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Communicative & Integrative Biology

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Ophiocordyceps unilateralis: A keystone for unraveling ecosystem functioning and biodiversity of fungi in tropical forests?

Harry C. Evans, Simon L. Elliot & David P. Hughes

To cite this article: Harry C. Evans, Simon L. Elliot & David P. Hughes (2011) unilateralis: A keystone species for unraveling ecosystem functioning and biodiversity of fungi in tropical forests?, Communicative & Integrative Biology, 4:5, 598-602, DOI: 10.4161/cib.16721 To link to this article: https://doi.org/10.4161/cib.16721

Copyright © 2011 Landes Bioscience

Published online: 01 Sep 2011.

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Ophiocordyceps unilateralis A keystone species for unraveling ecosystem functioning and biodiversity of fungi in tropical forests?

Harry C. Evans,1,* Simon L. Elliot1 and David P. Hughes2 1Department of Entomology; Universidade Federal de Viçosa (UFV); Viçosa; Minas Gerais, Brazil; 2Department of Entomology and Department of Biology; Penn State University; University Park; PA USA

phiocordyceps unilateralis (Ascomy- thus far4—this group of organisms still O cota: ) is a specialized receives relatively little press in terms of parasite that infects, manipulates and its biodiversity and the pivotal role it plays kills formicine , predominantly in ecosystem functioning. Recently, how- in tropical forest ecosystems. We have ever, the subject has been revisited within reported previously, based on a prelimi- the context of microbes associated with nary study in remnant Atlantic Forest beetles.5 Of the near one million species ©2011 Landesin Minas Gerais (Brazil), thatBioscience. O. uni- of insects that have been described, beetles lateralis represents a species complex. are by far the most numerous. However, in On each of the four species of infected terms of overall biodiversity, “beetles may carpenter (Camponotus) collected, represent just the tip of the iceberg,”5 since the —characterized macroscopi- “A single beetle species itself can house Do notcally by a singledistribute. stalk arising from the an entire community of associated [pre- dorsal neck region on which the sexual dominantly fungal] species.”5 The prem- structures (stromatal plates) are borne ise put forward is that microbial diversity laterally—can readily be distinguished and ecology have been neglected in favor both microscopically and functionally. of other disciplines, especially in the area Here, we describe and discuss the biol- of biological mutualisms and antagonisms ogy, life cycle and infection strategies of between macro- and micro-organisms.1,5 O. unilateralis s.l. and hypothesize that An illustration of Ophiocordyceps unilate- there may be hundreds of species within ralis on its ant host was chosen by the the complex parasitizing formicine ants latter authors to emphasize their point, worldwide. We then address the diversity that—for most of the insect-microbial within related hypocrealean fungi, with interactions—the ecology and, more Key words: zombie ants, particular reference to symbionts (mutu- pragmatically, the pharmacological prop- Ophiocordyceps, Hypocreales, tropical alists through to parasites), and argue erties of the microbes involved remain forests, fungal diversity, symbionts that the widely-quoted total of extant unknown. Submitted: 05/30/11 fungi (1.5 million species) may be grossly We have now shown6 that the inter- underestimated. actions between and its Accepted: 05/31/11 O. unilateralis Camponotus hosts are much more com- DOI: 10.4161/cib.4.5.16721 “How many species?” has been the ral- plex than has previously been supposed.7-10 *Correspondence to: Harry C. Evans; lying cry for systematists and ecologists, It had been posited that the morphologi- Email: [email protected] championing the interests of their partic- cal variation noted in global collections ular organismic group, since the question of this fungus from formicine ants “is Addendum to: Evans HC, Elliot SL, Hughes DP. was posed over 30 years ago.1,2 Although it undoubtedly a consequence of its wide geo- Hidden diversity behind the zombie-ant fungus 8 Ophiocordyceps unilateralis: four new species was answered soon after for the Kingdom graphic range,” and that this would best described from carpenter ants in Minas Gerais, Fungi3—with an estimated total in the be resolved at the varietal level, as with the Brazil. PLoS One 2011; 6:17024, PMID: 21399679; region of 1.5 million species, of which, less Ophiocordyceps species complexes associ- DOI: 0.1371/journal.pone than 100,000 (~7%) have been described, ated with myrmicine and ponerine ants.11,12

