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Russulaceae and Thelephoraceae form ectomycorrhizas with members of the Nyctaginaceae (Caryophyllales) in the tropical mountain rain forest of southern Ecuador Blackwell Publishing, Ltd.

Ingeborg Haug1, Michael Weiß1, Jürgen Homeier2, Franz Oberwinkler1 and Ingrid Kottke1 1

Spezielle Botanik und Mykologie, Universität Tübingen, Auf der Morgenstelle 1, D-72076 Tübingen, Germany 2Fakultät für Biologie, Abteilung Ökologie,

Universität Bielefeld, Postfach 100130, D-33501 Bielefeld, Germany

Summary Author for correspondence: Ingeborg Haug Tel: +49 70712978812 Fax: +49 7071295344 Email: [email protected] Received: 25 May 2004 Accepted: 21 September 2004

• Three members of the Nyctaginaceae, two Neea species and one Guapira species, occurred scattered within a very species-rich neotropical mountain rain forest. The three species were found to form ectomycorrhizas of very distinctive characters, while all other tree species examined formed arbuscular mycorrhizas. • The ectomycorrhizas were structurally typified according to light and transmission electron microscope investigations. The internal transcribed spacer (ITS) rDNA and part of the nuclear large subunit (LSU, 28S) rDNA of the mycorrhiza forming fungi were amplified and sequenced. Phylogenetic analyses were carried out. • Neea species 1 was found to form typical ectomycorrhizas with five different fungal species, Russula puiggarii, Lactarius sp., two Tomentella or Thelephora species, and one ascomycete. Neea species 2 and the Guapira species were associated with only one fungus each, a Tomentella/Thelephora species clustering closely together in an ITS-neighbour-joining tree. The long and fine rootlets of the Guapira species showed proximally a hyphal mantle and a Hartig net, but distally intracellular fungal colonization of the epidermis and root hair development. The ectomycorrhizal segments of the long roots of Neea species 2 displayed a hyphal mantle and a Hartig net around alive root-hair-like outgrowths of the epidermal cells. • The distribution and the evolution of ectomycorrhizas in the predominantly neotropic Nyctaginaceae are discussed. Key words: ectomycorrhiza, Guapira, molecular identification, Neea, neotropical mountain rain forest, Nyctaginaceae, Russulaceae, Thelephoraceae. New Phytologist (2005) 165: 923–936 © New Phytologist (2004) doi: 10.1111/j.1469-8137.2004.01284.x

Introduction The arbuscular mycorrhiza (AM) is the most ancient mycorrhizal association (Taylor, 1995) and is found with > 80% of extant land plant species (Smith & Read, 1997). The arbuscular mycorrhiza was replaced several times in a restricted number of plant species by the ectomycorrhizal association (Trappe, 1987). Ectomycorrhizas are exclusively formed in some families, for example Pinaceae and Fagaceae, but there are also several isolated ectomycorrhizal lineages in other plant families (Trappe, 1987; Brundrett, 2002). The Nyctaginaceae

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is one of the families where only few ectomycorrhiza forming species are known. There are ectomycorrhiza forming species in the genera Neea (Harley & Smith, 1983), Guapira (syn. Torrubia, Harley & Smith, 1983) and Pisonia (Ashford & Allaway, 1982). Ectomycorrhizal Neea species were found in the Amazonian rainforest (Singer & Araujo, 1979; Janos, 1980a,b) and in Peru (Alexander & Högberg, 1986), ectoand arbuscular mycorrhizal Neea species were reported from an Amazonian lowland rain forest (St. John, 1980a,b), from the south of Venezuela (Moyersoen, 1993) and from French Guiana (Béreau et al., 1997). Guapira sancarlosiana was found

