Echinodontium ballouii - Mushroom, the Journal of Wild Mushrooming

A Real American Ivory-billed Woodpecker:A long-extinct mushroom emerges in the dead of winterEchinodontium ballouii: the beast in the wild, covered with snow. Photo by Bill Neill.Fall 2007 Issue 97, Vol. 25, No. 4

Echinodontium ballouii: From Eyeballs to DNA, by Leon ShernoffFungal Classification: In theBeginningWay back in 1751, the Swedishbotanist Carol Linnaeus had great successwith a new system for classifyingplants – the system that is the basis ofthe one that we use today. Much ofLinnaeus’ success is often credited tohis invention of binomial nomenclature– the idea that each plant shouldbe referred to be a combination of agenus and species name. However, healso got a lot of mileage by categorizingplants according to how they rantheir sex lives – not just flower structure,as you might expect, but whethereach flower had both male and femaleparts or whether they were separated,and whether plants with separate maleand female flowers would bear themon the same plant or whether therewere separate male and female plants.He actually described these criteria invery provocative terms: as whether themale and female parts were in the samebed or the same marriage chamber ornot, and he was roundly attacked bythe fundamentalists of his time. Heusually responded by naming smallweeds after them.Any successful system demandsexpansion, of course, and Linnaeuswas soon extending his system fromthe green plants that he was originallyinterested in to fungi, lichens and evenanimals. Linnaeus really knew hisplants, and his focus on sexual systemsworked really well for them. Itworked much less well for fungi, buthe was pretty clueless about them (hisfirst classification of fungi includedonly about a hundred altogether) soit didn’t bother him that much. Bythat time, people were pretty sure thatmushrooms reproduced by means ofspores, so Linnaeus made a fungus’spore-bearing surface the basis of itsclassification and gave this surface thetypically provocative name of ‘hymenium.’All mushrooms that bore theirspores on gills went into the genusAgaricus. All mushrooms with a honeycombedhymenium on a stalk wentinto the genus Phallus (another typicalLinnaeus name), which sounds familiaruntil you realize that the morelswent in there, too. So the first name forthe morel under Linnaeus’ system wasPhallus esculenta, the edible phallus;no wonder the fundamentalists weredisturbed.Luckily, help was on the way. Inthe 1790s, Christian Hendrik Persoonstarted shaking things up a bit, andin 1821 Elias Magnus Fries redid theclassification of fungi in a major way.He separated the morels from the stinkhornsand liberated the chanterellesfrom the strange spot that Linnaeushad stuck them. Linnaeus had createdthe genus Merulius for fungi with anirregularly wrinkled hymenium – whenmany of you learned your mushrooms,you learned Merulius as a group ofwrinkly jelly-like things that lie flat(resupinate) on wood. Because chanterellesalso have a wrinkly hymenium,Linnaeus decided that they weresomehow closely related and it wasup to Fries to separate them. Fries alsoseparated the polypores from the boletes– Linnaeus had put all the “poredfungi” together – and Fries later supportedthe division of polypores intofurther categories like Fomes (for thehard, woody, perennial conks), using“polypores” as a larger-scale categorythat included all these new genera.Fries brought a lot of common sense tothe classification of fungi. But he wasstill working within the basic outlinesof Linnaeus’ system, just with furtherrefinements.So a hundred years ago, when Echinodontiumballouii was discovered,fungi were still primarily categorizedaccording to the structure of their hymenium– whether it was composedof gills, pores, spines, or somethingharder to categorize. Within thesebroad categories, mushrooms werethen broken down into genera on thebasis of whether they had a cap, a stem,or whether they were just resupinate(lying flat on what they were growingon).When Ballou sent Howard JamesBanker at the New York BotanicalGarden his first specimens of Echinodontiumballouii, Banker placed it inthe genus Steccherinum. Steccherinumwas a sort of catch-all genus for fungiwith a spiny hymenium and a capbut no stem. Other people disagreedwith Banker and placed Ballou’s findin other genera, and it didn’t end upin Echinodontium until 1964, whenHenry Louis Gross took a comprehensivelook at the genus Echinodontiumand pointed out how closely it fit in.The genus is based on E. tinctorium,which has some unique features – thethick cluster of spore-bearing spines,and the brightly-colored flesh – so ittook the work of Henry Louis Grossto point out that the other Echinodontiums– most of them less spiny andmore dully colored – were so similarto it microscopically.Morphology, then and nowWhat features merit inclusion ina genus? The mycological consensuson this has changed drastically in thelast fifty years. Linnaeus started us offwith an approach based on morphology:in broad terms, simply how themushroom is shaped. His approachwas very much based on the nakedeye – and, I might add, what the nakedeye can taken in quickly. My favoriteexample is an example from his firstclassification of the fungi: Agaricus(Russula) rubra, where Russula is asubgenus that was later raised to genuslevel. Yes, rubra means red, andhis treatment amounts to giving allred-capped Russulas, everywhere, thesame name. In fact, there are probablyalmost a hundred red-capped RussulasPage2Fall 2007, Mushroom the Journal

