Diversity and Ecology of Fungal Endophytes in Tropical Forests50 Biodiversity of Fungi: Their Role...

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48 Biodiversity of Fungi: Their Role in Human Life

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Zhdanova, N.N., Vasilevskaya, A.I., Lashko, T.N., Gavrllyuk, V.I. and Dighton, J. (1994).Changes in micromycetes communities in soil in response to pollution by long-livedradionuclides emitted in the Chemobyl accident. Mycological Research 98: 789-795.

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Diversity and Ecology of Fungal Endophytesin Tropical Forests

A. Elizabeth Arnold

Departmentof Biology,DukeUniversity,Durham,NC 27708E.mail:[email protected]

SUMMARY,~ _u_,- .

Fungal endophytes, ubiquitous among terrestrial plants, are especiallyabundant and diverse in asymptomatic foliage of tropical trees and shrubs_ Assuch, tropical endophytes are wjdely thought to comprise an importantcomponent of fl.!ngalbiodiversity at a global scale. Yet, neither the diversity ofendophytes associated with even a single individual plant, nor the potentialimportance of host preference and spatial heterogeneity to estimates ofendophyte diversity has been addressed in full. Here, several salientquestions regarding both endophyte diversity itseif, along with theImplications of Ejndophyterichness for plant ecology and evolution have beenexplored in the.context of tropical forests. Contributions of recent studies indiverse tropical sites have been highlighted in conjunction wjth an overviewof future directions in endophyte research.- - <.- < .

INTRODUCTION

Endophytes are microorganisms that colonize and cause unapparentinfections in healthy plant tissues (Petrini, 1991; Wilson, 1995). Ubiquitousamong terrestrial plants, endophytic fungi have been found withinasymptomatic tissues of non-vascular plants (Schulz et al. 1993), ferns(Fisher, 1996), conifers (Legault et al. 1989), and angiosperms (Petrini etal. 1982; Clay, 1988; Rodrigues, 1994; Faeth and Hammon, 1997). Thevast majority of fungal endophyte research has centered on rhizosphereendophytes, with a focus on mycorrhizal fungi (Rygiewicz and Andersen,

50 Biodiversity of Fungi: Their Role in Human Life Diversity and Ecology of Fungal Endophytes in Tropical Forests 51

1994; Redecker et al. 2000). Yet, endophytic fungi also occur in thephyllosphere, cryptically inhabiting aerial tissues such as leaves andstems of all plant species examined to date (Petrini, 1991; Saikkonen etal. 1998; Stone et al. 2000; Arnold, 2002).

Among endophytes associated with aerial plant tissues, the systemic,vertically transmitted endophytes in the family Clavicipitaceae(Ascomycota) have been relatively thoroughly studied (Clay, 1991;Saikkonen et al. 1998). These fungi may benefit the temperate grassesthey inhabit by conferring drought resistance, enhancing metal tolerance,and deterring both vertebrate and invertebrate herbivores (Clay, 1990;Hoveland, 1993; MalinoNski and Belesky, 1999; Cheplick et al. 2000; butsee Faeth, 2000). In contrast, horizontally transmitted endophytesassociated with angiosperms are poorly known, so that the nature of theirinteractions with host plants, their autecology, and their taxonomiccomposition have not been characterized in detail. Because angiospermsrepresent the majority of plant diversity, the ubiquitous, horizontallytransmitted endophytes associated with these host species are of specialinterest.

In this chapter, I will focus on three salient questions in endophytebiology, with special attention to horizontally transmitted endophytesassociated with foliage of angiosperms (hereafter, endophytes) in tropicalforests: (1) the scale of endophyte biodiversity; (2) the nature ofendophyte-host interactions; and (3) the evolutionary implications of thesesymbionts for terrestrial plants. Together, these topics are of relevance inresolving fungal biodiversity on a global scale (Hawksworth, 2001),understanding fundamental aspects of plant-fungus interactions in naturalsystems, and shaping knowledge regarding the formation, maintenance,and implications of asymptomatic microbial symbioses.

that definitions requiring or dismissing mutualism exclude the broadspectrum of interactions that characterize diverse endophyte-hostsymbioses (Carroll, 1988; Carroll, 1991; Saikkonen et a1.1998;Faeth,2000).

In 1991, Petrini provided a working definition for endophytes that hassince been widely accepted: endophytes comprise 'organisms inhabitingplant organs that at sometime in their life... colonize internalplant tissueswithout causing apparent harm to their host'. So defined, endophytesinclude microorganisms associated with the rhizosphere, phyllosphere,and vascular tissues of living plants. Endophytes may exhibit more thanone type of life history at distinct life stages; may include pathogenicorganisms that exhibit latency between infection and manifestation ofsymptoms; and may encompass microorganisms that existsimultaneously, or over a period of time, both upon and within planttissues. So defined, microbial endophytes writ large comprise a diverse,polyphyletic group encompassingan array of interactionswith host plants.

