 David Hansen and I are happy to be hosting Charlie O'Kelly who's here from Friday Harbor. He did his PhD at the University of Washington at Friday Harbor and after a number of stints in places like NSF and teaching in New Zealand and Australia and Canada. He's back at Friday Harbor. He's interested in biodiversity of protists and algae and has been starting to do more work on algal biofuels. Dave met him this summer while visiting Steve Stricke at Friday Harbor. They have a lot of common interests. We are starting a new program in biofuels funded through an NSF AppScore statewide program and so a lot of discussions in the last couple days on algal biofuels. Charlie was the NSF officer on my Pete grant funding when I was in grad school so we have a lot of connections in linkages. So he's going to talk today about tunnels in the history, algae that burrow into bore into carbon. They are not boring. Thank you Becky. Thank you Dave and I'm very very happy to have an opportunity to try not to bore you with some of the stuff that I do and some of the algae that I work with and it's my pleasure to give you an opportunity to introduce you to the various flavors of stuff that I do in my research world. I've been at this business for what 35 years now and the world of algae, protozoa and so forth never ceases to amaze me. As a matter of fact here's just a really quick snapshot of the kinds of stuff that I have been doing over the last several years. I really tell people that my place in biology is still to answer the very first question that ever comes to you when you were five years old and tripped over the tree root or something. What is it? You know so consequently I do an awful lot of looking at algae and protozoa and so forth. I grow them. I have a culture collection at the Friday Harbor Laboratories which I'm trying to make available for various classes of research, teaching, education, all this good stuff but involved in a lot of microscopy, a lot of DNA sequence stuff and increasingly getting involved in the ecology of some of these algae and especially the things that eat the algae and that's where I get into the biofuels type of game which I've been doing the last several years because once again what question is what is it? Why are we growing? You'd be surprised at the number of people who don't know what it is they're actually growing in their ponds. More to the point in my case what eats it? You know one of the principal elephants in the room for why we do not have algae biofuels in your gas tank right now is the crop disappears because of protozoa long before the crop actually gets to be harvested. So one of the things I'm doing right now in the biofuels realm is to ask what's eating the algae? Which algae are being eaten at what rate and how in hallelujah are we going to be able to figure out how to contain these things once we know what they are? But first what is it? And so a lot of my biofuels research right now is focused on that particular kind of question you know what are the contaminants what are they eating how fast what do we do about it? And even there I am coming into new questions of biodiversity for instance here is a freshwater protozoan that we have not yet formally named called retiaryllus because it throws a net around a prey cell and devours it. It's actually a very efficient eater of hematococcus which is where you get Estazanthin from the carotenoid that everybody's fond of right now and the Roman gladiator that used to catch prey by throwing a net around it was the retiaryllus little net gladiator and our preliminary analysis of where this thing exists in nature is that we don't know it's not closely related to anything are we dealing with a completely new lineage of eukaryotes. I think we finally just managed to send the samples to a lab this week so that we can get genome analysis on this puppy and see just what it is. So there in you know Twitter version is my career what is it what good is it right and that brings me to the topic of today and that is this whole group of algae that actually burrow into limestone calcium carbonate. How many of you have actually heard of such a thing before? I have a few of you okay here is a very attractive example these are two solitary corals that are living in shallow water in a fjord in Chile and their stalks have calcium carbonate like all other corals called clomial and solitary. Calcium carbonate is white correct well you've got a green alga on the left and a pink alga on the right and they are actually in the carbonate. You ran your fingernail across you will not get any color because everything's inside and so this is perhaps the most attractive picture I could come up with published in the literature about five years ago showing you just what a carbonate boring alga is and where it lives and they're pretty much everywhere if there is calcium carbonate in the sea water in fresh water in a bridge abutment that's been around long enough you will find these carbonate boring algae and the habitat has been around forever. There are fossils from the progerozoic era when carbonates were formed by chemical processes showing cyanobacteria forming boreholes in that calcium carbonate and of course since the beginning of the Paleozoic and later eras you have any number of animals especially in marine but also in fresh water environments that produce biogenic calcium carbonate and of course limestones and other sorts of things like concrete provide additional