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Pakaraimaea dipterocarpacea is ectomycorrhizal, indicating an ancient Gondwanaland origin for the ectomycorrhizal habit in Dipterocarpaceae Blackwell Publishing Ltd

Bernard Moyersoen 4 Place Ste Véronique, B−4000 Liège, Belgium

Summary Author for correspondence: Bernard Moyersoen Tel: +32 4 86752707 Email: [email protected] Received: 25 April 2005 Accepted: 29 June 2006

• The consistent association of Paleotropical Dipterocarpaceae with ectomycorrhizal (ECM) fungi suggests that ECM status is an ancestral character in the family. Despite its distinctive morphology, Pakaraimaea dipterocarpacea, a Neotropical Dipterocarpaceae endemic to the Guayana Region, is phylogenetically related to the Paleotropical Dipterocarpaceae. The confirmation of P. dipterocarpacea ECM status would indicate that Paleotropical Dipterocarpaceae and P. dipterocarpacea probably had a common ECM ancestor. • Mycorrhizal colonization of P. dipterocarpacea was assessed, and ECMs were recorded using histological and molecular methods. • P. dipterocarpacea was highly colonized by typical ECMs, and several ECM fungal taxa belonging to Clavulinaceae, Sebacinaceae, Cortinariaceae and Amanitaceae were identified. • This paper provides the first documented evidence of ECM in a neotropical genus of Dipterocarpaceae and indicates that ECMs possibly evolved in Gondwana in ancestors of Dipterocarpaceae before the separation of South America from Africa by the Atlantic, c. 135 million years ago. The observation of Sebacinaceae and Clavulinaceae suggests that broad host range fungi are important components of P. dipterocarpacea ECM communities. Key words: biogeography, Dipterocarpaceae, ectomycorrhiza, Guayana Region, Neotropical, Pakaraimaea dipterocarpacea. New Phytologist (2006) 172: 753–762 © The Authors (2006). Journal compilation © New Phytologist (2006) doi: 10.1111/j.1469-8137.2006.01860.x

Introduction The vegetation of the Guayana Region, in north-eastern South America, occurs on a land surface occupied by the very ancient, geologically stable Guayana Shield, overlain in extensive areas from south-eastern Colombia to western Guayana by the sedimentary rocks of the Precambrian Roraima Group. It is characterized by a flora with high species richness and endemicity adapted to nutrient-poor soils derived from highly weathered parent rocks (Maguire, 1970; Berry et al., 1995). Maguire (1970) hypothesized that many species in this region are ‘ancient’ or ‘relictual’ and that their evolution was restricted by their genetic and habitat isolation.

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Among the Guayana region endemics is Pakaraimaea dipterocarpacea, a taxon related with the mostly SE Asian Dipterocarpaceae (Maguire et al., 1977; Morton et al., 1999; Ducousso et al., 2004). Ectomycorrhizal (ECM) symbiosis is an important ecological feature of Dipterocarpaceae (Lee, 1990). Despite the evolutionary implications of the possible ECM habit in Pakaraimaea (Ducousso et al., 2004; Taylor & Alexander, 2005) there is no documented record of the mycorrhizal status of this genus. The taxonomic placement of the very distinctive genus Pakaraimaea has been controversial. Maguire et al. (1977), Maury (1978) and Maguire & Ashton (1980) related this genus to the Dipterocarpaceae, whereas Kostermans (1978)