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to be ecto- and arbuscular mycorrhizal in the south of Venezuela (Moyersoen, 1993). The presence of arbuscular mycorrhization is, however, questionable because Moyersoen (1993) neither found arbuscules nor vesicles, only thin and unseptate hyphae. Béreau et al. (1997) reported a low percentage of mycorrhization and never found arbuscules. Koske et al. (1992) found vesicles, coils and hyphae in Boerhavia repens sampled in Hawaii and classified the Nyctaginaceae Boerhavia repens, Bougainvillea spectabilis, Mirabilis jalapa, Pisonia umbellifera without AM functional infection. No mycorrhization was reported for Mirabilis jalapa, Boerhavia diffusa (Muthukumar & Udaiyan, 2000) and Boerhavia coccinea (Khan, 1974), and a low degree of mycorrhization with only vesicles and mycelium was reported for B. diffusa (Rachel et al., 1989). Thus, the Nyctaginaceae are considered a nonmycorrhizal family with isolated ectomycorrhizal species (Tester et al., 1987; Brundrett, 2002). However, a very low percentage of the c. 400 species (Daly & Roberts, 2004) of the family has been investigated. All the ectomycorrhizal genera belong to the tribe Pisonieae (Bittrich & Kühn, 1993). Most of the species of the Pisonieae occur in tropical and subtropical South America and the Antilles. One exception is Pisonia grandis, which is an abundant and widespread tree on islands throughout the Indian and Pacific Oceans. The Pisonia mycorrhizas have a unique structure with a hyphal mantle, Hartig net and transfer cells (Ashford & Allaway, 1982, 1985). A member of the Thelephoraceae (Basidiomycota) may be the fungal partner across its entire geographical range (Chambers et al., 1998). Here, the ectomycorrhizas of two Neea species and one Guapira species sampled in the species-rich neotropical mountain rain forest in South Ecuador were investigated in detail, and distinctive, unique mycorrhizal associations are shown. The mycorrhizal fungi were identified by molecular phylogenetic reconstruction. The hypothesized change from arbuscular mycorrhization to ectomycorrhization during evolution is discussed as a model in the Nyctaginaceae.

Materials and Methods Study site The study site is located on the eastern slope of the Cordillera El Consuelo in the Andes of southern Ecuador. The area of c. 1000 ha belongs to the Reserva Biológica San Francisco and borders the Podocarpus National Park half way between Loja and Zamora, Zamora-Chinchipe Province (3°58′S, 79°04′W ). The vegetation of the investigated area can be classified as montane cloud forest (according to Valencia et al., 1999) or as

montane evergreen forest (according to Balslev & Øllgaard, 2002). Different paths and plots were established in the primary forest on an altitudinal gradient between 1850 and 3200 m along the mountain ridges and the ravines. A thick organic layer (20–50 cm, Humic Cambisol) covers the weakly metamorphosed paleozoic sandstone on the mountain ridge, while a loamy, brown soil is found on the slopes of the ravine. Within 15 permanent plots, 1220 individual trees with a stem diameter of > 5 cm were identified and found to belong to 184 species out of 53 families (Homeier et al., 2002). The mycorrhizal state of 115 species was investigated, and 112 species formed arbuscular mycorrhizas (Kottke et al., 2004, and unpublished data), one tree (Graffenrieda emarginata) showed arbuscular and ectomycorrhiza (Haug et al., 2004). Sampling of mycorrhizas Root samples were collected from two Neea and one Guapira species, so far not identified to the species level. Neea species 1 is a multistemmed shrub reaching up to 5 m (similar to hazelnut) that was found on the border of the primary forest, close to the steep banks of the Rio San Francisco. Three specimens were sampled. Neea species 2 is a small shrub; one specimen was sampled in the ravine of the primary forest. Guapira sp. is a tree with stem diameters up to 25 cm and height of > 10 m. It only occurs within the ravines in the primary forest. Mycorrhizas were collected from four individuals, two of each in two ravines at a distance c. 500 m. Voucher specimens of fertile plants are kept in the National Herbarium of Ecuador (QCNE: Neea species 1 J.Homeier 387, Neea species 2 J.Homeier 1235, Guapira sp. J.Homeier 999), pictures of the three species can be seen on the internet at http://www.visualplants.de. The fine roots and very fine roots were sampled by tracing of single main roots from the trunks. From Neea species 1, 10 fine root systems from each specimen were collected, from Neea species 2, two fine root systems, and from the Guapira species, five fine root systems, respectively. The fine root systems were packed in plastic bags in order to prevent desiccation. Roots were cleaned under tap water the same day and all very fine root systems were collected. Ectomycorrhizas were recognized by the hyphal mantle and were selected using a dissecting microscope. Several tips of each ectomycorrhizal type were fixed in glutaraldehyde-formaldehyde 2.5% in Sörensen buffer (Karnovsky, 1965) to be used for ultrastructural investigations, and 2–3 tips were collected in 1.5 ml reaction tubes, dried and kept on silica gel for DNA sequencing. Part of the very fine root system was fixed in 50% ethanol for later light