FUNGALENDOPHYTES

MICROBIAL ENDOPHYTES

When first Introduced by deBary (1866), "endophyte" was broadly used torefer to any organism found within plant tissues (see Carroll, 1986).Various authors later narrowed the definition in accordance with particularresearch interests, leading to much confusion with regard to the breadthand application of the term (see Stone et al. 2000). Similarly, earlyinterests in endophytes as potential mutualists of their host plants led toworking definitions that necessitated evidence for mutualism, whereasothers required only evidence of asymptomatic infection (see Carroll,1986; Petrini, 1991; Wilson, 1995; Stone et al. 2000). Recent advancesindicate that taxonomically restrictive definitions exclude major lineages ofmicrobial endophytes, including bacteria and cyanobacteria, which may beabundant and diverse (Chanway, 1998). Moreover, it is now understood

Among terrestrial plants, endophytic fungi appear to be ubiquitous: everyplant species examined to date harbors at least one fungal endophytespecies (see Petrini, 1991; Stone et al. 2000; Arnold, 2001), and manyplant species may be associated with tens to hundreds of endophytespecies (Saikkonen et al. 1998; Stone et al. 2000). Within hosts, fungalendophytes may inhabit all available tissues, including leaves, petioles,stems, twigs, bark, xylem, roots, fruit, flowers, and seeds (Boddy et al.1987; Chapela and Boddy, 1988; Sieber, 1989; Clay, 1990; Bills andPOlishook, 1991; Bohn, 1993; Fisher et al. 1993; Lodge et al. 1996; Lupoet al. 2001; Meyer et al. 2001). As such, endophyte researchers mightagree that asymptomatic tissues of plants may well be viewed as complexhabitats filled with diverse, if unapparent, inhabitants.

Despite the broad occurrence of endophytes in plant organs, manyendophytic fungi appear specialized to particular host tissues (e.g. twigs.petioles, leaves). In particular, leaves and leaf-inhabiting endophytesrepresent a compelling focus for studies of endophyte diversity andecology. As the site of photosynthesis, they represent biochemicallydynamic habitats replete with microhabitats both upon their surfaces andwithin. As discrete islands, their boundaries are easily delimited formicrocosmal studies of ecology at the community level. Leaves providenatural, nested replicates within and among hosts; yet, they encompasssufficient diversity in chemical and structural components to allow testingof hypotheses regarding endophyte abundance, diversity, and hostpreference among and within host species. Finally, leaves interact with


herbivores and pathogens above the ground, where direct and indirectinteractions can be observed. Thus, leaves and their endophytes

represent an engaging arena for the study of biological diversity, as wellas a potential trove of insights into ecological relationships that are, atpresent, poorly known.

To date, two groups of fungal endophytes associated with foliage havereceived particular attention: the clavicipitaceous endophytes associatedwith temperate grasses (Clay, 1991; Saikkonen et al. 1998; Faeth, 2000),and the endophytes associated with leaves of temperate conifers

(Bernstein and Carroll, 1977; Carroll and Carroll, 1978; Stone, 1987, 1988;Legault et al. 1989; Rollinger and Langenheim, 1993). Horizontallyacquired endophytes associated with leaves of angiosperms in tropicalforests are poorly known in terms of diversity and interactions with hosts,and are the focus of this chapter. .

Diversity and Ecology of Fungal Endophytes in Tropical Forests 53

WHY TROPICAL FORESTS?

Most mycologists agree that fungal diversity on a global scale is greatestin tropical forests, where diversity of terrestrial plants also reaches its peak(see Hawksworth, 1991,2001). At present, however, the true scale oftropical fungal biodiversity is not known. Recent surveys of tropicalmacrofungi (e.g. polypores, Gilbert et al. 2002) have indicated thatdiversity of these relatively apparent fungi is much greater than waspreviously thought (see May, 1991). In turn, recent studies indicate that thesame is true for several guilds of cryptic microfungi, including saprophytes

(Lodge, 1997; Frohlich and Hyde, 1999; Aptroot, 2001), pathogens(Shivas and Hyde, 1997), and fungal endophytes associated with leavesof woody plants (Lodge et'al. 1996). Indeed, foliar endophytes in tropicalforests are thought to represent as much as an order of magnitude greaterdiversity than can be found in the temperate zone (Dreyfuss and Chapela,1994), but at present, relatively few quantitative data are available toestimate the diversity of endophytes in tropical forests. In particular, thescale of diversity of tropical endophytes-including both ecological andtaxonomic components; the nature of endophyte-host interactions in

tropical forests; and the evolutionary implications of these ubiquitoussymbionts-has not been established.

individual leaves and plants in tropical forests. Asymptomatic leaves oftropical woody angiosperms consistently contain multiple species offungal endophytes in lowland, moist forests (Lodge et al. 1996; Arnold,2002), and as many as 140 species of endophytic fungi have been foundin association with only th~ee individual plants in a wet tropical forest(Frohlich and Hyde, 1999). Similarly, Arnold et al. (2000) recorded morethan 400 morphotaxa of foliar endophytes from two host species in asingle site, and argued that tropical endophytes may be hyperdiverse.

