Mycorrhiza DOI 10.1007/s00572-015-0635-6

ORIGINAL PAPER

Preliminary findings on identification of mycorrhizal fungi from diverse orchids in the Central Highlands of Madagascar

Kazutomo Yokoya & Lawrence W. Zettler & Jonathan P. Kendon & Martin I. Bidartondo & Andrew L. Stice & Shannon Skarha & Laura L. Corey & Audrey C. Knight & Viswambharan Sarasan

Received: 8 August 2014 /Accepted: 26 February 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract The Orchid flora of Madagascar is one of the most damage and species loss. The diversity of culturable fungal diverse with nearly 1000 orchid taxa, of which about 90 % are associates, their density, and distribution within the Itremo endemic to this biodiversity hotspot. The Itremo Massif in the orchid hotspot areas will be discussed. Central Highlands of Madagascar with a Highland Subtropi- cal climate range encompasses montane grassland, igneous Keywords Orchidaceae . Fungal symbiont . Rhizoctonia . and metamorphic rock outcrops, and gallery and tapia forests. Tulasnella . Ceratobasidium . Sebacina . In vitro Our study focused on identifying culturable mycorrhizae from epiphytic, lithophytic, and terrestrial orchid taxa to understand their diversity and density in a spatial matrix that is within the protected areas. We have collected both juvenile and mature roots from 41 orchid taxa for isolating their orchid mycorrhi- Introduction zal fungi (OMF), and to culture, identify, and store in liquid nitrogen for future studies. Twelve operational taxonomic About 44 % of all vascular plant species are confined to 34 units (OTUs), of three known orchid mycorrhizal genera, global biodiversity hotspots (Mittermeier et al. 2005). were recognized by analysis of internal transcribed spacer Investing resources to the entire global hotspot could lead to (ITS) sequences of 85 isolates, and, by comparing with spreading of the resources too thinly on the ground, making GenBank database entries, each OTU was shown to have conservation of an entire hotspot area untenable, and there- closely related fungi that were also found as orchid associates. fore, conservation approaches must focus on selected areas of Orchid and fungal diversity were greater in gallery forests and maximum diversity and/or endemism (Murray-Smith et al. open grasslands, which is very significant for future studies 2009). Fenu et al. (2010) proposed the terms Bmicro-hotspots^ and orchid conservation. As far as we know, this is the first (i.e., endemism-rich areas analogous to biogeographic units) ever report of detailed identification of mycorrhizal fungi from and Bnano-hotspots^ (i.e., areas <3 km2 with an exceptional Madagascar. This study will help start to develop a pro- concentration of endemic taxa). By most estimates, Madagas- gramme for identifying fungal symbionts from this unique car is home to 4 % of the world’s plant and animal species all biodiversity hotspot, which is undergoing rapid ecosystem confined to just 0.4 % of the Earth’slandsurface,earningits reputation as one of the top five biodiversity Bhotspots^ (Tyson 2000). About 90 % of Madagascar’s natural vegetation : : : K. Yokoya J. P. Kendon M. I. Bidartondo V. Sarasan (*) has been cleared or permanently altered, and the remaining Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AB, UK 10 % is by no means secure. Roos et al. (2004) studied the e-mail: [email protected] vascular plants in the Malesian Islands and observed that spe- – L. W. Zettler : A. L. Stice : S. Skarha : L. L. Corey : A. C. Knight cies area relationships of families are dependent on species Department of Biology, Illinois College, 1101 West College Avenue, number; they found that island surface area is a predictor for Jacksonville, IL 62650, USA island percent endemism in the study area. Madagascar, the world’s second largest island, has 10,000–12,000 vascular M. I. Bidartondo Department of Life Sciences, Imperial College London, South plant species, of which roughly 1 in 10 (about 1000) are or- Kensington Campus, London SW7 2AZ, UK chids and about 90 % of which are endemic (Tyson 2000; Mycorrhiza

Moat and Smith 2007). The Itremo Massif within the Central flora and fauna. The area covered by the study represented Highlands is a Bmicro-hotspot,^ home to more than 50 orchid around 13 km2 both within and adjacent to the protected area taxa of which the majority are endemic and some species are (Fig. 1). locally endemic (e.g., Angraecum protensum, Angraecum In light of the ongoing demise of tropical ecosystems in magdalenae). Madagascar and worldwide, there is an urgent need to docu- Itremo Massif consists of a plateau of mixed igneous and ment and safeguard the full gamut of life forms in the land- metamorphic rock at an elevation of 1400–1923 m above sea scape, and to understand how these biotic agents interact with level. The average temperature is in the range of 18–21 °C, one another in this age of extinction. Among angiosperms, annual rainfall of 1416 mm with a 4–6-month dry season. orchids are particularly vulnerable given their dependency Savanna is the dominant habitat, with humid gallery forest, on other organisms—namely mycorrhizal fungi and insect remnant tapia (Uapaca bojeri) forest and rocky and montane pollinators—to complete their life cycles in nature (Swarts moorland habitats also present. The raising of cattle is com- and Dixon 2009). Thus, studying these connections becomes monplace in the region, and burning grassland is practiced crucial for orchid conservation. While orchids have received annually. While some advocate carefully controlled burning considerable study with respect to classification and phyloge- as a management strategy for tapia forest in Itremo and else- netic relationships, other important aspects (e.g., pollination, where in the Central Highlands (Alvarado et al. 2014), other propagation) have received less attention, and this is especial- habitats can be badly affected (Whitman et al. 2011). We ob- ly true of Orchidaceae in Madagascar (Cribb and Hermans served evidence of fire damage on two separate rocky ridges 2009), with a few exceptions (e.g., Nilsson et al. 1992a, b; and speculate that adjacent man-made grass fires were the Whitman et al. 2011). To date, virtually nothing is known cause. In addition to this, illegal mining for precious stones about the identity, distribution, and ecology of orchid mycor- is causing habitat degradation in some areas (Vorontsova et al. rhizal fungi (OMF) in Madagascar, or the physiological roles 2013). Following a proposal in 2008, a 273-km2 area was played by these fungi with native orchids. Madagascar’sre- given Protected Area status in 2012, thanks to Itremo’s unique mote location, coupled with its rugged terrain, continue to