in North America alone, and probablya dozen just in Linnaeus’ native Sweden.But to Linnaeus, they were all justred Russulas, Russula rubra. 1Persoon and Fries brought moresophistication to their classificationsystems. and during their lifetime moreand more microscopic features wereincluded in descriptions of fungi – extendingthe concept of morphology tothe microscopic level. Fries also triedto use developmental features as oneof the criteria for a genus. He didn’tdo a very good job of it, but by themid-20th century, some mycologistsadvocated taking microscopic anddevelopmental features into accountin fungal classification.When Gross monographed thegenus Echinodontium in 1964, he didsomething that was at that time quite“modern” and daring: he included thetwo species of the white crusty genusLaurilia. Why? There are a variety ofreasons. The illustrations below showsa microscopic view of the hymeniumof Echinodontium tinctorium and Lauriliasulcata, the species upon whichboth genera are based. These illustrationsare taken from Gross’ paper. BothMicroscopic view of Bill and Larry’s Echinodontium ballouii specimen,showing an encrusted thick-walled cystidium (long cell in the middle) . Thethe spores with slight bumpy amyloid ornamentation didn’t photograph aswell,fungi have thick-walled cystidia thatare encrusted with crystals. Cystidiaare cells that protrude from the mainbody of the fungus out into the hymenium.Since they seem to evolve quiterapidly, such a strong similarity incystidia has been considered a markerof evolutionary relatedness. The hyphaewith the thickened walls, by theway, are what stiffen the fruiting bodyand give it its woody (E. tinctorium)or tough (L. sulcata) texture. GrossThe hymenium of Echinodontium tinctorium and Echinodontium (= Laurilia) sulcatum, from Gross’ 1964 paper.In each illustration, a and b are thick-walled, crystal-encrusted end-cells, a protruding as cystidia and b gettingready to. C are the basidia, the spore-bearing cells of the hymenium, and d are the spores themselves, coveredin tiny amyloid bumps.1True, to many of us they’re also all just red Russulas! But we’re not posing as scientists when we feel that way.Fall 2007, Mushroom the Journal Page 3

also emphasized a developmentalfeature: that these fungi are perennial,and form each year’s new hymeniumby growing it directly on top of lastyear’s. If you cross-section each ofthese mushrooms, you’ll see a set ofgrowth layers outlined in the flesh, likethe growth rings on a tree, each parallelto the one before, outlining the sameset of teeth, or (in the case of Laurilia)the same set of random bumps andother irregularities. Gross noted thatthe spore-bearing spines of these fungithus get thicker each year, and in thecase of E. tinctorium the spines eventuallygrow into one another, startingfrom the base outward. The spines inthe specimen of E. tinctorium picturedhere seem to have grown together quitea bit. But, Gross said, if you cut theconk down the middle, you can stillsee the outlines of where each previousyear’s growth of each spine occurred,embedded in the now-solid flesh.All of this sounds like what manyother conks do: don’t the big Fomesand Phellinus species also form newlayers of pores each time they sporulate?Well, they do, and they alsoexhibit “growth rings” in cross section,but with them, the next layer ofpores comes from hyphae growing upthrough the previous layer of poresfrom the main body of the fungus.Gross emphasized 2 that after eachhymenium of a polypore sporulates, itbasically dies, so the next wave of poreformation comes from hyphae from themain body of the fungus growing downthrough the existing pore openingsand forming a new pore layer on topof them. In the Echinodontiums, theolder hymenium stays alive and growsa new one directly on top of itself. Healso emphasized 3 that in polypores,the hymenium will correct for previouserrors and try to present the poreopenings on a smooth horizontal plane.For instance, if the conk has to growaround a branch at some point, this maydistort the pore layers that enclose thebranch, but once it has grown past thebranch, the pore layers will becomeregular again. Not so in Echinodontiumand Laurilia – each irregularity of thehymenium is perpetuated in subsequentlayers.There were also more subtle similarities,difficult to put into words. Forinstance, when the Japanese mycologistRokuya Imazeki was describing his newfind, Echinodontium tsugicola in 1935,he mentioned that its white, tough andirregularly bumpy upper surface lookedexactly like that of Laurilia sulcata.It’s hard to formalize a similarity likethat, and make it part of a system, butit impressed Imazeki enough that hefelt obliged to mention it, and Grossof course felt that this was significantalso. 