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proposed an alternative relationship with the Tiliaceae. Later, the monotypic genus Pakaraimaea was classified in the subfamily Pakaraimoideae of the Monotaceae (Kostermans, 1989). A second subfamily, Monotoideae, within the Monotaceae includes the monotypic genus Pseudomonotes from Colombia (Londoño et al., 1995) and two genera, Monotes and Marquesia, from Africa (Kostermans, 1989). Recent phylogenetic studies based on rbcL have shown that Monotaceae and Dipterocarpaceae belong to a monophyletic group also including the Sarcolaenaceae (endemic to Madagascar) and the Cistaceae (Kubitzki & Chase, 2002). The phylogenetic study of Dayanandan et al. (1999) is consistent with the proposal of Maguire et al. (1977) to classify Pakaraimoideae and Monotoideae, together with Dipterocarpaceae s.s. within the same family Dipterocarpaceae s.l. An origin of Dipterocarpaceae in Gondwana was proposed to explain the transoceanic disjunct distribution of Dipterocarpaceae s.l. (Ashton, 1969; Maguire et al., 1977; MauryLechon & Curtet, 1998); several arguments, including fossil evidence (Ashton & Gunatilleke, 1987), and the poor dispersal ability of contemporary Dipterocarpaceae (Ashton, 1982), support this hypothesis. Pakaraimaea dipterocarpacea is probably a relict of Dipterocarpaceae ancestors that migrated westward from Africa to South America and remained isolated from the rest of the family after the separation of the continents (Dayanandan et al., 1999). All Dipterocarpaceae surveyed to date are associated with ECM fungi (Högberg, 1982; Alexander & Högberg, 1986; Högberg & Pierce, 1986; Lee, 1990), and fungal families with greatest diversity in SE Asian dipterocarp forests include the Russulaceae, the Boletaceae and the Amanitaceae (Watling & Lee, 1995; Watling et al., 1996; Lee et al., 2003). A peculiarity of Dipterocarpaceae is the hypothesized narrow host range of their fungal partners (i.e. the association of a fungus species with only one plant species; Smits, 1983). This trend was considered to be an important factor in the poor regeneration success of SE Asian Dipterocarpaceae (Ashton, 1981; Smits, 1983). Ectomycorrhizal symbiosis in Dipterocarpaceae was first discovered in SE Asia, where most of the diversity in this plant group is observed (Singh, 1966). Malloch et al. (1980) suggested that Dipterocarpaceae s.s. evolved the capacity to associate with ECM only recently and independently in the Asian tropics. Subsequently, Högberg (1982) and Högberg & Pierce (1986) reported the ECM status of Monotes and Marquesia. The recent discovery of ECMs in Sarcolaenaceae demonstrated the importance of ECMs as a taxonomic feature in taxa related to the Dipterocarpaceae (Ducousso et al., 2004). The consistent ECM status in Dipterocarpaceae, Paleotropical Monotaceae and Sarcolaenaceae indicated that the ancestor of these three plant groups probably had acquired the capacity to associate with ECMs in Gondwana, prior to 88 million years ago (Mya), before the separation of Madagascar from India-Seychelles block (Ducousso et al., 2004). The separation of South America from Africa is considered to

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have started just before 135 Mya in the early Cretaceous (Pitman et al., 1993). The presence of ECMs in Pakaraimaea would place the possible evolution of ECM habit in Gondwanan ancestors of Dipterocarpaceae in the early Cretaceous, before the splitting of South America from Africa. Pakaraimaea dipterocarpacea is distributed over scattered locations of the Guayana region, at mid altitude (between 400 m and 1100 m), with a definite short dry season, occurring in the Pakaraima range of Guayana and in the Caroní river basin (Cerro Guaiquinima, Icabarú, upper and lower Río Paragua), Venezuela (Maguire et al., 1977; Maguire & Ashton, 1980). Where present, it grows along marginal savannah woodland on white sand derived from the Roraima Formation sandstone (Maguire & Steyermark, 1981). Two subspecies have been described to date: ssp. dipterocarpacea from Imbaimadai savannahs in the Pakaraima mountains, and ssp. nitida from Venezuela (Maguire & Steyermark, 1981). In this study, the ECM status of P. dipterocarpacea ssp. nitida was confirmed using a combination of molecular and anatomical evidences. A preliminary below-ground survey of ECM fungi was made to relate the fungus flora associated with P. dipterocarpacea with fungal groups commonly observed in SE Asian dipterocarp forests. Nine fungal taxa belonging mostly to broad host range ECM fungal groups including Clavulinaceae, Sebacinaceae and Cortinariaceae were recognized.