substrate for these guys to exist and it turns out and in all of these things we will find these carbonate boring algae and even more interesting and this is where the tunnels into history come to play when you look at the fossil traces the boreholes made by these carbonate boring algae from 400 million years ago you find traces that are dead ringers for things that are alive today 400 million years ago was a period going to carbonate sea you know very very alkaline very very chalk weight-based ocean which generated a large radiation of calcifying marine invertebrates and looks right at the moment like our current biodiversity of carbonate boring algae at least in the sea co-rated with them and those lineages have survived all the extinction since then and persists to the present day so we are looking at what probably could be considered an order of this year carbonate boring floor here's what they look like in life on the left you take you know I used to joke that in order to actually see these things right you have to take a piece of coral rock right and in a sledgehammer because in order to get a microscope to see through a piece of coral rock you really need to make a small sliver so you know so I have people in the laboratory with sledgehammer it's like this the one on the right is the same organism but is a mold you take a piece of calcium carbonate with boreholes in it you pour liquid plastic into it slowly and then harden the plastic and dissolve away the calcium carbonate you end up with a mold of the borehole which of course mimics the morphology of the original algae so there's the living one on the left and the cast on the right and it's from these casts that we get much of our information on the morphology of these carbonate boring algae and indeed this is how we know that ancient or division algae in this habitat look almost exactly like the moderns furthermore people are taking habitats rich in carbonate boring algae carbonate like coral reefs and doing what amount to depth profiles for the different classes of borehole morphologies and being able to say all right this group is in shallow water this group is in deeper water and from this information being able to go to fossils and saying all right if we see this group of fossils we are dealing with a tropical reef shallow water environment if we see this group we're dealing with a tropical reef deep water environment and consequently you know geologists are using this kind of information to help date and characterize and paleo environmental terms the habitats in which these carbonate boring algae are being found so it becomes really important from the point of view of somebody doing biodiversity to be able to say here are here are a bunch of traces we give them names because say their species how many species are here really right there they're by their buying is the gist of my talk this afternoon now what does this mean to the price of house in other words okay it was a curious does it play a role in the environment well it turns out that if you're a parrotfish you care big time parrotfish you probably know our tropical reef fish that actually bite into coral rock why would they do something so dumb you know coral rock right it's okay it's an organic material but it's not what we mean by an organic material well the coral rock has lots of life and in this particular case literally in it because the primary productivity of that coral rock 40% of it is according to many estimates the actual wars so the parrotfish knows exactly what it's doing by biting into this rock and getting its nutrients from the things that are in as well as on that rock so it's a big deal there's also a commercial deal in that how many of you know about sushi wrapper right seaweed right you know that perhaps the sushi wrapper is a paper made out of a bladed seaweed which used to be called porphyra it's now called pyropia because we those of us who talk about what is it have just renamed the commercial porphyra right taxonomy strikes again that alga has a biphasic life history the blade is where you get the sushi wrapper from grows up on the nets form spores the spores germinate to form these little tiny filaments and they burrow into calcium carbonate and preserve of reservoir for spores that will be eventually be released and give you back the blades this phase lasts a few weeks this phase is forever and farmers will take shells or oyster shells with these filaments in them they know exactly what time of year those films will form spores so we'll have huge arrays of oyster shells and big large seawater tanks with a green house in a green house and under cold temperatures and short days the spores will be released on mass they drag ropes through the surface of the water put the ropes in the freezer and at the appropriate time put the ropes out on the farm the blades appear six weeks later you have the raw material for sushi wrapper for nori so being able to understand that you have a carbonate boring alga in the life history of this billion-dollar industry plant and knowing how to manage that is critical it was not even possible until 1947 when a woman by the name of Kathleen Drew Baker first described this life history the only woman in Japan to have a statue in her honor we're not done yet with what it matters because remember we are talking about calcium carbon we have calc carbon as a major player in greenhouse gases a major player in ocean acidification it's been estimated that