4All of this adds up to a pretty hefty setof similarities; why were the Echinodontiumand Laurilia species ever put in differentgenera? Because the two Lauriliaspecies have a “smooth” spore-bearingsurface, without regular structures ofthe types (such as gills, teeth, pores)that were then officially recognized.Linnaean/Friesian mycology, the firstsystematic approach to the classificationof the fungi, emphasized the shape ofthe hymenium and made its major divisionsof fungal classification accordingto its shape – gills versus pores versusspines and so on. Laurilia sulcata thusstarted out its scientific career in 1901as Stereum sulcatum, where Stereum isthe old general-purpose genus for fungiwhose hymenium is “smooth.” Now,in fact, its hymenium is anything butsmooth – it is covered with all sorts ofirregular bumps, protrusions and folds.But Edwin Angus Burt, who named it,felt that this should get recognition at thespecies level, not the genus level: sulcatameans “folded.”2and he may have been wrong in making such a sweeping generalization about the polypores3and here I think he was more correct in making a distinction4Gross p. 3.Does Morphology IncludeChemistry?At the time that Gross wrote hispaper, chemical features were beingadded to microscopic and developmentalones in determining which fungiwere related to which. For instance,around the same time as Gross’s paper,Rolf Singer placed the genus of hugepolypores, Bondarzewia, in with theRussulas and Lactarius in his classificationsystem, because all threegenera possess a unique type of spore:one having amyloid ridges or flanges.“Amyloid” is the fungal-speak term formushroom parts (usually microscopic)that have a starchy structural component,and so turn black when exposedto iodine. It’s not particularly rare forfungi to have an amyloid spore wall,but it is quite unusual for a fungus tohave an inamyloid spore wall withamyloid ornamentation, as these do.Amyloidity, as it has turned out, is animportant feature in the group of mushroomsthat includes Echinodontium.And at the same time, the Dutchmycologist Marinus Donk was ponderingwhether to include the delicatelyspinedgenus Hericium in the samegroup with Lactarius, Russula andBondarzewia, because some of thehyphae in Hericium contain a latexsimilar to that in Lactarius (althoughthe Hericium species never containenough to exude “milk”, the way theLactarius species do). In 1971, Donkwould be the first to propose that allthe fungi now known as the Russulalesbe grouped together because of theselatex-containing hyphae.But this sort of thing was slowto catch on. Already, in 1959, theCzech mycologist Zdenek Pouzardecided to create the genus Laurilia,because he felt that the fungi with encrustedcystidia and such an irregularspore-bearing surface deserved to beseparated from the more ‘ordinary’Page4Fall 2007, Mushroom the Journal

Spore-bearing spines, the ‘old view’ of fungal evolution. Starting at the top right and travelling clockwise, we can seethe spore-bearing spines of these fungi getting longer, thinner and more sophisticated. Alas, it’s not really so!Top right: Laurilia sulcata (photo courtesy of Scott Redhead and Jim Ginns) shows us the process just getting started– here we have a simple roughening of the fertile surface. Continuing clockwise, we have a photo of Echinodontiumryvardenii, taken by Cristina Spinelli. Here the spines, stalactite-like, are just beginning to extend downward fromthe cap. Lower right, the dense but thick and short spines of Echinodontium tinctorium, photo courtesy of Bill Neill.At top left, we see the thin, dense style of spore-producing spines that was thought to be ‘most highly evolved.’ It isclear that this style of spore-bearing creates much more surface area than the other fungi on this page do, but theDNA evidence says that they’re actually not more recently evolved than the other fungi on this page.. This photoof a Hericium was taken by John Denk only a few weeks before this magazine came out.Stereums. But he didn’t take the nextstep that Gross took and look to see ifthere was already another place to putthem – one where they fit accordingto microscopic and developmentalfeatures, but not according to sporebearingsurface. So most mycologistshave not gone along with Gross’ transferof Pouzar’s Laurilia species intoEchinodontium.An Attractive but IncorrectPicture of EvolutionIf one accepts Laurilia and Echinodontiumas being in the same evolutionarygroup of fungi, one can mapout a very smooth and convincing transitionfrom “primitive” to “advanced”spore-bearing surfaces, as you can seefrom the sequence of pictures above.From Laurilia, which is bumpy andirregular but doesn’t have anythingon it that you could call an organizedstructure, one moves to E. ryvardenii,which seems to have developed someof Laurilia’s little bumps into real stalactites.Then it’s on to E. tinctorium,which has a whole bunch of stalactites,and finally the fine-toothed Hericium,which would represent admirablemodern efficiency.However, now we also have DNAevidence, which has turned everythingon its head.Fall 2007, Mushroom the Journal Page 5