Fig. 1 Map depicting the study sites near Itremo in the Central Highlands of Madagascar. Circles Gallery forest dominated by epiphytic orchids, also terrestrial orchids on river banks; squares rocky montane grassland with lithophytic and terrestrial orchids; triangles montane moorland with terrestrial orchids; T terrestrial, L lithophyte, E epiphyte, L/T lithophyte/terrestrial, Cer Ceratobasidium, Seb Sebacina, Tul Tulasnella. Numbers 2, 3, 4, 5,and7 denote sites; solid line depicts core protected area. Site 1 is off the area of this map to the east. No Rhizoctonia-like fungi were isolated from collections at site 6. Image © 2014 Google Earth Mycorrhiza pose challenges to specialists seeking to gain access to orchid- labs in Europe (Kew) and North America (Illinois) will also rich habitats in search of such knowledge. This is especially be discussed. To our knowledge, this is the first report that true for mycologists faced with the burden of expediting fresh documents fungal associates from orchids of different life tissue samples harboring viable fungal material (e.g., forms and diverse ecosystems from Madagascar. pelotons) to the lab for further study. The main objective of this study was to understand symbi- otic relationships of orchids of the Itremo Massif in Madagas- Materials and methods car. This study was conducted to achieve the ultimate goal of producing symbiotic propagules for reintroduction by under- This joint study was conducted between Royal Botanic Gar- standing the role of mycorrhizal fungi in seed germination, dens Kew (Kew) and Illionis College with logistic and taxo- seedling development, and establishment of plants in the wild. nomic support from Kew Madagascar Conservation Centre While orchids throughout Madagascar require study, we chose (KMCC) and Parc Botanique et Zoologique de Tsimbazaza to focus on species inhabiting the Central Highlands—are- (PBZT). More than 40 taxa within 24 genera were selected for gion encompassing nearly 40 % of the island (Cribb and study. Root samples were shared between two partners, and to Hermans 2009)—and spontaneous seedlings in particular. facilitate the legal collection and international transport of The Itremo Massif has the largest area of exposed quartzitic orchid material from Madagascar to the UK and USA, a Con- substrate in Madagascar (du Puy and Moat 1996) with a mix- vention on International Trade in Endangered Species of Wild ture of ecosystems found nestled within a complex of hills and Fauna and Flora (CITES) permit was obtained, which allowed valleys separated by species-poor grasslands (Cribb and three tubes each containing seedling and mature roots per Hermans 2009). Several well-known, showy taxa are found species to be collected. Due to the limited availability of plants in the region, many of which persist as lithophytes on sun- in the wild, further restrictions were put in place locally to exposed rocks (e.g., Angraecum longicalcar). Others exist as collect from three each of juvenile and mature plants. This terrestrials of grasslands (e.g., Benthamia cinnabarina), moist was followed by a phytosanitary certificate, which was se- forests (e.g., Cynorkis purpurea), or well-drained soils (e.g., cured prior to departure from the country. For the import of Habenaria ambositrana), and a modest number cling to root samples (with soil) and seeds to the USA, two permits gnarled branches of host trees in open areas as epiphytes were needed from the US Department of Agriculture (USDA). (e.g., Bulbophyllum sp.). The US Government considers all OMF to be plant pathogens The orchid seed baiting technique initially developed by due to the ties of some to the Rhizoctonia complex. Rasmussen and Whigham (1993) remains the most widely used method to capture fungal symbionts that support germi- Study sites nation in situ. Although this technique has been applied to a large number of species with varied success, namely terres- Seven different sites within the Central Highlands were visited trials (McKendrick et al. 2000; Batty et al. 2006; Phillips et al. during 28 April to 3 May 2013 (Table 1). These sites were all 2011), the length of period required for successful baiting is within 5 km of one another with the exception of Analabeby one of its drawbacks, in addition to loss of baits (Gale et al. (Fig. 1). 2010). Given Madagascar’s large number of lithophytic taxa, affixing seed baits to exposed rocks also poses a serious prac- Collection and transportation tical challenge as we previously discovered. With these limi- tations, we opted to improve our odds for acquiring The collecting trip was conducted in April/May 2013 to col- germination-phase fungi by targeting spontaneous seedlings lect spontaneous seedling roots on orchid-rich substrates and on natural substrates. Additionally, we have collected roots mature roots shortly after the rainy season (December to from mature phase plants to identify the Rhizoctonia-like fun- March). Roots of orchids, of both mature plants and sponta- gi utilized by these orchids. In this paper, we report the my- neous seedlings where possible, were collected from 41 taxa corrhizal fungi cultured from spontaneous seedlings and ma- consisting of nine lithophytic, 17 epiphytic, and 15 terrestrial ture orchids in the Central Highlands of Madagascar using species, respectively. The collection of root samples was lim- morphological and molecular characterization (ITS sequenc- ited to three juvenile and three mature plants per species, ing). We also provide discussion on the distribution of these which was the maximum allowance as stipulated by our isolates in the landscape linked to the specific microhabitats of CITES permit. Depending on the species and availability, be- the orchids. Furthermore, we describe the molecular confir- tween one and five roots per specimen were collected. Spon- mation of the field identification of spontaneous seedlings taneous seedlings were putatively identified on site based on collected and propose it as a standard methodology for other subtle morphological features (e.g., presence of pseudobulbs collections of a similar nature. Collection and long-distance in Bulbophyllum sp.; Fig. 2) as well as proximity to mature transportation of fresh orchid material from Madagascar to plants on or near the same substrate; the identities were later Mycorrhiza

Table 1 General description of the seven orchid collection sites in the disturbance to the brittle root systems. Each root ball was then Central Highlands of Madagascar placed into its own separate plastic bag, and the bags were Site number/name General description then carefully packed into an insulated handbag for transport. Upon arrival at the KMCC base in Antananarivo, Madagascar 1. Analabeby Exposed marble outcrop 2–7 days after field collection, all root samples and root balls 2. Ambatoantrano Exposed rocks, occasional tapia trees were placed into a refrigerator (about 6 °C). Approximately 3. Ambatoantremo Exposed rocks, occasional gnarled small trees, 24 h before departure from the country, roots of terrestrial sandy stream bed orchids were lifted from the soil and rinsed off with UV- 4. Tsinahabeomby Open grassland, moist soil, occasional rocks irradiated and/or bottled water to remove soil particles and 5. Gallery Forest Reduced forest (canopy about 20 m) organic debris. Lateral branch roots, especially those that ex- 6. Soutrihotapaka Exposed ridges, montane vegetation hibited orange-yellow patches of coloration, were detached 7. Antsirakambiaty Dense shaded forest, downhill stream and placed over a premoistened cotton ball in a presterilized glass vial. Starch-filled tuberous roots were not retained be- cause previous studies (L. Zettler, unpublished data) have confirmed by DNA analysis as described later. To maximize shown that such roots are generally void of pelotons. The our chances for isolating viable pelotons, younger-appearing screw cap was then tightened firmly and wrapped with a strip roots were collected whenever possible. For epiphytic and of Parafilm BM.^ Likewise, caps on glass vials containing lithophytic orchids, these roots were those that were translucent roots of lithophytes and epiphytes were also tightened and to white in color, often with slight greenish pigmentation near wrapped with Parafilm BM^ at that same time (about 24 h the apex (Fig. 2). Upon detachment in the field, each root was prior to departure by air). All sealed glass vials were then placed over a small, premoistened cotton ball within housed in 50-ml plastic (shatter-proof) centrifuge vials, which presterilized glass vial with screw cap. To permit gas exchange were also firmly tightened and sealed with Parafilm BM.^ All leading up to departure from Madagascar (5–10 days after col- vials were repacked into insulated handbags and transported lection), the caps on each vial were tightened only slightly, then back to labs in the USA and UK as cabin baggage. placed within a 50-ml capacity centrifuge tube with screw cap. These tubes were then stored vertically within an insulated handbag for transport from field to shelter. Care was taken to Measurement of substrate acidity keep the handbag out of direct sunlight at all times so that the root samples would remain as cool as possible (15–25 °C). Substrate samples were collected at several locations and The root collection procedure for terrestrial orchids differed consisted of soil and humus in the case of terrestrial habitats, slightly in that soil containing intact root systems (root ball) and organic/inorganic debris on the surface and in the cracks was also collected. This permitted the roots to remain in a of rocks where lithophytic orchids were found. On return to seminatural state leading up to departure from Madagascar. KMCC, the collected samples were analyzed using LaMotte A trowel or small shovel was used to gently excavate the soil STH Series Combination Soil Testing Outfit (LaMotte, Mary- around individual plants and to lift the root ball with minimal land, USA).