the amount of carbon released into the biosphere by these carbonate boring algae is about one-fifth of the amount that we put out every year by burning fossil fuels so in a planet that is growing warmer and in an ocean that is growing more acidic one can predict that the activity of these puppies is getting larger releasing more carbonate and carbon into the atmosphere and into the biosphere can you say positive feedback loop right so if you're reading the IPCC report about having to keep all the fossil fuels in the ground in order to prevent the oceans from rising six feet in the next century what are we going to do about these algae right that's limestone is fossil carbon just like petroleum is or coal so maybe I'm overstating the case but we don't know here is a potentially significant source of loading about which we know next to nothing we don't even know how many species there are and whether those species contribute differentially for instance are there different species in the tropics in intemperate zones all we have are these boreholes and the boreholes suggest to us that these things are globally distributed come back to my principle question what is it and developing some indication that maybe even here we don't know what it is yet and so that's where I come up with the idea of trying to get at this principal basic what is it question I wish to know it's part of doing this investigation by the way I'm not by myself I'll just say me for the now but tell you about some of the other people later what is the global biodiversity and a hypothesis is that there is a lot more species diversity than we know about and that there are a lot of cryptic species hidden with any particular morphology and in terms of their performance in the environment are all carbonate boring algae created equal and hypotheses include that species do differ from each other in environmental tolerances cold water bottle water you know that species distributions of that reflect this kind of differentiation of physiology in other words no species is globally distributed even though the fossil people say you are and that you can sometimes make at least a little bit of correlation to morphology to species and to environment once you actually learn how to figure out which morphology is actually matter so that's what we're up to and this is how we do it you take a sample this is a mollusk shell from the vicinity of the same one islands you see is pink all that pink are carbonate boring algae in the shell there's probably at least three species in there so we take specimens take a chip you know I literally go in with a razor blade and the chips are like you know one millimeter you know tiny you don't want much because it's not for a lot other stuff there too you don't want to take the and put those into spot plate wells we have seawater put them in the incubator wait three months they don't grow very fast and you collect these cells that actually come out of these chips then and this is essential you take the isolated algae and you give them back to calcium carbonate and see if they bore you because believe me there's an awful lot of pretenders out there you might get 15 species of algae out of one of these chips only one of them is your target the rest of them won't bore into calcium carbonate so now by you only keep the ones that will do the borals then you can get at the microscopy of live and cast casts and start getting DNA sequences and with all of this you can start asking some of those key questions testing those hypotheses I put forward for you two case studies we'll illustrate where we're going with this one of these is perhaps one of the most commonly reported sign of bacteria amongst the carbonate borne algae that's originally described as the species Plectonema terraprans that was done by a couple of French fellows Borne and Lohot in 1889 they were the first to actually catalog carbonate boring algae for both freshwater and marine habitats so these two guys were the starting point they published a 60 page paper basically documenting all of their discoveries along these lines over five year period so sign of bacteria these are the pink ones in my solitary corals and they are basically filamentous non-heterocystis cyanobacteria that exhibits something called false branching we basically know very very a little more about them in the cast they look like this and for those of you who aren't familiar with fossil terminology a cast does not have the same name as a biological species it's got it's a very own nomenclature so that when you see a cast like this you will use this name Scolesia phyllosa so this is the name of the cast and this is the name of the biological species our question one of the many is this one species or many we don't know this is what we're trying to find out so can we grow them if so we'll make our own to carbonate and how many of us strain how many species do we get out of the ones that pass those first two tests one species in the literature you want biological species one ikno species that's the name for that fossil borehole and ikno species but there was a paper published a year and a half ago which they looked at algae from field collected samples to DNA samples and they didn't get one species they got six six different entities in one two well actually yep one group an isolated another group of all of these fit the Plectonema or the Scolesia phyllosa morphotype lots of different biological species but we don't have