We are lucky in that Echinodontiumryvardenii was discovered justin time to be included in a huge DNAsequencing project, Assembling theFungal Tree of Life (AFTOL). In fact,it arrived in time to be included in a2003 paper by Ellen Larsson and Karl-Henrik Larsson on the Russulales, thelarge-scale taxonomic group that alsoincludes Russula and Lactarius. A laterpublication in the official unveiling ofthe AFTOL results three years laterwas very much the same, and the monstrosityon the next page is a schematicview of their results, incorporatingfeatures from both papers. 5To make it easier to find the fewspecies that we’re really interestedin, I’ve put pictures of them by theirnames. The taxa are color-coded bytheir type of hymenium and fruitingbody:Resupinate formspores redspinesbluesmooth magentaForms with caps or branchespores orangespinesgills blackcoralloidgreenpurpleFrom this, you can see the greatdiversity of hymenial surface andfruiting body form in the Russulales.In this and the interweaving of differenthymenium and fruiting body typeswithin the same derived group, we findmore confirmation that the older viewof evolutionary relationships – thatthey are indicated simply by hymeniumtype – is incorrect. For example,not only are Laurilia sulcata and Echinodontiumtinctorium as close as canbe, but the two other fungi in the samegroup with them are Heterobasidionannosum, a hard, extremely fine-pored(i.e. sophisticated-looking) “polypore”that from looking at it appears to bejust a typical conk, and Bondarzewiaberkeleyii, a huge floppy thing that isalso pored but grows on the ground, isfleshy rather than hard, and has poresthat break apart and become maze-likein age. Three different types of hymenium,and a great difference in featuresbetween the two poroid species.Likewise, nutritional lifestyledoesn’t seem to be a good mid-scaleindicator of relatedness – the fungijust named are mostly wood-rotters,including the virulent “pest” of timber,Heterobasidion annosum; but Bondarzewiaberkeleyii is mycorrhizal,and a neighboring group includes themycorrhizal genus Albatrellus.The placement of names requiresa little explanation. As one moves tothe left in the chart, one is movingbackwards in time. The vertical blackline running down the left-hand sideof the chart is called the backbone.It connects to the presumed singleancestor (the black line leading off tothe left) of the group as a whole, andrepresents the initial radiating of thissingle ancestor into different species.These newer species are said to bederived from it, and the newer speciesthat have evolved from them are evenmore derived.Most of the branches coming offthe backbone lead to groups of varyingsize. For instance, near the top, besidethe picture of Echinodontium ryvardenii,is a node labeled “Gloeocystidiellum5”. This means that the originalchart has five species of Gloeocystidiellumin this position. Larger groupsusually contain more than one genus offungi; in this case, each genus is listedin turn, separated by a plus sign. Wherea species had no discernable relativesamong the other fungi sampled forthis project, it is connected directly tothe backbone. In cases where a genusappears in separate locations within agroup, it is listed only once but withslashes between the number of speciesat each appearance – for instance“Aleurodiscus 2/1” means that thereare two Aleurodiscus species presentat one point in the group, and anotherone by itself later on, with species fromother genera in between.Thus, another thing shown by thechart is how many of the more poorlyunderstood genera are really a randomcollection of unrelated species. Scytinostromellaand Wrightoporia seemto be the champions in that regard.These are resupinate genera – Scytinostromellahas a smooth hymeniumand Wrightoporia is pored – that aredefined by having dextrinoid thickenedstructural hyphae and crystallinebumps on their regular hyphae. Thiscombination of features seems to beabsolutely meaningless as a way toindicate evolutionary relationships.The champion for being scatteredamong different groups, however, isGloeocystidiellum, which appears infour different groups, and within oneof them in four different places. Itscriteria of prominent latex-containingcystidia is not meaningless, however,as the species fulfilling this criteriaare often grouped in clumps. What itdoes show is that prominent latex-containingcystidia seem to have evolvedindependently several times.Horizontal position in these chartsusually indicates when somethingevolved – the farther to the left a speciesis, the longer ago it evolved. Inthis chart, that dimension is almostmeaningless, since the relation of eachgroup to the backbone – and to eachother – is so poorly defined. The chartdepicts the larger groups as startingmore to the left, but that’s somethingthat’s forced by the fact that within5For those of you who wish to look at the original charts and original papers, the citations are provided at the end of this article. Youshould be able to find Mycologia (where most of these articles were published) in any college library; or, for those of you with an internetconnection, most of these papers (as well as many additional ones) can be downloaded from David Hibbett’s website: http://www.clarku.edu/faculty/dhibbett/publications.html. He was an author on almost all of them.Page6Fall 2007, Mushroom the Journal

Warning: Only the prefixes formany of these fungal names makesense, not the names themselves.Details are provided on page 7and 9....

