ectomycorrhizal fungi of the seychelles: diversity patterns

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Research

Blackwell Publishing Ltd

Ectomycorrhizal fungi of the Seychelles: diversity patterns and host shifts from the native

Vateriopsis seychellarum

(Dipterocarpaceae) and

Intsia bijuga

(Caesalpiniaceae) to the introduced

Eucalyptus robusta

(Myrtaceae), but not

Pinus caribea

(Pinaceae)

Leho Tedersoo

1

, Triin Suvi

1

, Katy Beaver

2

and Urmas Kõljalg

1

1

Institute of Botany and Ecology, University of Tartu. 40 Lai Street, 51005 Tartu, Estonia;

2

Plant Conservation Action Group. PO Box 392, Victoria, Mahé,

the Seychelles

Summary

• Ectomycorrhizal (ECM) fungi form highly diverse communities in temperate forests,but little is known about their community ecology in tropical ecosystems.• Using anatomotyping and rDNA sequencing, ECM fungi were identified on roottips of the introduced

Eucalyptus robusta

and

Pinus caribea

as well as the endemic


and indigenous

Intsia bijuga

in the Seychelles.• Sequencing revealed 30 species of ECM fungi on root tips of

V. seychellarum

and

I. bijuga

, with three species overlapping.

Eucalyptus robusta

shared five of thesetaxa, whereas

P. caribea

hosted three unique species of ECM fungi that were likelycointroduced with containerized seedlings. The thelephoroid (including the anamorphicgenus

Riessiella

), euagaric, boletoid and hymenochaetoid clades of basidiomycetesdominated the ECM fungal community of native trees. Two species of Annulatascaceae(Sordariales, Ascomycota) were identified and described as ECM symbionts of

V. seychellarum

.• The low diversity of native ECM fungi is attributed to deforestation and long-termisolation of the Seychelles. Native ECM fungi associate with exotic eucalypts,whereas cointroduced ECM fungi persist in pine plantations for decades.

Key words:

community structure of ectomycorrhizal (ECM) fungi, DNA barcoding,exotic forest plantations, host specificity,

Intsia bijuga

(Caesalpiniaceae), islandbiogeography,


(Dipterocarpaceae).

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© The Authors (2007). Journal compilation ©

New Phytologist

(2007)

doi

: 10.1111/j.1469-8137.2007.02104.x

Author for correspondence:

Leho TedersooTel/fax

:

+

372 7376222Email

:

[email protected]

Received:

12 February 2007

Accepted:

27 March 2007

Introduction

Ectomycorrhizal (ECM) fungi comprise an estimated 7000–10 000 species worldwide (Taylor & Alexander, 2005), mostof which remain undescribed. The majority of theseundescribed species likely inhabit poorly studied tropicalecosystems and/or form inconspicuous or no fruit-bodies (e.g.ascomycetes and members of the thelephoroid, sebacinoid

and athelioid clades of basidiomycetes). ECM fungi with suchinconspicuous fruit-bodies dominate fungal communities inboreal and temperate forest ecosystems (Horton & Bruns,2001). In temperate forests of the northern and southernhemispheres, ECM fungi form highly diverse communities ofup to a few hundred species per site (May & Simpson, 1997;Horton & Bruns, 2001; Bastias

et al

., 2006; Tedersoo

et al

.,2006).

Pseudotsuga menziesii

harbors

c

. 2000 species of ECM

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fungi throughout its range (Trappe, 1977), whereas a singletree may host more than 15 species (Saari

et al

., 2005). Sucha high fungal species richness most likely results fromdifferential preference for hosts and soil conditions (Bruns,1995). Some recent studies indicate that ECM fungi deliverspecies- and even strain-specific benefits to their host plants(Wong

et al

., 1989; van der Heijden & Kuyper, 2003). Suchfunctional diversity, especially in enzymatic activities (Courty

et al

., 2005) and nutrient acquisition strategies (van derHeijden & Kuyper, 2003), results in greater biomass andproduction of host plants with increasing symbiont richness(Baxter & Dighton, 2005).

In tropical ecosystems, the ecology and function of ECMfungi have remained poorly understood, and much of theinformation on community composition relies on fruit-bodysurveys. Despite some ecological limitations, fruit-body surveysindicate that tropical rainforests and savannas support highlydiverse communities of ECM fungi (Henkel

et al

., 2002;Verbeken & Buyck, 2002; Watling

et al

., 2002; Lee

et al

.,2003). A few preliminary below-ground studies support thesefindings in Dipterocarpaceae (Sirikantaramas

et al

., 2003;Moyersoen, 2006) and closely related Sarcolaenaceae (Ducousso

et al

., 2004). By contrast,

Pisonia grandis

R. Br. (Nyctaginaceae)hosts a few thelephoroid ECM symbionts in Australia (Chambers

et al

., 2005), whereas neotropical members of Nyctaginaceae(including

Pisonia

,

Neea

and

Guapira

spp.) seem to be associatedwith additional fungal lineages (Moyersoen, 1993; Lodge,1996; Haug

et al

., 2005).There is limited information on the biogeography of

ECM fungi, but most fungal genera are globally distributed,suggesting their antiquity and/or effective long-distance dis-persal. Tiny spores of fungi and other cryptogams are carriedby wind thousands of kilometers (Hallenberg & Küffer, 2001;Muñoz

et al

., 2004). For example, both Leptospermoideae(Myrtaceae) and associated

Pisolithus

spp. most probablycolonized New Zealand from the Australian continent vialong-distance dispersal (Moyersoen

et al

., 2003). In additionto dispersal, continental drift and climatic oscillations havedriven vicariance events and facilitated migration of entireplant communities and their associated microbes (Wilkinson,1998; Halling, 2001). More recently, humans have uninten-tionally facilitated the spread of pathogenic and symbioticfungi by importing forestry products and establishing exoticforest plantations (Mikola, 1969; Wingfield

et al

., 2001). Theintroduced pathogens have switched hosts and affected thelocal plant communities and populations detrimentally(Wingfield

et al

., 2001). There is little such information onthe invasion ecology of ECM fungi, because the naturalranges of individual species are poorly documented. A fewreview studies, however, suggest that two well-known fly agar-ics,

