, in terms of their complexity and frequency.


    Marty Martin  2:54  

    But frog eating bats also prefer these same calls, and they use them to locate and eat the frogs


    Cameron Ghalambor  3:00  

    Ah the interplay between sexual and natural selection


    Marty Martin  3:03  

    To get around this dilemma, some male frogs produce a complex call when there are a lot of other males around singing in a chorus, but then switch to a simpler call when they're alone and at greater risk of being eaten by a bat.


    Cameron Ghalambor  3:14  

    Our guest today, Robin Tinghitella, who's an associate professor at the University of Denver, and Dale Broder, who is an assistant professor at American University in Washington, DC, are exploring this fascinating interplay between sexual and natural selection in another very charismatic species, the Pacific field cricket.


    Marty Martin  3:33  

    Like túngara frogs, male field crickets use songs to attract females, but on the Hawaiian Islands where they live, there's also a parasitoid fly that haunts the crickets by listening for those songs.


    Cameron Ghalambor  3:44  

    This has resulted in rapid evolution of different song types and even the loss of singing completely.


    Marty Martin  3:49  

    We talk with Robin and Dale about the natural history of this fascinating system,


    Cameron Ghalambor  3:53  

    like how crickets produce songs using their wings, and how modifications of the wing structure alter the properties of the song.


    Marty Martin  4:00  

    We also talk about the evolutionary changes that have been happening across populations in just the past couple of decades.


    Cameron Ghalambor  4:06  

    And pay special attention during the breaks in our conversation. You'll get to hear some of the cricket songs that Robin and Dale are studying.


    Marty Martin  4:13  

    Dale and Robin are also very interested in science communication, and although we had hoped to talk to them about some of this work, we had so much fun talking about crickets that we ran out of time. Perhaps we'll have them on the show again in the future, but in the meantime, we'll put links to a few of their scicomm papers on Substack.


    Cameron Ghalambor  4:29  

    And one more housekeeping item before we get started. Thanks very much to the National Science Foundation for supporting Dale and Robin's research. A research grant to their labs also partly covered production costs for this show. Thank you, NSF.


    Marty Martin  4:43  

    I'm Marty Martin.


    Cameron Ghalambor  4:44  

    And I'm Cameron Ghalambor.


    Marty Martin  4:45  

    And this is Big Biology.


    Cameron Ghalambor  4:58  

    So Robin Tinghitella and Dale Broder, thanks so much for joining us today on Big Biology.


    Robin Tinghitella  5:03  

    Thanks for having us. We're so excited to be here with you all.


    Cameron Ghalambor  5:06  

    Yeah so we're looking forward to talking to you today about your research on Hawaiian crickets and the tension between sexual and natural selection and also about science communication. And for full disclosure, to start off, I should say that I've known both of you for quite a long time. I first met Robin when we overlapped at UC Riverside back in the early 2000s. Robin, you were finishing your or you were just starting your PhD, as I was finishing my postdoc, and then several years later, we both ended up in Colorado with with faculty positions, which was very exciting and and Dale, well, I was on your PhD committee, so we know each other quite well. I've known you since you were a graduate student, and we've collaborated on several papers together. And you know, it's super exciting for me to know you both like separately, and then to see you both collaborating together. And every time I read one of your papers, I just get super excited. You know that you guys are doing great, great work and being really successful together. So, so that's, that's, I guess, my start.


    Marty Martin  6:17  

    Well, let's, let's start talking about some crickets, specifically the, I guess, maybe some natural history about your favorite species, the Pacific field cricket. I guess I'm making that your favorite species based on what I've read. So apologies, if I've overstated. I think a lot of listeners are probably going to know classic examples of adaptive radiation, the happy face spiders, loads of Drosophila, the silverswords and another taxon in Hawaii, the honeycreepers, but I didn't until I read your work associate crickets in Hawaii in general, much less with respect to adaptive radiation. So why Hawaii? And why cricket radiations there?


    Robin Tinghitella  6:55  

    Well, I think a lot of evolutionary biologists think of islands as these sort of natural laboratories where, you know, we can learn a lot about the basics of how evolution works in these, sometimes small and, you know, isolated from one another, populations. And so I think that, for me, I actually grew up on an island. I was obsessed with islands, wanted to work on an island, and that's why I ended up pursuing graduate school working on this particular species and system in Hawaii, but really it's been this just wild playground of rapid evolutionary change that we have the privilege of watching in real time. And it's one of those study systems that just sort of sucks you in with how charismatic the players are and and how sort of almost deceptively simple. The interactions are. They make it easy to talk about and think about with lots of different audiences. And so we feel very lucky to get to watch this radiation in real time of, you know, cricket song as we're out there in the field.


    Cameron Ghalambor  7:56  

    So the cricket song, you know, for like maybe bird researchers who think about singing with a structure in the throat. Can you describe how crickets make their songs?


    Robin Tinghitella  8:09  

    Sure, so it's only males that produce these songs, and they're primarily using them to attract females from afar, but also to convince females to mate once they're in close proximity and in competitive interactions with other males. And the way that they do that is by rubbing their wings together in a particular pattern. So essentially, they're taking a sort of toothed file that's on the underside of one of their forewings and dragging that across a scraper that's on the edge of the opposite forewing, and that causes the wings to vibrate. And there are these modified wing veins that form structures or organs on the wings that together produce these calls that are at sort of a characteristic frequency or pitch, which in this cricket is somewhere around five kilohertz. So relative to bird song, they're actually more pure tone, typically, than a bird song might be.


    Cameron Ghalambor  9:04  

    So that description makes it sound almost like a harp, or, you know, another kind of like stringed instrument.


    Robin Tinghitella  9:13  

    Yeah, absolutely. There's even one of the organs on the wing is called the harp, and it's shaped like a harp. And if you can imagine the wings look sort of ridged. So these structures that are produced by the wing veins are sort of ridged looking. And any changes in the shape or size or location of those structures is changing sort of the spectral characteristics of the song.


    Marty Martin  9:37  

    Okay, how much cricket diversity is there in Hawaii. I have a sense that it's more than just the cricket you're focusing on, based on the papers that I read, but I mean, in general, you know, we're talking about adaptive radiations, or we're moving into that arena. How much radiation has happened?


    Robin Tinghitella  9:54  

    Yeah, so our cricket is definitely not the one that's best known for adaptive radiation in Hawaii, right? So there are Native Hawaiian crickets called Laupala that are a sort of classic example of an adaptive radiation where, you know, speciation happened really rapidly as they colonized island after island in Hawaii, as those islands were formed, and you get, you know, your sort of traditional isolation between populations and then reproductive isolation based on song differences and preferences for those song differences. So I'd say Laupala is really where it's at in terms of thinking about classic adaptive radiation within Hawaii. Our cricket is one of several introduced crickets in Hawaii. I don't know Dale if you want to say more about that?


    Dale Broder  10:41  

    Sure, well Robin's the one who really has done this work , but essentially, we think they've maybe arrived with Polynesian settlers. The timing seems to match, based on some of Robin's work, and even anecdotally, maybe were brought intentionally because they might have important meaning as representing the souls of ancestors, which is kind of beautiful. So we think they've been there for quite some time, anywhere from 700 to 1600 years ago, but they're quite established, and seem to be like the field cricket now.


    Cameron Ghalambor  11:10  

    And so where are they originally native to?


    Dale Broder  11:13  

    Australia. And they island hopped across this Pacific and kind of ended in Hawaii.


    Cameron Ghalambor  11:19  

    Oh, cool.


    Marty Martin  11:20  

    And they're, what's their current distribution? Are they sort of now limited to Australia and Hawaii? Do they make it into the States, South America?


    Dale Broder  11:26  

    They ended in Hawaii, but they also are all across the Pacific. So you know, French, Polynesia, Mo'orea, and we do sometimes visit those sites to study, kind of the ancestral pre-Hawaii sites, as well as Australia, which is kind of the original. And importantly, they always sing the same classic, and we kind of call it the ancestral song. So they sing this ancestral song everywhere in their range, only in Hawaii are there these new songs that we've been studying.


    Cameron Ghalambor  11:55  

    Cool, so there's another player in the system, this parasitoid fly. Can you tell us a little bit about it's like, kind of natural history, and what this parasitoid fly does to the crickets?