each group something had to come first, so in alarger group that first something gets shoved furtherback in line. The smaller groups could each be veryrecent or very ancient – there’s no way to tell fromthe data in the study. 6This use of “basal” and “derived” gets us awayfrom the older style of talking about more “primitive”and “advanced” fungi, which is preferable fortwo reasons: first of all, this isn’t obvious from mychart, which has horizontally compressed things abit, but in the original pretty much everything in itis about equally derived – in fact very much so – orof unknown placement. One way you can see thison the chart is that there’s no group of fungi withanother group coming out of it. So there isn’t reallya spread of taxa over enough evolutionary time tomake the older labels – “primitive” and “advanced”– meaningful.Second, you can see that the flat, resupinatehymenium that was formerly thought to be a “primitive”state (shown in magenta on the chart) is presentall over the place, in groups alongside fungi with amore structured one. So that old notion goes downthe tubes also.The first thing that strikes one is the poor quality– or more precisely, the non-usefulness – ofsome of the data. There are seven isolated speciesthat are connected directly to the backbone, whichmeans we have no idea whatsoever of their relatednessto anything else in the chart. If we add to thesethe two species of Aleurocystidiellum towards thebottom, which the authors especially single out asbeing unusually unrelated to all the other groups,and the strange duo of a Scytinostromella and aWrightoporia species in the next-to-last position,we have eleven species that are essentially floatingfreely within the chart, with no indication of theirrelations to any other taxa. This is just under 10%of the total number of species sampled.This is not a criticism of the quality of work thatthese scientists did – it is perfectly possible that the6Presumably vertical position in the chart means something,but it’s not clear what, and the authors do not discuss this.So it’s not clear, for example, among the species that areconnected directly to the backbone, why there’s a cluster offour of them at the top of the chart, and one at the bottom.There is only one by itself in the middle in the original chart– Pseudoxenasma verrucisporum. Scytinostromella cerinais mentioned in the original text as connecting directly tothe backbone, but the authors didn’t include it in their chart.I stuck it in at this arbitrary location because its presencesupports a couple of observations I want to make.127 species that make up the original tree are less than 1% of allthe currently existing Russulales; so it should not be surprising tosee that some of the taxa sampled have not had any near neighborsincluded. This work is preliminary, and even the follow-up workwill remain preliminary for some time.A more troubling problem is the poor resolution at the middlelevel of detail – between these small groups and the backbone.We have good resolution of the smaller groups that I have shownas single nodes in my schematic, and we are quite confident thatall the fungi shown belong in the Russulales. However, we havehardly any information at all about how each group relates to onethe other ones.This is a consequence of the two DNA areas that were sequencedto provide the data. These were two locations sequenced in thefungi’s ribosomal DNA: the first is the nuclear large subunit rDNA,which is thought to evolve fairly slowly, and so is used to broadlyposition a species in the fungal tree. The second is the so-calledITS region, which consists of two non-coding spacer regions andthe 5.8S rDNA gene. Since spacer regions don’t code for anything(they just separate coding genes), mutations are thought toaccumulate more rapidly so this locus is used to compare closelyrelated species. Unfortunately, the effect of using these two locationsis to give us good small-scale and whole-group resolution,but to leave the middle-ground almost blank. This is a shame,because the middle-ground should have been the most valuablepart of such a survey.There are those who will say, “No, no! It’s the small-scaledetail which is valuable,” but really, if we want to do small-scalework on the Russulas, we’ll sample more than five species, andundoubtedly at more than these two DNA locations.These are the same two locations that were used for the wholelarge-scale AFTOL project: a decision was made to limit the sequencingto these two, in order to sequence the fungi uniformlyacross the whole project. In addition to this desire for compatibilitybetween the results of all the branches of the project, there was alsoa practical consideration. First of all, these were two of the firstlocations discovered to be useful in sequencing fungal DNA, sothey have been used more than any others which means that they’vebeen obtained from more fungi. Work that was done a while agomeans less work to do now.Secondly, using only two locations made the project moremanageable, and even so it was five years late (and I think we stilldon’t have the results from the Polyporales). But it’s time to getworking on more locations, and get the middle-ground of the Russulalesresolved. As it is, the most valuable results of the survey areto show that many of these resupinate genera are badly defined, andthe function of including things like those five Russula species isin effect to calibrate the system – to show that the procedure doesin fact produce the results that we were dead certain of already, sothe surprising ones are probably correct also.Page8Fall 2007, Mushroom the Journal