Amanita muscaria

and

A. phalloides

, can become invasivein native communities (Orlovich & Cairney, 2004; Pringle &Vellinga, 2006). Establishment of exotic forest plantationswith pregrown tree seedlings is the main source of cointroduc-

tion of mycorrhizal fungi (Mikola, 1969). Such cointroducedfungi, in turn, facilitate invasion of natural communities by

Eucalyptus

spp. in Spain (Diez, 2005). In addition, exoticfungi may deplete soil resources (Chapela

et al

., 2001). Nativeorganisms of small, isolated islands are especially vulnerable toexotic pathogens and invasive species (Lomolino

et al

., 2006).The granitic islands of the Seychelles represent mountain

tops of the largely submerged Mahé microcontinent, whichwas separated from the Indian subcontinent (Deccan plate)

c

. 65 million yr ago (mya; Briggs, 2003). Long-term isolationof the Seychelles has resulted in the development of endemic-rich biota. Fleischmann

et al

. (2003) considered 34% of thenative higher plant species endemic. Such a high degree ofendemicity, substantial deforestation and the small area renderthe Seychelles highly vulnerable to invasive species. Afterhuman settlement in 1770, up until the early 20th century,native forests were gradually cut down and replaced mainly byintroduced cinnamon (

Cinnamomum verum

), which wasspread by birds. Coconut plantations (

Cocos nucifera

) replacedlow-altitude forest during much of the 19th and 20th centuriesas the main economic crop. Further deforestation occurred inthe early 20th century when commercial distillation of cinnamonoil required huge amounts of wood fuel. Other introducedtrees, shrubs and lianas have since invaded the indigenousplant communities. Native vegetation still exists in remote, lessaccessible mountain regions and small uninhabited islands,providing invaluable sources for taxonomists and biogeographers.Among the native plants of the Seychelles, representatives ofthree families, Dipterocarpaceae, Caesalpiniaceae and Nyctagi-naceae form ECM associations (Alexander & Lee, 2005). Onroots of

P. grandis

(Nyctaginaceae), two unidentified species ofECM fungi were previously documented in the Seychelles(Ashford & Allaway, 1985).

The aims of this study were to document the above- andbelow-ground diversity of ECM fungi of both the indigenous

Intsia bijuga

(Caesalpiniaceae) and the endemic

Vateriopsisseychellarum

(Dipterocarpaceae) as well as the introduced

Eucalyptus robusta

and

Pinus caribea

in the Seychelles. Usingmolecular sequence data, it is shown that ECM communitiesof the native plants are species-poor and overlap with symbiontsof the introduced

E. robusta

, but not

P. caribea

.

Materials and Methods

ECM host plants

Intsia bijuga

(Colebr.) Kuntze is an indigenous, highly valuedtimber tree that inhabits particularly the coastal areas (up to

c

. 200 m) of the granitic islands of the Seychelles.

Intsia bijuga

is distributed from Southeast Asia to the Mascarenes,Tanzania, New Guinea, Fiji and Samoa (ILDIS, 2001).Several isolated patches of a few clumped trees have remainedin the Seychelles.


Heim, a Seychellesendemic hardwood, was virtually extinct by the early 20th


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century as a result of extensive logging. Later, a small naturalstand was discovered at L’Abondance in a less accessiblemountainous area. From this site, several seedlings werecollected and outplanted at Casse Dent, where a single treehas survived. More recently, seedlings from this single tree werecollected, pregrown in nurseries, and outplanted in mountainslopes at Le Niol and Sans Soucis, but with little success.

To prevent erosion and to increase timber production,experimental plantations of exotic forestry trees were establishedin 1977. Seeds of

Eucalyptus robusta

Sm. were introduced fromAustralia and sown in containers in the Seychelles. Contain-erized

Pinus caribea

L. seedlings were imported from Kenya,where they most likely received their ECM symbionts. Seed-lings of both the introduced and native trees were raised ina nursery at Sans Soucis Forestry Station (W. Andre, pers.comm.). Subsequently, a few small plantations of

E. robusta

and

P. caribea

were established in eroded mountain slopes.Both species form declining stands and display no evidence ofnaturalization.

Sampling

Details of study sites and sample size are given in Table 1.ECM fungi associated with

V. seychellarum

and

I. bijuga

werestudied in most major native stands in Mahé (Anse Major,L’Abondance and North Point) and Praslin islands (Vallée deMai) (Supplementary Material, Fig. S1). To estimate thecontinuity of ECM fungal communities, a few experimentalplantations of

V. seychellarum

(Le Niol, Sans Soucis) weresampled. In addition, a single planted

V. seychellarum

adultand surrounding seedlings were studied in a tea plantation atCasse Dent. The introduced

P. caribea

and

E. robusta

weresampled in the largest plantation at Le Niol Forestry Stationto document potential host shifts of ECM fungi between thenative and introduced trees.