    Robin Tinghitella  12:09  

    Yeah, we can tell you what we know. They're a little bit of a mystery. They're one of those animals that you only encounter if you've got the right thing going, which happens to be cricket song. So you don't run into this fly on a day to day basis if you're not out there broadcasting songs from traps the way that we do. So essentially, what happened was that sometime after these crickets were introduced to Hawaii, they started interacting with this parasitic fly that's called Ormia ochracea. And it's, if you imagine, like a house fly sized fly, but white with these really striking red eyes, kind of exactly what you might imagine a parasitic fly to look like if you made one up in your head.


    Robin Tinghitella  12:50  

    The fly is actually native to North America, and like the cricket, it was introduced to Hawaii, but we don't know yet when or how. The first record of it there is from 1989 so these two, the cricket and the fly, have co-occurred in Hawaii for at least the last 35 years. And, you know, we mentioned that this cricket produces these long distance songs that are loud and kind of tonal to attract females. In Hawaii, though, that song also attracts this parasitic fly. So what happens is, when female flies are ready to have babies, they actually hunt for a male cricket host by listening in on those songs. So they're eavesdroppers, and when they get up close, they sort of spray larvae on the crickets that will burrow into the body cavity of the animal and essentially eat them from the inside out over the next seven to ten days. So a bit of a gnarly situation for a singing male, right?


    Marty Martin  13:51  

    Wow It's perfect timing for us to be talking about this a week before Halloween. So and this behavior, this relationship is the same in North America. These flies are hunting down crickets, and only crickets in North America?


    Robin Tinghitella  14:06  

    Yeah, that's right, some other orthopterans, if I'm remembering correctly, Dale?


    Dale Broder  14:12  

    I think it's largely crickets that, yeah, but don't quote us, I guess. But yeah, I know there are 17 known hosts throughout North America and into Mexico. And one thing that's important to note is that all of those hosts produce songs with a peak frequency right around five kilohertz, which is the same frequency as ours. So you know, when the fly, you know, host switched to the specific field cricket when they first overlapped in Australia, the temporal components are different, but the kind of dominant frequency information wasn't a big shift for these flies.


    Marty Martin  14:44  

    Okay


    Robin Tinghitella  14:44  

    Yeah. And one of the cool things about this fly, like, we don't know a whole heck of a lot about their natural history. You know, in terms of, like, do they live in trees or grass? Like, we don't know, what do they eat? Other than a cricket, we don't know. But we do know a lot about their hearing. So the flies are actually a model system for hearing aids. Their hearing is extremely well studied, in part because they have this really cool ability to get around that sort of Cocktail Party Problem. So they have this like, very directional hearing that allows them to hone in on the host that they are seeking, regardless of what's going on around them. So it's a there's a lot known about the flies' hearing ability, and not so much about aside from, you know, hunting for crickets, what they do on a day to day basis in their natural lives.


    Marty Martin  15:35  

    Yeah, this is amazing. It's such a recurring theme. Last episode, we talked to Aubrey Kelly about spiny mice, and they're known unbelievably well for a few things, but then really conspicuous things that field biologists would ask, no idea, no idea whatsoever.


    Cameron Ghalambor  15:51  

    Yeah, the natural history. So, okay, so, so this parasitic fly shows up on Hawaii, and what happens to the cricket song? Can you describe the sort of evolutionary changes that have happened initially?


    Robin Tinghitella  16:08  

    Sure. Yeah so this interaction between crickets and flies, like you said, really places these males in kind of an evolutionary bind, right? They should sing because it's attractive to females, but they're simultaneously risking attracting this deadly eavesdropper. So about 20 years ago, when I was a new graduate student, we started to find some pretty wild evolutionary changes in cricket songs and the wing structures that make them, in Hawaii in particular. And what all of those changes have in common is that they seem to protect the crickets from this parasitic fly.


    Robin Tinghitella  16:42  

    So the first thing we found in the late 1990s was that there was a genetic mutation that we ended up being able to map to the X chromosome that changed male's wings by eliminating most of those ridged structures on the wings, the organs that produce sound, which meant that they went completely silent, so obligately silent, they couldn't produce sound if they wanted to. They would rub their wings together as much as they wanted. They actually even do that in the same pattern that an ancestral male producing a song would, and they do it as much as a male who's actually producing sound. But that silencing mutation meant that nothing came out, right? No song at all. And that might seem like a really bad thing for a cricket that can't, you know, sing to attract mates, but that mutation actually spread through multiple populations, one of them incredibly quickly, from, you know, none, or at least very few males in the late 1990s to somewhere between 90 and 95% of males being entirely silent by 2003, so extremely rapid evolution. That's about 20 cricket generations. And of course, the reason we think it spreads so quickly and to so many individuals within these populations is that silence, of course, protects them from the parasitoid.


    Dale Broder  18:00  

    Well, can I chime in? Because there's another piece to the story, and this is Robin's work from her PhD. So how can these males mate if they don't have a song? And I think if you want to mention, Robin discovered that actually the males just hang out next to ancestral males and intercept the females that are coming to mate with the ancestral male.


    Marty Martin  18:23  

    Wow!


    Cameron Ghalambor  18:24  

    Whoa so, so these males at some cognitive level level, they're aware that they are unable to produce any kind of sound. So they go and sort of hang out by these other males, is that...?


    Robin Tinghitella  18:47  

    We actually don't know if they are aware that they're not producing any sound, right? Like, like we said before, they will stridulate, rubbing their wings together all the time, and nothing comes out, which seems like a big waste of energy, if nothing else, if you are aware that no sound is coming out. So what we think is actually going on is that this tendency to act as what we would call a satellite, so a male who hangs out next to another male, actually predates the evolution of silence, and it was something that existed in these populations in Hawaii and even elsewhere in the Pacific, in situations where males hadn't had recent mating success. So you can actually kind of induce satellite behavior. So if they haven't, you know, mated recently have been signaling with no success, they're not running into females as they're out walking around, you can kind of mimic reduced densities or stop playing songs around them, and that will actually increase the tendency to act as a satellite. So we think it was a pre-existing behavior that was really important for facilitating the success of this silent type. And that also meant that for a while, in order locate mates at all, there had to be some singing ancestral type males in these populations. So for a long time, things were sort of, you know, like very cool, but static, right? Where about 90% 95% of males were silent, but some small proportion of the males in the population were still capable of singing. It's not that way anymore, but for a long time, this is what we thought was happening.


    Marty Martin  20:24  

    Is there any role for the male song? I mean, is there any direction towards other male? How do males sort of respond to each other? Is it solely about female courtship, or is there a competitive dimension?


    Dale Broder  20:37  

    I mean, they do produce. So they have three types of songs, one for long distance made attraction, one for close courtship encounters, and a third, one for aggressive encounters with other males, or perhaps even with females. It's really fascinating that they use the same instrument to produce all three of those really distinct songs. And so the aggressive song is a very short, quick chirp- that's just like, "leave me alone." We don't really study that one much in our lab, because, you know, we're interested in sex.


    Robin Tinghitella  21:07  

    Yeah, yeah. We like it when they actually mate. There is one paper, though, on male male competition in sort of dyads where one or both of the males are silent. And I think the answer that they are, what they found in that paper was super cool. I think David Logue is the first author. What he found was that if you have contests between males where at least one of the males is silent, or both of the males is silent, they actually escalate to bigger fights. So without that signal saying like, Hey, get out of here you end up getting more like direct grappling, fighting interactions between males.


    Marty Martin  21:46  

    Interesting. So can I ask one more question about, you know, sort of these pairings where the silent one ends up next to the singing one? I mean, how much variation is there among males that are singing in the population, such that one should really be preferable to go hang out by this buddy because his song is so sexy that I have an increased chance of him getting parasitized and me finding a mate.


    Robin Tinghitella  22:10  

    Yeah, that is a great question. So one of the things that will become important later in this conversation is that there is some variation among ancestrally singing males in, you know, how loud their song is, or what their calling effort is, seems to be really important. So you know, how much are they stridulating in a given 10 minutes or half hour or hour? But there's not a lot of variation in other components of that song. So the temporal patterning of the song is, you know, set by what's happening in the brain that is very characteristic of the species. It's always in that same temporal pattern. The frequency of the song, as Dale was saying, is set by the structures on the wings. And it's this one instrument that's producing all of those different types of song. And so the pitch of the song is fairly canalized, there's not a lot of variation among males there. But females are really cool and do really interesting, complex things, right? And so there are subtle differences that they have preferences for, for sure. So if you look like across the crickets range, there are differences, slight differences, in temporal patterning. And, you know, these other characteristics that we've talked about between Australia and Hawaii, let's say, and they do have preferences for certain components. And there's some evidence too, that, you know, components of the courtship song, for instance, might have, might be sort of indicator traits of immune function. And the calling song seems to be more like a hey, I'm over here. Come find me sort of, sort of signal.