Fig. 2 a Spontaneous seedlings of Angraecum species affixed to outer in the palm of a hand (b, top right). c An advanced stage seedling of bark of a host tree adjacent to a foliose lichen from Itremo in the Central Angraecum protensum displays a young healthy root system with green Highlands of Madagascar. b A small seedling, just past the protocorm tipped apex (arrow). Younger roots of spontaneous seedlings were stage(denotedbythearrow), was discovered beneath loose bark shown targeted for collection Mycorrhiza

Fungal isolation, initial identification, and deposition performing the cycle sequencing reactions on a thermocycler for 25 cycles of 10 s at 96 °C, 5 s at 50 °C, and 4 min at 60 °C. Immediately upon arrival at Kew and Illinois 24–48 h after Reaction products were purified by ethanol precipitation and departure from Madagascar, all root samples were placed in analyzed using Applied Biosystems 3730xl DNA Analyzer refrigeration (4–6°C)foraperiodlastingupto1week,during (Life Technologies, Carlsbad, CA, USA). which time fungal isolations took place. Fungi were isolated All sequence analyses were performed using Geneious® following the method of Zettler et al. (2003). Colonization of software package (Biomatters, Auckland, New Zealand). The fungi in the cortical region of root sections was scored as per- forward and reverse sequences were checked for accuracy and centage of cortical cells containing pelotons for all three life consensus, and compared to database sequences using BLAST forms. Clumps of macerated cortical cells containing pelotons (National Center for Biotechnology Information, Bethesda, were immersed in fungal isolation medium (FIM; Mitchell MD, USA). Sequences that matched Rhizoctonia-like fungi 1989) containing streptomycin sulfate (Clements and Ellyard were aligned and grouped in to operational taxonomic units 1979) and incubated at 18 °C. After 1–4 days, hyphal tips that (OTUs)basedonaconservativesimilaritythresholdof95%. were observed emerging from cortical cells and/or pelotons un- Representative sequences of each OTU were used to requery der a dissection microscope were subcultured to FIM (Kew) or the GenBank database using BLAST. Phylogenetic trees were potato dextrose agar (PDA; Difco™, Becton, Dickinson and separately constructed for each of the three genera, Tulasnella, Co., Sparks, MD, USA) (Illinois) using a sterile scalpel. Cultures Ceratobasidium,andSebacina, together with closely matched likely to be OMF were initially distinguished from common sequences from GenBank database. The sequences were molds using previously published descriptions (Zettler et al. aligned with CLUSTALW algorithm, and neighbor-joining 2003). Those that yielded cultural characteristics (e.g., monilioid trees were made using Tamura–Nei genetic distance model. cells) resembling basidiomycetes in the Rhizoctonia complex (e.g., Tulasnellaceae, Ceratobasidiaceae) were retained for fur- Molecular confirmation of species identification of orchid ther identification by ribosomal DNA internal transcribed spacer seedlings (rDNA ITS) amplification and sequencing. Colony growth rates were recorded during early subculture. To safeguard these The DNA of orchid seedlings that yielded Rhizoctonia-like fungi strains for the purposes of future work (e.g., symbiotic seed was extracted and sequenced in order to confirm their species germination) and long-term conservation, fungi isolated in Illi- identification by matching the sequence of the chloroplast DNA nois were deposited into the University of Alberta Microfungus region trnL-F to that of an identified plant. DNA was extracted Collection and Herbarium (UAMH), Edmonton, Canada, and from root tissue using Sigma Extract-N-Amp™ Plant PCR Kit Kew for permanent safekeeping. (Sigma Aldrich, St. Louis, MO, USA) or from desiccated leaf tissue with a modified cetyltrimethylammonium bromide Molecular identification of fungi by ITS sequencing (CTAB) protocol (Doyle and Doyle 1987)followedby chloroform/isoamyl alcohol (24:1) extraction and precipitation To identify each fungal sample, DNA was extracted and se- in isopropanol. The trnL-F sequences were amplified using quenced. Genomic DNA was isolated from mycelia using Sig- primer combinations c with d for the trnL intron, and e with f ma Extract-N-Amp™ PlantPCRKit(SigmaAldrich,St.Louis, for the trnL-F intergenic spacer (Taberlet et al. 1991) and using MO, USA). ITS sequences were amplified using primer combi- Reddymix™ PCR master mix (Thermo Scientific ABgene, Pitts- nations ITS1F with ITS4, and ITS1 with ITS4-tul (White et al. burgh, PA, USA) with additional 150 mM trehalose, 200 μg/ml 1990; Gardes and Bruns 1993;TaylorandMcCormick2008) bovine serum albumin, and 0.2 % Tween-20 (Samarakoon et al. and using Sigma Extract-N-AmpTM PlantPCRKit(SigmaAl- 2013). The reaction was performed on a programmable drich, St. Louis, MO, USA). The reactions were performed thermocycler for 28 cycles of 1 min at 94 °C, 1 min at 48 °C, using a programmable thermocycler for 35 cycles of 30 s at and 1 min at 72 °C. The PCR products were cleaned using 94 °C, 35 s at 53 °C, and 1 min at 72 °C, with the extension QIAquick® columns (Qiagen Inc., East Crawley, UK) and se- step increased by 5 s per cycle. Amplification was verified on quenced as described above for fungus ITS sequencing. 2 % agarose gels containing 0.5 μg/ml ethidium bromide in 1× TBE (89 mM Tris, 89 mM boric acid, and 2 mM EDTA) or 1× Endophytic efficacy testing SB (50 mM boric acid, pH 8.5 with sodium hydroxide). The amplified DNA samples were sequenced by BigDye® To assess the mycorrhizal ability of some of the fungi that (Life Technologies, Carlsbad, CA, USA) Sanger sequencing were acquired, preliminary in vitro symbiotic seed germina- with both forward and reverse primers. The PCR products tion experiments were carried out using standard protocols were cleaned using exonuclease I and shrimp alkaline phos- (Dixon 1987; Zettler et al. 2013). Mature seed collected dur- phatase (Affymetrix, Santa Clara, CA, USA) to remove resid- ing the same trip were surface-sterilized and sown onto oat ual single-stranded primers and remaining dNTPs before meal agar (2.5 g oats, 7 g agar, 1 l deionized water, pH 5.4 Mycorrhiza prior to autoclaving; Dixon 1987) in Petri plates (about 100– (Angraecum longicalcar and Oeceoclades calcarata)had 300 seeds per plate). Each plate was then inoculated with one roots that were aerial or adherent on rock surfaces, of which of the 12 fungal isolates (Table 2), sealed with Parafilm BM^ many had reached and grown into the accumulated debris in and incubated in the dark at 18 °C for 5 months. Seeds were the cracks of the rocks. These debris were noticeably basic, sown on same medium as above, without fungal isolates, as with three separate samples having pH values of 7.8, 8.0, and the control. Plates were periodically inspected for germina- 8.2, although the soil taken from the surrounding grassland tion. Germination is defined as the stage when the seeds have had a pH of 5.8. Debris collected from among the rocky out- reached the protocorm stage. Protocorms were exposed to crops at Ambatoantrano (site 2) had a pH of 5.4, while the illumination provided by full spectrum bulbs (Sylvania Hg/ marshy, wet soil at Tsinahabeomby (site 4) had a pH of 5.0. 32W Octron 410 0K, F032/841/ECO, 80 μmol m2 s−1,12h light/12 h dark). To verify the establishment of a mycorrhizal Recovery and sequencing of fungal isolates association in the seedling stage, roots from three Tylostigma nigrescens seedlings inoculated with fungus OTU tul6 (from Percent of colonization with fungi of the root cortical regions of T. nigrescens) were detached, macerated using a sterile scal- 23 orchids is represented in Fig. 3. Most colonization was pel, and immersed in FIM as described previously. Plates were found in terrestrial taxa followed by lithophytes and epiphytes. then incubated at 18 °C for 48 h and inspected for actively Although colonized in smaller percentages, epiphytes yielded growing pelotons. Resulting fungal colonies and hyphae were more mycorrhizae than lithophytes. Of the 41 orchid taxa col- then compared to the original isolate for verification using lected within seven collection sites in the Itremo region of cen- light microscopy and visual comparisons. tral Madagascar, Rhizoctonia-like fungi were recovered from 11 orchid taxa (Table 2). This determination was based on morphological features matching Rhizoctonia-like fungi and Results confirmed by sequence data (Fig. 4). Many isolates fit the typ- ical profile of OMF in pure culture on FIM and/or PDA. In Analysis of substrate general, these isolates had cream-colored to yellowish orange colonies and usually modest to fast growth rates at 18 °C. Col- The pH of substrates collected from three collection sites was ony margins were often entire and submerged slightly with measured. At Analabeby (site 1), lithophytic orchids raised aerial mycelia towards the (older) colony center