Fruit-body surveys and root tip sampling were performedsimultaneously once or twice in each stand between 28 Februaryand 15 March, 2006. To find resupinate and hypogeous fruitingtaxa, dead wood and litter were carefully turned over. To studythe effect of microtopography on ECM fungal communitycomposition, root samples (15

×

15 cm to 5 cm depth;

n

=

5–24 per site) were taken from different microsites (i.e. humus,mineral soil, woody debris and decayed coconuts). From eachsample, roots were manually separated from adhering soil andcut into approx. 3 cm fragments. Twenty randomly selectedfragments per root sample (comprising

c

. 200–400 root tips)were mounted into 10 ml 1% CTAB extraction buffer(100 m

M

Tris-HCl (pH 8.0), 1.4

M

NaCl, 20 m

M

EDTA, 1%cetyl trimethyl ammonium bromide) to preserve the DNAand integrity of ECM roots. Within 6 wk, ECM root tipswere assigned to morphotypes and anatomotypes followingAgerer (1991), and photographed using Axiovision 4.0 soft-ware (Carl Zeiss Vision, Munich, Germany). Plan views wererecorded at

×

10 to

×

50 magnification and mantle structure

was recorded at

×

1000 magnification with Normarski differ-ential interference contrast.

Molecular typing

At least two root tips of each anatomotype per study site andpieces of selected fruit-bodies (Table 2) were subjected toDNA extraction using a High Pure PCR Template PreparationKit for Isolation of Nucleic Acids from Mammalian Tissue(Roche Applied Science, Indianapolis, IN, USA). The rDNAinternal transcribed spacer (ITS) and nuclear large subunit(nLSU) were amplified as described in Tedersoo

et al

. (2006)using primers ITS1F (5

′

-cttggtcatttagaggaagtaa-3

′

) and TW13(5

′

-ggtccgtgtttcaagacg-3′). DNA extracts producing multiplePCR products were reamplified using the primer ITS1F anda newly designed homobasidiomycete-specific primer ITS4B1(5′-caagrgacttrtacacggtcca-3′), which is a modification fromITS4B (Gardes & Bruns, 1996). PCR products were purifiedusing Exo-Sap enzymes (Sigma, St Louis, MO, USA). Sequencingwas performed using primers ITS4 (5′-tcctccgcttattgatatgc-3′)and/or ITS5 (5′-ggaagtaaaagtcgtaacaagg-3′) for the ITSregion; and ctb6 (5′-gcatatcaataagcggagg-3′) and/or TW14(5′-gctatcctgagggaaacttc-3′) for the nLSU. Contigs wereassembled using Sequencher 4.0 (GeneCodes Corp., AnnArbor, MI, USA). A value of 97.0% ITS region identity(excluding flanking 18S and 28S rDNA sequences) was usedas a DNA barcoding criterion (Tedersoo et al., 2003). Allunique sequences were submitted to EMBL (accession nosAM412253-AM412304). BlastN and fasta3 searches wereperformed against public sequence databases NCBI, EMBLand UNITE (Kõljalg et al., 2005) to provide at least tentativeidentification for the ECM fungi.

Phylogenetic analyses

To determine the phylogenetic affinities of two ECMascomycetes from the Seychelles, 75 nLSU sequences ofidentified fruit-bodies or cultures were downloaded from theNCBI database. In addition, an nLSU sequence of NothofagusECM from Tasmania (Tedersoo et al., 2007) was included.The sequences were aligned using Mafft ver. 5.681 (Katohet al., 2005) and corrected manually. The final matrixcomprised 78 sequences and 1442 characters. Parsimonyanalyses were performed using PAUP ver. 4.0d81 (Swofford,2002) with 1000 heuristic search replicates, tree bisection-reconnection (TBR) branch swapping and gaps as missingcharacters. Representatives of Orbiliomycetes and Pezizomyceteswere used as outgroup taxa. Bootstrap support was calculatedbased on 10 random sequence addition replicates and 500bootstrap replicates. Bayesian analyses were performed asimplemented in MrBayes version 3.1.1 (Ronquist &Huelsenbeck, 2003) with 200 000 generations, GTR + I + Gsubstitution model (as retrieved from MrModeltest ver. 2.2;Nylander, 2004) and 20 000 as the ‘burn in’ value.

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Table 1 Characteristics of study sites in the Seychelles (ectomycorrhizal hosts are indicated in bold)

Study siteaGeocode,altitude (m asl) Vegetation Soil type nb

Anse Major steep coastal shrubland

4º37.3′S, 55º23.5′E; 40 m

Intsia bijuga (Colebr.) Kuntze (six scattered trees, 20–50 yr), Humus patchily accumulated in cracks of granite bedrock

6 (10)Nephrosperma vanhoutteana (Wendl. ex van Houtt) Balf.,Chrysobalanus icaco L., Alstonia macrophylla Wallich ex Don,Dicranopteris linearis (Burm.)