    Cameron Ghalambor  23:44  

    So I'm, I'm really curious about, you know, you have this very cool ability to look at the morphology and structure of the wing, and I guess, make some predictions then about what the sound should be like. But it's also interesting that the genetic basis for the change in the wing is decoupled from the genetic basis of the behavior to rub your wings. And so is that something that like have you seen any patterns over time? For example, where the these quiet, silent males don't spend as much time rubbing their wings as they used to, and, you know, engage in other kinds of behaviors.


    Dale Broder  24:33  

    Yeah. I mean, that's something we're working on. So anecdotally, we're finding, we think, that, oh well, we haven't even gotten into all of the novel morphs yet. But in addition to the silent cricket Robin just talked about, we're going to tell you more about some other male morphs, which do, we think, maybe sing a bit less, spend less energy producing songs though we haven't can't say that for sure yet. And as far as we can tell the temporal piece seems to not be changing. That seems to be pretty, like Robin said, kind of neurologically based, and isn't something that is changing at the moment, but we'll keep following that.


    Robin Tinghitella  25:11  

    Although there is this really cool thing that we're working on with a postdoc in the lab now, and that Marlene Zuk has actually worked on a bit in the early 90s too, where the temporal pattern of the individual song seems to be set, but the timing throughout the day is something that has evolved. So in Hawaii, before these silent crickets evolved, Marlene found that the males in Hawaii would actually start calling later in the evening than males elsewhere. So in Australia, you might hear this same Pacific field cricket singing at, you know, three in the afternoon, four in the afternoon, five in the afternoon. In Hawaii, they really don't start singing until after the sun has completely gone down, and that's because the fly is active, or most active at dusk. That's when they're hunting. So this is another sort of temporal release sort of strategy to get out of some of that risk of parasitism, which I think is really cool. And we're starting to look at how that is, whether that's truly circadian, and whether those kind of patterns of when you call and when your phonotactic are things that are evolving and coming to differ between these different types of males that Dale was mentioning.


    Marty Martin  26:26  

    Well, in this back up, and Dale maybe tell us what you almost told us there. So it's not just the absence of the call and or the timing of the call that Marlene found. There's altogether different calls, multiple varieties of calls that, or songs that you've observed.


    Dale Broder  26:42  

    Yeah and I will say Robin's been doing a lot of the talking, because this is Robin's been studying the system for a very long time. But this is actually the moment when I enter the scene. So I was a postdoc in Robin's lab, actually doing some kind of pedagogic and diversity, equity, inclusion types of research, and happened to be there when Robin discovered a new cricket morph. So not to steal your thunder here, Robin, but I was in the perfect place at the perfect time to then join Robin in this big investigation, which has produced much more than we could have ever imagined. But I'll let Robin talk about how she made this discovery.


    Robin Tinghitella  27:18  

    Alright, I think the stories behind science are really important, you know, like it contextualizes everything. So


    Marty Martin  27:18  

    Agreed.


    Robin Tinghitella  27:20  

    All right, so I'll set the stage for you. Here. I was in graduate school when we found silent crickets. That was amazing. So fast forward about 15 years. I'm now a faculty member in Colorado. I'm studying how human generated noise impacts singing crickets, and I'm a new faculty member, so I'm desperately seeking funding, as we all do, right? So I decided, in my infinite wisdom, that I was going to try and get some funding from National Park Service, but that meant that I would have to work on my study organism in a national park and I did not do that. And I had recently learned about Kalaupapa National Historical Site, which is on Molokai, and had been like cold calling people trying to figure out if they would let me in to look for my cricket there. And I finally got this invitation to go into and stay in the park overnight, which requires special permission there.


    Robin Tinghitella  28:19  

    And as Dale said, this is exactly when Dale had joined the lab as a postdoc. So while my research team is elsewhere on Hawaii, I took this ridiculously expensive, tiny puddle jumping flight into Kalaupapa, which is like the least visited National Historic Site in the country. I was there for less than 24 hours, and after it got dark, I went out walking with the deputy director of the park for several hours. We searched and searched and searched through the lawns there, looking for our favorite cricket, but we kept coming up empty handed. We weren't hearing anything, and eventually we made our way to this one little lawn at a place called the bishop house, and I started finding Pacific field crickets there. None of them were singing, so I figured this must be another silent population, but that was equally as exciting. So I collected a bunch of these animals, and I took them back to my room with me, and I set them up in a little Tupperware container where I hoped that they would lay some eggs in moist cotton so that I could take those back to my lab with me.


    Robin Tinghitella  29:26  

    And then I got ready for bed, and I'm like, laying down, starting to do some work, and about a half hour later, I woke up to the sound of this very annoying cat purring in my room, which was super jarring, right? Like you're in this national park, you've never been there before, you're in a weird dorm room, you don't know anybody there, and this cat is like, waking you up from your slumber. So I eventually, like, got up out of the bed and started to look for this cat so I could kick it out, and realized fairly quickly that it was actually my box of crickets that was purring.


    Robin Tinghitella  30:01  

    So these crickets on Molokai, it turned out, were producing an entirely different sounding song. It has that same characteristic pattern that we talked about, and we think, Cam, to your point earlier, that having that sort of vestigial trait where you still rub your wings together really set the stage for these new signals to evolve in a cool way. And there were really, you know, this song I could recognize as the right species, but it had very different qualities because of the shape of the wing that had again mutated, this time in a way that still allowed the crickets to produce audible song, but a song that was quieter, more attenuated, and had, you know, different pitch and and broadband sort of characteristics than the ancestral song.


    Cameron Ghalambor  30:50  

    Wow.


    Marty Martin  30:50  

    So we're going to try to stick some of the songs in, but, you know, Cam and I, I don't know that I've heard them before. We can't hear them now. I'm not going to ask you to mimic them, unless you want to how different do they sound? Because, you know, I have in my mind the iconic sound of a cricket, but this, you used the word purring, that seems so very different than a typical way of thinking about a song.


    Dale Broder  31:15  

    Yeah, so one of the first things we did is get the crickets back to the lab and record them, actually in a professional recording studio at the University of Denver, which was so fun. So we're working with sound tech engineers, and it's pitch black with just red lights, because crickets can't see red light. And we're, you know, talking to the sound engineers, like, okay, record. And you know, it was such a surreal experience for everyone involved, but we got these fantastic recordings and confirmed, in fact, yes, these songs are very distinct from ancestral songs, and some of the biggest differences were like broad bandedness. So a pure tone song would be like a whistle, which is what is kind of characteristic of the ancestral song, and a broad band song would be closer to white noise. So white noise is the most broad band where you have all the frequencies equally represented across amplitudes, so like, so this cricket is more like instead of, and so that's why it can kind of have more of a purring sound like. And also, what I, you can tell me if you agree, Robin. But to me, the purring is really more characteristic of the courtship song, whereas the calling song has kind of different features, which are also quite different.


    Robin Tinghitella  32:23  

    Yeah so what I heard in my room was this, like group of purring males that were all courting females. And so, you know, the reason that we call this a per is just historical contingency, right? I thought it was a cat. It doesn't necessarily have sound characteristics that are super in common with cat's purrs. But when you've got a bunch of very quiet males producing this sound all in close proximity, that's what it mimicked so.


    Dale Broder  32:51  

    I should say it was super funny. When this first came out, there was quite a bit of press, and I remember Robin even, like some little magazines in Hawaii picked it up the story and news articles. And there were a lot of headlines that said Hawaiian crickets are now purring and becoming cats, you know, just like science communication fail . Like, oh God. Like, that's not what we're saying at all.


    Robin Tinghitella  33:14  

    We even had one that said climate change was causing Hawaiian crickets to purr. And Dale and I were like, what have we done?


    Big Biology  33:23  

    Oh, wow.


    Cameron Ghalambor  33:26  

    So one thing that's like, super cool about the system is that you have the ability to study these very early stages of divergence in traits, which is something I always harp on, is like, you know something we really don't know that much about in nature of what happens during the very early stages of of divergence. So, Robin, you were lead author on a 2021 paper in Nature Communications, where you focus on these different sources of selection, on the purring call, and you tested how female crickets would respond to this new song, the purring song, and also how the flies responded. So can you describe a little bit of the first of all the methods, which I think are really interesting for like how you test that, and also like what the sort of take home message was?