Fig. 1 Micrographs of mycorrhizas (a) Russula puiggarii–Neea species 1; (b) Lactarius sp.–Neea species 1; (c) Tomentella/Thelephora species 1–Neea species 1; (d) Tomentella/Thelephora species 2–Neea species 1; (e) Ascomycete–Neea species 1; (f) Tomentella/Thelephora sp.–Neea species 2; (g & h) Tomentella/Thelephora sp.–Guapira species; (g) overview: long roots partially with hyphal mantle (arrows), partially with root hairs and lacking a hyphal mantle (*); (h) one root in detail: distal part with root hairs (*), proximal part with hyphal mantle (arrows). Scale: a, b, f, h, 1 mm; c–e, 0.5 mm; g, 5 mm.

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microscopical studies. Vouchers are kept as resin-embedded and as ethanol fixed material in the Botanical Institute, Systematic Botany and Mycology, University of Tübingen, Germany. Sampling of fruitbodies One to two specimens of Russula puiggarii (det. R. Krettek) were sampled close to Neea species 1 in the years 2001, 2002 and 2003. Voucher specimens are kept in Tübingen, Systematic Botany and Mycology. Thelephoraceae fruitbodies were found in the area (E. Langer, personal communication) but not in close connection to the sampled trees and not yet identified. Arbuscular mycorrhization of the fine roots A microscopical investigation of ethanol fixed fine roots and mycorrhizas was done to obtain information about arbuscular mycorrhization. Staining was carried out according to Grace & Stribley (1991). Rootlets were cleared in KOH 10% solution in a water bath at 60°C overnight, rinsed two times in tap water, exposed to HCl 10% for 2 min, and stained in Methyl blue (Merck, Darmstadt, Germany) 0.05% solution in lactic acid for 2 h at 60°C. The samples were mounted on microscopical slides in lactic acid and examined under a Zeiss Phot III microscope. Light and transmission electron microscopy of ectomycorrhizas Ectomycorrhizas and adjacent parts of the roots were selected from the glutaraldehyde-fixed material, washed six times in Sørensen buffer and fixed in 1% osmium tetroxide for 1 h in the dark. After six washes in ddH2O, samples were dehydrated in an acetone series at 10%, 20%, 50%, 70%, 85%, 95% and three times 100% for 10 min. Samples were embedded in ERL (low viscosity, longer pot-life formulation; Spurr, 1969; Kottke & Oberwinkler, 1988). Semithin sections were cut by use of a glass knife, stained with neofuchsin crystal-violet, mounted in Entellan and studied by a Zeiss Phot III microscope. Ultrathin sections were cut by use of a diamond knife, mounted on Formvar-coated copper grids and stained with 1% uranyl acetate (40 min) and lead citrate (20 min). Selected parts of rootlets were examined by use of the transmission electron microscope Zeiss TEM 902.

Molecular methods DNA extraction, PCR, and sequencing DNA was isolated from dried mycorrhizal samples and fruitbodies using the DNAeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Part of the nuclear LSU gene was amplified by the polymerase chain reaction (PCR) with the primer-pairs NL1