Although striking, these values represent only alpha diversity, i.e. localdiversity, and thus are of limited use in estimating endophyte diversity atthe landscape or global scale. In ecological terms, the missing dataneeded for extrapolative estimates of endophyte biodiversity include: (1)evidence for consistency in endophyte abundance and richness amongphylogenetically diverse hosts and at diverse tropical sites; (2) evidencefor host preference among tropical endophytes; and (3) evidence forspatial structure in endophyte assemblages. Consistency of endophyteabundance and richness has been confirmed for a phylogeneticallydiverse group of host taxa in Panama (Figure 4.1; Arnold, 2002) and isbeing established at a global scale through an accumulation of studies indiverse tropical sites (see Frohlich and Hyde, 1999; Arnold et al. 2000;Guo et al. 2001; Gamboa and Bayman, 2001; Suryanarayanan et a!. 2002;but see Cannon and Simmons, 2002). Here, I will focus on host preferenceand spatial structure among tropical endophytes.

BIODIVERSITY OF FOLIARENDOPHYTES:ECOLOGICAL FACTORS

A number of recent studies (Lodge et al. 1996; Frohlich and Hyde, 1999;Arnold et al. 2000; Arnold et al. 2001; Gamboa and Bayman, 2001) provide

strong evidence that fungal endophytes are highly diverse on the scale of

HOST PREFERENCE OF TROPICAL ENDOPHYTES

Several recent studies have compared endophyte assemblagesassociated with different host species in single sites to determine whethertropical endophytes demonstrate host preference (Arnold et al. 2000,2001; Gamboa and Bayman, 2001; Arnold, 2002; Arnold et al. 2003).Although restricted to relatively small numbers of host species andrelatively few sites, several of these studies have indicated that althoughmany tropical endophytes are generalists, at least a subset demonstratehost preference (but see Cannon and Simmons, 2002). Such affinity istypically demonstrated by low similarity values for comparisons ofendophyte assemblages from different hosts, as calculated using theabundance-based Morisita-Horn index (Gamboa and Bayman, 2001;Arnold et al. 2003), variously calculated versions of S0rensen's index(Arnold et al,. 2001), or Jaccard's index, which is based on presence/absence data alone (Arnold et al. 2000). Although intuitively useful, theseindices suffer from a lack of explicit statistical significance, and fromdebate regarding the appropriateness of using index values as data forstatistical tests.

54 Biodiversity of Fungi: Their Role in Human UfeDiversity and Ecology of Fungal Endophytes in Tropical Forests 55

Ceratophyllales

Laurales

Magnoliales

Piperales

(Monocots)Ranunculales

Proteales

Caryophyllales

Saxifrigales

Santalales

Geraniales

Oxalidales

Malpighiales

Fabales

Rosales

FagalesCucurbitales

Myrtales

Malvales

Sapindales

Brassicales

Ericales

Comales

Garryales

Gentianales

Lamiales

Solanales

Aquifoliales

Apiales

Asterales

Dipsacales

Recently, several authors have begun to use randomization tests tocounter the above-mentioned problems, and have found strong evidencefor significantly non-random distributions of tropical fungi with respect tohost species (see Arnold et al. 2000; Gilbert et al. 2002). Although verycompelling, randomization tests also warrant caution in their use. Becausethey are based on isolation frequencies of given taxa, these tests requirecareful definition as to what constitutes an endophytic individual. Suchanalyses must also be restricted to non-singleton taxa, which may restricttheir utility in highly diverse endophyte communities. Further, such testscould confound spatial factors such as local dispersal with apparent hostpreference, thus leading to potentially faulty conclusions from survey data.The last difficulty could be resolved if survey data were coupled withempirical assessments of potential mechanisms for host preference(Arnold and Herre, 2003), but such examples are rare. '

To date, even general mechanisms for such preference have not beenexplored in detail for tropical endophytes. However, studies of hosts in thetemperate zone suggest that substrate chemistry and associated host-recognition mechanisms are likely to be important as determinants of hostaffinity. For example, Toti et al. (1992) have shown that adhesion of sporesof Oiscula umbrinella, a common endophyte of beech, is greater on hostsurfaces relative to non-host surfaces. Similarly, Chapela et al. (1990)showed that xylariaceous fungi, which include many endophytes oftropical plants (see Bayman et al. 1998), employ a complex mechanismfor' host recognition that acts quickly following fungus/plant contact.Further, Todd (1988) has provided evidence that the genotype of hosttrees influences infection success by fungal endophytes in the case ofconiferous hosts. However, the generality of these effects has not beendetermined for the tremendous richness of tropical endophytes. whichincludes representatives of no fewer than five classes of Ascomycota(Arnold, 2002). .