Casse Dent tea plantation 4º39.3′S, 55º26.4′E; 410 m

Camellia sinensis (L.) Kuntze; Vateriopsis seychellarum Heim (a single 40-yr-old tree with tens of seedlings), no herb layerby recalcitrant leaves of V. seychellarum

Ploughed soil (formerly yellow laterites) patchily covered

Old tree, 10 (10),seedlings, 5 (5)

L’Abondance undisturbed submontane forest

4º41.3′S, 55º29.1′E; 480 m

V. seychellarum (c. 15 trees, 20–200 yr), Pandanus spp., Northea hornei (MM Hartog) Pierre, Cinnamomum verum J.Presl, abundant mosses

Thin litter and humus layer on coarse quartz sand

16 (16)

Le Niol eucalypt plantation 4º37.5′S, 55º25.9′E; 210 m

Eucalyptus robusta Sm. (c. 200 trees, 29 yr), C. verum, Dillenia Strongly eroded yellow lateritic soils with a thin humus horizon

10 (12)ferruginea Heim, C. icaco, D. linearis

Le Niol pine plantation 4º37.5′S, 55º25.8′E; 180 m

Pinus caribea L. (c. 1000 trees, 29 yr), Sandoricum koetjape Merrill, sparse herb layer

Yellow lateritic soils with a thin humus horizon and thick litter layer

11 (12)

Le Niol Vateriopsis plantation 4º37.6′S, 55º25.8′E; 180 m

S. koetjape, V. seychellarum (c. 20 trees, 2–20 yr), Pandanus spp., Paraserianthes falcataria (L.) Niels, C. verum, sparse herblayer

Eroded brown soil with patches of organic matter

Saplings, 9 (12)seedlings, 5 (5)

North Point coastal forest 4º33.7′S, 55º26.3′E; 30 m

I. bijuga (five clumped trees, 25–50 yr), Albizia lebbeck (L.) Benth., C. verum, A. macrophylla, Calophyllum inophyllum L.,Cocos nucifera L.

Eroded yellow to yellow-brown soils with patchy humus layer

18 (24)

Sans Soucis forestry plantation in steep slope

4º38.4′S, 55º26.9′E; 300 m

S. koetjape, V. seychellarum (two trees, 15 yr) Eroded red-brown soils with little organic matter

5 (10)

Vallée de Mai, Unesco World Heritage Site

4º20.0′S, 55º44.0′E; 180 m

Lodoicea maldivica (JF Gmelin) Persoon, Pandanus spp., I. bijuga (14 trees in 5 clumps, 25–80 yr), Psidium cattleianumSabine. For more details, see Fleischmann et al. (2005)

Yellow to red lateritic soils with patchyhumus and litter layers

19 (24)

aAll sites are on Mahé island, except Vallée de Mai, which is on Praslin;bn, number of root samples that included living ectomycorrhizas. The number of root samples actually taken is indicated in parentheses.

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Statistical analyses

A computer program EstimateS, version 7. (Colwell, 2004),was used to calculate species accumulation (rarefaction)curves and the minimum richness estimators, Chao2 andJackknife2 (Gotelli & Colwell, 2001). These functions wereused to predict the sufficiency of sample size and to estimatethe proportion of unseen species at different sites and in theSeychelles in general, using root samples and sites as samplingunits, respectively. The sampling units were sampled randomlywithout replacement using 1000 replications. Confidenceintervals were calculated for the rarefaction curve at the wholecommunity level as implemented in Colwell (2004).

To study the relative effect of soil microsites on ECM fungalcommunity composition, detrended correspondence analysis(DCA) was performed using individual root samples, binary-transformed species and downweighted rare species. Theeffects of host species, host population size, soil type, altitudeand geographical distance on the ECM fungal communitycomposition were determined at the site level. Above- andbelow-ground data were pooled for the latter analysis andbinary-transformed. Both ordination analyses were per-formed in PC-Ord (McCune & Mefford, 1997).

Results

Diversity of ECM fungi

Anatomotyping integrated with sequencing revealed 30 speciesof ECM fungi on root tips of the Seychelles’ native trees(Table 3). Only Scleroderma sp1 and sp3 (North Point) wereindistinguishable based on anatomical characters. Additionalfruit-body surveys in native forests revealed 13 species(including eight Tomentella spp.), of which Tomentella sp.

(TU103641) and Tomentella fibrosa (Berk. & M.A. Curtis)Kõljalg remained undetected below ground (Table 2). V.seychellarum and I. bijuga hosted 18 and 15 fungal species onroot tips, respectively, whereas the introduced E. robusta andP. caribea were associated with seven and three species,respectively (Table 4). P. caribea shared no ECM fungalspecies with other hosts, whereas E. robusta had three speciesin common with V. seychellarum and two species in commonwith I. bijuga. Despite greater sampling effort, the two nativehosts shared only three fungal species. All species associatedwith native trees had ITS sequence identities of less than90.4% to any published sequences in databases (Table 3).

The minimum richness estimators Chao2 and Jackknife2predicted that 51.2 and 57.4 species, respectively, of ECMfungi are associated with native trees of the Seychelles (Fig. 1).The Chao2 estimator curve was descending, whereas theJackknife2 curve was steeply rising when all plots were sam-pled in random order. Because of the large variation in Chao2richness estimates, probably resulting from a relatively smallnumber of samples and species, its values are considered lessreliable and hence are not reported further. The only naturalstand of V. seychellarum at L’Abondance supported the highestnumber of ECM fungal species (15 spp. in 16 samples; Table 4).Jackknife2 estimated the minimum richness of 29.5 speciesfor this site. Jackknife2 and rarefaction curves were steeplyrising with increasing numbers of samples at L’Abondance(Fig. 2), indicating insufficient sampling effort. At other sites,however, the observed species richness ranged from one toeight, resulting in the leveling off of both rarefaction andestimator curves (Fig. 2).

Small plantations of V. seychellarum were established byreplanting current-year seedlings from Casse Dent. At thissite, seedlings were colonized by four species of ECM fungi intotal (mean, 1.4; range, one and two species per seedling),

Table 2 List of fruit-bodies found in the Seychelles and sequenced for this study

Species Herbarium codea Locality EMBL accession no.