Table 2 Operational taxonomic units (OTUs) of mycorrhizal isolates, and their identities based on ITS sequences

OTU Source species Closest informative matches in GenBank Identity (%)

Ceratobasidium OTU cer1 Aerangis punctata (A),a Aerangis sp. 1a HQ687894 uncultured Ceratobasidium from Spiranthes spiralis,Italy 89.5 OTU cer2 Aerangis ellisii (A)b JF691362 uncultured Ceratobasidiaceae from Habenaria chloroleuca, Reunion 95.3 OTU cer3 Aerangis sp. 2 (B)a HQ630980 Ceratobasidium sp. from Miscanthus giganteus,USA 94.4 OTU cer4 C. purpurea (A)c FJ788724 uncultured Ceratobasidiaceae from Pterygodium catholicum, 94.8 S. Africa Sebacina OTU seb1 C. purpurea (A)c HQ154302 uncultured Sebacina from Veratrum album, Germany 96.5 Tulasnella OTU tul1 Angraecum magdalenae (A)b EU909232 uncultured Tulasnella from Riccardia palmata,Germany 88.1 OTU tul2 B. cinnabarina (m)c KC291643 Tulasnella sp. from Hadrolaelia jongheana, Brazil 97.6 OTU tul3 Angraecum protensum (A)b, C. purpurea (A)c JF691348 uncultured Tulasnellaceae from Angraecopsis parviflora, Reunion 99.5 OTU tul4 C. purpurea(A)c, T. tenellum (m)c JF691517 uncultured Tulasnellaceae from A. ramosum, Réunion 98.8 OTU tul5 Tylostigma sp. (m)c JF691515 uncultured Tulasnellaceae 95.6 OTU tul6 T. nigrescens (m)c JF691303 uncultured Tulasnellaceae from Aeranthes arachnites, Réunion 98.7 OTU tul7 Aerangis punctata (A)a JF691324 uncultured Tulasnellaceae from Aerangis punctata, Réunion 99.8

Plant host identity confirmed: (m) as mature specimen, by morphology, (A) to species level by trnL-F sequence match, and (B) to genus level by trnL-F sequence match a Epiphytic orchids b Lithophytic orchids c Terrestrial orchids Mycorrhiza

Fig. 3 The maximum observed percent colonization of cortical cells of Highlands of Madagascar. Data were not recorded for all collections. root sections by pelotons in lithophytic (first eight taxa), epiphytic Letters C (Ceratobasidium), T (Tulasnella), and S (Sebacina) represent (second group of seven taxa) and terrestrial (last group of eight taxa) Rhizoctonia-like fungi that were isolated from those taxa. Plain bars orchid roots collected from study sites near Itremo in the Central denote no fungi of the Rhizoctonia complex among the isolates cultured

(Fig. 5). Some isolates of Sebacina and Ceratobasidium of 85 Rhizoctonia-like fungal isolates revealed the presence of yielded noticeable concentric zonation along the surface of 12 OTUs: four Ceratobasidium,oneSebacina, and seven the plate. Upon examination by light microscopy, many of the Tulasnella (Table 2). A phylogeny tree was constructed, sep- isolates produced ovoid to barrel-shaped monilioid cells in sin- arately for each genus, which included closely matched exam- gle or sparsely branched chains (Fig. 5). In some cases, numer- ples from the GenBank database (Figs. 6, 7,and8). ous monilioid cells were evident within 1 week of subculturing. All 12 OTUs had orchid-derived fungi among their closest A few isolates, however, failed to yield monilioid cells, even matches from GenBank. In the Tulasnella phylogeny tree aged (>2 months) cultures. Isolation of Rhzoctonia-like fungi (Fig. 8), representative sequences from Girlanda et al. (2011) was unsuccessful in 30 taxa due to very low colonization were also included, as the authors described two distinct clades (Angraecum coutrixii, Graphorkis concolor, and few other spe- of Tulasnella from terrestrial orchids. Similarly to Jacquemyn cies), high rate of digested pelotons (Satyrium trinerve)orin et al. (2012), all seven Tulasnella OTUs in the current study other cases, such as Bulbophyllum spp., the roots were extreme- matched with clade A sensu Girlanda et al. (2011). The ly thin and difficult to isolate pelotons. In some other cases, Ceratobasidium phylogeny tree (Fig. 7) was also well repre- dark septate endophytes (Angraecum rutenbergianum, sented by orchid-derived fungi, although somewhat less than Angraecum sp.) and other fast growing fungi made the isolation with Tulasnella; particularly noticeable was that some branches and culture of slow growing fungi difficult. Overall, epiphytes of the tree also had a number of fungi from Rosaceae and/or yielded less isolates compared to lithophytes and terrestrial. Poaceae. In the Sebacina phylogeny tree (Fig. 8), fungi found Most of the Rhizoctonia-like fungi that were isolated were in orchids were clustered on some of the branches, while other assignable to Ceratobasidium, Sebacina,orTulasnella branches were represented by fungi found in plants mainly of (Table 2) based on BLAST searches of their ITS sequences the Ericaceae and Aneuraceae. At the time of root collecting, against the GenBank database. Analysis of the ITS sequences seedling material was not available for collecting in majority of

Fig. 4 a Number of orchid species with isolates of different OTUs of the five ecosystem types from three mycorrhizal genera, Ceratobasidium, Rhizoctonia-like fungi found within five ecosystem types near Itremo in Sebacina,andTulasnella the Central Highlands of Madagascar. b Number of OTUs found within Mycorrhiza

Fig. 5 Morphological and cultural characteristics of selected from roots of Tylostigma nigrescens that grew on moist seepage mat in Rhizoctonia-like fungi collected from Itremo in the Central Highlands full sun at site 4. b Isolate OTUtul3 (UAMH 11789) acquired from roots of Madagascar on Potato Dextrose Agar (PDA) >3 months incubation of Cynorkis purpurea in site 7. c Isolate OTUcer1 from roots of an at 22 °C in 9 cm diameter Petri dishes. a Isolate OTUtul6 (UAMH 11781) Aerangis sp., an epiphyte in forest at site 7 terrestrial taxa. However, the root materials collected from ma- of 12 OTUs, three were isolated from mature orchid roots, two ture plants were colonized by Tulasnella OTUs (Table 3). Out from Tylostigma roots, and one from B. cinnabarina.