the organisms so we don't really know if any of these are truly boring into the carbonate or just sitting on the surface what we do know there's a lot more than meets the eye so here are samples from the collections we made two different morphotypes one with very short cells one with very long cells from temperate waters around San Juan island in the Washington state and from Massachusetts we got 11 of these strains two like this nine like that low and behold the two with the short cells were not burrowing into calcium carbonate the nine with the longer cells work so already we know two species but we reject one because it is not a bore we do the DNA in 16 s uh reversal RNA genes and all gores cluster together they are not identical as in they are not a hundred percent synonymous but according to the rules of bacteriological nomenclature they are close enough to be considered one species and there's one environmental sequence from the Australian great barrier reef that falls in the same cluster here are the two species that do not bore into carbonate they are different from each other and different from the carbonate boring group so that small difference in morphology turns out to be a significant difference in the DNA sequence data we have at least two species possibly three and only one of them is a carbonate bore so the carbonate boring strains form a clade non-carbonate boring strains do not belong to it and both of those clades not shown on this graph but it's I alluded to it here they're unrelated to the clades that were found in those intertidal carbonates by the colleague in 2012 so now instead of there being like six groups we have seven and this one we confirm is carbonate boring we don't know about the other six yet because we don't have samples now this taxon had been known as plectonema it also sometimes got known by the genus named lektolingbia well there's plectonema lektolingbia down here totally unrelated to these things up here and if I expended the tree down through the flooring into the next story you would find the species of plectonema and lektolingbia distributed all through this tree these genera are polyfiletic and none either of them includes the carbonate just demonstrated carbonate boring cyanobacteria that we found in washington and massachusetts can we grow them yes well they burrow into carbonate well we found a group that did we've got one species but that's one species different from all the other ones that have been found so far and because it is from temperate zone waters which is very close to the waters around lecroixique in france which is the type locality for this this might be the bona fide plectonema terra brands for which we'll need a new genus so we still have a lot of unanswered questions about the things that while this same morphology believe in intertidal tropical habitats subtitle tropical habitats they may be the same they may be different at least now we have a reference point that's case study one case study two is perhaps the second most abundant representative of the carbonate boring flora in tropical and temperate marine waters and unlike the first one which is a pink cyanobacterium this one is a green green it's a number of the chlorophytes and it's referred to by the name of osteobium kiketii kiket was a collector of seaweeds in the 19th century and osteo osteobium comes from austria which is one of the genera of edible oysters obviously the thing was first found in oyster shells it's a very unusual organism this is the one that has the dates back at least to the orificium it's one of the oldest fossil tax that we have if you know about green algal morphology and i say the word syphonus they might need something to you this albeda does not have cross walls all of this network is a single ununpeded tube like things like codium and halamida eudotia and some of those others calarpa some of the largest tropical green seaweeds osteobium has that same morphology it just does it and i micro alga instead of a proper macro fight seaweed once again it is considered to be universally distributed here is its single ikno species the fossil name and here is the single biological species name that has been assigned to all of these things you find them in the um sub Antarctic and arctic waters you find them in the tropics and everywhere in between and they are famous for their association with living corals here is the cast of one of these just to give you an idea of what it will look like same questions by and large as one we asked with Plectonema will they burrow into calcium carbonate it was in fact determined group that demonstrated this for species in the north sea and how many species among the things that we can demonstrate will burrow in calcium carbonate i showed you that the literature says one but three years ago there was a study published by a group that went to one location in the Gulf of Ilaat collected a series of corals from that one location came back did DNA surveys from it and found at least seven very distinct taxon groups that they assigned to osprey opi so there is evidence from that perspective that australian is not a single species but even in one single location is a bunch of species these are environmental sequences so we really do not yet know how to interpret some of the branch links in some of these clusters whether those are actual different sequences or whether that represents some insecurity in the actual sequences that were generated the authors