(5′-GCATATCAATAAGCGGAGGAAAAG-3′)–NL4 (5′GGTCCGTGTTTCAAGACGG-3′; O’Donnell, 1993) or NL1–LR6 (5′-CGCCAGTTCTGCTTACC-3′; Vilgalys & Hester, 1990). The internal transcribed spacer (ITS) within the ribosomal RNA genes was amplified using the primers ITS1F and ITS4 (White et al., 1990; Gardes & Bruns, 1993). PCR reaction volume was 50 µl, with concentrations of 1.5 m MgCl2, 200 µ of each dNTP (Life Technologies, Eggenstein, Germany), 0.5 µ of each of the primers (MWGBiotech, Ebersberg, Germany), 1 U Taq polymerase (Life Technologies, Eggenstein, Germany), 10% amplification buffer (Life Technologies, Eggenstein, Germany), and an empirically determined dilution of the DNA extract. Good results were achieved at diluting the DNA extract 1 : 25 or 1 : 100. PCR conditions were chosen as follows: a touchdown profile with annealing temperatures between 60 and 50°C for the LSU and between 55 and 45°C for the ITS. For details see Haug (2002). The PCR products obtained were purified using the QIAquick protocol (Qiagen). Direct sequencing of PCR products was performed using the PCR primers as sequencing primers. Cycle sequencing was conducted using the ABI PRISM Big Dye-Terminator Cycle Sequencing Ready Reaction Kit v.3.1 (PE Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s protocol, except that reaction volumes were reduced by half and the kit was diluted 1 : 4 with double distilled water. Electrophoresis and data sampling were performed on an automated sequencer (ABI 3100, Applied Biosystems). Both strands of DNA were sequenced. Sequence editing was done using the program SEQUENCHER, version 4.1 (Gene Codes Corporation, Ann Arbor, MI, USA). The sequences have been deposited at the National Center for Biotechnology Information (NCBI, GenBank: http://www.ncbi.nlm.nih.gov) under accession numbers AY667411–AY667427. Sequence similarities were determined using the BLAST sequence similarity search tool (Altschul et al., 1997) provided by GenBank. Phylogenetic analysis LSU sequences with closest BLAST matches in the Thelephoraceae were aligned with other published sequences of the Thelephorales using CLUSTAL X (Thompson et al., 1997). Further visual alignment was done in Se-Al version 2.03a (Rambaut, 1996). The corresponding ITS sequences were aligned with the closest matches of GenBank BLAST searches and with identified Thelephora and Tomentella species. To derive the phylogenetic position of the samples with LSU BLAST affinity to the Russulales we used the data sets of Miller et al. (2001; matrix accession no M1305 in TreeBASE, http://www.treebase.org/treebase/) and Larsson & Larsson (2003, kindly provided by E. Larsson), together with the closest GenBank BLAST hits. To estimate phylogenetic relationships we used *, version 4.0b10 (Swofford, 2002):


Research Table 1 Ectomycorrhizal types of Neea species 1 Fungal partner

Colour

Hyphal mantle structure

Reference specimens

Sequences

Russula puiggarii (Fig. 1a)

whitish to light yellowish

plectenchymatous

305-R, 305–6, 415

Lactarius sp. (Fig. 1b) Tomentella/Thelephora sp.1 (Fig. 1c) Tomentella/Thelephora sp.2 (Fig. 1d)

whitish, cottony brown, smooth

plectenchymatous plectenchymatous

403, 450 401, 413

AY667425 fruitbody AY667426 mycorrhiza AY667427 AY667411 AY667418

yellowish-brown, partially woolly with reddish hyphae black, partially woolly with black hyphae

plectenchymatous and irregularly synenchymatous

405, 414

AY667412 AY667419

plectenchymatous

408

no sequences

Ascomycete (Fig. 1e)

Assignment to fungal genera or species was done using molecular identification, for details see the text and Figs 2–4. Detailed descriptions with figures of longitudinal sections are available in the electronically supplementary material.

neighbour-joining analyses (NJ; Saitou & Nei, 1987) were done with Kimura 2-parameter genetic distances (Kimura, 1980), combined with bootstrap analyses (Felsenstein, 1985) from 1000 replicates. If several sequences of the same species were present in GenBank and if they clustered together, only one sequence was included in the final tree. After preliminary analyses using the complete Russulales alignment as described above (data not shown), we restricted further phylogenetic analysis to the Russulaceae s. str. (Russula, Lactarius, and related gasteroid taxa), which appeared as a monophyletic group in our preliminary analyses (Fig. 2).

Results Mycorrhizas of Neea species 1 The root system consisted of long roots and short roots. On the surface of the long roots extraradical hyphae and vesicles/ spores were occasionally found. Arbuscules were not observed. Ectomycorrhizas were formed on short roots (diameter 0.2– 0.6 mm). Five different mycorrhizal morphotypes were distinguished and the fungi identified at the species or genus level (Table 1; Fig 1a–e). The mycorrhizas of Russula puiggarii were identified comparing the ITS and LSU sequences of mycorrhizas and of fruit bodies which were collected several times in the vicinity of the shrubby tree. The identification of the Lactarius sp. mycorrhiza was done with molecular phylogenetic analyses (Fig. 2): the LSU sequence of the mycorrhizal fungus (403) clustered with Lactarius panuoides, L. volemus, L. piperatus, L. uyedae and L. deceptivus. A BLAST search of the LSU sequences of the thelephoracean mycorrhizal fungi (401, 405) and a NJ tree (Fig. 3) clearly showed the membership within the genera Thelephora/Tomentella. An ITS–NJ tree with all known Thelephora and Tomentella sequences from GenBank (Fig. 4) showed no close relationship to any of the so far sequenced species of Thelephoraceae. The amplification of the ITS and LSU region of the ascomycetous mycorrhizal fungus failed but TEM investigations clearly showed simple pores with Woronin bodies.