Fig. 6 Neighbor-joining phylogeny tree of aligned sequences of the seven Tulasnella OTUs (tul1–tul7) from orchid roots collected from Itremo in the Central Highlands of Madagascar. Also included in the tree are five closest BLAST matches in GenBank for each OTU, other close matches, and one representative of each of 10 OTUs from clades A and B described by Girlanda et al. (2011)(tulA1–4, tulB1–6). Sequences of clade B sensu Girlanda et al. (2011) were used to root the tree. Bootstrap percentages >50 %, after 1000 replicates, are shown Mycorrhiza

Fig. 7 Neighbor-joining phylogeny tree of aligned sequences of the four Ceratobasidium OTUs (C1–C4) from orchid roots collected from Itremo in the Central Highlands of Madagascar. Also included in the tree are five closest BLAST matches in GenBank for each OTU and other close matches. Sequence of Sebacina isolate OTU seb1 was used to root the tree. Bootstrap percentages >50 %, after 1000 replicates, are shown

Roots of C. purpurea, collected from moist soil adjacent to When the maturity of the plants that gave rise to the isolat- a clear stream in a shaded forest in Antsirakambiaty, yielded ed OTUs was considered, it appears that roots of seedling- the most diverse assemblage of isolates spanning all three stage orchids harbored more Rhizoctonia-like fungi, both in Rhizoctonia groups (Ceratobasidium, Tulasnella,and lithophytic and epiphytic orchids (Table 3). On the other hand, Sebacina). The moist dense gallery forest had four orchids terrestrial orchids, as a whole, seem able to yield Rhizoctonia- that yielded culturable Rhizoctonia-like fungi, as had the like isolates from either mature or juvenile plants. With re- moist open grassland (Fig. 4a, b). The open grassland had stricted sample sizes due to stipulations on our collection per- more Tulasnella OTUs than other sites, all of which were mit, statistical tests of significance were not carried out on the isolated from terrestrial orchids. Site 5 (Small Gallery Forest) data. gave three culturable OTUs, while the exposed rocky areas of sites 2 and 3 had two species with culturable OTUs. The dense Endophytic efficacy testing moist gallery forest had the most diverse collection of culturable fungi from the collection of root samples compared Seeds of nine orchid species collected in the trip were avail- to any other habitat. Sites 4 and 5 yielded only Tulasnella and able for symbiotic germination tests. Apart from C. purpurea, Ceratobasidium. The epiphytic orchid Aerangis punctata had none of these orchid species were able to develop to the an individual plant that harbored more than one OTU (cer1 protocorm stage on oat meal agar medium alone, while and tul7). No other individual plant yielded more than one C. purpurea developed protocorms but stopped short of fungal OTU of the Rhizoctonia complex. In addition, pelotons attaining leaf development in the absence of fungi. On the in our samples also yielded non-Rhizoctonia fungi; in partic- other hand, nine out of the 12 OTUs helped seeds to progress ular, slow-growing dark-pigmented colonies that were assign- towards the protocorm stage and/or allowed C. purpurea able to Toxicocladosporium, Cladophialophora,and protocorms to develop into seedlings within a period of Lophiostoma were isolated from more than one collection. 12 weeks, even in combination with orchid species other than Mycorrhiza

Fig. 8 Neighbor-joining phylogeny tree of aligned sequences of the Sebacina OTU seb1 found from orchid roots collected from Itremo in the Central Highlands of Madagascar. Also included in the tree are close BLAST matches in GenBank. Two representative sequences of Sebacinales subgroup A sensu Weiss et al. (2004) were used to root the tree. Bootstrap percentages greater than 50 %, after 1000 replicates, are shown

its original host species. The OTU tul2 isolated from mature tul4) were isolated more than once, from different collections roots of the terrestrial B. cinnabarina induced germination of of a species (Tables 2 and 3), suggesting a certain level of another terrestrial taxon, S. trinerve, which was also collected specificity in the association within a background of a diver- from the same site (site 4). Late stage leaf-bearing seedlings sity of fungi that are apparently available to an individual were produced from three terrestrial orchid species orchid plant. Although this observation could alterna- (T. nigrescens, C. purpurea,andH. ambositrana)fromour tively suggest that the fungi are restricted to specific preliminary symbiotic seed germination experiments. Roots habitats that coincide with the preferred habitats of their of three T. nigrescens seedlings cultured in the presence of host orchid species, it is very noteworthy that OTU tul3 fungus OTU tul6 (isolated from mature T. nigrescens)yielded was isolated from different habitats: from lithophytic actively growing pelotons evident in FIM, and the resulting Angraecum protensum and terrestrial C. purpurea.This cultures matched morphology of the original fungus culture. indicates that at least some of the fungi are able to Three OTUs (cer4, tul4 and tul7) did not help protocorm or survive in, and colonize, a range of orchid species in seedling development of any of the orchids tested. a variety of habitats, but that there is an element of selection by the orchid. Some orchids harbored a surprisingly rich assemblage of different Rhizoctonia-like fungi; Discussion C. purpurea had a Ceratobasidium, Sebacina, and two OTUs of Tulasnella. With the exception of C. purpurea and Aerangis Twelve OTUs, of three known orchid mycorrhizal genera, punctata (OTUs cer1 and tul7), all other orchids yielded only were recognized by analysis of ITS sequences of 85 isolates, one type of Rhizoctonia-like fungus. and, by comparing with GenBank database entries, each OTU The phylogeny tree is based on isolates from a limited was shown to have closely related fungi that were also found number of root samples due to extremely low number of as orchid associates. Of the 12 OTUs, three (cer1, tul3, and plants available for collecting; therefore, this does not Mycorrhiza