It has been proposed that distributions of endophytes associated withtropical woody angiosperms may be strongly and generally influenced byleaf defensive chemistry (Arnold and Herre, 2003). Tropical treesgenerally bear both higher quantities and a greater diversity of secondarychemicals than do their temperate zone counterparts, and interspecificdifferences in quantity and quality of defenses are strong (Coley andBarone, 1996). Preliminary research has indicated that integration of leafextracts into growth media induces differential growth in endophytic fungi(Arnold and Herre, 2003), and pairwise interactions on these media differfrom trials conducted on standard nutrients (malt extract agar). Further, invitro trials have shown that for endophytes examined thus far, growth onmedia containing extracts from hosts from which the given morphotaxa I

are frequently isolated leads to more rapid growth in culture, relative to

Fig. 4.1 Endophytesare ubiquitous among angiosperm trees and shrubs in moisttropical forests in lowland Panama (Amold 2002). Mature leaves of up to five species ineach order (black circles) were surveyed for endophyte infection. In all the cases,endophytes were present in 100% of the sampled leaves, and in the majority of leaf area(>95%). Consistency in endophyte abundance and richness was not influenced bystructural or defensive traits of leaves. Additional studies at other sites have foundendophytes in association with leaves of additional groups (e.g. monocots), such thatendophytes appear to be ubiquitous among tropical host species. Tree is redrawn inmodified form from Judd et at (1999).


extracts from hosts in which given endophytes are rarely encountered(Arnold et al. 2003; Herre et al. unpublished data). These results suggesta compelling link between growth responses in vitro and occurrence ofendophytes in planta, and are worthy of further study.

SPATIAL STRUCTURE OF TROPICAL ENDOPHYTEASSEMBLAGES

Studies of individual host species in different sites have suggested thattropical endophytes demonstrate heterogeneity at various spatial scales(Bayman et al. 1998; Frohlich and Hyde, 1999; Arnold et al. 2000; Gamboaand Bayman, 2001). To date, no mechanism for spatial structure has beenproposed; however, it appears that processes related to dispersal arelikely to play an important role in shaping the spatial heterogeneity ofendophyte assemblages. Horizontally transmitted endophytespreferentially sporulate from senescent tissues, and because many foliarendophytes do not grow systemically (see Lodge et al. 1996), sporedispersal is the primary means by which endophytes encounter newtissues for colonization. Spores of many forest endophytes may bedispersed among hosts via air currents (Arnold and Herre, 2003), whichmay move fungal spores only a few meters in densely vegetated areas(McCartney and Bainbridge, 1984) and could, therefore, contribute tospatial heterogeneity of endophyte assemblages on a local scale. Further,Monk and Samuels (1990) showed that feces of Orthoptera in an Indo-Malayan rainforest contained viable propagules of endophytic fungi,suggesting that herbivory (and, therefore, mycophagy) may be animportant mechanism of local dispersal for some foliar endophytes aswell.

Such factors would be expected to contribute to the spatial structureof endophyte assemblages at small scales, and therefore, to spatialheterogeneity at landscape scales. For the most part, however, datademonstrating spatial structure at large geographic scales are lacking.Large-scale heterogeneity among tropical endophytes has beenchallenged by Suryanarayanan et al. (2002) who found similar endophyteassemblages in four forest types in India. In contrast, Frohlich and Hyde(1999) have found evidence for differences in endophyte assemblagesassociated with palm species in Australia vs Brunei. Similarly, Arnold et al.(2003) found strong evidence for spatial structure of endophytesassociated with a rainforest tree (Theobroma cacao, Malvaceae) at fivesites in Panama, ranging over an area of 20-325 km. In that study,endophyte assemblages in more proximate sites were more similar to oneanother than to assemblages in more distant sites. Notably, this pattern

I .


was consistent regardless of overt ecological, biotic, and. abiotic

differences among sites (rainfall regime, duration of dry season, etc.).However, Gamboa and Bayman (2001) found a disparity in the richnessof endophytes with regard to study site in their surveys of Guarea guidonia,and suggested that factors such as land use patterns may influenceendophyte richness, and by inference, species composition. To date,therefore, the degree to which endophytes of woody angiospermsdemonstrate spatial structure at landscape and global scales has not beenresolved with certainty.