    Robin Tinghitella  34:24  

    Yeah for sure. And this, this is really a partner in crime paper with Dale, so I think we're technically co-first authors. I don't remember. We tend to bounce back and forth, and it's a coin toss, who ends up where. Anyhow, so yeah, we, you know, having found what essentially was a brand new, novel sexual signal in real time, we started doing a longitudinal study, essentially, where we went out to the field every two cricket generations, or every six months. And we've done that for about the last six years. And when we go out, we are measuring a series of things. In this particular paper, what we were reporting on was something that we called the time zero data, essentially right after this song had emerged for the first time, what were the conditions that sort of facilitated it being successful or not, and sticking around or not? And one of the things that inspired that was that we started finding purring crickets on different islands, so it wasn't just Molokai. We had never been to Molokai before, so we didn't actually know when purring crickets evolved there, or, you know, how long they'd been there, interacting with this parasitoid fly. But on other islands, they're very well studied by multiple different labs, and so we knew that purring was not there, but we now had purring crickets in some of these long studied populations where it had never existed before.


    Robin Tinghitella  35:48  

    So we had this really cool opportunity to look across sort of replicate islands within Hawaii where purring was brand new, and to ask: How do female crickets respond to this new sound? And how do parasitoid flies respond to that new sound? And the way we do that is pretty low tech. So we go out to the field. We wanted to do this all in the field so that we weren't disturbing these small, rapidly evolving populations. So we collect crickets using, you know, headlamps at night, just the way I described on Molokai. And then we bring them into these sort of field station, you know, laboratories, where over a period of, you know, 10 hours each night, from dusk until dawn, we can ask animals what they think about these sounds. And to do that, we essentially, like, roll out a yoga mat that has, like, it's really low tech. We roll out a yoga mat. It's got like a little wall around the four sides, and we take a speaker and place it in the center of that mat, and we broadcast songs from that speaker to female crickets that we release at one end of that arena. And we measure things that are pretty straightforward, that indicate whether or not the female can hear that sound and if she likes it. So if she does like a sound, she'll do this really characteristic, what we call phonotaxis, where she moves toward that singing male. And their ears are on their elbows, essentially, so you can tell that a female is phonotactic because she's making this sort of winding S shaped track to get to that sound. So we measure, you know, does she go to the sound that we're playing? How fast does she get there? Does she actually contact the speaker, as if she were trying to contact that male?


    Robin Tinghitella  37:34  

    And we do the same thing for parasitoid flies. You can set up traps out in the field, which are just like two liter soda bottles with the top inverted. So they're super fancy. We stick a speaker inside of those and do the same thing, like, from, you know, some sort of Bluetooth contraption, play these different songs to parasitoid flies, and they'll come into those funnel traps and sort of get stuck inside of the funnel trap. And we can go back and count, you know, how many were attracted to each of the different songs that we played.


    Robin Tinghitella  38:05  

    So the first thing that we were really interested in in this paper was understanding differences in how purring crickets were balancing natural and sexual selection, with respect to ancestral crickets that produce that typical song and you know, some negative controls, like white noise or silence. And when we did that, we found this really sort of remarkable pattern where about 20% of crickets, on average, will locate a purring song from, you know, a relatively far distance, which means that purring is much less attractive than that ancestral song, but still much better than white noise, and certainly better than silence at attracting females, right? So purring crickets are doing something that silent crickets aren't- they're managing to attract these females. But for flies, we caught like 47 flies at traps that we set out in the field that were playing ancestral song, but we only ever caught a single fly at a trap that was playing a purring song, and no flies were attracted to our negative controls. And that result on its own, we thought was really, really cool, because it suggests that purring might be sort of a private mode of communication that allows crickets to communicate with one another without attracting this deadly eavesdropper.


    Marty Martin  39:26  

    So one thing that you mentioned when you were describing the study design that I think I should have asked before, you found the purring relatively recently on Molokai, and now you found it in other places, but you sort of suggested that you know when it first showed up, wherever it first showed up. So how do you know, and where was that?


    Robin Tinghitella  39:46  

    So we don't know when they showed up or, you know, like, evolved on Molokai. We actually don't know which of those happened, because we didn't work there prior. But in other locations, on the islands of Oahu and Kauai, Hawaii and the Big Island of Hawaii, there are long term research sites that are places that my lab, Dale's lab, now, Marlene Zuk's lab, Nathan Bailey's lab, all work in these, these very kind of distinct fields. So these crickets are weirdly site-specific. They just live in grassy, disturbed areas. But there's something about these specific lawns that they are really attracted to, and that keeps them in these relatively small areas, even though the lawn right around that looks perfectly good to us, we have no idea why the cricket is not interested in it. We look all the time, right? You'll go to all these surrounding locations, and it's just very clear that the crickets are not interested in the lawn at that part. They only like this part.


    Robin Tinghitella  40:46  

    So there are these very kind of isolated sites where these crickets are found, and those are the places that all of these labs have to go to collect their animals. And so because we're out there, you know, once a year, twice a year, doing field surveys of crickets and flies and looking at wing morphology and listening to songs, we know that purring was absent in these places at least until 2017 and that's when we start to see purrs pop up. And again, we don't know, you know if purring males are moving from island to island, or if like silence, this is something that's actually relatively easy to evolve. Silence actually evolved convergently three times in different locations in relatively quick succession over  a matter of just a couple of years, and spread to, you know, different kind of proportions of the population in each of those places. So purring could be like that, or it could be that, you know, we not us personally, but humans are moving these animals around, or they're floating from island to island, or getting blown from island to island fairly regularly.


    Marty Martin  41:53  

    Yeah, but I mean, does that, in your experience, does that seem very likely for a cricket over that short period of time to move in a new population to be established. I mean, it almost, I'd put my money on the sort of recurrent evolution and all of the different places. Even though we don't have any idea, I don't have any idea what the means might be, the likelihood that you're going to have, you know, some innovation showing up and then populating the whole archipelago doesn't seem very likely.


    Robin Tinghitella  42:18  

    It doesn't to me either. And Nathan Bailey's lab has done some really cool work looking at things like the actual genetic isolation of these populations and gene flow. So there is regular gene flow between locations. But despite that gene flow these different populations are evolving really independently from one another, even populations that are like, you know, 10 minutes away, a 10 minute drive from each other on the same island, can have completely different morph compositions. You know?


    Marty Martin  42:47  

    Yeah, wow


    Robin Tinghitella  42:47  

    Yeah, it's, it's really wild. So they really do seem to be evolving independently.


    Dale Broder  42:51  

    And also, one of our close collaborators, Jay Gallagher, actually phenotyped purrs across the Hawaiian Islands and found that already they've diverged. So purrs across islands are already different from each other.


    Marty Martin  43:06  

    Wow


    Dale Broder  43:06  

    Which could mean that, you know, it arose once and has quickly diverged, or that it's, you know, evolved a couple of different times.


    Cameron Ghalambor  43:15  

    Yeah, it seems like you should be able to do some kind of like population genetic analysis and maybe see if it originated once and spread, or if it has popped up independently.


    Robin Tinghitella  43:28  

    Yep


    Dale Broder  43:29  

    Yeah, Robin's doing that.


    Robin Tinghitella  43:30  

    Yeah I have an awesome PhD student who is headed to Scotland next week to do exactly that


    Cameron Ghalambor  43:36  

    Nice. Cool well so that makes me wonder, so you'd mentioned that outside of Hawaii, you don't see a lot of variation in the ancestral song. And so do you think that, like these kind of mutations that alter wing shape probably happen with some relatively high frequency, and then those individuals just have very low fitness, and they get eliminated because they have very low ability to attract females. Is that kind of the thought for like, why you don't see the variation outside of Hawaii?


    Robin Tinghitella  44:18  

    Yeah, absolutely. I think you hit it right on. So there are two things I want to pick up on there. One is that we have talked with other folks who work on crickets, different species, who have seen things like that silencing mutation pop up in their lab populations and then go nowhere. I've done breeding experiments, and Nathan Bailey's done the same breeding experiments to look at the mode of inheritance of these traits, and both of us had spontaneous mutation to the flat wing type in those breeding experiments. So I think these types of mutations are not rare. I think it's relatively easy to disrupt the song in various ways. Has given changes in wing morphology, whether that's because of, like, genomic hotspots or, who knows, like, what that reasoning is, but yeah, I don't think they're very rare.