598 Communicative & Integrative Biology Volume 4 Issue 5 article addendum

©2011 Landes Bioscience. Do not distribute. Figure 1. Most common carpenter ant species infected with Ophiocordyceps s.l. in Atlantic rain forest in Minas Gerais, Brazil. (A) Sample area, Mata do Paraíso, near Viçosa, a small (300 ha) reserve of remnant forest; (B) Camponotus rufipes showing early stage in development of Ophiocordyceps camponoti-rufipedis with a single asexual synnema arising from the dorsal pronotum, the ant had died biting into a plastic label identifying a disease- outbreak site, (bar = 1.5 mm); (C) Camponotus balzani with mature Ophiocordyceps camponoti-balzani, showing the elaborate, branching, antler-like synnema/clava bearing scattered ascostromata with prominent semi-erumpent ascomata (compare E, bar = 1.0 mm); (D) Camponotus rufipes in more typical death position biting into leaf margin, with unbranched synnema/clava producing groups of stromata (arrow, bar = 4 mm); (E) inset, shows the ascomatal necks with the ascomata buried within the stroma (bar = 0.4 mm).

However, working in remnant fragments of arthropod population dynamics and, holis- fungal pathogen must attach securely Atlantic Forest (Mata Atlántica) in Minas tically, in ecosystem functioning. to the arthropod exoskeleton and pen- Gerais, Brazil,6 we discovered a far more etrate it—avoiding or suppressing host complex scenario: one that will necessitate The Host-Pathogen System: defences—then, control the behavior of a re-evaluation of the diversity of species A Diversity of Interactions the host before killing it; and finally, it within Ophiocordyceps unilateralis s.l., as must protect the cadaver from microbial well as in the Ophiocordyceps and As has been emphasized previously in and scavenger attack. What is not gener- related genera of Hypocreales—and, eventu- reference 2 and 5, insect-microbial asso- ally realized—certainly not by drug com- ally, within the kingdom fungi—with par- ciations have been a source of useful panies—is that such coevolved fungi are ticular reference to tropical forests. These drugs: although, “The pharmacologi- pleiomorphic, with well-defined parasitic, ecosystems harbor a hyperdiversity of cal properties of the ant-infesting fun- necrotrophic and saprophytic phases: fungi, especially when we consider they are gus [O. unilateralis s.l.] have yet to be each one morphologically, genetically found as symbionts (ranging from mutual- investigated.”5 What can we expect from and physiologically distinct. Only now, ists to parasites) of the extremely diverse such specialized fungi, and from related are we beginning to try to understand the flora and entomofauna, and even of other entomopathogenic Hypocreales, in gen- mechanisms and the biochemical path- fungi. Based on such studies, it may also eral? Throughout the life cycle, there are ways that drive these systems. be possible to better interpret their role in unique challenges that must be met by Our initial studies are focused on equally unique metabolic activities. The investigating the form and function of