ISSUES RELATED TO METHODS OF STUDY

Even when questions of host affinity and spatial structure are laid aside,the true scale of endophyte diversity for even a single individual at a singlesite is not yet clear. This lack of clarity derives, in part, from a lack ofunderstanding regarding the efficacy of" methods used to isolate andenumerate endophytic fungi. Previous work by Carroll (1995) and Gamboaet al. (2003) has underscored the importance of leaf segment size inestimating endophyte diversity, arguing that a greater species richness will

be inferred from studies using smaller leaf fragments. Similarly,implications of using specific nutrient media to isolate endophytes aregenerally not known. Most studies of endophyte diversity use malt extractagar, potato dextrose agar, cornmeal agar, or other plant-derived nutrient

media in original isolations (Lodge et al. 1996; Frohlich and Hyde, 1999;Arnold et al. 2000). Preliminary data from Magnolia grandif/ora in thesoutheastern USA suggest that apparent species composition,abundance, and diversity may differ with regard to media when individualleaves were cultured on malt extract, potato dextrose, corn meal and

agars (Arnold and Lutzoni, unpublished data), with a greater diversity ofendophyte species, genera, and orders recovered on malt extract.However, the generality of this pattern across diverse host taxa is notcertain.

An additional challenge to endophyte research lies in the need to use

morphotaxa as functional taxonomic units for sterile cultures. Endophytesurveys, especially in tropical forests, consistently encounter myceliasterilia, which cannot be identified due to a lack of reproductive structures.Many authors designate these cultures as morphotaxa, yet criteria for

delineating morphological groups are rarely published. Recent work byArnold (2002) and Lacap et al. (2003) has provided support formorphospecies designations based on nrDNA markers, and Arnold (2002)has explicitly explored the plasticity of vegetative characters with regard tonutrient source, illumination, and temperature in vitro. Arnold (2002)concluded that morphotaxa may represent a surprisingly conservative


estimate of species richness, and that cultural differences may prove to bemore useful for separating some taxa into representative units thanothers. For example, differentiation among putative species ofColletotrichum was especially difficult based on the three media used byArnold (2002), which included malt extract agar, molasses agar, and V8medium. Notably, this study further showed that estimates of diversityusing the same morphospecies concept differed with regard to isolationmedia and colony age, thereby suggesting that standardization within andamong collections is necessary in order to make accurate comparisons.

Finally, it is important to note that the degree to which leaves of tropicalangiosperms harbor uncultivable endophytes has not been established.Bridge and Spooner (2000) argued that fewer than 17% of fungi can beisolated and maintained in culture. If this value holds true for tropical

endophytes, then a tremendous diversity of endophytic fungi remainsundocumented. Environmental sampling techniques represent an

important tool for assessing the diversity of unculturable fungi. Forexample. studies of Pinus taeda (loblolly pine) in the southeastern USA(Eells et at unpublished data) demonstrate a marked disparity in thediversity and species composition of endophytic fungi when culturalsurveys are compared against environmental samples fromasymptomatic, surface-sterilized foliage. Similar results are emerging forstudies of mycorrhizal fungi as well (see Husband et al. 2002). Althoughsuch methods are prone to certain difficulties (chimeric sequences andprimer biases), direct PCR represents an important future direction forendophyte research.

BIODIVERSITY OF FOLIAR ENDOPHYTES: TAXONOMICPATTERNS

Because tropical endophytes represent an understudied group, relativelylittle is known regarding the broad taxonomic patterns that underlieendophyte species diversity. Surveys in tropical sites consistently yield asubset of common genera (Colletotrichum, Xylaria, Fusarium,Botryosphaeria, .and Diaporthe; see Frohlich and Hyde, 1999; Arnold,2002), but whether these represent truly common endophytes, or simplyendophytes that can be cultured easily, is.not known. Because endophytesurveys may involve rapid accumulation of numerous isolates (Arnold,2002), and because many endophytes may remain sterile in culture (Guoet al. 2001), identification of all endophytes obtained in biodiversityinventories may be impossible due to both logistics and to a lack oftaxonomically informative characters. Therefore, establishing theprevalence of endophytism among major lineages of fungi through culturalstudies alone may prove difficult. In the absence of such data, it would be


of use to determine: (1) whether particular traits of major fungal lineagesindicate frequent occurrence of endophytism among fungi in those clades;and (2) the degree to which our understanding of endophytism can beincreased by current knowledge regarding other mycobiont-photobiontsymbioses.