did not choose to present us with the actual sequence data so we don't know so we don't know whether group a actually contains one species or four so there's an awful lot of there's something going on here but no cultures we don't know to which organism these sequences belong and a further question that was a tropical site osteoviums are found in temperate zones the type specimen is from a temperate ocean are the temperate species the same as the tropical ones or are they different so what we did we went back and got some more rocks and we got some more slivers and waited three months for things to grow up and collected 28 strains most of them from washington area we did get one from massachusetts we had a couple of others they died oh well so all of them were in a carbonate which was very fortunate so we were very confident that we had you know representatives of osteoporosis and here is one from the salish sea and one from the salish see that they look the same or different now different yeah that's how that's that's what we thought this one actually has what i call cables so there are some main axes that actually have a little bit of differentiation as opposed to here where the main axes are very much the same diameter as the lab rooms about the only difference that we found among all of these 28 strains some of them have cables some of them don't everything else they look exactly alike one species one morphological species that's what the DNA tells us there are at least eight and i put on the tree a series of taxa in the uh chowler palies or bryocidalis the distances in many of these taxon groups among these temperate osteoleumes are at least as great as those that separate genera in the rest of the bryocidalis it makes sense after all these things have fossil records dating back at least to the orubitium it suggests to me that there has been a considerable amount of cryptic evolution leading to several different species that have formed within this one morphological type and this happens to be a tree based on ribulosevis phosphate carboxylase oxygen a sequences those based on tough a are exactly kind of and the bootstraps are pretty good add to this tree the sequences found in the Gulf of a lot and there is exactly zero conference the tropical entities and the temperate entities are clearly distinct this tree is based on short sequences because the published sequences are very short and that's why i didn't put very many bootstrap values on here because the sequences are too short for a robust tree generation however no matter how you do the analysis these same groups appear the order might shuffle but the same groups are retained and we had a further cross check on that because we did get samples from Hawaii and we have samples with full length sequences from the Philippines published by a colleague the same relationship obtains no group of algae that we isolated from temperate zone waters correlates with any representative of austriolium that we've gotten from tropical waters as it turns out the tropical things we've gotten from white don't match the things that goodner hawk and fine found in the Gulf of a lot so it looks like we haven't even come close to saturating the biodiversity curve i wouldn't expect to with data from four sites but it remember we are dealing with things that people have said represents one species and we've also come up with things like this which are carbonate boring members of this borax adelian lineage that are not austriolium they belong to some other group we don't have any clue yet what those are so answering our questions yep austriolium will indeed burrow into calcium carbonate the ones that we've got and instead of having one species and one in the genus we have at least 11 and at least three more things that are somewhere else in the perhaps a day of a huge amount of cryptic biodiversity in this series of algae and it looks to me as if we have very distinct flores there are as a temperate flora of austriolium and a tropical flora of austriolium if you are taxonomist you're saying which one of these is austriolium caquetti i will at the moment until we go to france and go to the quasi collect oyster shells culture from there and determine which of any of the algae there matches sequences from where we've got we won't know what a spirobin caquetti i is in the meantime we have all these other taxes that if we were going to name them i'm not really nervous about that yet but if we're going to name them it all need different names and the fossil people are still screaming at me because they basically say well all of these are it no reticulina elegance how are we going to make any distinguishing marks about them when i say i don't know maybe there are if you look carefully at the fossils you might say some that have capils and some that don't and that might give you some indication i've been trying to do more for metrics on these things and i've not come up with any kind of statistically significant this determination amongst all the castes we've got so so those are my two key stays as it turns out there are several different groups of these carbonate boring algae which i've not yet touched on have touched on plectanema have touched on austriolium we're beginning to get a bit of a picture and my colleagues are adding a lot more tropical samples to this so that we hope in another few months years hopefully not years we will have a global phylogeny for things placed in this austriolium