Back to the Fungus at HandWell, enough of this analysis of thestudy. Let’s see what help it gives uswith the fungi we’re most interestedin at the moment.First of all, it takes that tidily linkedevolutionary chain that I outlined inphotos a while ago and blows it outof the water. Two of the fungi thatwe’ve been discussing as being partof that sequence, Laurilia sulcata andEchinodontium tinctorium (towardsthe bottom), turn out to be sister taxa,at exactly the same level of evolutionaryderivedness. 7 Of the other fungiin our model evolutionary sequence,Hericium actually looks like it’sslightly more basal, but this is justan artifact of it being part of a largergroup. Echinodontium ryvardenii, onthe other hand, is way up at the top ofthe chart, and is connected directly tothe backbone, which means that nothingdirectly related to it was included inthe survey. Larsson and Larsson reportthat “despite having [a whole bunch ofanatomical similarities that we haven’teven discussed here,] it is not related tothe other Echinodontium species. 8 Itstrue affinities are unclear.” It could beextremely basal, with no descendents;it could be extremely derived, with allits close relatives extinct; or it could bein pretty much the same position as theother fungi in the chart, but no relatedfungi were included in the sampling.We just don’t know. 9So is there any physical similarityacross the whole Russulales? Well,there turn out to be two. Unfortunatelyfor you and me, they turn out to be (usually)microscopic. The one absolutelydiagnostic feature – all the Russulaleshave it, and no fungus outside the Russulalesdoes – is hyphae that contain alatex. Only three groups of Russulales(to my knowledge) produce enoughlatex to exude it when cut – the generaLactarius is of course named for thisability; some Stereums also exudelatex; and Bondarzewia berkeleyiiwill do so when young. 10 However,whether or not they have enough latexto exude, all the Russulales turn out tohave hyphae that are filled with thesecompounds. There has been little formalresearch into their function, butthe AFTOL paper on the Russulalesmentions the hypothesis that “basedon the chemistry of the fluid containedin hyphae from Lactarius velutinus,Camazine and Lupo (1984) suggestedthat the laticiferous 11 hyphae functionedas a storage depot for precursorsof pungent dialdehyde compounds.These compounds are largely unstableand change rapidly from nontoxic totoxic form, leading to the hypothesisthat they are chemical defensive agentsprotecting the spore-producing structuresfrom mycophagy.” 12 To which Iwould add that because most of thesewould-be mushroom grazers are tinybugs, the fungi don’t need to produceamounts of these chemicals that arevisible to you and me to be an effectivedeterrent against the mushroom’s mostimportant pests.The other diagnostic feature, somewhatless reliable, is amyloidity, the reactionwhere a starch compound reactswith iodine to turn blue-black. Readersof field guides know that many gilledmushrooms have amyloid spores(for instance, two of the more toxicsections of the genus Amanita), sosimple amyloidity is not the exclusiveprivilege of the Russulales. However,a layer of amyloid ornamentation on anotherwise inamyloid spore does seemto be, and having cystidia or someother bodily feature that are amyloid isvastly more common in the Russulalesthan outside them.If you look at the chart, you’llsee that almost all the names that aretotally unfamiliar to you start withamylo- or gloeo-, 13 indicating thatthis is a resupinate fungus with somepart (usually microscopic) that waseither amyloid or latex-bearing. 14 Theabundance of such names serve as anindication of the completeness withwhich those two features cover theRussulales. It could mean that suchfeatures were present in the presumedsingle ancestor of this large group offungi, or that they’ve simply evolvedindependently multiple times. Or both– they could have been lost in somefungi and then re-evolved. We simplydon’t know at this point.This isn’t to say that closely relatedspecies don’t share these sorts of features.We’ve already mentioned howLaurilia sulcata and Echinodontiumtinctorium both have thick-walled,crystal-encrusted cystidia. But theother two genera in the same littleclade, Heterobasidion and Bondarzewia,have nothing of the sort.Meanwhile, the next genus up fromthem is Aleurocystidiellum, which hasminutely spiny, amyloid spores and7One of the little perks of being a mycologist is being able to use words like “derivedness” in the face of all the grammar you learned inhigh school.8That’s “species” in the singular. They mean that it isn’t related to E. tinctorium, which is the only other species of Echinodontium in thechart.9The later, official AFTOL chart uses something called “neighbor joining”, which mushes adjacent branches together and makes thingslook more related. One effect of this is to mush E. ryvardenii together onto a branch with the neighboring species that are also connecteddirectly to the backbone, a result that is probably just an artifact of the neighbor-joining process.10I don’t know about B. montana. Perhaps some of our Western readers can help us out with that one.11My mind truly boggles in considering how this ridiculous word was formed. Surely it should be “lactiferous”, “milk-bearing.” “Laticiferous”comes out as bearing some sort of lattice or network; but it’s intended to mean “latex-bearing.” Oy.12Miller et al, “Perspectives in the new Russulales,” Mycologia 98(6), 2006, pp. 960–969.13“Gloeo-“ literally means oily, but is used as myco-speak for something involving latex.14I didn’t just leave them in to confuse you!Fall 2007, Mushroom the Journal Page 9

Two top photos:E c h i n o d o n t i u mb a l l o u i i ,photographed byB i l l N e i l l . R i g h tmiddle and below:E c h i n o d o n t i u mryvardenii,photographed byCristina Spinelli.Note in the two righthandphotos above,how big the tree is,and think also howvery slow-growingjuniper and Atlanticwhite cedar are.Photo immediatelyabove: the brightorange interior fleshof Echinodontiumtinctorium, used byNative Americansas a face and bodypaint.