Several studies have shown that the majority of endophytic fungi areAscomycota, including many mitosporic fungi traditionally classified asDeuteromycota (Alexopoulos et at 1996; Frohlich and Hyde, 1999).Ascomycota represent an ancient group (400 my - 1.2 byo; see Berbeeand Taylor. 1993; Heckman et al. 2001) whose members have interactedclosely with plants for at least 460 my (Berbee, 2001). Although recentmolecular analysis have left'the directionality of endophyte evolution anopen question (see Kuldau et al. 1997), Clay (1988) proposed that theendophytic habit of grass symbionts may reflect an evolutionary transitionfrom plant parasitism to mutualism, in accordance with theory regardingevolution of species interactions (Bull 1994). Several authors posit this tobe true for horizontally transmitted endophytes also (see Saikkonen et al.1998).

If it is generally true that endophytism has arisen from parasitism,groups of fungi containing large numbers of plant-pathogenic species maybe predisposed to contain large numbers of endophytic taxa as well. Thefact that most endophytes appear to occur within the Pezizomycotina(filamentous Ascomycota) is, therefore, not surprising: this subphylumcomprises> 3000 genera, including the majority of ascomycetous fungipathogenic to plants (Berbee, 2001). However, neither theTaphrinomycotina nor Saccharomycotina has been shown to containendophytic taxa, despite the sister relationship of the latter to thePezizomycotina (Eriksson et al. 2001), and the prevalence of plantpathogens among the former (Berbee, 2001). Moreover, theBasidiomycota contain a large number of plant pathogens (Alexopoulos etal. 1996), and, as a sister group to the Ascomycota. are also an ancientgroup; yet, basidiomycete fungi are much less frequently isolated asendophytes than are filamentous ascomycetes (see Petrini, 1986; Frohlichand Hyde, 1999). Prevalence of plant-associated fungi within a fungallineage thus may be one factor among many indicating prevalence ofendophytism at a general level. However, it is not sufficient in itself toidentify endophyte-rich clades.

Endophyte surveys in tropical forests typically encounter diverseSordariomycetidae, Dothideomycetidae, and Leotiomycetes (see Lodgeet al. 1996; Frohlich and Hyde, 1999; Arnold, 2002). Occurrence ofendophytes within these lineages is expected, as endophytes have been

encountered frequently within each group in previous studies. More I

60 Biodiversity of Fungi: Their Role in Human Ufe

surprising is the observation of tropical endophytic fungi among theChaetothyriomycetidae and Eurotiomycetidae (= traditional Plectomycetes).These clades appear to contain relatively few known endophytes,although the chaetothyrialean genera Phaeococcus (Johnson andWhitney, 1992) and Rhinocladiella (Petrini and Fisher, 1988), and theeurotialean genera Aspergillus (Southcott and Johnson, 1997) andPenicillium (Larran et al. 2001), have been recorded as endophytic inhabit. Both lineages contain species that interact closely with terrestrialphotobionts: members of the Chaetothyriomycetidae often fruit on rottenwood or on plant surfaces, and species of Eurotiomycetidae often attackfruits and other plant materials after harvest (Berbee, 2001). Interestingly,both classes appear to have descended from lichen-forming ancestors,having derived from one of several evolutionary losses of the lichen habitamong Ascomycota (Lutzoni et al. 2001). Thus, their historical associationwith living photobionts (Le., their former photosynthetic symbionts) mayfurther predispose these clades to endophytism. Given that these fungiare highly diverse, the rarity with which they are isolated as endophytesrepresents an unresolved question.

Relative to other, better-known mycobiont-photobiont symbioses,taxonomic patterns of diversity among tropical endophytes appear quitedistinct. For example, although arbuscular mycorrhizal fungi (AM F)colonize roots of the vast majority of terrestrial plant taxa (>70%,Alexopoulos et al. 1996), they comprise a single group (Glomeromycetes)within the phylum Zygomycota. Although taxonomic revisions may alterthe level of organization at which AMF are united, it is clear that they forma unique group represented by ca. six recognized families (Eriksson et al.2001). Species-level diversity among the Glomales/Glomeromycetes hasbeen thought to be comparatively low, especially relative to the largenumber of host species with which AMF are associated (but see Bever etal. 2001). In contrast, lichen symbioses are formed by ca. 20% of allknown, extant fungi, including> 13,000 species representing 15-18 ordersof Ascomycota (Lutzoni et al. 2001). Although most lichen-formingAscomycota belong to a single class (Lecanoromycetes; see Lutzoni et al.2001; Berbee, 2001), lichens are highly diverse at the species level andat small spatial scales. For example, Aptroot (2001) recorded ca. 200lichen and saprophyte species on a single tree in Papua New Guinea, andLOcking and Matzer (2001) have found >30 lichen species on individualtropical leaves. Like endophytes, lichen-forming fungi are primarilyPezizomycotina; yet the class that contains most lichen-formingAscomycota contains neither known plant pathogens (Berbee, 2001) norendophytes.

"


ENDOPHYTE-HOST INTERACTIONS

.