Table 2 Occurrence of ectomycorrhizal types with different specimens of Neea species 1 Neea species 1

shrub A

Russula puiggarii Lactarius sp. Tomentella/Thelephora sp. 1 Tomentella/Thelephora sp. 2 Ascomycete

x x x x

shrub B

shrub C

x x x

x x x

Each of the three investigated individuals of Neea species 1 had 3–4 mycorrhizal types (Table 2). Russula puiggarii mycorrhizas were only found at one shrub, also the ascomycete, Lactarius sp. was found at two neighbouring shrubs, the two thelephoraceous species were found on all individuals (Table 2). Mycorrhizas of Neea species 2 (Figs 1f and 5) The root system of Neea species 2 consisted of brown straight long roots, which had bright, unramified fine roots. No arbuscular mycorrhiza was detected in the stained rootlets. The fine roots (diameter 0.3–0.6 mm) were partially covered by a hyphal mantle (Fig. 1f ). Only one morphotype was detected. Morphological characters: hyphal smooth (Fig. 1f ).

mantle

brown

and

Anatomical characters: hyphal mantle plectenchymatous throughout, outer mantle layers loosely plectenchymatous (Fig. 5a), middle and inner layers compactly plectenchymatous (Fig. 5b), prominent Hartig net between root-hair-like outgrowths of the epidermal cells (Fig. 5c–e), root-hair-like outgrowths attached to the root surface (Fig. 5d,e), appear as longish cells with a round base in tangential sections (Fig. 5c), in median sections connections of the outgrowths with the


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Fig. 2 Phylogenetic relationships of Russulaceae and fungal sequences from the mycorrhizas of Neea species 1. Neighbour-joining analysis of partial nuclear rDNA coding for the ribosomal large subunit, using Kimura 2-parameter genetic distances, combined with a bootstrap analysis from 1000 replicates (bootstrap values < 50% not shown). The tree was rooted with the Gloeopeniophorella sequences.



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Fig. 3 Phylogenetic relationships of Thelephorales and fungal sequences from the mycorrhizas of Neea species 1, Neea species 2 and Guapira species. Neighbour-joining analysis of partial nuclear rDNA coding for the ribosomal large subunit with Kimura-2parameter genetic distances, combined with a bootstrap analysis from 1000 replicates (bootstrap values < 50% not shown). The tree was rooted with Hymenochaete rubiginosa and Inonotus nodulosus.

epidermal cells can be seen (Fig. 5d, *), sometimes they touch each other, reminiscent of a septum (Fig. 5d); epidermal cells, cortical cells and hyphae plasmatic; partially intracellular hyphae in the epidermal and cortical cells, hyphae with clamps (Fig. 5f). DNA analysis: Different specimens from the sampled mycorrhizas showed the same sequence. A GenBank BLAST search of the LSU sequence and an NJ tree (Fig. 3) clearly showed a membership within the genera Thelephora/Tomentella. In an ITS NJ tree with all Thelephora and Tomentella sequences present in GenBank (Fig. 4), the fungal sequence of Neea species 2 mycorrhizas clustered together with the thelephoracean sequence from the Guapira mycorrhizas. The closest matches from GenBank were Tomentella ellisii (AF272913) and a mycorrhizal fungus from Populus tremula (AJ510270).


Reference specimens: 925, 926 (Systematic Botany and Mycology, Tübingen). GenBank accession numbers: AY 667417 (LSU), AY667424 (ITS). Mycorrhizas of the Guapira species (Figs 1g,h and 6) The fine root systems of Guapira species consisted of long roots only. Light microscopic investigation of stained rootlets revealed few extraradical hyphae and vesicles/spores, few inter- or intracellular hyphae, few intracellular vesicles and no arbuscules were found. Proximally the finest roots (diameter 0.3–0.6 mm) were covered by a hyphal mantle and distally there were root hairs but no hyphal mantle (Fig. 1g,h). Sometimes segments with and without hyphal mantle were alternating twice on one long root (Fig. 1g). Root tips covered with a


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Fig. 4 Phylogenetic relationships of Thelephoraceae and fungal sequences from the mycorrhizas of Neea species 1, Neea species 2 and Guapira species. Neighbour-joining analysis of the ITS (including ITS1, 5.8S and ITS2) with Kimura-2-parameter genetic distances, combined with a bootstrap analysis from 1000 replicates (support values < 50% not shown). Abbreviations: Th. := Thelephora, To. := Tomentella.