thickened hyphae that terminate inthe hymenium as encrusted cystidia.In other words, its hymenium, sporesand hyphal system are exactly the sameas the L. sulcata and E. tinctorium;apparently the only reason that no oneseems to have ever thought about thembeing related is that Aleurocystidiellumis a cup fungus – it produces disc-likeflat fruiting bodies. Unfortunately, thislittle group of Aleurocystidiellums connectsdirectly to the backbone, leavingits relationship to any of the othergroups undefined, as we’ve seen alreadywith Echinodontium ryvardenii.So the presence of encrusted, thickwalledcystidia is a tantalizing featurein some of these fungi; however, it hasproved to be an unreliable indicator ofrelatedness.But What About Echinodontiumballouii?Well, Manfred Binder, one of themycologists who did a lot of the sequencingwork on the AFTOL project,also sequenced a bit of Bill and Larry’sE. ballouii, using the same two genesas the rest of the AFTOL project, and itcame up extremely close to E. ryvardenii;so first of all, everything we’vebeen saying about the evolutionaryposition of E. ryvardenii also appliesto E. ballouii. Second, the rediscovery(and sequencing) of E. ballouii putsanother data point into this widelyisolated area of the chart. True, it nowmakes for two linked, very close pointsthat are collectively isolated from everythingelse, but it’s a start. We needmore data – more sampling of the otherspecies currently placed in Laurilia andEchinodontium.What does all this do for Echinodontium‘sstatus as a living fossilfungus? Well, as far as E. tinctoriumis concerned, it’s case is busted: itseems to be a recently-derived fungus– or at least more recently-derivedthan many others. However, as far asE. ryvardenii and E. ballouii are concerned,the jury’s still out. Connectionstraight to the backbone of the largergroup like this has been used as anindicator of basalness in other largescalegroups, but here the backbone isstill very poorly defined, so we reallycan’t use the DNA evidence make anyinferences about the large-scale relationshipsbetween any of the speciesclusters in the Russulales.So Where Does This LeaveUs?With both DNA evidence andmorphological evidence ruled out,this leaves us only with ecology. Theworld polypore expert Leif Ryvardennotes that these fungi all occur on prepinegymnosperms. Pines – with theirbig, woody cones and large, nut-likeseeds – are the most recently-evolvedgymnosperms. All the other conifersare much older. Since Echinodontiumsonly occur on these older types oftrees, he concludes “Thus the genusmust be a very old one.” His case isactually strengthened by the separationof E. ryvardenii and E. ballouii – perhapswe can call them the paleo-Echinodontiums– from the more moderngroup represented by Echinodontiumtinctorium and Laurilia sulcata. Themodern pair occur among a widevariety of old-growth conifers, whilethe paleo-Echinodontiums are eachlimited to a single species of host in theCupressaceae, the basal (oldest) familyfor the Pinales (the largest groupof conifers).Their situation reminds me of anunusual but recurring scenario in theevolution of other kingdoms, where anearly family of organisms is displacedby a more specialized one, but retainsa hold on its most inhospitable niches,which the newcomer for one reasonor another is unable to take over. Forinstance, the aardvark, armadillo, andSouth America’s giant anteater areall members of a group of mammalscalled paenungulates, which used to beprimarily herbivores (think elephants,which are also paenungulates) andwere a very populous and diversegroup in the southern hemisphere.However, they were outcompeted intheir primary niche by more specializedgrazers, and these three animalsended up getting more and more oftheir nutrition from insects, whichhad played a minor role in their dietup until that point. Gradually, they gotmore and more specialized as nocturnalraiders of insect nests, and endedup in the forms that they have today.As for the newcomers (gazelles andhorses and so on), the very specializationthat allowed them to take overthe local grazer niche also preventedthem from competing with these survivingpaenungulates for the role ofinsect-eater.Likewise, the unique plant Welwitschiagrows in the Namibian desert, thehottest and driest climate on the planet.Presumably at one point there were lotsof related plants, from which this onespecies evolved to live in the desert.The ones that lived in more hospitableplaces were outcompeted by newcomers,but none of the more recent plantsmade the jump into the desert. My ideahere is that the Chamaecyparis wood,which is so tremendously resistantto rot, may here serve in the role ofthe inhospitable environment that theolder fungus managed to adapt to, andthe younger ones have not caught upwith it yet.Greg Mueller, the head mycologistat the Field Museum, strongly objectsto these speculations. He emphasizesthat there’s no actual evidence placingthese paleo-Echinodontiums at anearlier point in history than any otherfungus, and that furthermore what littlebackbone evidence there is for thelarger-scale basidiomycete group hintsthat the Polyporales/Thelephoralesgroup is a little older than the Russulales/Agaricales/Boletalesgroup, soodds are that there were “mainstream”polypores around prior to these paleo-Echinodontiums. He may be right. Theemergence of these particular fungi asthe dominant attackers of this ancientgroup of trees may just be one of thoserandom things.Fall 2007, Mushroom the Journal Page 11