At present, very little is known regarding the costs and benefits ofendophyte infection for tropical plants. However, the ubiquity,abundance,and diversityof tropical endophytesstrongly suggest that endophytic fungiand host plants may interact in ecologically meaningful ways. In theabsenceof experimental insights, theory regardingevolution of symbiosesmay prove useful for inferring general aspects of endophyte-hostinteractions. In particular, patterns of symbiont transmission and diversityare often correlated with life history characteristics, and can provide aframework for hypotheses regarding costs and benefits for hosts of anendophyte infection.

Horizontally transmitted endophytes are, by definition, transmittedamong hosts via contagious spread. Further, endophytic fungi may behighly diverse within individual host tissues, host individuals, and hostspecies. Together, these traits frequently correlate with antagonisticinteractions (see Bull, 1994);yet plants containing endophytes are overtlyasymptomatic. Recent advances in endophyte exclusion (Arnold andHerre, 2003) and selective inoculation (Mejra et al. unpublished results)have set the stage for experiments to elucidate the costs and benefits ofendophyte infection in tropical trees. Here, I will briefly elaborate on theexisting hypotheses regarding the costs and benefits of endophytesymbioses for host plants.

In tropical trees, endophytes may be neutral inhabitants of their hosts.This inference is consistent with the hypothesis that endophytic fungifound in leaves of trees are simply transient and ecologicallyunimportant-or only incidentally important-inhabitants of host tissues.However, endophytes are obligately heterotrophic, and they denselyinhabit tissues of plants that may be carbon-limited (e.g. plants in thetropical forest understory). This leads to an alternative inference:endophytes may be mildly parasitic to hosts, subtly costing their hostplants in ways not yet understood. Evidence from monocotyledonoushosts in tropical regionssupports this view: for example, Pintoet al. (2000)demonstrated that endophyte infections of maize and banana wereassociatedwith decreasedratesof photosynthesisin individual leaves andat the whole-plant level. Although effects of endophyteson photosyntheticrates for woody angiosperms have not been assessed, Arnold andEngelbrecht (unpublished data) have found strong evidence thatendophytes associated with a tropical tree may negatively influence hostwater relations. However, the generality of such costs is not known. Assuch, cryptic costs of endophyte infection are worthy of further study.

Although neutralismand mild parasitismmaydescribe the interactionsbetween endophytes and hosts for some taxa, evidence suggests that

62 Biodiversity of Fungi:Their Role in Human Life

these may not be universal for endophyte associations in tropical forests.This evidence takes the form of a paradox: in the tropical forestunderstory, plants that are well defended against fungal pathogens (seeColey and Barone, 1996) consistently accumulate dense endophyteinfections within healthy leaves. Why would diverse endophytes, many ofwhich are closely related to pathogens, escape these defenses? Thisraises a third hypothesis: endophytes may be either neutral in terms ofcost, or mildly parasitic; but they may also confer upon their hosts a benefitthat compensates, at least under certain conditions, for costs ofendophyte infection. Evidence for such benefits is lacking, althoughpreliminary data suggest that endophytes. associated with Theobromacacao (Malvaceae) may positively influence host resistance to a majorfoliar pathogen (Phytophthora sp.; Arnold et al. 2003). The generality ofthis result in woody plants, the mechanism by which it operates, the roleof endophyte abundance, diversity, and species composition in hostdefense, and the ecological relevance of endophyte-mediated protectionin the field have yet to be established. These represent useful directionsfor future research.

EVOLUTIONARY IMPLICATIONS OF ENDOPHYTISM

The ubiquity of endophytic fungi among plants and within plant tissues,and the observation that fungi have been associated with plants since thefirst colonization of land (Heckman et al. 2001; Berbee, 2001), suggestthat plants and endophytes are likely to share a long and intimate history.However, without an understanding of general costs and benefits ofendophyte infection, or of the plasticity and diversity of endophyte-hostinteractions, the evolutionary importance of endophyte symbiosesremains unclear. Indeed, whether the asymptomatic nature of endophyteinfection represents plant-mediated control of the interaction (Faeth, 2000;Redman et al. 2001); competition among endophytes within plant tissues(Herre et al. unpublished data), or a general pattern of evolution away frompathogenicity (see Kuldau et al. 1997) is not yet known. If endophytes ofwoody plants are similar to mycorrhizal, lichenized, and clavicipitaceousfungi in terms of influencing responses of photobionts to biotic and abioticstressors (Clay, 1991; Rygiewicz and Andersen, 1994; Faeth, 2000), thenendophytes are likely to have played - and continue to play-an importantrole in plant evolution.