www.landesbioscience.com Communicative & Integrative Biology 599 ©2011 Landes Bioscience. Do not distribute. Figure 2. Variations on a theme. (A) Ascospore of Ophiocordyceps camponoti-rufipedis germinating to produce secondary spores (capilliconidia) on needle-like capilliconidiophores (bar = 30 μ); (B) Ascospore of O. camponoti-melanotici producing true phialide with swollen base tapering to a needle-like neck (bar = 15 μm); (C) Ascospore of O. camponoti-balzani becoming swollen with bead-like cells and the formation centrally of an appressorial-like structure (bar = 30 μm); (D) Ascospore of an un-named Ophiocordyceps sp. from Camponotus atriceps producing numerous swollen side branches of unknown function (bar = 30 μm); (E and F) Two undescribed, specialized mycoparasites on Ophiocordyceps illustrating the biodiver- sity and complexity associated with the system (E, bar = 2 mm; F, bar = 2 mm). the weaponry deployed by the pathogen ant activity is at its peak and the potential as the ecological significance of the world- (O. unilateralis s.l.) to breach the ant host’s targets are in range and, therefore, that wide collections of the zombie-ant fun- formidable defenses. We suppose that the microclimatic factors (light, temperature, gus, especially of the somewhat bizarre, heavy armament “of choice” is the stroma- humidity) will play a crucial role. What we multiple asexual stages (synanamorphs) tal plate (the sexual stage or teleomorph) do know, from our preliminary study in described on Polyrhachis ants infected produced laterally on a stalk or clava reference 6, is that there is a species com- with O. unilateralis s.l. from West Africa.8 emerging from behind the head of the plex of macroscopically-similar pathogens From studies of other entomopatho- ant—the emblematic feature of the zom- (O. unilateralis s.l.) producing not only genic fungi,13 we know that there are also bie-ant fungus—buried within the stroma sexual stages (teleomorphs), but also asex- critical host-recognition factors to trigger are the flask-shaped, thick-walled asco- ual stages (anamorphs), that differ widely the formation of the limpet-like appres- mata producing sac-like structures called in form and function, each equipped with sorium: the “beach-head” from which the asci. Each of these is fitted with a special- in-built insurance mechanisms to increase infective hypha drills through the com- ized firing cap containing the missiles: the chances of success if the primary mis- plex multi-layered exoskeleton using a large (from 80 > 200 μm long) multisep- siles fail to hit their targets. combination of mechanical pressure and tate, sexual spores (ascospores). These are Currently, we are working on docu- enzymes (chitinase, lipase and protease). released under pressure from the launch menting the diverse strategies employed We now have circumstantial evidence pad—the ant cadaver—typically, affixed by this newly-delimited complex of from our work with freshly-released asco- to the underside of a leaf on an under- Ophiocordyceps species to reach new spores of Ophiocordyceps from different storey forest shrub. We also assume that hosts. Thus, we will be in a better posi- Camponotus hosts, which suggests that the system is designed to operate when tion to re-interpret the taxonomic, as well there are also sophisticated signals between