Materials and Methods Study site The 8.5 × 10 m study plot was located at 4°20′N, 61°48′W, 500 m altitude, near Icabarú, in the Caroní headwaters, Estado Bolivar, Venezuela. The location of this plot was selected on basis of the previous report of a stand of P. dipterocarpacea ssp. nitida Maguire & Steyermark in the same area (Maguire & Steyermark, 1981). The sampling expedition was conducted on 25 November 2003. No flowers or fruits were observed on P. dipterocarpacea adult trees, and individuals were identified on the basis of vegetative characters including leaves morphology and coppicing habit described by Maguire et al. (1977) and Maguire and Steyermark (1981). A total of 15 adult individuals of P. dipterocarpacea dominated the plot. Fine root sampling Fine roots were traced from four P. dipterocarpacea trees. They were also sampled in 15 10 × 10 × 20 cm soil cores collected near the 15 adult trees. Nine ECM systems recognized by eye from traced root, each one including approx. 10 tips, were directly fixed in 2% cetyltrimethylammonium bromide (CTAB) buffer in the field. The time between root tracing and fixation was less than 1 h. Additional fresh material, including

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pooled traced fine roots and the soil cores, were stored in polythene bags, transported to the laboratory and stored at 5°C before further processing. Transit was 5 h to the field laboratory and 12 h to Caracas. Evaluation of mycorrhizal colonization Mycorrhizal colonization was evaluated in Caracas, in 8-d-old traced fine roots. A subsample of fine roots of approx. 1 m total length selected randomly from pooled traced roots was cleared and stained using a phenol-free modification of Phillips & Hayman’s (1970) method to evaluate both ECM and arbuscular mycorrhizas (AM) colonization as described in Moyersoen et al. (1998). Stained root pieces were laid on microscope slides and per cent root colonization was scored using the magnified intersections method (McGonigle et al., 1990) by inspecting intersections between the microscope eyepiece cross hair and roots at ×100 magnification. ECM morphotyping Direct fixation of ECMs in the field altered their ‘habit’ (ECM mantle and attached mycelium) and prevented their morphological description. Morphotypes were recognized on basis of their habit in traced roots stored in polyethylene bags for < 1 d, using a dissecting microscope in the field laboratory. The ECM roots from soil cores stored in Caracas for maximum 14 d were carefully washed from a 27 cm3 subsample and morphotyped as above. Between six and 11 morphotypes were recognized in traced roots and in soil cores, respectively. The habit of each morphotype stored in water at 5°C for maximum 3 d was recorded using a dissecting microscope (Leica MZ6) fitted with a camera (Leica MPS60) and the anatomical features were described from mantle peels using Agerer’s (1991) method. The presence of an Hartig net was confirmed in 5 µm root sections of each morphotype previously fixed in FEA (formaldehyde, acetic acid, 70% ethanol) (5 : 5 : 90) and embedded in paraffin. A voucher of each morphotype is stored in the University of Liège Herbarium (LG). Fixation of ECM morphotypes for further molecular typing Time elapsed between root sampling and fixation in 2% CTAB buffer was less than 1 d for morphotypes from traced roots and 14 d for morphotypes from soil cores. Success rate of fungus DNA amplification was greatest for ECM samples fixed directly in the field, followed by samples stored for 1 d before fixation.