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Fig. 5 Longitudinal sections of mycorrhizas of Neea species 2: (a) Outer hyphal mantle:loose plectenchyma; (b) middle and inner hyphal mantle: dense plectenchyma; (c) tangential section in the epidermal layer: epidermal outgrowths (*) and Hartig net; (d & e) median section through mycorrhiza: hyphal mantle, epidermal cells with outgrowths (*) and Hartig net; (f) hyphal mantle, Hartig net and intracellular hyphae (with clamps (arrow)) in epidermal and cortical cells (scale: a–f, 15 µm).

hyphal mantle were rare, many roots were without a hyphal mantle (Fig. 1h). Only one morphotype was distinguishable. Morphological characters: hyphal mantles dark brown with silvery patches on the surface (Fig. 1g,h), colourless emanating hyphae with clamps.


Anatomical characters: hyphal mantle plectenchymatous throughout, outer layers loosely plectenchymatous (Fig. 6a), middle layers densely plectenchymatous (Fig. 6b), prominent Hartig net on the epidermal layer (Fig. 6c–e); root segments without hyphal mantle with root hairs, loose hyphae and intracellular infections in the root hairs and in the epidermal cells (Fig. 6f,g).


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Fig. 6 Longitudinal sections of mycorrhizas of the Guapira species: (a) Outer hyphal mantle:loose plectenchyma; (b) middle hyphal mantle: dense plectenchyma; (c and d) tangential sections of the Hartig net layer adjacent to the outer walls of the epidermal cells; (e) median section through mycorrhiza: hyphal mantle and prominent Hartig net; (f) tangential section of the epidermal layer: intracellular hyphae; (g) median section: intracellular hyphae in epidermal cells and root hairs; (h and i) transmission electron micrographs of hyphal mantle hyphae, intracellular hyphae in cortical cells and root hairs (scale: a–g, 15 µm, h–i, 2.5 µm).



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As ultrastructural details are the same as in the hyphae of the Hartig net and hyphal mantle (Fig. 6h,i), we conclude that the intracellular infection is due to the ectomycorrhizal fungus. DNA analysis: All samples obtained from 4 specimens showed the same ITS and LSU sequences. A GenBank BLAST search of the LSU sequence and a NJ tree (Fig. 3) clearly showed a membership within the genera Thelephora/ Tomentella. In an ITS–NJ tree with all present Thelephora and Tomentella sequences from GenBank (Fig. 4), the Guapira sequences cluster together with the thelephoracean sequence from Neea species 2 mycorrhizas. The closest named species are Tomentella ellisii and an ectomycorrhizal fungus from Populus tremula. Reference specimens: 424, 508, 971, 979 (Systematic Botany and Mycology, Tübingen). GenBank accession numbers: AY 667413–AY667416 (LSU), AY 667420–AY 667423 (ITS).

Discussion The Nyctaginaceae are distributed mostly in the tropics and subtropics of the New World and only very few species are found in the Old World (Bittrich & Kühn, 1993). This family was placed in the order Caryophyllales (APG, 1998), an order that contains many non-mycorrhizal species (Trappe, 1987; Kottke, 2003), and the Nyctaginaceae were also considered to contain a high percentage of non-mycorrhizal plants (Trappe, 1987). Up to now, no species with arbuscules was described within this family (Rachel et al., 1989; Koske et al., 1992; Moyersoen, 1993; Béreau et al., 1997). Neea, Guapira and Pisonia are genera in the tribe Pisonieae within the Nyctaginaceae (Bittrich & Kühn, 1993). The ectomycorrhizal state of several Neea and Guapira species and of Pisonia grandis has been documented (Singer & Araujo, 1979; Janos, 1980a,b; St. John, 1980a,b; Ashford & Allaway, 1982, 1985; Alexander & Högberg, 1986; Tester et al., 1987; Moyersoen, 1993; Béreau et al., 1997). No detailed description of the mycorrhizas of Neea and Guapira species was, however, given, and the fungi were rarely identified. Our studies revealed the typical features of ectomycorrhizas, a hyphal mantle and a Hartig net in all the mycorrhizal morphotypes from the three tree species. Plectenchymatous hyphal mantles are common for the ectomycorrhizas of Thelephoraceae (Agerer, 1995), but there are also pseudoparenchymatous mantles (Raidl & Müller, 1996; Jakucs & Agerer, 1999, 2001). A heterogenous assemblage of mantle and rhizomorph types exists in the Russulaceae; characteristic of the genus Lactarius are smooth mantles with lactifers (Agerer, 1995). The ectomycorrhizas Lactarius sp.–Neea species 1 showed abundant emanating hyphae and no distinct lactifers