    Robin Tinghitella  45:09  

    And I think the other thing that you said, Cam, that is like a hallmark of what's going on in Hawaii that really we're digging into a lot now, that facilitates all of this variation sticking around there is some differences in female preferences in Hawaii versus other places in the crickets' range. So if you're in Australia and you stick, we've done this, you stick a silent or a purring or a rattling, talk about that later, any of these morphs, these new types with a female, nine times out of ten, they are rejected so completely, you know it's a female choice system. The females have to mount the males for mating to happen without an attractive song. They often don't find them, but also they reject them at really, really high rates if they're not producing that ancestral courtship song. But if you go to Hawaii and do that same experiment, what happens is completely different. So more than 50% of females in some Hawaiian populations will accept a male who doesn't produce a song at all, and the acceptance of purring males is at least that high, if not higher. So there's something really importantly different about the way that females are making mating decisions in Hawaii that's allowing this variation to get a foothold in some of these populations.


    Marty Martin  46:30  

    Wow, that's really that's amazing. Do the, I don't think you've mentioned it yet, but what about the volume of these songs? I mean, I know that in the field, that's probably ridiculously difficult to know, but how does that come into play?


    Dale Broder  46:41  

     Yeah, the amplitudes are definitely quieter in these novel morphs, for sure.


    Robin Tinghitella  46:45  

    Yeah, we just did some really cool work to think about how far away they can hear these different songs from, for the fly, not for the cricket, but it's a dramatic difference. So the ancestral song a fly can hear from, you know, 200 meters away, a purring song they can hear from about two meters away.


    Marty Martin  47:04  

    Wow.


    Robin Tinghitella  47:05  

    And we think that's similar. Just, you know, based on our data from all of these trials that we've done in the field, it looks like females are likely to use a purring song in relatively short distance mate location and then in courtship interactions, but they're not going to be able to hear and find that song from, you know, five meters away, which means that there are some other things going on in the field that facilitate them finding each other too, which is also cool. There's some other behavioral differences that allow them to find one another without these loud, kind of pure tone, long distance songs.


    Cameron Ghalambor  47:37  

    So one of the results that was, I think, really important was there was still this preference, a stronger preference, for the ancestral call than the purring call. But also that when you look across females, the sort of preference function was more or less flat so meaning that whatever variation there was, either in the call or in the females, you didn't see preference for a particular type of purring and so I think that has some interesting implications about whether, like in guppies, for example, females tend to prefer novelty, and that doesn't seem to be the case here. And then these calls are also at frequencies that would sort of suggest that it's not biased towards what the females prefer. Is that, is that? Right?


    Dale Broder  48:36  

    Yeah, maybe I'll start and then you can take over, Robin.


    Robin Tinghitella  48:38  

    Sure


    Dale Broder  48:39  

    I don't know if we've mentioned this. But one thing that's cool about the purrs is that we have an incredible amount of variation in frequency and in all these other components of songs. So we mentioned the ancestral song is always around five kilohertz, but we can have purrs as low as two kilohertz and all the way up into ultrasound. So like huge variation in frequency components, which is really interesting, because there's a lot of theory about anytime you have a new signal, it's expected that there should be a lot of variation at the beginning. There's also a lot of theory about how preferences should kind of align, so that you need variation in preferences, or that you need kind of coupling between preference and signal, or maybe one comes first, and so there's kind of a lot of interesting hypotheses to test. You also mentioned, kind of like a pre-existing bias. So yeah, we were curious, do these female crickets like purrs that are around that ancestral frequency of five kilohertz? Or do they just like, you know, the new ones or the rare ones or the uncommon ones? Or do they have a preference at all? So, yeah, essentially, like you said, Cam, we found that at the population level, preference functions were flat, so they really didn't have any preference at all. So no specific purr was being selected for, however, individual females did have preferences. So some females really liked low frequency, some liked high frequency, some liked those intermediate. And so it's really fascinating that I'm not sure if this is something that other people had observed, that this incredible variation and also kind of low threshold for acceptance might be what's really important in facilitating the origins of a novel signal, rather than having like, a coupled preference signal.


    Marty Martin  50:18  

    Yeah  well, that's why, when I first read your preference functions are flat statement. I, you know, had to go back and dig through that because, you know, at the population level, that's definitely what you would see, just based on the how the average is calculated. But what you just said, Dale, I mean, it makes it both more fascinating and staggeringly complicated.


    Robin Tinghitella  50:38  

    Yes


    Marty Martin  50:39  

    Because you either have preference for ancestral or are females just like whimsically picking something that's nothing like ancestral. I mean, why should that ever be preferred? That's just, what are your ideas? Rob,


    Robin Tinghitella  50:52  

    Yeah, you know, it is pretty wild. I actually, I was giving a talk about this paper once, and a graduate student kind of pulled me aside after the talk, and was like: "Wow. I think it must have just really been, you know, a big conversation in your lab when you guys found essentially negative results." And then were like: "Hey, we should send that to Nature Communications." And I kind of like, I hadn't thought about it that way, that yeah, they were right, right? Like, we went out looking for this preference, and what we found was nothing. Like this, like absolutely flat preference function. But I think what we were super excited about is what you just said, like, this is incredible variation that is so, so cool amongst females. And how do you get to that point, right? And I think over the last 20 or 30 years, we've been paying a lot more attention as evolutionary biologists, as behavioral ecologists, to variation amongst females, right? And rather than thinking about that as noise that complicates what your preference looks like, we are interested in why that exists, and so you can imagine lots and lots and lots of reasons for individual variation in preferences, right?


    Robin Tinghitella  52:06  

    Remember, we're doing this out in the field, so we're not in common garden. We've done this kind of thing in common garden too, but we're doing this out in the field, so every female has a different experience that you know might shape what that preference function looks like. Some of them may have had positive sexual experiences with males that have these traits before. So there's potential for learning of a preference. Some of them may have had no recent experience at all, like maybe they haven't mated, and so their threshold is just, you know, relatively low across all those purrs, they're willing to accept whoever they run into. I think it's super variable, and, yeah, it's, it's a really interesting problem. But it, I think it's so cool from the perspective that it, you know, matches what I found with silent crickets 20 years ago, where this huge proportion of females in Hawaii was accepting of males who didn't produce a sexual signal at all. So I think another thing that is underlying all of this is that there's been essentially 20 years of crazy, relaxed selection in Hawaii because of that silent male type that came to dominate in a bunch of different populations. So there's, you know, no selection that's been maintaining sort of wing structures and song characteristics. There hasn't been selection that's maintaining female preferences in a particular shape or type, and that relaxed selection might be sort of allowing females to sort of go in different directions and to have expressed different preferences.


    Marty Martin  53:45  

    Yeah, Dale, you said, though, I mean, and both of you alluded to this, that there are individual preferences. So it's sort of difficult to get your head around the individuals doing something, but the population looks different when you have pictures. These things are easier to look at. But do you see consistent preferences in individual females? I mean, have you, have you asked about that? And there's their variation among individuals in their consistency? Now I'm really going to make everyone's head hurt.


    Dale Broder  54:10  

    I don't think we've published that, but I think we have done that in the lab and found yes.


    Marty Martin  54:16  

    So if they're picky for low frequency, they're always preferring low frequency. Is that how it goes?


    Robin Tinghitella  54:22  

    Yeah, there's more consistency within individuals. So there's more variation between individuals than there is within individuals. So we have looked at like repeatability of preferences, and, you know, there's some variation there. But again, we're, we're doing this out in field conditions, right? So, yeah, so they are repeatable, but they're also malleable. They're super plastic, and Dale's done a bunch of really cool work on that too. So


    Dale Broder  54:49  

    Yeah, I know we're not really going to talk about that today, but that's ongoing work that we've been doing where we're letting females in the lab from different morph types. So purring females, rattling females, ancestral females, Australian females, letting them grow up hearing different songs, like novel songs or ancestral songs or silence, to see how that shapes their preferences. And essentially, in our preliminary data, we can see that if a purring female hears a purring song, I think there's like 90% acceptance of purring males, but if a purring female hears ancestral song, over half will reject a purring male.


    Marty Martin  55:26  

    Wow.


    Dale Broder  55:26  

    So it seems like plasticity is going to be really important, and that's something we're working on now,


    Marty Martin  55:32  

    Right I mean it's almost as if there's an existing template, though, and the plasticity is there's some kind of interplay with learning versus what's already there. Wow.