Plant Mini Kit (Qiagen SA, Courtaboeuf, France) from ECM samples including a maximum of three tips belonging to the same ECM system or morphotype. For the molecular identification of fungi, the internal transcribed spacer (ITS) region of nuclear rDNA was amplified using the primers ITS1f, ITS4b (Gardes & Bruns, 1993) and ITS1f, ITS4 (White et al., 1990). The PCR reactions were performed on a GenAmp PCR 9600 thermocycler (Perkin Elmer, Norwalk, CT, USA), using 25-µl reaction volumes each containing: 2 µl DNA template, 3 µl of 10× amplification buffer (Q.BIOgene, Montreal, Canada) containing 1.5 mM MgCl2, 0.6 µl dNTPs mix (10 mM), 0.6 µl of each primer (10 µM), 0.2 µl Taq DNA polymerase (5 U µl−1; Q.BIOgene). Cycling parameters were 1 cycle of 94°C for 3 min, 30 cycles of 94°C for 30 s, 50°C for 30 s and 72°C for 2 min, with a final extension at 72°C for 10 min. Amplification products were electrophoresed in 1% agarose gels stained with ethidium bromide and visualized under UV light. 1kb + DNA ladder (Invitrogen, Carlsbad, CA, USA) was used as marker. Controls with no DNA were included in every set of amplifications. For the molecular identification of the host plant, rbcL DNA was amplified using the primers rbcLN and rbcLR (Käss & Wink, 1997). PCR reactions were performed using 25 µl volume reaction volumes each containing: 5 µl DNA template, 3 µl of 10 × amplification buffer (Q.BIOgene, Montreal) containing 1.5 mM MgCl2, 0.6 µl dNTPs mix (10 mM), 0.6 µl of each primer (10 µM), 0.2 µl Taq DNA polymerase (5 U µl−1, Q.BIOgene, Montreal). Cycling parameters were following Käss and Wink (1997) with 1 cycle of 94°C for 2 min, 30 cycles including 94°C for 30 s, 45°C for 30 s, and 72°C for 1 min, with a final extension at 72°C for 4 min. Amplification products were visualized as for ITS products and controls with no DNA were included in every set of amplifications. rbcL restriction fragment length polymorphism (RFLP) To confirm that host identity was identical in traced roots, rbcL DNA RFLP patterns were compared between amplified ECMs. Hinf1 was chosen as the restriction enzyme (using Sequencher 4.2; Gene Codes Corporation, Ann Arbor, MI, USA) on the basis of its capacity to produce several distinctive bands on P. dipterocarpacea different from the ECM tree species Aldina, which can also be present in the same area. Ten units of the endonuclease were used to digest 20 µl of the amplified rbcL product for 1 h at 37°C. Restriction fragments were separated by electrophoresis in a 4% agarose gel stained with ethidium bromide before visualization under UV light. 100 bp DNA Mass Ladder (Invitrogen) was used as marker.

DNA extraction and polymerase chain reaction (PCR) To identify both fungus and plant partners on the same ECM tip, a combination of fungus and plant specific primers were used. Genomic DNA was extracted using QIAGEN Dneasy

ITS and rbcL sequencing and sequence analysis The ITS and rbcL PCR products were purified on a 96-well filtration system (Multiscreen-PCR plate; Millipore Corporation,

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Billerica, MA, USA). The primer pairs ITS1f, ITS4b and ITS1f, ITS4 were used to generate both strands of the ITS region. A segment of approx. 560 bp of single-stranded rbcL DNA was sequenced twice per sample using rbcLN. The sequencing reactions were run on the automated multicapillary CEQ 8000XL DNA sequencer (Beckman Coulter, Fullerton, CA, USA), using the CEQ Dye-labelled Dideoxy-Terminator Cycle Sequencing kit (Beckman Coulter), as recommended in the manufacturer’s instructions. Gels were tracked using the CEQ 8000XL sequencing program and raw data were edited using the CEQ 8000XL sequence analysis program. The ITS strands generated from both reverse and forward primer pairs were assembled with SEQUENCHER 4.2 (Gene Codes Corporation, Ann Arbor, MI, USA). Single strands generated either from forward or reverse primers were edited with SEQUENCHER 4.2 and ambiguous extremities were disregarded for posterior sequence analysis. Searches for similar ITS and rbcL sequences were conducted using BLAST at the NCBI/GenBank (http:// www.ncbi.nlm.nih.gov/). The results were compared using BLAST with UNITE database (Kõljalg et al., 2005). Singlestranded rbcL DNA was aligned with a P. dipterocarpacea sequence kindly supplied by the author (Dayanandan et al., 1999).