we also have another group of cyanobacteria which are heterocyst forming forms called mastoclecholius which are actually very very fast growing relatively fast growing cyanobacteria that form and once again here's a case in which we have a single species but we have evidence already that there are probably three or four of them and the thing that has been described as being the epitite for this probably will not match the actual type which is from cold water in sweet we're still trying to get samples from sweet and so that we can test that hypothesis and then another very abundant green something called pheophilid and droides we have a lot of data on that that's again a thing that's very very worldwide distributed and in that case we know that there are at least five different species all encompassed within this morphology and then two groups we haven't talked about mainly because the studies are still very much in their infancy one is the whole porphyra complex there are probably hundreds of species in the vangiles porphyra vangia pyropia vildemania i can't list them all i don't my memory won't hold them all each of them has an alternate stage and and that is a carbonic boring alternate stage and so that's going to require a massive grant maybe we get the japanese farmers to fund a grant to try and work out what the different carbonic boring stages of all these look like in the field we are in fact trying to work on this now for pyropia in hawaii where people are interested in warm water pyropias that can be turned into sushi wrapper and we have a study in the progress which tells us that instead of there being one species of pyropia in hawaii there are five and maybe more and lots more cryptic diversity and then a whole group of cyanobacteria that sort of are classified as a pseudo filamentous these are the ones that predominate in intertidal zones in terrestrial concretes and things like that probably hundreds of species there too and sorting that out might take longer than i've got on this planet but there they are this this is the basically the biodiversity checklist for the scientific carbonate boring algae in marine environments and pretty much takes care of the freshwater and terrestrial ones too especially these high elastolentina types of things so lots of work yet to be done but i think that the bottom line remains the bottom line and that is we still you know we're asking the first question what is it we don't know we're finding out that many of these things have many cryptic species in case within a particular morphology we may never get to the point of being able to tell the paleobiological community how to divide up all the cryptic species from the morphotypes they're seeing in fossil strata and that will complicate matters of trying to reconstruct paleo environments using these bore fossils and that's my story i do obviously have a whole lot of people that have been involved with this i'm especially thankful for undergraduates like Geneva, Jeff, Angela, and Lauren all of whom have contributed either DNA work or cast work to this stuff people that's one of the great benefits of working in the Friday Harbor Labs you get a lot of people coming through they have a short period of time they get enthused about this kind of stuff we have the scanning electron microscope there we have all the DNA stuff and they come in they work for a few months Geneva i actually had for 18 months and we're able to do a whole bunch of solid research contributing to this particular study colleagues from kawaii and from France Aline was in both Hawaii and France and scored some money from the French government to help get samples and process some of the DNA sequences and so forth and so on and the Washington State Department of Natural Resources funded the boat trips from which we got samples from the Salish Sea and then a whole lot of other people who have helped along the way you know this every but every project there's like one name on it actually should have about 40 but it's the same with me and it was everyone else and a lot of this we remember a scientist from the University of Hawaii who was interested in the biogeochemistry of these boring algae who was the supervisor for Aline Triple A who was very very helpful with us visited the labs started to work on the physiological issues and then tragically died of a heart attack at the beginning of the year so we in the morning okay that's my story I'm sticking to it thank you that was what we were going to start doing with marlin before he passed away um I can tell you that these things generally grow quite slowly um but the Saras rates are concerned we weren't interested in that because of the nature of the study we were asking yes no questions but by and large I can tell you that if you take a um piece of clamshell besides your thumbnail and put some algae that will bore into it into that dish the clamshell will become fully covered in like four months so they do not grow at all rapidly and the whole question of rates some of the new grow more fast faster than others and then carbonate the solution rates will be proportional to that so the capacity is the capacity to dissolve the carbonate is that based on an enzyme encoded by the algae or is it maybe mediated by endosymbiotics or other like I guess associations with microbial communities inside the algae I'm just wondering if like your your protocol right you just put the chip into the well and then you wait for months and