    Cameron Ghalambor  55:40  

    So what are your thoughts then about the sort of coevolution between the male signal and the female preference? Because I think it was many years ago Anne Houde and  John Endler, maybe, published a paper where they selected on male coloration and found a correlated evolutionary response in the female preference, suggesting that, you know those, those are really locked together. And maybe that happens in some cases, but in the it sounds like in the cricket example, you have both genetic variation for the signal in the male and separately you have variation in the female preference. And then there might be some lag time between when the signal changes and when the preference for that signal catches up. Is that a fair description?


    Dale Broder  56:39  

    I think that's exactly what we've been thinking. So we actually recently thought about this with some other brilliant scientists, and kind of did a think piece about where new signals, how they arise. And, you know, do they have to be coupled? Does one have to come first? And I do think our system is a really unique opportunity to test some of those ideas. And like you said, it seems like, at least at the beginning, what we're seeing is a lot of variation in both the sender and the receiver, and that at the moment, it doesn't appear to be coupled, but that's something that theory would suggest should happen over time, and that, you know, things like plasticity and other mechanisms that we're exploring maybe explain kind of this important variation at time zero.


    Robin Tinghitella  57:22  

    Yeah and NSF, if you're out there listening, we're sending you a grant to do exactly that in the next couple of months. Yeah one of the things that I think is so cool about this system is how tractable it is, both in the lab and in the field, right? So I mentioned that when we started writing that NatComms paper, we were talking about data at time zero, as close to the origin of purring crickets as we could possibly find. But we've been tracking preferences of female crickets, preferences of parasitoid flies, hearing ability of both of those players, the morph composition of different populations and how they're producing these songs, so what the wings look like what the songs sound like over the last five years, six years now,


    Dale Broder  58:05  

    I think it's like, I think it's almost eight.


    Robin Tinghitella  58:07  

    Is it? Okay, how old am I? Anyhow, so we, we think there's a lot of value in continuing to track this for the reasons that you're describing, like we could actually pick up on the very earliest stages of coevolution between a novel signal and a female preference, or between, you know, a novel signal and parasitoid preference, so those host parasite interactions. And we're, you know, we're starting to see, like Dale was saying, evolution of some of these songs, we've actually found that across populations, there are some differences in preferences that are starting to show up. So for instance, Molokai females are more accepting of purrs than females from other locations. Yeah so there's a lot that can be gained, I think, from continuing this, like detailed longitudinal approach to data collection.


    Dale Broder  59:02  

    Yeah. And in fact, in doing that, we haven't explicitly stated this yet, but we've discovered what like four more morphs after purring.


    Robin Tinghitella  59:10  

    Yeah


    Dale Broder  59:10  

     Just in the past seven years by using this approach.


    Marty Martin  59:22  

    So yeah, Dale, you mentioned the other morphs. You've given them very creative names of rattling and small wing and curly wing and probably others. What are these things and how do they sound?


    Dale Broder  59:33  

    Yeah so working with someone from Robin's lab, a collaborator, Jay Gallagher, helped essentially characterize all of the songs that we were recording by doing really detailed analysis of the songs and morphological analyses of the wings to really show that there's a new one which was rattling, which was kind of the first one that we worked on. And this one is different from both purr and ancestral. It has more of like a rattling quality to me. It sounds like, almost like a playing card stuck in the spoke of a bicycle, kind of clicky. And that one's a little bit louder. And I'm just gonna keep it short here. The TLDR is that we showed that, like purring, this one also appears to be a private mode of communication, because I think something like, is it 40 or 50% of females are attracted to this rattling song, and again, largely protected from flies. And then the Bailey lab actually characterized these curly wing ones, which are a little bit curled up at the tips, making it hard for the wings to come in contact. And in some of our work, it seems like that one, in some cases, is almost equivalent to silent as far as its function in attracting females and then the small wing, it's literally just shrunken, which makes it higher frequency, like thinking of a violin compared to a cello, which is pretty fun, but yeah, I don't know if you want to chime in on like, the mechanism of rattling, Robin?


    Robin Tinghitella  1:00:59  

    Yeah sure. So as Dale said, Jay, the former graduate student in the lab, was hearing these things that he couldn't explain in the data, and that's how he found that rattling was a thing. But he also found this really cool morphological pattern where, if you look at the top of the wings, which is what we usually do, and do sort of traditional landmarking morphometric approaches, there's actually no difference between an ancestral wing and a rattling wing. So initially, we were really puzzled about like, how these guys were producing this categorically different song that sounds all clicky, but with the same structures on the wings. And then Jay had this bright idea to actually stick it under the scope and look in more detail. What he did was flip the wing over and look at the file, which is on the underside of the wing, so the thing that has all those little teeth in it, and he found that 100% of these rattling males actually had these characteristic gaps in the teeth of the file. S o places that there should have been teeth, but there was essentially just gunk, no teeth developing. So it's almost like something genetically is turning off and on and creating teeth, and then no teeth, and then teeth, and then no teeth. And it's that morphological difference that results in this song having completely different, you know, characteristics in terms of how loud it is and what the pitch is and how broadband It is, yeah, and


    Dale Broder  1:02:23  

    Yeah and  these kind of large scale differences across wing morphology are more characteristically seen across species, not within a species. So that's been really wild to see.


    Cameron Ghalambor  1:02:33  

    So given what we were talking about earlier, variation in female preference, then do you see within populations that certain females are more attracted to the rattling call versus a more purring call?


    Robin Tinghitella  1:02:52  

    That's a really good question. I don't think that I have seen anything like that yet. So we do often ask females to respond to multiple different songs, right? So we'll play a series of songs in random order and look at the behavior of the female to different songs. And I think that more often than not, it's that, you know, a greater proportion of females overall responds to, you know, ancestral followed by rattling, followed by purring, and then nobody responds to silence or white noise. So I don't know that there are females that have sort of intrinsic preferences for one morph over and discriminates against another. I think my gut is that it has more to do with things like the power in certain frequencies and how that stimulates the sensory system of the female. So if you play these different purring songs that have this incredible variation in, you know, the peak frequency or dominant frequency of their songs, you do get females that respond differently to them, some will go toward one and not to the other, and the opposite will happen with a different female. But I think they also, both of those females, might be likely to go to a rattling song because it's louder and the dominant frequency is closer to the ancestral and sort of fits that just gestalt of what their sensory system is most attuned to. We don't know that for sure. We're doing some neurophysiological work to look at that with some collaborators, but that's what our sort of biological gut says when we watch them behaving,


    Dale Broder  1:04:34  

    Yep, and I will say in some of the plasticity experiments that I'm doing, s o this is purely anecdotal, but there are a handful of individual females who, if they're reared with a purring song, they will go to a purring song and not go to a rattling song, even though rattling is louder. So there could be some cool stuff going on that we're currently kind of uncovering. Yeah,


    Cameron Ghalambor  1:04:52  

    Well, because it to me, it sounds like the conditions are being kind of set up for the potential for there to be some kind of like frequency dependent mechanisms at play, and you know if, especially if females do develop these kinds of preferences. But then it also makes me wonder if you've seen any kind of like fitness, differences in fitness associated with different calls. Like do these females mate multiply? Is there like a spermatophore or...And how does that play out? Like, if a female could do, will they mate with multiple males? And what happens if they mate with two different morphs? I don't know these are all just random questions.


    Marty Martin  1:05:45  

    Cam is writing the next six grand proposals, it sounds like.


    Robin Tinghitella  1:05:48  

    I know. Yes, yes and yes, I think is the answer to all those questions. They do mate multiply, sometimes very multiply. Males can produce a new spermatophore in a matter of 20 minutes to an hour. So males also mate multiply. It's a female choice system where, essentially, the female has to accept the male as a mate by climbing on top of him, so she mounts the male, and then he, like, weaves this spermatophore into her genital tract, and she can then hop off and take that with her. There's some potential for differences in fitness, like post-mating, too, where females can actually remove spermatophores of non-preferred males, so they might mate, get a little bit of sperm, but decide he's not really what she was going for. And Dale's doing some cool stuff, watching courtship interactions pre- and post- mating to see you know whether that actually happens. But I think a lot of these are still just open questions that we're really, really excited about.