600 Communicative & Integrative Biology Volume 4 Issue 5 pathogen and host in this species complex tens if not hundreds of species still to be pockets in fragile cloud forest systems since recognizable infection structures delimited within the complex. Moreover, on the western border of Ecuador and (appressoria) are never produced in vitro, there are similar Ophiocordyceps com- Colombia. Searches to find the centre of only the secondary (“insurance”) spores plexes within tropical ants, in general, yet origin of a devastating disease of cocoa mentioned above: sticky-capped conidia to be investigated.10 So, we will be hoping (Theobroma cacao) revealed a complex on hair-like extensions (capilliconidio- to address the question: to what degree associated with its rare indigenous for- phores) for species with delicate, filiform does this diversity scale up when we step est host (T. gileri) and the causal fungal ascospores; thick-walled, resting spores back and look at a fragment of forest or pathogen (Moniliophthora roreri).19 Not (chlamydospores) for the larger, more at a biome, such as the Atlantic Forest, only is there a guild of novel mycopara- robust ascospores. Once the infective and imagine all of the host-symbiont sites colonizing the pathogen, but also hypha breaches the exoskeleton, avoiding interactions, focusing on the - a suite of unusual invertebrate natural the cellular and humoral defences of the lean fungi? But how does this sit with the enemies. One of these, a bizarre dipteran ant, the fungus grows in the hemocoel oft-repeated mantra that arthropods are larva—equipped with a hooked abdo- as free-living yeast cells. However, time the true “kings of the jungle,” especially men to cling onto the substrate as it feeds periods are not clear cut, but we suppose when species diversity is the parameter of on fungal spores covering the pods high that in a matter of days the yeast phase interest? It has been proposed that there is in the canopy—proved to be difficult to colonizes the hemocoel and then produces an intermediate body size at which organ- identify. Attempts to follow the life cycle, the nerve toxins to alter the behavior of isms are globally most species diverse,14 in order to obtain adult flies for taxonomic the ant causing it to climb and bite onto effectively a humped distribution of spe- studies, were thwarted since all the pupae vegetation, often choosing the adaxial leaf cies diversity that is concentrated some- were hyperparasitized by a wasp belonging midrib and a specific orientation—hence, where between small and large insects. to a genus described over a century ago but the zombie-ant epithet6—where it soon According to this view, smaller organisms not collected since (A. Polaszek, Natural dies. During this phase, we expect—based (i.e., microbes) have cosmopolitan distri- History Museum, London, pers. comm.). on work from other systems—that there butions. However, much of the data pre- Moreover, tissue samples taken from is a complex©2011 interplay of protein expres Landes- sented concern predominantly Bioscience. free-living healthy pods and stems of T. gileri revealed sion between fungal parasite and manipu- organisms and even for these, the view has a rich diversity of endophytic fungi, with lated host. It would come as no surprise to been challenged.15 It is becoming increas- many representatives in the Hypocreales,20 discover that much of the host-pathogen ingly apparent that microbes such as fungi including a new clade of interaction occurs prior to protein expres- may be more influenced by biogeography spp.21 Molecular examination of this novel sion, via RNA silencing orDo suppression ofnot than previously distribute. thought, and the symbi- endophytic clade in the commercially- silencing. Hyphal outgrowths from the otic lifestyle can be an important factor in important genus Trichoderma, is reveal- body orifices and joints further secure the speciation.16 ing only now the presence of seven new ant to the substrate whilst, internally, the Not only does this make the question species within the T. harzianum com- saprophytic mycelium, consisting of lipid- of “how many species” appear imponder- plex alone (G.J. Samuels, ARS-USDA, rich storage bodies, colonizes and mum- able—and, thereby, makes the original Washington DC, pers. comm.). mifies the cadaver. The logistics governing estimated figure of 1.5 million fungal spe- production of the fruiting stalk appear to cies redundant, even though this was later Perspectives be controlled to some extent by external still held to be a viable working hypoth- conditions, but, probably in a matter of esis17—it also raises basic questions about The reciprocal question to that posed in weeks, rather than days for the synana- the role of these pathogens in ecosystem the introduction to this paper, is: “Why morphs, the stromata are fertile and the functioning, particularly in tropical for- should we care how many species there cycle is completed. ests where ants are the drivers.18 We have are?”2 As highlighted before,2,5 microbial focused here on Ophiocordyceps as the ecology has been neglected, and we need Diversity and Importance of Fungi flagship genus for a re-examination of to know how these systems function and in Tropical Forest Ecosystems fungal diversity. However, there are many their microbial species composition: the similar fungal: macro-organism associa- pragmatic incentive is that there could The zombie-ant fungus has a pantropi- tions in tropical forest systems that we are be “products of great use to humans.”22 cal forest distribution on formicine hosts, only beginning to uncover, let alone inves- The latter authors have recently made an and may also be found in warm-temperate tigate in any depth. Another example, also important contribution in addressing this forest systems. However, like the ants, it involving hypocrealean fungi, should help largely conjectural analysis, and provided is far more common in tropical forests. to emphasize the biodiversity, food webs evidence that “novel biology [of tropical Our study,6 in an outlying fragment of and multi-trophic interactions involved in forest endophytes] will indeed yield novel the fast-disappearing Atlantic rainforest, such systems. chemistry of potential value.”22 Since has revealed unexpected diversity within Theobroma gileri, the only species of tropical forest systems are fast disappear- O. unilateralis s.l., which, if repeated on the chocolate-tree genus found west of the ing, there is an added urgency if we are “to a global scale, could mean that there are Andean Cordillera, occurs in restricted understand the structure and functioning