were observed in the hyphal mantle. Henkel et al. (2000) described a similar morphology of the ectomycorrhizas Lactarius panuoides–Dicymbe altsonii from Guyana’s western border with Brazil. According to the anatomy of the ectomycorrhiza the species of Russula can be divided in two groups: a group with plectenchymatous outer mantle layers with cystidia and a group with pseudoparenchymatous outer mantle layers without cystidia (Beenken, 2001). The ectomycorrhizas of Russula puiggarii possess a plectenchymatous outer mantle layer. However, cystidia were not observed. Ectomycorrhizas on short roots were only found on Neea species 1, resembling the mycorrhizal short root systems with monopodial ramification of Neea obovata described previously (Moyersoen, 1993). The other Neea species 2 and the Guapira species show a fine root system of only long roots. A similar root system was described previously on Neea robusta (Moyersoen, 1993), and Pisonia grandis (Ashford & Allaway, 1985). The differences of the root systems correspond to a different degree of mycorrhization. While mycorrhization was close to 100% of rootlets on species forming short root systems (Neea species 1, Neea obovata, Guapira sancarlosiana; Moyersoen, 1993 and this study), species with long root systems showed an incomplete development of the hyphal mantle and no suppression of root hair formation (Pisonia grandis, Neea robusta, Neea species 2, Guapira species; Ashford & Allaway, 1985; Moyersoen, 1993, and this study). The latter species were associated with only one fungal taxon (Alexander & Högberg, 1986; Moyersoen, 1993; Chambers et al., 1998, and this study), which is again very untypical for ectomycorrhiza-forming plants. Five different fungi were found associated with the short root-forming Neea species 1, and Moyersoen (1993) mentions seven ectomycorrhiza-forming fungi in the surroundings of the nyctaginacean trees, but ectomycorrhiza-forming trees in the temperate regions have a much higher number of associated fungi (Molina et al., 1992; Haug & Pritsch, 1992). The tree species that were associated with only one fungal taxon formed mycorrhizas with a member of the Thelephoraceae (Chambers et al., 1998, and own results). The fungi associated with Neea species 2 and the Guapira species from our plots cluster together in the phylogenetic tree, but the thelephoracean fungus identified from Pisonia grandis (Chambers et al., 1998) is not closely related (Fig. 4). Chambers et al. (1998) hypothesize that only this thelephoracean species may be associated with P. grandis across its entire geographical range. This idea is supported by our findings of one fungal taxon on several Guapira specimens growing separately in two ravines c. 500 m apart. The described situation of long root systems that are only partly transformed into ectomycorrhizas by only one fungus, not suppressing root hair formation, partly with intracellular penetration of hyphae, sometimes forming transfer cell like structures, has not been described from any other plant family. We hypothesize that the situation displays an early evolutionary