Results Assessment of mycorrhizal colonization Ectomycorrhizal colonization could be easily recognized in stained traced roots, and colonization was extensive. Among the 70 microscope fields scored for mycorrhizal colonization, 91% were colonized by ECMs and 9% were uncolonized. No AM structures were observed. Molecular identification of the host plant A total of 23 ECM samples were analysed to identify both host plant and fungus partners. The rbcL sequencing was performed in three of these samples. Partial rbcL sequence (528 bp) of two traced root samples (Clavulinaceae 2) (Table 1) aligned (uncorrected ‘p’ = 0.2%) with P. dipterocarpacea ssp. nitida (Ashton 001) rbcL gene sequence (Dayanandan et al., 1999, http://clone.concordia.ca/ Department/course/faculty/daya_sequence.html). Ashton 001 P. dipterocarpacea plant material was collected in the Icabarú area (P. Ashton, pers. comm.). Partial rbcL sequences from P. dipterocarpacea ECM samples are deposited in GenBank (accession numbers DQ406586, DQ406587). The third sample (collected from a soil core, without tracing of the root) aligned with Aldina latifolia: identities 388/414 (93%). A single rbcL/RFLP pattern corresponding to 75 bp, 104 bp, 145 bp, 233 bp, 269 bp and 403 bp was

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obtained from eight successfully amplified traced roots, including a sequenced P. dipterocarpacea sample. The remaining traced ectomycorrhizas that could not be amplified using rbcL primers are assumed to belong to P. dipterocarpacea. Description of ECM fungi A total of 12 different morphotypes were recognized from traced roots fixed after 1 d, and from soil cores. Six of these morphotypes, including the Aldina root sample were observed only in soil cores. Taking into account the co-occurrence of two host plants in the study plot and the difficulty of confirming host identity in most ECMs from soil cores, only the nine ECMs observed in traced P. dipterocarpacea roots (Table 1) are reported concisely here. The fungus associated with Aldina and an additional ECM sample collected in a soil core aligned best with the ITS1/ 5,8S/ITS2 region of a Tomentella sp. J54 (Tedersoo et al., 2003): identities 641/725 (88%) and 524/588 (89%). Sequence matches and morphological and anatomical descriptions of traced ECMs are described concisely in Table 1 and Figs 1 and 2. Seven ECMs could be allocated to fungal groups. Although the best sequence matches of Clavulinaceae 1 and 2 were with temperate Clavulinaceae, significant alignments were also obtained with C. dicymbetorum, C. caespitosa and C. monodiminutiva from Guyana (data not shown). No DNA could be amplified from the two unidentified ‘morphotypes 1 and 2’. Anatomical characters of unidentified ‘morphotype 1’ (cottony habit (Fig. 1e)), plectenchymatous mantle with thicker walls in outer than in deeper layers, thick-walled, simple septated, emanating hyphae, outer mantle layer with ramified lobed hyphae and elongated cystidia) were similar to Sebacinoid ECM (Selosse et al., 2002; Urban et al., 2003). Unidentified ‘morphotype 2’ (Fig. 1f) was similar to Ascomycete ECMs (Danielson, 1984; Agerer, 1988, 1999; Danielson & Pruden, 1989; Ingleby et al., 1990; Jakucs et al., 1998) for the presence of a voluminous envelop of thick-walled, simple septated, often ornamented emanating hyphae. The rhizomorphs, the presence of a superficial hyphal net and the brown colour were also similar to Telephoroid ECMs (Jakucs & Agerer, 1999, 2001). The four sequenced ECMs that could be studied morphologically (Fig. 1a–d) and anatomically presented an organized mantle. The Hartig net was complete (cf. Fig. 2 for Sebacina sp.) in all morphotypes, except for the Clavulinaceae.

Discussion ECM status of P. dipterocarpacea This study is the first demonstration of ECM habit in a neotropical Dipterocarpaceae taxon. Among the six ECM

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Research Table 1 Sequence matches and morphological and anatomical descriptions of ectomycorrhizas associated with Pakaraimaea dipterocarpacea traced roots Best aligned fungal sequence (Accession No.) (overlapping basepairs/total aligned bases) Fungal ID (Collection No.)