www.landesbioscience.com Communicative & Integrative Biology 601 3. Hawksworth DLH. The fungal dimension of biodi- 13. Samson RA, Evans HC, Latge JP. Atlas of ecosystems, particularly tropical eco- versity: magnitude, significance and conservation. of Entomopathogenic Fungi. Berlin: systems, much better than we do. And Mycol Res 1991; 95:641-55. Springer-Verlag 1988. 4. Kirk PM, Cannon PF, Minter DM, Stalpers JA, Eds. 14. Finlay BJ. Global dispersal of free-living microbial we cannot hope to do this if we do not Dictionary of the Fungi, 10th edition. Wallingford, eukaryote species. Science 2002; 296:1061-3. even know what there is there, and why UK: CAB International 2008. 15. Green J, Bohannan JM. Spatial scaling of microbial tropical diversity is what it is?”2 Fungi 5. Berenbaum MR, Elsner T. Bugs’ bugs. Science 2008; diversity. Trends Ecol Evol 2006; 21:501-7. 322:52-3. 16. Giraud T, Refrégier G, Le Gac M, de Vienne DM, underpin tropical forest systems. If we 6. Evans HC, Elliot SL, Hughes DP. Hidden diversity Hood ME. Speciation in fungi. Fungal Genet Biol only know a small fraction of the extant behind the zombie-ant fungus Ophiocordyceps uni- 2008; 45:791-802. fungal species,4 we have a lot of catching lateralis: four new species described from carpenter 17. Hawksworth DL. The magnitude of fungal diversity: ants in Minas Gerais, Brazil. PLoS ONE 2011; the 1.5 million species estimate revisited. Mycol Res up to do. The task has suddenly become 6:17024. 2001; 105:1422-32. even more daunting with the very latest 7. Evans HC. Natural control of arthropods, with 18. Hölldobler B, Wilson EO. The Ants. Cambridge, estimate of the number of fungi being put special reference to ants (Formicidae), by fungi in MA: Harvard University Press 1990. the tropical high forest of Ghana. J Appl Ecol 1974; 19. Evans HC, Holmes KA, Reid AP. Phylogeny of at 5.1 million species, based on molecular 11:37-49. the frosty pod pathogen of cocoa. Pl Pathol 2003; data.23 Prospects are not bright, however, 8. Evans HC, Samson RA. species and 52:149-60. as we are rapidly losing the tropical forest their anamorphs pathogenic on ants (Formicidae) 20. Evans HC, Holmes KA, Thomas SE. Endophytes in tropical forest ecosystems. II. The Camponotus and mycoparasites associated with an indigenous for- ecosystems, as well as the mycologists who (Formicinae) complex. Trans Br mycol Soc 1984; est tree, Theobroma gileri, in Ecuador and a prelimi- can contribute to understanding them. 82:127-50. nary assessment of their potential as biocontrol agents 9. Samson RA, Evans HC, Hoekstra ES. Notes on of cocoa diseases. Mycol Progress 2003; 2:149-60. entomogenous fungi from Ghana. VI The genus 21. Samuels GJ. Trichoderma: Systematics, the sexual Acknowledgments Cordyceps. Proc K Nederl Akad Wetensch, Ser C state and ecology. Phytopath 2006; 96:195-206. H.C.E. is a visiting scientist in the 1982; 85:589-605. 22. Smith SA, Tank DC, Boulanger LA, Bascom-Slack 10. Evans HC. Entomopathogenic fungi associated with CA, Eisenman K, et al. Bioactive endophytes warrant Postgraduate Program in Entomology, ants (Formicidae): a review. In: Misra JK, Horn intensified exploration and conservation. PLoS One funded by the Brazilian National BW, eds. Trichomycetes and Other Fungal Groups. 2008; 3:3052. Council for Research (CNPq, grant no. Enfield, USA: Science Publishers 2002:119-44. 23. Blackwell M. The fungi: 1, 2, 3… 5.1 million species? 11. Evans HC, Groden E, Bischoff JF. New fungal Am J Bot 2011; 98:426-38. 401610/2009-8). S.L.E. is in receipt of a pathogens of the red ant, Myrmica rubra, from the CNPq scholarship (300920/2010-5). UK and implications for ant invasions in the USA. Fungal Biol 2010; 114:451-6. 12. Evans HC, Samson RA. Cordyceps species and ©2011References Landestheir anamorphs pathogenic on antsBioscience. (Formicidae) 1. May RM. How many species are there on earth? in tropical forest ecosystems. I. The Cephalotes Science 1988; 241:1441-9. (Myrmicinae) complex. Trans Br Mycol Soc 1982; 2. May RM. How many species? Phil Trans R Soc Lond 79:431-53. B 1990; 330:293-304. Do not distribute.

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