months until you do your texture functional tests are these particular you know isolates able to dissolve the carbonate is it possible that it is due to some other symbiotics with all their microorganisms you lose that capacity because endosymbiotics have left their party or am I just bringing that this is absolutely we don't know there is exactly one paper on the subject published by the fellow in Garcia if you can't even come up with his name anymore uh Farron Garcia Pichel who was at Arizona State yeah and he has determined that the boron burrowing function in mastococcolius testarum is based on a calcium-dependent ATPase yeah so and that calcium-dependent ATPase requires photosynthesis to function because it doesn't work in the dark and that is literally as far as we've got however the mastococcolius was in azenic culture so there were no other bacteria around so that least in that one experimental system it was the sound of bacterium and no partners that were dealing with it and I'm pretty convinced that that will be the explanation but we have a long long way to go before we have a fuller understanding of how the boring mechanism occurs so there's no like phylogemetric studies on that particular ATPase to compare ATPase in a different unless Farron has it and hasn't published it yet yeah well there you go well uh this is the kind of thing that we could be doing with the genomic stuff um I am presuming that hydrogen pumps are involved somehow because I would have to it's always tip growth for these things and also some lateral growth as boreholes expand but mostly it's tip growth and so if I were doing membrane physiology on this sort of thing I might expect to be have a concentration of hydrogen pumps at the domains where the boreholes are being formed um but that's sort of like you know pying the sky hypothesis at this point and I think we're a long way away from getting to the point where we can test the genomic approaches maybe we get closer faster but entirely possible um I don't know enough to be able to that's um good question and I don't I simply don't know the answer um it's possible there's enough phylogenetic distance between these things in the fungi to suggest that there may be something completely different in um some of Farin's inhibitor studies where he talked about the inhibit inhibiting the calcium-dependent ATPases in some of the algae he tested other than mastic acolias you got function in some cases and not function in others so that's a preliminary indication that there may be multiple mechanisms involved but I quite literally don't know anymore I favor the latter hypothesis but again um data are pretty lacking um my explanation which is a just so story for the ecologists out here is that the carbonate serves as a hiding place it's a matter of if you are a small alga it's growing very slowly in a world full of gastropods your lunch but if you are embedded in the calcium carbonate not only are you not lunch but the snails are cleaning your windows for you yeah because you've got things if oftentimes you will get that you will get a gastropod with a strong enough radula to rasp off the top half millimeter but the layers are frequently one to two millimeters deep and so they just simply you know get some filaments growing further down of the carbonate and others spreading out so they take advantage of any new surface that they can find so I think that the explanation for that is almost entirely ecological and any benefit of carbon from the dissolution may be well gravy but test it see so if they're boring from the tip and they need both synthesis to bore how they deal with attenuation of light because they mostly grow laterally very rarely will they grow down and I would assume that there is a certain level but low which they can't reach before attenuation does become fatal so um the what happens obviously is that as you bore away the calcium carbonate is eroded away and so that you if you break one of these things open you'll find that if the shell is that thick the algae are here and that's a pretty constant relationship for any particular environment so so that I think that's that's how they deal with attenuation they don't they they avoid it whenever possible you deal with um depth of sampling for your your you know assessing biodiversity of these different locations it seems really possible that with your limited samples I mean and the amount of time and resources it takes for you to test these different questions you could be missing out on a lot of the diversity that is actually at those locations so then to try and you know make global uh yeah us or global conclusions about diversity in these experiences is kind of hard you are correct and in fact I would argue that the methods that we've used here are inadequate and will always be inadequate for a global biodiversity study I reckon that this kind of study is doing two things one since giving us a starting point and the other is that it's giving us raw materials that we can do other experiments on such as investigating rolled carbon such as investigating attenuation such as investigating what actually is the mechanism for boring without them we don't have any tools for that but one way I can envision proceeding has been used some of the data we've got from these culture studies as sort of like reference points for like global biodiversity genomic work and so you make a lot of samples and you can then probe for the total inventory of