GenBank

UNITE

Habit

Anatomy

Clavulinaceae D44 (AJ534710) (248/266, 93%) Clavulicium delectabile (AJ534708) (387/420, 92%) Sebacinaceae sp. A (AY296253) (520/575, 90%)

Clavulina rugosa (UDB000051) (143/145, 99%) Clavulina cristata (UDB000073) (244/248, 98%)

Not recorded

Not recorded

White, smooth or woolly

Sebacina sp. (UDB000773) (464/514, 90%)

Yellowish to orange, woolly

Plectenchyma on top of a pseudoparenchyma, simple septa only, emanating hyphae (nonramified, with thick, rough wall), Hartig net incomplete Plectenchyma (net on top of a plectenchyma), simple septa only, rhizomorphs, emanating hyphae, Hartig net paraepidermic

Cortinarius sp.* (BM03M4) Inocybe 1*° (BM03M20) Inocybe 2+° (BM03M2)

Cortinarius sp. (AY040712) (320/343, 93%) Inocybe sp. (AF325665) (241/251, 96%)

C. neofurvolaesus (UDB001359) (304/319, 95%) I. aurea (UDB000612) (226/236, 96%) Inocybe petiginosa (UDB000766) (188/192, 98%)

Whitish or orange, end bent or sinuous, cottony

Amanita sp.* (BM03M21)

Amanita sp. (AB015696) (413/448, 92%)

Amanita rubescens (UDB000038) (553/605, 91%)

Not recorded

Clavulinaceae 1* (BM03M19) Clavulinaceae 2X∆ (BM03M1,22) Sebacina sp.δ° (BM03M3)

Not recorded

Whitish, often pinkish at very tip, smooth, rhizomorphs

Unidentified 1 (BM03M5)

White, nonramified, densely cottony or woolly

Unidentified 2 (BM03M7)

Brown, silvery, densely woolly

Plectenchyma, clamp connections, frequent rhizomorphs (hairy, ramified) and emanating hyphae, Hartig net paraepidermic (Fig. 2) Not recorded

Plectenchyma covered by a loose net, rhizomorphs with smooth margin, clamp connections (large, with hole) only in rhizomorphs, Hartig net paraepidermic Not recorded

Plectenchyma (net on top of a plectenchyma), emanating hyphae, elongated cystidia, simple septa only, Hartig net paraepidermic Plectenchyma or pseudoparenchyma, rhizomorphs and emanating hyphae, hyphal walls brown orange, simple septa only, Hartig net paraepidermic

Sequencing with: ITS1f*; ITS 1f, 4bX; ITS 1f, 4δ; ITS 4b+. Host identity confirmed after: rbcL sequencing∆, rbcL RFLP matching°.

morphotypes from P. dipterocarpacea traced roots that could be studied anatomically, five presented a typical Hartig net, diagnostic of ECM symbiosis (Harley & Smith, 1983). The seven sequenced fungus species identified from P. dipterocarpacea roots belonged to typical ECM fungal groups (Cortinariaceae, Amanitaceae; Hibbet et al., 2000), fungal groups that include ECM taxa (Sebacinaceae; Weiss et al., 2004) or in which ECM habit is uncertain (Clavulinaceae; Hobbie et al., 2001). The great ECM colonization suggested that ECMs are important in P. dipterocarpacea ecology. The roots of nonECM plant species co-occurring in the same plot were heavily colonized by AM fungi, and several AM fungal taxa were observed (G. Cuenca, unpublished). The lack of AM colonization in P. dipterocarpacea was not related to a lack of AM inoculum, suggesting that this species is obligately ECM. Whether ECMs are also present in the remaining neotropical Monotaceae, Pseudomonotes, still has to be reported.

Evolutionary and biogeographic implications of ECM status in P. dipterocarpacea This study on Pakaraimoideae and Ducousso et al.’s (2004) study on Sarcolaenaceae confirmed that ECM habit is an ancestral character in Dipterocarpaceae s.l. It is commonly recognized that ECM symbiosis (or the propensity to form it) evolved at least twice, once in the branches leading to the Pinaceae and once in the clade that include the Rosids and Asterid lineages of Chase et al., 1993) (Fitter & Moyersoen, 1996). The oldest fossil evidence of an ECM association involving a Pinus with a Suillus fungal species only dates from the middle Eocene (c. 50 Mya; Lepage et al., 1997). By contrast, evolutionary analysis using rDNA sequences suggest that holobasidiomycetes containing the majority of lineages with ECM fungi radiated from the early Cretaceous (c. 125– 130 Mya) after angiosperms had become an important part of the flora (Berbee & Taylor, 1993). Although the high species