    Robin Tinghitella  1:06:51  

    There are for some of the morphs for silence, we know more about silent, flatwing males than we do about these other morphs. But there are some other characteristics, other traits that are correlated with being a flat-wing, silent male. So there are changes in things like the cuticular hydrocarbons, you know, what these guys smell like, that are rather feminized that impact whether females pay attention to them, like them. So there are lots of things that are probably correlated with these wing characteristics that are likely to impact fitness differences across morphs.


    Robin Tinghitella  1:07:25  

    The other super cool thing is that there are different combinations of morphs in different populations across Hawaii. So these are like replicate populations, in the sense that cool things are evolving all the time in each of them, but the composition of one population in terms of whether there are, you know, small wing or curly wing or ancestral or silent crickets in them is different in every location that we go to, so there's this very cool opportunity to watch that process potentially play out differently across populations that are isolated from one another and evolving independently.


    Marty Martin  1:08:02  

    Nice


    Dale Broder  1:08:03  

    Yeah, and Cam, you asked about fitness, in terms of response of females to these different morphs. But one thing we've been doing is we've just been going out and measuring morph composition in these populations just every two generations, and we can look at, you know, which morph is winning, and kind of think of that as a proxy for fitness, especially because, although we don't completely understand it, we do know there's a genetic basis for these morphs, because we can breed them in the lab, and they're true breeding for multiple generations. And so, for instance, in this one population, there are so rattling, curly,  small wing and ancestral are all four present. And so we've kind of tracked how those dynamics fluctuate over time, and they are fluctuating. And so we're essentially watching evolution in action. And at the moment, we don't have a clear, you know, winner, but it would be really interesting to see if, you know, one morph, you know, disappears or you know, so our polymorphisms maintained? And just watching that process in real time, like Robin said, across replicate island populations, where morphs are with different other morphs. It's a really amazing, just natural experiment.


    Marty Martin  1:09:11  

    Yeah, well, it's also intimidating, because that doesn't include the fly, right? And you could end up with some sort of crazy Rock Paper, Scissors, Hammer, Saw. Depending on how much diversity you're gonna have in this toolkit, it can get crazy quickly.


    Robin Tinghitella  1:09:26  

    Yeah, we've tried designing that experiment. We have one location where a former postdoc set up these like super cool mesocosms in the field that we can stock with different types of crickets, right? So mimicking what's happening in these different populations, and then let it play out and measure fitness. You know, look at who has babies, who has more babies who doesn't make it, who gets out competed? What happens if you let the fly in or don't let the fly in? So how does that parasitoid pressure impact things? And it's just so incredibly difficult to kind of imagine actually running that experiment with all of these different iterations that we need to think about, right? Like it is one thing when it's rock, paper and scissors, but then you throw in all these other tools, and it's like the number of mesocosms and people that you need just becomes outrageous and time


    Marty Martin  1:10:17  

    and time always time. Okay? One other question about this system, and then we want to, Cam has a big picture question. But I think we didn't address it. Do the females ever get infected? And how and when?


    Robin Tinghitella  1:10:32  

    Very rarely. So we think that females and silent males actually get infected about the same rate, and it seems to be because they're in the wrong place at the right time, right they're hanging out next to a singing male. So if you are attracted to a male who is attractive to that fly, they sometimes get parasitized because they are nearby, and the larvae sort of get sprayed on and around the male, like on the grass, so they can pick those up as they're walking in close proximity or mating with a male who was recently parasitized, those kinds of things.


    Marty Martin  1:11:06  

    And have you done experimental infections with the female? Are they much more susceptible? Are they more quickly to sort of have the parasites come out? Are they? Are they better or less protected without that evolutionary, at least direct evolutionary interaction?


    Dale Broder  1:11:19  

    I think we infected some females for our host switching study, Robin. And I think from the purpose of the parasitoid, it's just having enough nutrients, so I think the host is a host for them.


    Marty Martin  1:11:34  

    Yeah. Okay, so differences between male and female immune systems are inconsequential for this parasite. That's sort of what you're suggesting.


    Robin Tinghitella  1:11:42  

    Yeah, there's not a heck of a lot that a cricket can do, unfortunately for them, to you know, fight this particular thing off these, like, the size of the parasitoid fly maggots when they come out, is just wild, like you can, you know, artificially or naturally, infest a male and he'll have, you know, between one and four larvae inside of him, but they come out and they're, like, nearly the size of the cricket, right? Like everything is gone inside. So there are some components of the immune system that have responded, and, you know, vary across locations that have the fly and don't have the fly, but I haven't, like actually gone in and compared males and females, but like Dale said, the females do a very good job of carrying the parasitoids until they emerge. So,


    Marty Martin  1:12:32  

    Yeah okay, okay.


    Cameron Ghalambor  1:12:33  

    But there's sort of one kind of big question related to the parasitoid, I guess, that we haven't touched on, which is now that all of the crickets seem to be evolving away from the ancestral call, why haven't the flies gone extinct?


    Dale Broder  1:12:49  

    Yeah, I'll take this one, because I kind of led this work. But like you said, we were puzzled. Why are flies present? So we have at least three long studied populations where ancestral has not been there for about eight years. So why is the fly still there? We showed that these novel morphs are protected from the fly. And then actually Robin and I went to Kalaupapa, which is where Robin first discovered purring crickets. And we actually found in 2021 that our cricket had gone locally extinct. So our cricket was gone in this place, and we still found flies. So at this point, we had to explore the possibility that they have adopted alternative host species.


    Dale Broder  1:13:28  

    And so essentially, we searched and found three good candidates that were inhabiting kind of the same spaces as our cricket, and two of them are actually common pet store crickets that have been introduced, and we think they've pretty recently become established a common pet store cricket from North America and one from Europe. And then a third one, which is in kind of a small, very no one knows anything about it, this tiny cricket with two papers published about it in Hawaii. But interestingly, two of these species are about half the size of our cricket, so they're really small, and two of them have songs that are much higher in frequency than any known host song of this fly. So no one really thought these were viable hosts, but we had to explore this.


    Dale Broder  1:14:10  

    And so we recorded their songs, played them. And indeed, we did collect flies to all of these other host species songs in both long distance and short distance experiments. And in fact, some of the songs were even better at attracting flies than the ancestral Pacific field cricket song.


    Marty Martin  1:14:26  

    Wow.


    Dale Broder  1:14:27  

    Yeah. And so, of course, the next thing we had to demonstrate is, can they support a fly, especially these really tiny crickets. So we artificially infested, which is a little bit crue, by placing a larvae on the cricket allowing them to burrow inside. And indeed, all of them could host the fly and produce adult flies. So yeah, one thing that's kind of cool is we don't actually know when the fly started using these because the first documentation of the fly in Hawaii is 1989 and actually, all three of these species we know were there then. Or it could be that they recently shifted, because their favorite host, they can't detect them anymore. I don't know if we'll ever know the answer to that, but regardless, it's really interesting that this changes how we think about this system, because we've been thinking about it as this kind of tight evolutionary story of a single host and a single eavesdropper, and now we kind of have opportunity to think about this from a more community perspective and ask really different types of questions.


    Cameron Ghalambor  1:15:24  

     Yeah, super cool. So Marty alluded to the fact that I had some like philosophical questions. It's not really that philosophical, but one thing that we talked about earlier was just how cool it is to be able to study the early stages of these traits diverging, you know, the signals and preference and the morphology, etc. But, you know, in reading the papers, and in discussing the results, one term that comes up a lot is, is novelty. And so, you know, it was, it kind of made me think like, well, what's, what's the definition of novelty? Like, in this case, like the crickets obviously have a new signal. They've gone from like an ancestral call to something that is either purring or silent or, you know, some of the other morph types that we've described, but it's still variations, I think, on the same theme, as opposed to, you know, a cricket all of a sudden, you know, busting out the top hat and doing a little dance, which might be really different, and something that is completely outside of the


    Marty Martin  1:16:36  

    It might be really different? Where are these crickets you're seeing?


    Cameron Ghalambor  1:16:40  

    Thinking back to the those cartoons when I was a kid, where there was, like, the dancing frog that would only dance for the guy.


    Marty Martin  1:16:42  

    Oh the Warner Brothers frog, yeah.


    Cameron Ghalambor  1:16:52  

    And then when he whenever he wanted him to play for other people, he would just not do anything. But anyways, back to the question, I guess, yeah, so is there a distinction between novelty versus like, new or different, and is that, is there a continuum there or like? Where do we draw the line between saying like, something is like, novel versus something is new or different?