Boletellus sp.b TU105073 Vallée de Mai, Praslin AM412293Coltriciella dependensb TU103611 Casse Dent, Mahé AM412254Coltricia aff. oblectansb TU105089 Vallée de Mai, Praslin AM412245Coltricia aff. oblectansc TU103621 Le Niol, Mahé AM412246Scleroderma sp.b,c TU103614 Le Niol, Mahé AM412304Thelephoraceae sp.c TU105081 Danzil, Mahé AM412302Tomentella fibrosa TU105118 Vallée de Mai, Praslin AM412301Tomentella sp.b TU103690 Anse Major, Mahé AM412294Tomentella sp.b TU105090 Vallée de Mai, Praslin AM412295Tomentella sp.b TU105130 Vallée de Mai, Praslin AM412296Tomentella sp.b TU103595 North Point, Mahé AM412297Tomentella sp.b TU105060 North Point, Mahé AM412298Tomentella sp.b TU105058 North Point, Mahé AM412299Tomentella sp.b TU103582 North Point, Mahé AM412300Tomentella sp. TU103641 North Point, Mahé AM412303

aTU, acronym for the Herbarium of the Natural History Museum of the University of Tartu, Estonia.bAlso found below ground.cFound only in a nonnative stand.

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including the three most common species from the mothertree (five species in 10 samples; Table 4). Saplings replanted atisolated sites were colonized by the same fungal speciespresent at Casse Dent, except for hymenochaetoid sp3, whichwas shared with L’Abondance, and thelephoroid sp8, a uniquespecies. However, the replanted saplings had retained onlyone to three ECM fungal species per plantation, indicatinglocal extinction of several taxa.

Community composition of ECM fungi

On a fine scale, the dominant fungal species occurred in mostroot samples of a particular site, independent of microtopo-

graphy (nonsignificant Fishers’s exact tests, not shown).Accordingly, DCA failed to detect any effect of forestmicrosite on ECM community composition (not shown). Atthe site level, ECM host species, altitude, soil type andlongitude were the parameters most influential on ECMfungal community composition. These variables were stronglycorrelated with the main axes of DCA (Fig. 3).

Different fungal species dominated the study sites (Table 4).Russuloid sp. was the most frequent taxon on V. seychellarumat L’Abondance and in the E. robusta plantation. Sclerodermasp2. dominated both the adult V. seychellarum and its seedlingsat Casse Dent, and 2-yr-old seedlings at the Le Niol forestryplantation. Thelephoroid sp14, sp11 and sp7 were the most

Table 3 Identification of ectomycorrhizal fungi on root tips based on blastN or fasta3 queries against the public sequence databases, EMBL, NCBI and UNITE (all sequence matches are based on full-length ITS region, unless otherwise indicated)

Species EMBL accession no.

Best ITS match in public sequence databases

Species % identities

Hymenochaetoid sp1a AM412255 Coltricia perennis DQ234559 77.0b

Hymenochaetoid sp2 AM412260 Coltriciella pusilla AY059060 97.8c

Hymenochaetoid sp3 AM412258 Coltricia perennis DQ234559 78.9b

Hymenochaetoid sp4a AM412257 Coltricia perennis DQ234559 72.1b

Boletellus sp.a AM412261 Boletellus mirabilis AF335451 73.3b

Boletoid sp3 AM412262 Boletellus mirabilis AF335451 73.8Boletoid sp5 AM412263 Boletus aereus DQ131619 75.9b

Boletoid sp6 AM412264 Boletus erythropus DQ131634 76.4b

Cantharelloid sp1 AM412265 Clavulina cristata AJ889929 79.7Cortinarius sp1 AM412266 Cortinarius teraturgus AF389151 89.8Cortinarius sp3 AM412267 Cortinarius teraturgus AF389151 90.9Cortinarius sp4 AM412268 Cortinarius acutovelatus AY083175 90.3Cortinarius sp5 AM412269 Cortinarius caninus U56024 83.2Gomphoid sp1 AM412270 Ramaria sp. DQ36508 76.8b

Inocybe sp1 AM412271 Inocybe lanuginosa DQ357905 78.8Pisolithus sp1 AM412272 Pisolithus tinctorius AF374634 85.7Rhizopogon sp1 AM412273 Rhizopogon succosus AF062933 95.9Rhizopogon sp2 AM412274 Rhizopogon fuscorubens AF058313 98.5Russuloid sp1 AM412275 Gymnomyces fallax AY239349 90.1Scleroderma sp1 AM412276 Scleroderma cepa DQ453694 75.7Scleroderma sp2a AM412277 Scleroderma cepa DQ453694 87.1Scleroderma sp3 AM412278 Scleroderma cepa DQ453694 87.5Sordariomycetes sp1 AM412279 Thielavia hyrcaniae AJ271581 79.7Sordariomycetes sp2 AM412280 Chaetosphaeria preussii AF178561 80.7Thelephoroid sp1 AM412281 Thelephora penicillata U83484 87.2Thelephoroid sp3a AM412288 Tomentella sublilacina AJ889976 87.2Thelephoroid sp7a AM412289 Tomentella sublilacina AJ889976 89.5Thelephoroid sp8 AM412290 Tomentella sublilacina AJ889976 89.3Thelephoroid sp9 AM412291 Tomentella bryophila AJ889981 87.5Thelephoroid sp10 AM412282 Tomentella bryophila AJ889981 87.5Thelephoroid sp11a AM412283 Tomentella bryophila AJ889981 86.7Thelephoroid sp14a AM412284 Thelephora caryophyllea AJ889980 87.6Thelephoroid sp15a AM412285 Tomentella bryophila AF272908 86.3Thelephoroid sp16 AM412286 Tomentella bryophila AJ889981 87.4Thelephoroid sp18a AM412287 Thelephora caryophyllea AJ889980 85.6

aAlso found as fruit-bodies.bBased on partial ITS match.cBased on nLSU match.