To resolve the evolutionary implications of endophytic symbioseq forhost plants, ecological data regarding endophyte-host interactions shouldbe integrated with phylogenetic analyses of endophytic fungi in the contextof Ascomycota evolution. Because a large proportion of leaf endophytesremain sterile in culture, and because morphological characters may be

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plastic with regard to nutrient media and some environmental conditions(Arnold,.2002), molecular data will be of particular use in phylogeneticanalyses. A multilocus approach synthesizing data from multiple,independent regions of the genome is likely to prove most insightful inreconstructing endophyte evolution, and subsequently inferring ancestralstates, origins of endophytism, the interplay of pathogenicity andavirulence in an evolutionary timescape, and the potential for unexploredclades to contain endophytic fungi. Studies of this sort that focus ontropicalendophytesare likely to be extremely useful, for they will capture-and add to the fungal tree of life-a richness of fungi at low and hightaxonomic levels. This will, in turn, inform our understanding of theevolutionary implications for plants of endophyte-host interactions, andadd to the growing body of knowledge regarding general patterns ofsymbiont-host evolution.

PERSPECTIVE

The current state of knowledge regarding diversity, host preference,spatial structure, isolation techniques, and endophyte-host interactions intropical forests indicates several useful directions for future research.First, samples at diverse geographic locations are needed to assessendophyte diversity at a global scale. Such sampling will benefit frommolecular analysis, which will help to discern the genotypic diversity ofendophytes in different sites. Second, further assessment of leafchemistry as a. mechanism for host affinity is needed. Selectivereinoculations using endophyte-free plants will be especially useful in thisregard. Third, assessing interactions among endophytes will be necessaryto understand the degree to which apparent patterns of host affinity aredictated by endophytes, host plants, or endophyte-endophyte interactions.Fourth, concurrent isolation of endophytes from individual leaves ontomultiple nutrient media will be important for assessing biases imposed bynutrient sources. Such studies would be especially insightful provided the

diversity of endophytes in hClst tissues were known, a priori, by usingleaves inoculated with a known number of endophyte taxa. Fifth,interactions between hosts under diverse biotic and abiotic conditions willbe needed to understand the costs and benefits of endophyte infection.Sixth, environmental sampling techniques will be critical for assessing theoccurrence and diversity of unculturable endophytes within healthy planttissues. Finally, these studies can be united by integration of endophyticfungi into phylogenetic analyses of the Ascomycota. Together, theserepresent a few of many research directions that will enhance ourunderstanding of tropical endophyte diversity and its implications forestimates of fungal biodiversity at a global scale, as well as the importanceof endophyte-host symbioses in plant communities.

64 Biodiversity of Fungi: Their Role in Human Life Diversity and Ecology of Fungal Endophytes in Tropical Forests 65

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68 Biodiversity of Fungi: Their Role In Human Life

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CHAPTERr

15I

Halotolerant and Halophilic Fungi

Nina Gunde - Clmerman 1, Jens C. Frlsvad2, Polona Zalar1, andAna Plemenltai3

'Unlversity of Ljubljana, Biotechnlcal Faculty, Biology Department, Veena pot 111,. SI-1oooLjubljana, Slovenia.

E.mail: [email protected] E.mail: [email protected]

~echnical University of Denmark, Biocentrum-DTU, Building 221, DK-2800 Kgs.Lyngby, Denmark. E.mail: [email protected]

3University of Ljubljana, Medical Faculty, Institute of Biochemistry, Vrazov trg 2,SI-1000 Ljubljana, Slovenia. E.mall: [email protected]

SUMMARY

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Hypersaline ~nvironments provide special living conditions formicroorganisms. They are extreme environments because of highconcentrations of NaCI and other salts, and sometimes occasional rapidchanges in water activity (aw), low oxygen concentration, and l1igh UVradiation. It has been a common belief that eukaryotic organisms, with fewexceptions, are unable to adapt to these extrfilme conditions ana that such IIenvironments are populated almost exclusively by bacteria, Archaea and one

reukaryotic species, the alga Duns/ie/Is ss/ins.AII fungi growing on low wateractivity substrate~ were termed xerophiles, due to the b,eliefthey reflected ageneral xerophyllcphenotype, determined primarily by the water potential ofthe medium arid not by the chemical nature of the solute. It was assumed thatfungi do not popplate natural hypersaline environments: Only a few reportsde.scri~ed isolatiqn of fungi from natural, moderately saline environments

: such as salt marshes, saline soil and sea water. These isolates were mainlycategorized as ubiquitous fungi, not specifically adapted to saline

. environments or halotolerant flush. But recently, a considerable diversity ofhalophilic and halotolerant fungi, belonging to several genera of melanizedand non-melanized filamentous fungi and yeasts, is becoming evident innatural hypersaline environments. Further, it is becoming increasingly clearthat this indigenous halophilic mycobiota is adapted to long-term survival and

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