Table 3 List of orchid species from which roots were collected, Growth Species Site Growth phase OTUs UAMH numbers and the Rhizoctonia-like fungi habit isolated that they yielded Lithophytic Aerangis ellisii 4 Seedling cer2 (11) 6 Mature plant6 Angraecum coutrixii 2, 3 Seedling 3 Mature plant Angraecum longicalcar 1 Mature plant Angraecum magdalenae 3 Tiny seedling tul1 (14) Angraecum protensum 2 Seedling tul3 (4) 2 Mature plant Angraecum rutenbergianum 3, 4 Seedling Angraecum sororium 3 Tiny seedling Bulbophyllum sp. 1 6 Mature plant Oeceoclades calcarata 1 Mature plant Epiphytic Aerangis sp. 1 7 Seedling cer1 (4) Aerangis sp. 2 5 Tiny seedling cer3 (8) 5 Mature plant Aerangis ellisii 6 Mature plant Aerangis punctata 5 Seedling cer1 (7), tul7 (2) Aeranthes sp. 7 Mature plant Angraecum sp. 1 5 Tiny seedling Angraecum sp. 2 7 Seedling Angraecum coutrixii 7 Mature plant Angraecum protensum 2 Seedling Angraecum rutenbergianum 5 Mature plant Bulbophyllum sp. 2 3 Seedling Bulbophyllum sp. 3 7 Mature plant Bulbophyllum sp. 4 7 Mature plant Bulbophyllum sp. 5 7 Mature plant Jumellea densefoliata 5 Seedling Polystachya concreta 3 Mature plant P. cultriformis 7 Tiny seedling Terrestrial Benthamia sp. 2 Mature plant B. cinnabarina 4 Mature plant tul2 (2) B. glaberrima 3 Mature plant B. rostrata 4 Seedling Calanthe sp. 7 Mature plant Cynorkis gibbosa 7 Mature plant Cyn. purpurea 7 Seedling cer4 (1) 11787 seb1 (2) 11791 tul3 (13) 11783, 11786, 11788, 11789, 11790, 11792, 11793 The maturity level of the plants tul4 (2) 11784, 11785 that yielded the roots was noted at Disa incarnata 3, 4 Mature plant collection. For some species, roots from both mature and Eulophia macra 1 Mature plant juvenile specimens were collected Graphorkis concolor 7 Mature plant with minimum of three roots per Habenaria sp. 2 Mature plant collection. Numbers in Satyrium trinerve 4 Mature plant parentheses indicate the number of isolates identified as belonging Tylostigma sp. 4 Mature plant tul5 (5) to that OTU. Some isolates have T. nigrescens 4 Mature plant tul6 (4) 11779, 11780, 11781, 11782 been deposited at UAMH T. tenellum 7 Seeding tul4 (2) (Edmonton, Canada) Mycorrhiza represent the full genetic diversity of the orchids within the general profile. The Ceratobasidiaceae was also well repre- remit of this study. However, this preliminary study suggested sented in Madagascar root samples, paralleling reports mostly that a fungus seems able to associate with different types of from the New World (e.g., Richardson et al. 1993; Zettler and orchids and some are specific to only certain orchids. Very Piskin 2011), and Sebacinaceae fungi were also present notable was that the closest GenBank match of OTU tul7 among our isolates. Representatives of both Tulasnellaceae (from Aerangis punctata) was a fungus that was identified in and Ceratobasidiaceae were isolated from several orchid spe- Aerangis punctata in Réunion (JF691324 and JF691326, cies encompassing terrestrial, lithophytic, and epiphytic Martos et al. 2012), a neighboring island to Madagascar but habits, but more epiphytes yielded Ceratobasidiaceae fungi some 800 km to the east. OTU cer4, from the terrestrial while terrestrials had mostly Tulasnellaceae fungi (Table 2). C. purpurea, was found to be closely matched with sequences It is interesting to note that gallery forest and the moist open of fungi from Pterygodium catholicum (FJ788724, FJ788725, grassland had the most hits for culturable Rhizoctonia-like and FJ788812, Waterman et al. 2011), another African terres- fungi, predominantly from terrestrial taxa. The open grassland trial orchid. It is planned to further investigate such indications and adjacent lithophytic habitats are subjected to man-made of specificity; future collections will focus on roots of a greater fire (Whitman et al. 2011). Seedling recruitment and survival number of individual plants and from several different sites for are likely to be adversely affected; therefore, conservation of species of high conservation and scientific interests. Fungus– orchids from these areas warrants immediate attention. Gal- orchid relationships of selected orchids will also be studied by lery forests are also affected, therefore, mapping the orchid genomic analysis of mycorrhizal fungi in root sections with diversity and their fungal symbionts need urgent study. commonly used genetic markers in addition to ITS. Results of soil/substrate analysis showed that most habitats To our knowledge, this is the first report documenting where orchids were collected in this study showed an acidic pH culturable, potentially mycorrhizal endophytes from orchids of 5.0–5.8. One exception to this was the lithophytic habitat at of different life forms from diverse ecosystems from Mada- Analabeby (site 1) where the pH of the accumulated substrate gascar, and one of the few from the African region (e.g., between the rocky outcrops, within which fungal symbionts Jonsson and Nylund 1979; Mugambi 2001; Bonnardeaux might be expected to exist until they encounter their hosts, et al. 2007; Waterman et al. 2011;Martosetal.2012), most was consistently basic at 7.8–8.2. Analabeby (Fig. 1) collection of which have used culture-independent molecular methods to sites feature rocky outcrops consisting mainly of marble, detect and identify the fungi. Our success at isolating these whereas the outcrops at the other study areas were of sandstone, fungi from remote and inaccessible locations suggests that our granite, and quartzite, which explains the difference in pH of method used to collect and transport fresh root samples may the immediate environment of lithophytic plants. It was partic- have practical merit for other researchers faced with the daunt- ularly noticeable that the surfaces of the rocks at Analabeby ing task of long-distance field work. For the terrestrial orchids were devoid of lichen, in contrast to its abundance on the rocks in particular, the removal of a root ball (roots with surrounding at Ambatoantrano, where the debris among the rocks had a pH soil), as opposed to immediate root detachment, followed by of 5.4. Lithophytic habitats at other collection sites also had transport from the field to Antananarivo days later, may have profuse growths of lichen, suggesting that these locations also contributed to the high number of isolates we recovered. For had similarly acidic substrates. Rhizoctonia-like fungi were all samples, especially roots of small epiphytic seedlings successfully isolated from lithophytic roots collected at prone to desiccation, placing fresh tissues over a moist cotton Ambatoantrano (site 2), Ambatoantremo (site 3), and ball, coupled with (cool) incubation in darkness during trans- Tsinahabeomby (site 4), whereas in contrast, none were isolated port, also may have contributed to the favorable outcome. from lithophytic roots collected from Analabeby. Epiphytic or- These, and numerous other, studies support the hypothesis chids were also often found in association with lichen; four that at least some tropical orchids, like their temperate coun- collections from epiphytic habitats yielded OTUs of terparts, associate with basidiomycetes in the Cantharellales Rhizoctonia-like fungi. These observations could be indicating and Sebacinales (Bidartondo et al. 2004; Selosse et al. 2004). that fungal associates of the lithophytes at Analabeby may have Within this group, the overwhelming majority of isolates fall a different physiology as a consequence of having adapted to a under the category of BRhizoctonia-like fungi^ (Arditti 1992; different environment, and they may therefore have different Otero et al. 2002; Rasmussen 2002; Dearnaley 2007), culture requirements. It could even be possible that orchids in encompassing teleomorphic genera Ceratobasidium these environments have symbiotic associations with types of (anamorphs=Ceratorhiza), Tulasnella (anamorphs= fungi other than those of the Rhizoctonia complex. It may be Epulorhiza), and Sebacina (Warcup and Talbot 1980). necessary to adjust the isolation medium composition if mycor- Tulasnelloid fungi, in particular, have been recovered from rhizae are to be successfully isolated from orchid roots from orchids with regularity throughout the world (e.g., such environments. Nontachaiyapoom et al. 2010; Zettler et al. 2013; Zi et al. As an alternative to in situ seed baiting, we made a con- 2014), and the orchids of Madagascar appear to fit the same scious effort to collect roots from young orchid plants, Mycorrhiza including protocorms and very small seedlings. These juvenile a good source of isolates (Table 3). Regardless of whether plants were identified in the first instance by local botanical these fungal OTUs are the same fungi that first allowed the specialists based on morphology, habitat, and the proximity of successful germination of the orchid seed and establishment of mature specimens that could be the likely parents. We set out to the protocorm, the relative ease with which juvenile roots of bolster our confidence in the identities of the seedlings by seek- some orchid species yielded Rhizoctonia-like fungi may be ing a definitive identification using DNA sequence analysis to indicative of a greater dependence upon mycorrhizal symbio- compensate for the lack of mature morphological features. By sis at particular stages of the life cycle. Older plants may be matching DNA sequences of the seedlings to those of collec- less dependent on Rhizoctonia-like fungi and/or may be tions of mature plants and/or GenBank entries, we have been switching to different fungi upon maturity. With the focus on able to confirm the identities of the putatively identified seed- Rhizoctonia-like fungi in this study, there is scope for further lings. As one of our aims is to investigate the specificities in the investigations into the possibility of fungal taxa other than orchid–fungus relationship, we consider the molecular confir- Rhizoctonia-like fungi that may be OMF. Roots that were mation of the seedling identity to be of critical importance and heavily colonized by fungi, as evidenced by a large number propose the methodology as standard procedure for our future of visible pelotons, yet failed to yield Rhizoctonia-like isolates collections of fungal associates from juvenile plants in Mada- may have harbored different and/or difficult to culture fungi gascar, with its diversity of orchid species. that were missed in this study and demands further attention. Targeting spontaneous seedlings offers greater flexibility, Furthermore, as the collection trip for this study was made at compared to seed burial (baiting) technique, with positive the start of the dry season and possibly at the start of plant identification being done within weeks instead of months or dormancy, a collection made during wet season, when the years of collecting the material. With the help of fingerprinting