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step in ectomycorrhiza formation. The Nyctaginaceae may be considered as a rather derived family (Savolainen et al., 2000) that radiated in the neotropics. Several genera and many species of the Thelephorales occur in the neotropics, including the lowlands (Singer et al., 1983), although the main descriptions come from the temperate and cool regions (Kõljalg, 1996; Kõljalg et al., 2000, 2002). Moyersoen (1993) did not mention Thelephoraceae in his list of ectomycorrhizal fungi in the Amazon Caatinga, but it may be that the inconspicuous fruit bodies have been overlooked. Thelephoracean fruiting bodies are formed on the ground, often on rotten wood, and spores may therefore be dispersed in the tropical rain forest. It is remarkable that in the neotropics two species of the tribe Pisonieae, and in the Australian-pacific region one species of the Pisonieae, are ectomycorrhizal with only one thelephoracean fungus, respectively. This may be indicative of a close phylogenetic relationship of the respective tree species, but molecular phylogenetic studies have still to be carried out to clarify the systematic relationships in the tribe Pisonieae. Probably in a second evolutionary step the typical ectomycorrhizal state was reached as found in the short root forming nyctaginacean species. Besides thelephoracean species, we found two russulacean species and one ascomycete forming ectomycorrhizas. Moyersoen (1993) found fruit bodies of seven ectomycorrhiza-forming fungi, among them three Russula species. Fungi belonging to Russula and Lactarius are obligate symbionts and can only be distributed over larger areas if the ectomycorrhizal tree is also spreading. We hypothesize that Russula puiggarii and the Lactarius species we found within the arbuscular mycorrhizal community of the mountain rain forest have come into this area together with the mycorrhizas of Neea species 1. The migration might have followed the river side, as the Rio San Francisco flows into the Amazonas some way down from the Venezuelan lowland forest, where the other Neea species were discovered. Alternatively, the ectomycorrhizal Nyctaginacean species in the mountain rain forest of the Northern Andes may be relicts from the time before the elevation of the Andes, when the lowland forest covered also this part of the country (van der Hammen, 1989). The study of the mycorrhizal status of further species in the family Nyctaginaceae of South America and the identification and molecular phylogeny of the associated fungi will be a future challenge.

Acknowledgements The research was generously supported by the Deutsche Forschungsgemeinschaft (DFG project FOR 402). We thank the Fundacíon Científica San Francisco, Ecuador, and the NCI for providing research facilities and the Universidad Técnica Particular de Loja (UTPL) for kind personal help and access to laboratories. We also thank Roman Krettek for the


determination of Russula puiggarii and Ellen Larsson for providing us with her Russulales LSU alignment. The skilful technical assistance of Jutta Bloschies is gratefully appreciated.

Supplementary material The following material is available as supplementary material at http://www.blackwellpublishing.com/products/journals/ suppmat/NPH/NPH1284/NPH1284sm.htm Supplementary text Detailed description of the mycorrhizal types of Neea species 1. Fig. S1 Russula puiggarii – Neea species 1. a. Micrograph of the mycorrhiza, b.–f. micrographs of longitudinal sections, b., c. outer hyphal mantle: loose plectenchyma with matrix, d., e. middle and inner hyphal mantle: dense plectenchyma, hyphae arranged in parallel, f. median section through mycorrhiza: hyphal mantle and epidermal cells (scale: a. 1mm, b.–f. 15 µm). Fig. S2 Lactarius sp. – Neea species 1. a. Micrograph of the mycorrhiza, b.–f. micrographs of longitudinal sections, b. emanating hyphae: straight, septa without clamps, c. outer hyphal mantle: hyphae with larger and irregular diameter, d., e. middle and inner hyphal mantle: dense plectenchyma, f. median section through mycorrhiza: hyphal mantle and cortical cells with epidermal Hartig net (scale: a. 1mm, b.–f. 15 µm). Fig. S3 Tomentella/Thelephora species 1 – Neea species 1. a. Micrograph of the mycorrhiza, b.–f. micrographs of longitudinal sections, b. outer hyphal mantle: net like arrangement of the hyphae, c., d. middle mantle layers: irregular synenchyma, e. inner hyphal mantle: dense plectenchyma, f. median section through mycorrhiza: hyphal mantle and epidermal cells with Hartig net (scale: a. 0.5 mm, b.–f. 15 µm). Fig. S4 Tomentella/Thelephora species 2 – Neea species 1. a. Micrograph of the mycorrhiza, b.–f. micrographs of longitudinal sections, b. outer hyphal mantle: loose prosenchyma, hyphae with clamps, c., d. middle mantle layers: irregular synenchyma, e. inner hyphal mantle: dense plectenchyma, f. median section through mycorrhiza: hyphal mantle and cortical cells with epidermal Hartig net (scale: a. 0.5 mm, b.–f. 15 µm). Fig. S5 Ascomycet – Neea species 1. a. Micrograph of the mycorrhiza, b.–f. micrographs of longitudinal sections, b., c. outer and middle hyphal mantle: star like arrangement of the hyphae, d. tangential section through hyphal mantle and adjacent cortical cells with Hartig net, e. median section through mycorrhiza: hyphal mantle and cortical cells with epidermal Hartig net, f. TEM micrograph of a simple porus with Woronin body (scale: a. 0.5 mm, b.–e. 15 µm, f. 250 nm).

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