austriobiums or hyellas or whatever if you don't have that starting point I argue then you'll get a lot of these sequences but you'll really have any clue who belongs to what um and especially with the sign of bacteria where we saw that the trees are really quite um you know the trees are really all over the place then you've got um you have a very very hard time interpreting your data with some of these culture-based studies you have some reference points and then your genomic studies will be a little bit more informed but I do think that it's that way that will get the diversity not this way so do you have a do you know if it's is this um the boring morphologies uh the primary or or one of every few methods of assessing sort of past sea depths or origin depths I mean is this losing this data do we really just have no idea of the depths of the oceans or shallow seas and the no there are certainly several other techniques this happens to be a convenient one for the people who do this kind of stuff but I would argue that people have other ways of going about this but and they will find them if they're not already using them in terms of like isotope dating and isotope characterizations and things like that this just happens to be handy and people have argued that yes it is a possibility I'm arguing that we don't lose all the data but we just have to be as usual very careful about over interpretation so and I also have to be honest with you this is not a really heavy-duty component of paleo ecological assessment it's the kind of a tiny backwater and I'm not giving anybody any evidence to make anything more than a tiny backwater yes Charlie you mentioned all these species in molestine shells what about anything outside of mollusks that are born into just rocks I know there's bandias that attached to rocks are they boring into the surface of these rocks or is there something special about the calcium carbonate in these molestine shells well I hope I didn't give you the impression there was something special about molestine shells they just happen to be available as far as these boring algae are concerned if it's calcium carbonate it's so it's a subject for boring calcium you know for an experimental point of view the mollusk shells especially in areas where you don't have coral are a convenient substrate no way it's going to be counting how many mollusks I'm like coral animals right you sample a coral animal in Hawaii you got 14 bureaucrats trying to record every piece you've taken mollusk shells I don't care so much so they and since calcium carbonate is calcium carbonate you can go ahead and make use of that substrate the one that really gets to me is literally coral reef or other kind of limestone rock ancient stuff which will also have these things in so if it's carbonate it'll get bored into so don't please take away that mollusk shells or anything special they're just convenient yeah the way I think you have another seminar coming up in a couple minutes okay how long functionally how long do these take to establish in terms of boring patterns because functionally could theoretically if you have a higher concentration of these algae understand they're very slow growing but could you possibly re-establish some coral reefs with enough algae to I'm trying to think of functional ways to apply this information well I think one of the big deals is that we're talking about degradation rather than assembly so certainly when you have a large mass of these algae some of these especially the faeophila they're the first recruits to any new carbonate surface that comes available so if you have a lot of those algae around and a lot of bare rock then you will get considerable amounts of these dissolutions and we don't have a really good idea of the succession patterns what we do know is that there's a lot of diversity in shallow water less diversity deeper water my point about the ocean acidification and global warming thing is that since there are a lot of these things out there remember 40 percent of the primary productivity of coral reef rock if you add temperature if you lower pH you're probably going to degrade these rocks faster than they already are being degraded and we need better measurements and I would argue that knowing what species you're dealing with and having a better idea eventually of which species are doing most degradation fastest will help add that piece of information to the global carbon budget picture I guess you're getting these from different types of water you know temperature, pH, is that not? But when you culture them you allow is it maintaining those natural conditions or giving them something that they all like? Yeah I'm giving them something that they all like because I didn't have the resources to try and do several different kinds of conditions so the only thing I think they're very tolerant sorry does that mean that they're very tolerant to any kind of well? I didn't make the point very strongly but in general if I isolate it from temperate waters it will tend not to grow at tropical temperatures if I isolate it from tropical waters it will tend not to grow at temperature so at 23 degrees something isolated in the tropics won't grow at 15 something isolated from the temperate zone won't grow at 23 so we have a preliminary indication that these sorts of things do matter we didn't get there that was what Marlon and Nui were going to do before he passed away so so yeah but all those are open lots lots to do