orchids and fungi are anticipated to be participating in active techniques as we used here, identifying the seedlings has been symbiosis, will be investigated for future study. Collecting made more reliable. As orchid populations continue to decline more material to obtain larger sample sizes would be needed worldwide, it is conceivable that cross-pollination between for statistically meaningful survey data, which the authors are unrelated individuals will diminish resulting in lower seed reluctant to recommend as a routine protocol to study excep- viability. As a result, it may be necessary for future workers tionally rare orchid species as reported here. Still, it would also to rely more on spontaneous seedlings and less on the standard be of interest to study the levels of fungal associations found seed baiting techniques as a means to acquire early germina- on a single established plant over time, collecting samples at tion phase fungi. For critically endangered taxa, use of the different times of year, and over a period of multiple years. seed baiting technique may also be viewed as too wasteful This will help understand the nature and significance of fungi considering that few seed packets typically yield seedlings to orchid growth, recovery, and development, and its flux (protocorms) once retrieved. Nevertheless, we recommend through the seasons and the orchid’s life cycle. that seed baiting techniques aimed at recovering protocorms not be abandoned, as the specific fungi supporting initial seed germination may actually be replaced by other types of fungi Conclusions and future studies around the time that leaves are initiated in the seedling stage (fungal succession). Thus, it is conceivable that some of the As the first detailed investigation into the identification of fungi isolated from spontaneous seedlings may not be the culturable orchid endophytes in Madagascar that are potential same as those that initiated germination in situ. However, OMF, our findings will form the foundation of our future based on preliminary results involving in vitro symbiotic seed approach in understanding how the orchid–fungus relation- germination using the fungi isolated in this study (research in ship relates to their native environments. In addition to progress), majority of these fungi are indeed capable of initi- planned further collections from different locations, different ating germination beyond the protocorm stage. This suggests orchid species, and at different times of the year, in vitro ger- that spontaneous seedlings—at least in part—harbor fungi of mination tests will be carried out to reveal the specificity/ some physiological significance, and are not merely carriers of generality of the symbiosis. It is expected that the sum of benign fungal assemblages. A thorough investigation into the knowledge will lead to a better conservation strategy that is possible mycorrhizal ability, or lack thereof, of the different designed to protect vulnerable orchid taxa by taking into ac- fungi during orchid germination and subsequent growth is count of their need for symbiotic partners. Furthermore, this essential to better understand the diversity of fungi that have will help to develop a phylogentic analysis of mycorrhizae, so far been isolated. their evolution across the land mass of Madagascar, and how Our tally of Rhizoctonia-like fungi suggested that seedling this affects natural regeneration of orchids in pristine and roots of some lithophytic and epiphytic taxa were able to pro- fragmented ecosystems. vide good quality pelotons for isolating Rhizoctonia-like iso- Erratic pollination and low seed set were identified in lates, whereas mature root material of terrestrial taxa was also Aerangis ellisii (Nilsson and Rabakonandrianina 1988; Nilsson Mycorrhiza et al. 1992a, b), but there may be similarly affected taxa in map its distribution and to assess its conservation status. In: Madagascar. In the case of Aerangis ellisii,wehavenoticedvery Lourenço WR (ed) Proceedings of the International Symposium on the ‘Biogeography de Madagascar’, Paris, September 1995, pp low or no germination of mature seeds by asymbiotic methods 205–218 (unpublished data). To develop genetically diverse stocks for Fenu G, Mattana E, Congiu A, Bacchetta G (2010) The endemic vascular species restoration projects is a huge challenge as in vitro flora of Supramontes (Sardinia), a priority plant conservation area. – asymbiotic germination alone is not an effective tool. If mycor- Candollea 65:347 358 Gale SW, Yamazaki J, Hutchings MJ, Yukawa T, Miyoshi K (2010) rhizal seed germination can be applied to improve not just seed Constraints on establishment in an endangered terrestrial orchid: a germination but seedling survival in cases otherwise impossible comparative study of in vitro and in situ seed germinability and may be a good step in the right direction. Although data are not seedling development in Nervilia nipponica. Bot J Lin Soc 163: – available at the moment regarding fungal diversity and density 166 180 Gardes M, Bruns TD (1993) ITS primers with enhanced specificity for for a specific habitat as part of this study, symbiotic seedlings basidiomycetes: application to the identification of mycorrhizae and can be used for reintroduction. Detailed studies to identify the rusts. Mol Ecol 2:113–118 mycobiont populations benefit the resilience of reintroduced Girlanda M, Segreto R, Cafasso D, Liebel HT, Rodda M, Ercole E, orchids in Madagascar and other biodiversity hotspots areas. Cozzolino S, Gebauer G, Perotto S (2011) Photosynthetic Mediterranean meadow orchids feature partial mycoheterotrophy This will be developed as a major research area to contribute and specific mycorrhizal associations. Am J Bot 98:1148–1163 to ecosystem services, conservation, and phylogenetic studies. Jacquemyn H, Deja A, De hert K, Bailarote BC, Lievens B (2012) Variation in mycorrhizal associations with tulasnelloid fungi among Acknowledgments We kindly thank the financial support received populations of five Dactylorhiza species. PLoS One 7(8):e42212 from Sainsbury Orchid Project, Bentham and Moxon Trust, and Margaret Jonsson L, Nylund JE (1979) Favolaschia dybowskyana (Singer) Singer A Cargill Foundation. We acknowledge the invaluable assistance re- (Aphyllophorales), a new orchid mycorrhizal fungus from tropical ceived from Gaëtan Ratovonirina and Landy Rajaovelona (KMCC), Solo Africa. New Phytol 83:121–128 Rapanarivo (PBZT) for field support during the collection; Korrie Ed- Martos F, Munoz F, Pailler T, Kottke I, Gonneau C, Selosse MA (2012) wards (Illinois College), and Helen Sandford, Margaret Ramsay, and The role of epiphytism in architecture and evolutionary constraint Edward Jones (Kew) for technical assistance; Connie Gibas and Lynne within mycorrhizal networks of tropical orchids. Mol Ecol 21:5098– Sigler (UAMH) for deposition of isolates; Stuart Cable (Kew) and Hanne 5109 Rasmussen for helpful suggestions; and Mike Fay and Robyn Cowan McKendrick SL, Leake JR, Taylor DL, Read DJ (2000) Symbiotic ger- (Kew) for their advice on genetic fingerprinting of plant samples. mination and development of myco-heterotrophic plants in nature: ontogeny of Corallorhiza trifida and characterization of its mycor- rhizal fungi. New Phytol 145:523–537 Mitchell RB (1989) Growing hardy orchids from seeds at Kew. References Plantsman 2:152–169 Mittermeier RA, Robles PG, Hoffman M, Pilgrim J, Brooks T, Mittermeier CG, Lamoreux J, da Fonseca GAB (2005) Hotspots Alvarado ST, Buisson E, Rabarison H, Rajeriarison C, Birkinshaw C, revisited: earth’s biologically richest and most endangered terrestrial Lowry PP (2014) Comparison of plant communities on the Ibity ecoregions. University of Chicago Press, Chicago and Itremo massifs, Madagascar, with contrasting conservation his- Moat J, Smith P (2007) Atlas of the vegetation of Madagascar. Royal – tories and current status. Plant Ecol Divers 7:497 508 Botanic Gardens, Kew Arditti J (1992) Fundamentals of orchid biology. Wiley, New York Mugambi GK (2001) Ensuring survival of Kenyan orchids: the ex situ Batty AL, Brundrett MC, Dixon KW, Sivasithamparam K (2006) In situ conservation interventions. International Orchid Conservation symbiotic seed germination and propagation of terrestrial orchid Congress I (Conference Proceedings), p 102 – seedlings for establishment at field sites. Aust J Bot 54:375 381 Murray-Smith C, Brummitt NA, Oliviera-Filho AT, Bachman S, Moat J, Bidartondo MI, Burghardt B, Gebauer G, Bruns TD, Read DJ (2004) Lughadha EM, Lucas EJ (2009) Plant diversity hotspots in the Changing partners in the dark: isotopic and molecular evidence of Atlantic coastal forests of Brazil. Conserv Biol 23:151–163 ectomycorrhizal liaisons between forest orchids and trees. Proc R Nilsson LA, Rabakonandrianina E (1988) Hawk-moth scale analysis and Soc Lond B 271:1799–1806 pollination specialization in the epilithic malagasy endemic Bonnardeaux Y, Brundrett M, Batty A, Dixon K, Koch J, Aerangis ellisii (Reichenb fil) Schltr (Orchidaceae). Bot J Linn Sivasithamparam K (2007) Diversity of mycorrhizal fungi of terres- Soc 97:49–61 trial orchids: compatibility webs, brief encounters, lasting relation- Nilsson LA, Rabakonandrianina E, Pettersson B (1992a) Exact tracking ships and alien invasions. Mycol Res 111:51–61 of pollen transfer and mating in plants. Nature 360:666–668 Clements MA, Ellyard RK (1979) The symbiotic germination of Nilsson LA, Rabakonandrianina E, Rotaharivelo R, Randriamanindry JJ Australian terrestrial orchids. Am Orchid Soc Bull 48:810–816 (1992b) Long pollinia on eyes: hawk-moth pollination of Cynorkis Cribb P, Hermans J (2009) Field guide to the orchids of Madagascar. Kew uniflora Lindley (Orchidaceae) in Madagascar. Bot J Linn Soc 109: Publishing. Royal Botanic Gardens, Kew 145–160 Dearnaley JDW (2007) Further advances in orchid mycorrhizal research. Nontachaiyapoom S, Sasirat S, Manoch L (2010) Isolation and identifi- Mycorrhiza 17:475–486 cation of Rhizoctonia-like fungi from roots of three orchid genera, Dixon KW (1987) Raising terrestrial orchids from seed. In: Harris WK Paphiopedilum, Dendrobium,andCymbidium, collected in Chiang (ed) Modern orchid growing for pleasure and profit. Orchid Club of Rai and Chiang Mai provinces of Thailand. Mycorrhiza 20:459–471 S Australia, Inc, Adelaide, pp 47–100 Otero JT, Ackerman JD, Bayman P (2002) Diversity and host specificity Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small of endophytic Rhizoctonia-like fungi from tropical orchids. Am J quantities of fresh leaf tissue. Phytochem Bull 19:11–15 Bot 89:1852–1858 du Puy D, Moat J (1996) A refined classification of the primary vegeta- Phillips RD, Barrett MD, Dixon KW, Hopper SD (2011) Do mycorrhizal tion of Madagascar based on the underlying geology: using GIS to symbioses cause rarity in orchids? J Ecol 99:858–869 Mycorrhiza