    Robin Tinghitella  1:17:16  

    Yeah, we doubt ourselves about this all the time, we struggled with this one a lot. And we've, you know, we've gone to other people and said that carefully, like, do we count? Is this actually novel? So if you go and do research on this, in the literature, there are hundreds of definitions of what counts as novel. You know, what someone has considered novel, how they defined it in their own paper, or how they want the field to think about novelty. And I think that- ah, man, yeah, Dale and I have had a lot of this philosophical conversation over the years. I think that part of what made us feel like we were dealing with something novel when we found purring was that it was sort of categorically different. So if you think about the variation that existed before in, you know, in the ancestral song, there's variation along multiple different sort of axes of, you know, sound in that song. And there's geographic variation and things that contribute to sort of where that variation comes from. And if you were to, like, draw a cloud that encompassed all of that variation, purring would fall way the heck outside of that cloud in multiple like all of the dimensions that we've measured, right? And there's a categorical difference in wing morphology that creates that sound. And each of these new things that we've called morphs, we think are in this in a similar sense, categorically different, because they completely expand the acoustic space that existed, right? Like they fall outside of that multi-dimensional acoustic space that was there 20 years ago. But I think like they are still the sort of directions that things are evolving are not along a single axis. And to us that that was more of a sort of hallmark of, yes, it is a different thing. It is a novel thing. But, you know, they're not doing it in a different way. Sometimes definitions of novelty are like they have to do the thing in some other way. These guys are still just rubbing their wings together. The mutations that are responsible for these changes are probably really simple, right? Like silence and purring seems to also sort of map to the chromosome, those are single gene differences, right? Like they're relatively simple evolutionary changes to have happen. Yeah, I don't know, Dale, if you have other thoughts about this?


    Dale Broder  1:19:46  

    Yeah, definitely. So like I said, we did kind of think about this with a group of people recently, and like, a perspective piece, and lots of different opinions, lots of different definitions, like Robin said. And so some people think it really is about the mechanism, that's the critical piece. Other people think it's about the function. So there's different ways people are thinking about it. So we try to think about all of that as we think about our system. And other people think about that if it's just kind of continuation of what's going on, like a tail of a bird gets longer and longer and longer and longer, that wouldn't be novelty, because it's kind of along the same axis of just getting longer and longer. And since we're having these complete-so each sound is produced by completely novel wing structures. So like a file mutation versus the loss or gain of a scraper versus completely shrinking a wing, versus changing the 3D structure of a wing, so the wings are so different that the mechanisms producing these are completely different. And like Robin said, almost like moving into unique morpho space. There's no way you could take an ancestral wing and get it to go to this place without, you know, taking away a scraper or modifying a file in a dramatic way. But something Robin and I've been thinking a lot about in the current grant we're working on, is we're kind of starting to see a shift towards more continuous variation, and that's changing how we're thinking about this whole system.


    Robin Tinghitella  1:21:03  

    Yeah. So as time goes on, these, you know, these morphs exist in populations where there are other different types. Females have really variable and dramatically different preferences. And what we're starting to see is evidence of things like hybridization between morphs, back crosses, expression of multiple different mutations on a single genetic background that then cause even other changes. So we went from, you know, this everybody singing to a dichotomous system for 15,  16, years, where you were singing or you were silent, and then more started to pop up. And now it's almost getting to the point where we have near continuous variation in song characteristics. And so, yeah, where do you go? Will we, you know, end up with getting to watch sort of evolutionary dead ends, where some of these things just don't work and they go locally extinct? Or are we going to end up with this, like, very, you know, kind of clean, continuous variation in song that can get molded by, you know, the preferences and the other selective pressures out there.


    Cameron Ghalambor  1:22:09  

    Yeah, that's super cool, that has all kinds of really interesting implications. And we could, I think we could. We could spend a lot more time talking about that.


    Marty Martin  1:22:37  

    I'm also holding my tongue on this novelty thing. There's a half an hour there for sure, too.


    Robin Tinghitella  1:22:43  

    I know every time we go in a room with other biologists, we want to ask them, you know, like, what do you think? Does it count? Like, what should we be talking about?


    Marty Martin  1:22:51  

    Well, one of my favorite conversations, just very briefly, I'm not going to say anything about it, but I'm going to ask you guys, or point you to somebody that has said super cool things. In my opinion, Karl Friston. And Cam knew I was going to say his name, because I always do. He's a neuroscientist, and he talks about surprise, so he's a theoretical physicist, and has a really nice quantitative way of describing novelty in the context of surprise. So it's sort of the departure of expectations of a system.


    Robin Tinghitella  1:23:19  

    Cool


    Marty Martin  1:23:20  

    And I mean, in your particular context, the females have expectations of the signals, and so there's potentially a nice parallel there.


    Robin Tinghitella  1:23:27  

    That's so cool.


    Marty Martin  1:23:28  

    I'm happy to send you the paper, if you want. We talked a lot of when we had him on the show. We talked a bunch about it, but definitely not in the context of crickets, and not, obviously, even in the context of novelty. But yeah, it's been pretty, pretty powerful for me, and thinking about what's reasonably called novelty.


    Marty Martin  1:23:56  

    Is there anything that we didn't prompt you to talk about with your plans for the system or cool discoveries that you made, you know, that we just never got to? Is there anything else that you want to say?


    Robin Tinghitella  1:24:38  

    One I think I can mention, because it's not accepted yet, so we're not embargoed on it yet. We have started, like I mentioned, looking at sort of the neural underpinnings of the responses that female crickets and female parasitoid flies have to these different songs. Things that are evolving. And we've just found our very first compelling piece of evidence that the flies might be fighting back through evolutionary time. So we've found some new, it seems, in Hawaii, at least, changes in what the tuning of the fly's ear looks like, so the frequencies that they are most attentive to and hear best, and those changes in fly hearing correspond with the dominant frequencies of two of our novel cricket songs, which is really cool. So we might have the beginnings of that sort of coevolutionary story where flies are actually able to do something about detecting novel males.


    Cameron Ghalambor  1:25:44  

    Wow.


    Marty Martin  1:25:44  

    Well, that's  amazing, because, you know, after you, Dale, you were just talking about the many other hosts that are infected, I wouldn't have expected that to have happened if they've got plenty of other choices. But it must be that your crickets are particularly tasty.


    Dale Broder  1:26:00  

    Yeah. Well, I guess another thing, I don't know if we even talk about it in our paper, Robin, but some of the alternative hosts have kind of higher frequency songs, and like purring is a higher frequency song. And so we're finding that in some of the higher frequencies, Hawaiian flies are more responsive to those frequencies compared to like mainland flies, so they might have evolved kind of more sensitivity in these ranges that would be beneficial for a number of hosts in Hawaii.


    Cameron Ghalambor  1:26:26  

    So I wonder if the flies are sort of like the crickets, in the sense that mutations that alter the tuning also maybe relatively common, at least on the time scale that you know you've been looking at, at the system. But again, you know, most of those mutants would be eliminated because they're maladaptive to the calls of the cricket, until the cricket changed their calls, and now all of a sudden, you might see really rapid evolution, you know, favoring these, these new flies.


    Robin Tinghitella  1:27:02  

    Yeah potentially, I think it's a totally reasonable hypothesis, and something we're really excited to, you know, have the privilege of following.


    Marty Martin  1:27:10  

    No shortage of work. This is good. This is a good system.


    Cameron Ghalambor  1:27:15  

    Yeah, really, really, really fascinating. Great stuff. Thanks so much for talking, taking time and talking to us about it all today.


    Marty Martin  1:27:24  

    Yeah, thank you


    Robin Tinghitella  1:27:25  

    Of course,


    Dale Broder  1:27:26  

    Thanks for inviting us. We love talking about what we do


    Marty Martin  1:27:43  

    Thank you for listening. If you like what you hear. Let us know via X, Facebook, Instagram, Tiktok, LinkedIn, wherever, or leave a review wherever you get your podcasts. And if you don't, we'd love to know that too. Write to us at info@bigbiology.org


    Cameron Ghalambor  1:27:54  

    Thanks also to Steve Lane, who manages the website, and Molly Magid for producing the episode.


    Marty Martin  1:27:55  

    Thanks also to Dayna de la Cruz and Carolyn Merriman for their social media work. Keating Shahmehri produces the cover art for each show.


    Cameron Ghalambor  1:28:06  

    Thanks to the College of Public Health at the University of South Florida, our patrons and donors and the National Science Foundation for support


    Marty Martin  1:28:14  

    Music on the episode is for Podington Bear and Tieren Costello.


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