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frequent taxa on I. bijuga at North Point, Vallée de Mai andAnse Major, respectively. Pisolithus sp1. was the most frequentspecies on root tips of P. caribea. In general, thelephoroid cladewas the most species-rich taxon on I. bijuga and V. seychellarum(seven and six spp., respectively), followed by the boletoid cladeon I. bijuga (five spp.) and the euagaric and hymenochaetoidclades on V. seychellarum (three spp. for both). V. seychellarumhosted two species of ascomycetes, which had a thin, blackpseudoparenchymatous mantle (Fig. S2, Text S1). Several roottips produced identical sequences, and phylogenetic analysesplaced these taxa within Sordariomycetes, particularly Annu-

latascaceae (Fig. 4). It is noteworthy that V. seychellarumformed an additional ECM anatomotype (sequence accessionAM412286) with large, capitate cystidia that correspond tothe description of ‘conidiophores’ in the enigmatic anamorphgenus Riessiella (cf. Jülich, 1985). Fasta3 searches against theEMBL sequence database suggested its placement within thegenus Tomentella (Table 3), which was supported by phylo-genetic analyses (not shown, but available upon request).

Of the 11 spp. that were shared between at least two studysites, four taxa displayed intraspecific ITS variation within asite (mean, 0.14%; range, 0–0.52%) and six had ITS sequence

Table 4 Proportion of root samples colonized by species of ectomycorrhizal fungi in study sites in the Seychelles

Species

Intsia bijuga Vateriopsis seychellarumEucalyptusrobusta

Pinus caribea

AnseMajor

NorthPoint

Valléede Mai L’Abondance

Casse Dent

Sans Soucis

Le Niol

40 yr 1 yr 20 yr 2 yr Le Niol Le Niol

Thelephoroid sp7 0.67a Fb 0.16a

Boletellus sp1 0.50 FBoletoid sp3 0.33Cortinarius sp1 0.17 0.05 0.10Hymenochaetoid sp2 0.17Thelephoroid sp14 0.61a 0.63a

Thelephoroid sp18 0.56a 0.06 0.30 0.77Thelephoroid sp3 0.17a

Scleroderma sp1 0.06Scleroderma sp3 0.06 0.10Thelephoroid sp11 F 0.68a

Hymenochaetoid sp4 0.26a 0.06Thelephoroid sp9 0.16 0.19 0.40 0.40 1.00 1.00Thelephoroid sp15 0.05a

Boletoid sp6 0.05 0.80Russuloid sp1 0.94 0.70 0.20Cortinarius sp4 0.44Cortinarius sp5 0.31Sordariomycetes sp1 0.25Thelephoroid sp1 0.25Cantharelloid sp1 0.19 0.20Hymenochaetoid sp1 F 0.06 0.10a

Thelephoroid sp16 0.06Hymenochaetoid sp3 0.06 0.11Boletoid sp5 0.06Thelephoroid sp10 0.06Sordariomycetes sp2 0.06 0.10a

Scleroderma sp2 0.80 0.60 0.77Inocybe sp1 0.20Thelephoroid sp8 0.60 0.30Gomphoid sp1 0.10Cortinarius sp3 0.73Pisolithus sp1 0.18Rhizopogon sp2 0.09Rhizopogon sp1Thelephoroid sp20 FTomentella fibrosa FColtricia aff. oblectans F

aFound also as fruit-bodies.bF, found only as fruit-bodies.


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variation among sites (mean, 0.23%; range, 0–1.70%). Inparticular, the ITS sequences of ECM fungi in the E. robustaplantation were invariable (up to 0.18% sequence variation)and always identical to the ITS sequences of conspecific taxaassociated with native trees, providing further evidence fortheir common origin.

Discussion

Low ECM fungal diversity was demonstrated in the Seychellescompared with temperate forests (Horton & Bruns, 2001)and tropical forests of Dipterocarpaceae (Sirikantaramas et al.,2003; Moyersoen, 2006) and Sarcolaenaceae (Ducousso et al.,2004) that potentially support hundreds of fungal species.Neither I. bijuga nor V. seychellarum formed ECM associationswith narrow fungal lineages, in contrast to P. grandis, whichinhabits extremely nutrient-rich coral cays (Chambers et al.,2005). DCA suggested that host species, soil type, altitudeand longitude affected the community composition of ECMfungi most strongly at the site level. However, these variableswere strongly related, rendering the actual causal mechanismsuncertain. In this study, we were unable to detect micro-habitat preference among ECM fungi, which is in contrast to

Fig. 3 Detrended correspondence analysis (DCA) ordination demonstrating the relative effects of host, soil and geographical variables on the ectomycorrhizal (ECM) fungal communities of the study sites. Axes 1 and 2 explain 31.6 and 2.0% of the variation, respectively. Open triangles, Vateriopsis seychellarum adults; inverted triangles, V. seychellarum seedlings; closed triangles, Intsia bijuga; open circle, Eucalyptus robusta. Pinus caribea was not included in this analysis, because it shared no ECM fungal species with other hosts. For clarity, placement of species is not shown. The effects of latitude and host population size are not shown because they lie below the threshold of 2%.