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Fig. 1 Habit of Pakaraimaea dipterocarpacea ectomycorrhizal (ECM) morphotypes. (a) Clavulinaceae 2; (b) Sebacina sp.; (c) Cortinarius sp. (d) Inocybe 2; (e) unidentified 1; (f) unidentified 2. Bar, 4 mm.

diversity of dipterocarps in the Far East suggested that this plant group originated on the Eurasian plate (Merrill, 1923), the presence of dipterocarps across the Atlantic, together with their poor dispersal capacity, is better explained by an origin in Gondwana (Maguire et al., 1977; Ashton, 1982; Dayanandan et al., 1999). The endemic status of P. dipterocarpacea, which is basal in the Dipterocarpalean clade (Dayanandan et al., 1999; Kubitzki & Chase, 2002), corroborates the second hypothesis. On basis of the ECM status of Sarcolaenaceae, Ducousso et al. (2004) hypothesized that Dipterocarpaceae ancestors were already ECM in Africa before 88 Mya. The clear ECM status of P. dipterocarpacea suggests that ancestors of Dipterocarpaceae already had the capacity to associate with ECM fungi before the separation of South America from Africa by the Atlantic, c. 135 Mya. The timing of evolution of ECM habit in Dipterocarpaceae ancestors estimated on the basis of biogeography coincides with the beginning of the radiation of fungal taxa involved in this symbiosis. Any phylogenetic relationship of fungal associates of P. dipterocarpacea and Paleotropical Dipterocarpaceae ECM fungi has yet to be established. Pakaraimaea dipterocarpacea and other members of Dipterocarpales might share ECM

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fungal taxa with relictual disjunct distribution as a consequence of comigration trend. Such phylogenetic and biogeographic relationships were demonstrated for the narrow host-range fungus Suillus and Pinus species (Wu et al., 2000). Disjunct fungal distribution patterns were also proposed by Halling (2001) for host generalist ECM fungal taxa with ancient genome. The observation, for the first time in this study, of a species in the Sebacinaceae with a Dipterocarpaceae is particularly relevant taking into account the basal position of this fungal group in the Hymenomycetes and the wide range of host species from different plant families (Weiss & Oberwinkler, 2001; Weiss et al., 2004). The association of P. dipterocarpacea with Clavulinaceae, a cosmopolitan fungal group (e.g. Corner, 1950, 1970; Roberts, 1999) observed in SE Asian dipterocarp forests (Roberts & Spooner, 2000; Lee et al., 2003), is also significant. Although Cortinariaceae are typical ECM associates of a wide range of plant species in diverse environments (Hibbett et al., 2000; Peintner et al., 2001; Halling & Mueller, 2005; Matheny, 2005), only few Cortinarius and Inocybe fruit body species have been recorded in association with SE Asian Dipterocarpaceae (Lee et al., 2003). These results contrast with the importance of Inocybe ECM morphotypes observed in dipterocarp ECM

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Fig. 2 Longitudinal section in Sebacina sp. + Pakaraimaea dipterocarpacea. E, epidermis; HN, Hartig net; M, mantle. Bar, 20 µm.