Rasmussen HN (2002) Recent developments in the study of orchid my- Warcup JH, Talbot PHB (1980) Perfect states of Rhizoctonias associated corrhiza. Plant Soil 244:149–163 with orchids III. New Phytol 86:267–272 Rasmussen HN, Whigham DF (1993) Seed ecology of dust seeds in situ: Waterman RJ, Bidartondo MI, Stofberg J, Combs JK, Gebauer G, a new study technique and its application to terrestrial orchids. Am J Savolainen V, Barraclough TG, Pauw A (2011) The effects of Bot 80:1374–1378 above- and belowground mutualisms on orchid speciation and co- Richardson KA, Currah RS, Hambleton S (1993) Basidiomycetous en- existence. Am Nat 177:54–68 dophytes from the roots of neotropical epiphytic Orchidaceae. Weiss M, Selosse MA, Rexer KH, Urban A, Oberwinkler F (2004) Lindleyana 8:127–137 Sebacinales: a hitherto overlooked cosm of heterobasidiomycetes Roos MC, Kessler PJA, Gradstein SR, Baas P (2004) Species diversity with a broad mycorrhizal potential. Mycol Res 108:1003–1010 and endemism of five major Malesian islands: diversity–area rela- White TJ, Bruns TD, Lee S, Taylor JW (1990) Amplification and direct tionships. J Biogeogr 31:1893–1908 sequencing of fungal ribosomal RNA genes for phylogenetics. In: Samarakoon T, Wang SY, Alford MH (2013) Enhancing PCR amplifica- Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols: a tion of DNA from recalcitrant plant specimens using a trehalose- guide to methods and applications. Academic, San Diego, pp 315– based additive. Appl Plant Sci 1:1200236 322 Selosse MA, Faccio A, Scappaticci G, Bonfante P (2004) Chlorophyllous Whitman M, Medler M, Randriamanindry JJ, Rabakonandrianina E and achlorophyllous specimens of Epipactis microphylla (Neottieae, (2011) Conservation of Madagascar’s granite outcrop orchids: the Orchidaceae) are associated with ectomycorrhizal septomycetes, in- influence of fire and moisture. Lankesteriana 11:55–67 cluding truffles. Microb Ecol 47:416–426 Zettler LW, Piskin KA (2011) Mycorrhizal fungi from protocorms, seed- Swarts ND, Dixon KW (2009) Terrestrial orchid conservation in the age lings and mature plants of the Eastern Prairie Fringed Orchid, of extinction. Ann Bot 104:543–556 Platanthera leucophaea (Nutt.) Lindl.: a comprehensive list to aug- Taberlet P, Gielly L, Pautou G, Bouvet J (1991) Universal primers for ment conservation. Am Midl Nat 166:29–39 amplification of three non-coding regions of chloroplast DNA. Plant Zettler LW, Sharma J, Rasmussen F (2003) Mycorrhizal diversity. In: MolBiol17:1105–1109 Dixon KW, Kell SP, Barrett RL, Cribb PJ (eds) Orchid conservation. Taylor DL, McCormick MK (2008) Internal transcribed spacer primers Natural History Publications (Borneo), Kota, pp 185–203 and sequences for improved characterization of basidiomycetous Zettler LW, Corey LL, Jacks AL, Gruender LT, Lopez AM (2013) orchid mycorrhizas. New Phytol 177:1020–1033 Tulasnella irregularis (Basidiomycota: Tulasnellaceae) from roots Tyson P (2000) The eighth continent: life, death and discovery in the lost of Encyclia tampensis in south Florida, and confirmation of its my- world of Madagascar. William Morrow (Harper Collins) Publishers, corrhizal significance through symbiotic seed germination. New York Lankesteriana 13:119–128 Vorontsova MS, Ratovonirina G, Randriamboavonjy T (2013) Revision Zi XM, Sheng CL, Goodale UM, Shao SC, Gao JY (2014) In situ seed of Andropogon and Diectomis (Poaceae: Sacchareae) in Madagascar baiting to isolate germination-enhancing fungi for an epiphytic or- and the new Andropogon itremoensis from the Itremo Massif. Kew chid, Dendrobium aphyllum (Orchidaceae). Mycorrhiza 24:487– Bull 68:1–15 499