Fig. 1 Minimum species richness estimates and species accumulation curves of ectomycorrhizal (ECM) fungi on native trees with increasing sample size. Open circles, Chao2 richness estimates; closed circles, Jackknife2 richness estimates; triangles, species accumulation (rarefaction) curve; dotted lines, 95% confidence interval of the species accumulation curve. Sites were used as sampling units and sampled randomly without replacement (n = 1000).

Fig. 2 Minimum species richness estimates and species accumulation curves of ectomycorrhizal (ECM) fungi in study sites with increasing sample size. (a) Species accumulation (rarefaction) curves; (b) Jackknife2 curves. Open triangles, Vateriopsis seychellarum; inverted triangles, seedlings; closed triangles, Intsia bijuga; open circles, Eucalyptus robusta; closed circles, Pinus caribea. For clarity, site names are not shown. Root samples were used as sampling units and sampled randomly without replacement.


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boreal forests (Goodman & Trofymow, 1998; Tedersoo et al.,2003). This further indicates that specialist fungi areuncommon in the Seychelles. The lack of niche different-iation supports the coexistence of fewer species (Lomolinoet al., 2006). The low observed alpha diversity (one to 15species per site) probably results from low overall gammadiversity (species pool), which could be related to therestricted area of host populations and the Seychelles’ long-

term isolation from neighboring continents as predictedby the equilibrium theory of island biogeography (MacArthur& Wilson, 1967; Lomolino et al., 2006).

In agreement with the low observed richness, the rarefac-tion curves suggest that most sites were exhaustively sampled,except for L’Abondance, where at least twice as many speciesprobably occur. Similarly, the site-based rarefaction curve didnot level off, indicating low fungal species overlap between

Fig. 4 Bayesian tree demonstrating the phylogenetic placement of three ectomycorrhizal (ECM)-forming Sordariomycete species (in bold) among euascomycetes. Parsimony bootstrap support (above branches; threshold, 70%) and Bayesian posterior probabilities (below branches; threshold, 95%) are indicated.


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isolated stands. Based on recent, more widespread distributionof ECM host trees before deforestation and during periods oflow sea level (Fleischmann et al., 2003), we hypothesize thatmany ECM fungi became extinct during deforestation in the19th and early 20th centuries. Thus, we believe that the esti-mated value of 51–57 species likely represents a substantialunderestimate of the historical species richness.

Certain lizard and snail taxa rapidly diversified in the graniticislands of the Seychelles during the loss of land connection inthe Pleistocene (Radtkey, 1996; Gerlach, 1999). Contrary to thesefindings, the ITS region provided no evidence for speciationof ECM fungal populations among islands and host plants,except for thelephoroid sp9, which had 1.70% ITS sequencedifference in Mahé vs Praslin islands. The low intraspecificITS diversity in the Seychelles’ ECM fungi is comparable tothe low ITS variation in temperate woodlands of the northernand southern hemispheres at the local scale (Glen et al., 2001;Horton, 2002). Moreover, the interspecific ITS sequencevariation was high (at least 12.0%) and sister relations amongtaxa were not detected. Notable exceptions include Sordario-mycetes spp. (Fig. 4) and Scleroderma spp. (M. Binder, pers.comm.) that are, however, poorly covered with sequence data.We believe that the lack of speciation in ECM fungi is bestexplained by widespread distribution of host trees before therise of sea level and deforestation. Alternatively, sister species mayhave gone extinct during the loss of habitat and decline inpopulation size. The biogeographic relations and dispersal vsvicariance origin of the Seychelles’ fungi remain to be establishedwhen DNA sequence data accumulates from related continents.

Intsia bijuga and V. seychellarum shared only three fungalspecies, which may be attributed to either host or habitateffects. Namely, I. bijuga occupies coastal areas with seasonalclimate up to c. 200 m above sea level, whereas V. seychellarumhas persisted and is planted in more humid submontanehabitats. Nevertheless, Alexander et al. (1992) demonstrated thatECM fungi from adult dipterocarps colonized and benefitedthe seedlings of Intsia palembanica Miq. in Malaysia, indicatingthat at least some fungal species are functionally compatiblewith both plant families. The ECM Caesalpiniaceae, Diptero-carpaceae and Uapacaceae co-occur in miombos and seasonalforests of continental Africa (Lawton, 1978), where they havelikely evolved and subsequently dispersed to other continentsor drifted away during the break-up of Gondwana (Dayanan-dan et al., 1999; Lavin et al., 2005). Extremely poor dispersalabilities and presence of ECM symbiosis explain best thetransatlantic disjunction of Dipterocarpaceae, suggesting theirdevelopment in the Early Cretaceous (135 mya; Moyersoen,2006). Based on dated phylogenies and fossil data, Caes-alpiniaceae emerged in the Latest Cretaceous or Early Tertiary,whereas ECM lineages evolved around 30 mya and later(Lavin et al., 2005). Because of the shared habitats and at leastsome shared fungal species, we hypothesize that Caesalpiniaceaeand Uapacaceae likely obtained most of their ECM fungifrom dipterocarps in Africa.