communities (Ingleby et al., 1998; Brearly et al., 2003). Further below-ground studies are needed to quantify Cortinariaceae in P. dipterocarpacea ECM communities. Only the Amanita sp. observed on P. dipterocarpacea belonged to a fungal group with many fruit body species in SE Asian mixed dipterocarp forests (Watling et al., 1996, 2002; Lee et al., 2003). Amanitaceae have also been described with African Monotaceae (Sanon et al., 1997) and in association with the Sarcolaenaceae (Ducousso et al., 2004). This preliminary below-ground survey has demonstrated that several fungal groups, including broad host-range fungi are associated with P. dipterocarpacea. The verification that the disjunct distribution of the Dipterocarpaceae is reflected below ground would corroborate the hypothesis of a common Gondwanan ECM ancestor. Smits’s (1983) hypothesis about Dipterocarpaceae ECM specificity remains to be tested. Pakaraimaea dipterocarpacea isolation from paleotropical Dipterocarpaceae might have favoured fungal sharing with host plants from different families in the same area. The two Caesalpiniaceae ECM genera Aldina and Dicymbe are also components of Guayana shrublands and forests on white sand (this study; Steyermark et al., 1995; Henkel et al., 2002). Among these two genera, comparisons between Dicymbe and P. dipterocarpacea ECM communities are particularly relevant. Dicymbe is the only neotropical genus in the mostly African clade Amherstieae of Caesalpiniaceae (Herendeen et al., 2003). Alexander (1989) demonstrated the importance of ECM habit in Amherstieae

and the recent discovery of Dicymbe ECM status (Henkel et al., 2002) confirmed the consistency of this feature in this clade. As for Dipterocarpaceae, biogeographic, molecular and species trait arguments suggest a Gondwanaland origin of ECMs in Amherstieae (Henkel et al., 2005b). Ectomycorrhizal fungal communities including the Sebacinaceae (Henkel et al., 2004), Clavulinaceae (Henkel et al., 2002, 2005a; Thacker & Henkel, 2004), Cortinariaceae (Henkel et al., 2002; Matheny et al., 2003) and Amanitaceae (Simmons et al., 2001; Henkel et al., 2002) have been described under Dicymbe in Guyana. The possible long-term coexistence of neotropical Dipterocarpaceae and Caesalpiniaceae Amherstieae might have favoured a ‘host jump’ between these two plant groups. This below-ground study of ECMs, together with the studies of Supaart et al. (2001) and Ducousso et al. (2004), have shown that a modern approach, using a combination of morphological and molecular methods, can be used successfully in a tropical environment to identify precisely both ECM fungus and plant partners. To date, data on ECM fungal diversity associated with Dipterocarpaceae were mostly based on fruitbody surveys in SE Asian mixed Dipterocarp forests (Watling & Lee, 1995; Watling et al., 1996; Roberts & Spooner, 2000; Lee et al., 2003). This approach is particularly useful for fungal taxonomy which is based on sexual features. On the other hand, connections between fruit body and ECMs are often difficult to trace, making it difficult to

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demonstrate specific host–fungus associations in forests where several ECM tree species co-occur. Fructifications are also erratic (Watling, 1995) and resupinate and/or cryptic sporocarps produced by ecologically important species such as Thelephoraceae (Kõljalg et al., 2000) and Sebacinaceae (Weiss et al., 2004) can easily be overlooked. The ECM morphotype studies on Dipterocarpaceae (Jülich, 1985; Smits, 1994; Watling et al., 1995; Lee et al., 1997; Ingleby et al., 2000; Brearly et al., 2003; B. Moyersoen, unpublished) are scanty and only few comprehensively described ECM morphotypes with precise identifications are available (Jülich, 1985; Watling et al., 1995; Lee et al., 1997). Further belowground studies using modern methods of identification are needed to test hypotheses about the diversity and specificity of ECM fungal species associated with Dipterocarpaceae.

Acknowledgements G. Cuenca invited me to Venezuela, and her team collaborated in field work. Field laboratory facilities were provided by the Autoridad Gran Sabana at Parupa Scientific Station, and root processing and morphotyping were performed at the Venezuelan Institute of Scientific Research (IVIC), Centro de Ecología, Laboratorio de Ecología de Suelos, Caracas. Molecular work was performed in collaboration with F. Martin’s team at Centre INRA de Nancy, Unité mixte de recherche interactions arbres microorganismes, using the DNA sequencing facilities financed by INRA Région de Lorraine and the European Commission. Laboratory equipment for anatomical observations was provided by the University of Liège, in the laboratories of C. Périlleux and of J.C. Bussers. I thank C. Delaruelle for support in molecular analyses and I.J. Alexander, P.E. Berry, V. Demoulin, A.H. Fitter, T.W. Henkel and O. Huber for comments on the manuscript.

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