As Gross presciently noted, “thefamily may prove to be phylogeneticallyimportant because it is a smalltaxonomic group containing charactersproposed to be both primitive andadvanced.” 15 Distinguishing betweencharacters that are “primitive” andthose that are “advanced” – or, as weprefer to say nowadays, characters thatare basal and those that are derived –is an issue that continues to bedevilus today.For the FutureMuch basic work remains to bedone – for instance, we still don’treally know whether the Laurilia/Echinodontium taxodii from southeasternNorth America is really the samething as the L./E. taxodii of east Asia.Let’s get all these things sequenced –and at more than two DNA locations!And let’s see some more attention totheir continued survival in the wild.Gross, for instance, notes that only afew collections of E. japonicum haveever been made. There are cultures ofit listed in the stock of various culturebanks, but it’s not clear if it’s presentin the wild anymore. Perhaps a programof inoculating seedlings withthe rarer of these fungi can be undertaken.If nothing else, it will at leastdemonstrate whether these culturesare viable or whether something likeE. japonicum is gone for good. And ifthe fungus does fruit on the saplings, itwill likewise demonstrate if the cultureis of the correct mushroom! And itwill hopefully somewhat increase thevigor of the cultures, which (accordingto the CBS notes on them) seemto be fading away on their refrigeratedPetri dishes.There are all sorts of other connectionswaiting to be made – for instance,the orange-red flesh of E. tinctorium isa striking feature, and definitely rare –and it is also present in E. tsugicola.Gross also notes that it fruits at branchstubs. In hindsight, this all seems significant– and it may also be significantin another genus.Chemistry, AgainThe other genus of fungi I’d liketo see attacked is Pyrofomes. Gross 16notes that Mildred Nobles, the greatAmerican expert on fungal cultures,felt that there was a great similaritybetween E. tinctorium and Fomes juniperinus.F. juniperinus turns out tohave been renamed Pyrofomes demidoffii;17 it is a woody, perennial conkwith bright orange-red flesh that growsonly on juniper. Sound familiar? Furthermore,this pigment, which seemsto be one of the defining features ofthe genus Pyrofomes, 18 seems to bethe same in both Echinodontium andPyrofomes: the abstract from an articlein the structural chemistry journalTetrahedron notes that “An orangepigment named echinotinctone wasisolated from the wood-rotting polyporesEchinodontium tinctorium andPyrofomes albomarginatus.” And ifthat weren’t enough, the pigment isfairly unique in the entire biosphere:not only is echinotinctone “the firstsimple fluorone pigment from fungi,”it is also “the first naturally occurringpigment [anywhere!] with a simplefluorone chromophore.” 19 Since fluoroneis a fairly simple compound – aset of three carbon rings with an oxygenatom at part of the central ring anda double bonded oxygen atom attachedto the side of one of the outside rings– I’d say that there’s some explainingthat needs to be done about whythis unique pigment is found only inthese two (groups of?) fungi. 20 The15p. 2316p. 2417Okay, get it out of your system – we’ll all groan and roll our eyes together.18See? There is some point to these modern genera after all!explanation need not be one of simplederivedness 21 – maybe this pigment isa metabolic by-product of the specializedchemistry needed to feed on thewood of these tough-to-decay ancientconifers, and has evolved independentlyin two unrelated groups offungi that both feed on the same sortsof trees. Either way, the story shouldbe interesting. And that holds for thegroup as a whole.BibliographyS. Camazine and A. T. Lupo, “Labiletoxic compounds of the Lactarii:the role of the laticiferous hyphae as astorage depot for precursors of pungentdialdehydes,” Mycologia 76, 1984, pp.355-358.Marinus Anton Donk, “A conspectusof the families of Aphyllophorales,”Persoonia 3, 1964, 199–324.Marinus Anton “Progress in thestudy of the classification of the higherBasidiomycetes,” In: Petersen R.H.,ed. Evolution in the higher Basidiomycetes,1971, Knoxville, p 3–25.David Hibbett, “A phylogeneticoverview of the Agaricomycotina,”Mycologia 98(6), 2006, pp. 917–925.Ellen Larsson and Karl-HenrikLarsson, “Phylogenetic relationshipsof russuloid basidiomycetes with emphasison aphyllophoralean taxa,” Mycologia95(6), 2003, pp. 1037-1065.Steve Miller, Ellen Larsson, Karl-Henrik Larsson, Annemieke Verbekenand Jorinde Nuytinck, “Perspectives inthe new Russulales,” Mycologia 98(6),2006, pp. 960–969.Yang Yea, Ingrid Jostena, NorbertArnolda, Bert Steffana and WolfgangSteglich, “Isolation of a fluorone pigmentfrom the Indian paint fungusEchinodontium tinctorium and Pyrofomesalbomarginatus,” Tetrahedron52:16, 1996, pp. 5793-5798.19“Isolation of a fluorone pigment from the Indian paint fungus Echinodontium tinctorium and Pyrofomes albomarginatus,” Yang Yea,Ingrid Jostena, Norbert Arnolda, Bert Steffana and Wolfgang Steglich, Tetrahedron 52:16, 1996, pp. 5793-5798.20And now that we know what to look for on a chemical basis and not just by color, perhaps related but less colorful compounds will befound in the closely related species.21In fact, I don’t even know if Pyrofomes, at this point, is considered part of the Russulales.Page12Fall 2007, Mushroom the Journal