The introduced Eucalyptus and Pinus hosted few ECMsymbionts in the Seychelles, although these genera associatewith hundreds or even thousands of ECM fungi in theirnatural range (May & Simpson, 1997; Horton & Bruns,2001). Dunstan et al. (1998) listed 99 species of ECM fungiassociated with exotic pine plantations worldwide, but in thepresent study P. caribea hosted only three species of ECMfungi, two Rhizopogon and a Pisolithus species, that were neverobserved on other hosts in the Seychelles. P. caribea mostlikely obtained these host-specific fungi (Molina & Trappe,1982; Martin et al., 2002) from a nursery in Kenya. Based onfruit-body surveys, Rhizopogon spp. are among the most com-mon introduced fungi in exotic pine plantations (Mikola,1969; Dunstan et al., 1998; Giachini et al., 2004). With a fewexceptions (Valenzuela et al., 1999; Orlovich & Cairney, 2004),fruit-bodies of pine-associated ECM fungi are not usuallyobserved in native plant communities. Moreover, Pinus spp.seem unable to associate with native fungi in exotic habitats,resulting in death when cointroduced ECM fungi are lacking(Mikola, 1970). In contrast to pine, E. robusta most likelyreceived all its ECM symbionts from the native trees in thenursery at Sans Soucis or from other native stands severalkilometers away. Namely, E. robusta was introduced as seedsand its plantations shared most of the ECM fungal specieswith either V. seychellarum or I. bijuga in native and plantedstands. In addition, none of the ECM fungi on E. robusta atLe Niol are known from other exotic eucalypt plantations(Bougher & Lebel, 2001; Giachini et al., 2004; Diez, 2005)or from Australian eucalypt woodlands (May & Simpson(1997); Bougher & Lebel, 2001). Compared with pines,probably greater similarity in physiological features renderseucalypts more compatible with fungi native to dipterocarpsand caesalpinioids. Associations with indigenous ECM fungimay potentially facilitate the invasion of eucalypts in nativeplant communities elsewhere, but this remains to be proven.

The thelephoroid, boletoid and euagaric clades were themost species-rich taxa on root tips of the Seychelles’ nativeplants. This supports the results of Sirikantaramas et al. (2003),who found thelephoroid fungi dominating on roots of Dipte-rocarpaceae in Malaysia. The high observed abundance ofresupinate fruit-bodies of thelephoroid fungi at the beginningof the dry season suggests their relatively high drought tolerance.Because of the formation of resupinate fruit-bodies on theunderside of debris, thelephoroid fungi are often overlooked.On the contrary, members of the boletoid and euagaric cladesare strongly represented in tropical fruit-body surveys inAfrica and Southeast Asia (Verbeken & Buyck, 2002; Watlinget al., 2002), suggesting their commonness in the tropics.Among the euagarics, however, Cortinarius spp. are consid-ered rare in tropical forests (Peintner et al., 2003), which is incontrast to the finding of four Cortinarius species in thisstudy. The hymenochaetoid clade includes five species ofColtricia/Coltriciella in the Seychelles (Tedersoo et al., 2007).Although seldom reported in below-ground ECM fungal


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communities, Coltricia and Coltriciella are common in thetropics, particularly in Southeast Asia and Australasia (Cor-ner, 1991), but also in South America (Aime et al., 2003). TheECM mantle structure of two sordariomycete species resem-bled most the black unidentified ascomycetes found on Salixsp. in Washington state, USA (Trowbridge & Jumpponen,2004; J. Trowbridge & A. Jumpponen, pers. comm.) and onShorea leprosula in Malaysia (type T12; Lee et al., 1997). Sur-prisingly, the sebacinoid, athelioid and russuloid clades ofbasidiomycetes and Cenococcum geophilum (Ascomycota), whichare common members of ECM fungal communities in thenorthern hemisphere, were rare or lacking in the Seychelles.In particular, the russuloid clade is most diverse in tropicalforests based on fruit-body surveys (Verbeken & Buyck,2002; Watling et al., 2002). We hypothesize that similarly tothe low alpha and gamma diversity, peculiarities in fungalcommunities can be attributed to the long-term isolation ofthe Seychelles and small host populations that permit thepersistence of the most easily dispersed, stress-tolerant andgeneralist species.

In conclusion, communities of the Seychelles’ ECM fungiare species-poor and the community composition is biasedcompared with other tropical and temperate ecosystems. Thisprobably results from small host population sizes and thelong-term isolation of the Seychelles. To conserve the biodi-versity of ECM fungi and associated microbes, all nativeECM stands should be protected. Attempts to propagatenative ECM trees should focus on coinoculation of soil fromnative stands to improve seedling establishment (Mikola,1970). Low host specificity and high risk of cointroduction ofsoil microbes make native microbial community studies ofhigh importance. Functional compatibility and microevolu-tion between introduced plants and native fungi (and viceversa) require further studies.

Acknowledgements

W. Andre, L. Chong-Seng, D. Dogley, C. Küffer, J. Mougaland staff of the Forestry Section (Ministry of Environmentand National Resources, the Seychelles) are thanked for helpduring preparation of the project, information, and supportin the Seychelles. We thank M. Binder, A. Jumpponen, U.Peintner and J. Trowbridge for discussions on certain taxa.Constructive comments from three anonymous referees andF. Martin greatly improved the manuscript. The study wasfunded by Estonian Science Foundation grant no. 6606.

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Supplementary Material

The following supplementary material is available for thisarticle online:

Fig. S1 Geographic position of granitic islands of the Seychellesand location of study sites in Mahé and Praslin islands.

Fig. S2 Ectomycorrhizas of Sordariomycetes on Vateriopsisseychellarum at L’Abondance, the Seychelles.

Text S1 Descriptions of Sordariomycete ectomycorrhizas.

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ectomycorrhizal fungi of the seychelles: diversity patterns

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