fungal biology reviews 27 (2013) 83e99

journal homepage: www.elsevier.com/locate/fbr

Review Lineages of ectomycorrhizal fungi revisited: Foraging strategies and novel lineages revealed by sequences from belowground

Leho TEDERSOOa,*, Matthew E. SMITHb,** aNatural History Museum and Institute of Ecology and Earth Sciences, Tartu University, 14A Ravila, 50411 Tartu, Estonia bDepartment of Plant Pathology, University of Florida, Gainesville, FL, USA article info abstract

Article history: In the fungal kingdom, the ectomycorrhizal (EcM) symbiosis has evolved independently in Received 29 April 2013 multiple groups that are referred to as lineages. A growing number of molecular studies in Received in revised form the fields of mycology, ecology, soil science, and microbiology generate vast amounts of 10 September 2013 sequence data from fungi in their natural habitats, particularly from soil and roots. How- Accepted 17 September 2013 ever, as the number and diversity of sequences has increased, it has become increasingly difficult to accurately identify the fungal species in these samples and to determine their Keywords: trophic modes. In particular, there has been significant controversy regarding which fungal Biogeography groups form ectomycorrhizas, the morphological “exploration types” that these fungi form Ectomycorrhizal symbiosis on roots, and the ecological strategies that they use to obtain nutrients. To address this Evolutionary lineages problem, we have synthesized the phylogenetic and taxonomic breadth of EcM fungi by us- Exploration types ing the wealth of accumulated sequence data. We also compile available information about Internal Transcribed Spacer (ITS) exploration types of 143 genera of EcM fungi (including 67 new reports) that can be tenta- Phylogenetic diversity tively used to help infer the ecological strategies of different fungal groups. Phylogenetic analyses of ribosomal DNA ITS and LSU sequences enabled us to recognize 20 novel line- ages of EcM fungi. Most of these are rare and have a limited distribution. Five new lineages occur exclusively in tropical and subtropical habitats. Altogether 46 fungal genera were added to the list of EcM fungal taxa and we anticipate that this number will continue to grow rapidly as taxonomic works segregate species-rich genera into smaller, monophyletic units. Three genera were removed from the list of EcM groups due to refined taxonomic and phylogenetic information. In all, we suggest that EcM symbiosis has arisen indepen- dently in 78e82 fungal lineages that comprise 251e256 genera. The EcM fungal diversity of tropical and southern temperate ecosystems remains significantly understudied and we expect that these regions are most likely to reveal additional EcM taxa. ª 2013 The Authors. Published by Elsevier Ltd. Open access under CC BY-NC-ND license.

* Corresponding author. Tel.: þ372 56654986; fax: þ372 7376222. ** Corresponding author. Tel.: þ1 011 352 2732837; fax: þ1 011 352 3926532. E-mail addresses: [email protected] (L. Tedersoo), trufflesmith@ufl.edu (M. E. Smith). 1749-4613 ª 2013 The Authors. Published by Elsevier Ltd. Open access under CC BY-NC-ND license. http://dx.doi.org/10.1016/j.fbr.2013.09.001 84 L. Tedersoo, M. E. Smith

1. Introduction ectomycorrhizal symbionts as well as devastating fungal plant pathogens (Veldre et al., 2013). Several lineages within The ectomycorrhizal (EcM) symbiosis has evolved multiple Pezizales are associated with tree roots in truffieres where times both in plants and fungi. The fungal kingdom includes they compete for space and resources with the valuable truffle at least 66 independent lineages of EcM fungi, mostly mem- “crop” species (Bonito et al., 2011, 2012; Rubini et al., 2011). bers of the Basidiomycota and Ascomycota (Tedersoo et al., However, most species of Pezizales are saprotrophic and a 2010). In the past it has been challenging to unambiguously few are pathogenic (Hansen and Pfister, 2006). The rapid shift determine whether some fungal groups are ectomycorrhizal from root tip-based studies to soil fungal community studies or not because of limited or ambiguous evidence and alterna- necessitates discriminating between mycorrhizal and non- tive interpretations (Rinaldi et al., 2008; Tedersoo et al., 2010; mycorrhizal fungi. Molecular studies of soil increasingly use Comandini et al., 2012; Ryberg and Matheny, 2012). However, sophisticated second and third generation sequencing tech- community studies of ectomycorrhizal fungi have become nologies to generate millions of reads. In contrast to EcM roots more sophisticated over the past two decades and a variety and fruit-bodies that can be stored as vouchers and morpho- of techniques have been used to determine EcM status logically examined in the future, soil-based studies cannot and to delimit groups of EcM taxa, including experimental provide morphological or ultrastructural information to infer synthesis trials, field observations combining anatomical ecological interactions. These high-throughput sequencing and molecular techniques, stable nitrogen and carbon isotope methods are an easy and cost-effective way to study mycor- signatures, and phylogenetic analyses. Unfortunately, many rhizal ecology in situ but currently our ability to adequately EcM fungal taxa detected as environmental sequences do identify fungal DNA sequences and interpret the ecological not match sequences of fruit-body vouchers or pure cultures role of these species is lagging behind our ability to produce (Tedersoo et al., 2010). Although reference sequences from sequence data. The phylogenetic and functional breadth of fruit-bodies are accumulating at an exponential rate, many fungi in soil and other complex substrates poses a great chal- sequences from EcM root tips remain unmatched to their sex- lenge for taxonomic identification as well as functional char- ual stages. This suggests that much of the EcM fungal diversity acterization. Because of the large volume of sequence data is indeed cryptic. Nevertheless, many described species and and the large number of fungal taxa involved in the EcM sym- even genera lack publicly available sequence data (Tedersoo biosis, researchers would benefit from a well-annotated refer- et al., 2010) and this underscores the need to produce DNA se- ence database from which they can automatically extract quences from identified herbarium specimens (Brock et al., information on ecology and taxonomy of fungal taxa (Koljalg~ 2009). et al., 2013). The rapidly growing DNA sequence data in public reposi- Within EcM fungi, there are major differences in ecological tories, in conjunction with recently developed sequence anno- strategies of dispersal (Ishida et al., 2008), metabolic activity tation tools (e.g. PlutoF workbench e Abarenkov et al., 2010; (Trocha et al., 2010) and in relative benefits to plant hosts Tedersoo et al., 2011a), provide an invaluable source of meta- (van der Heijden and Kuyper, 2003; Nara, 2006). This is at least data about the host plants, isolation sources, and geographic partly ascribed to differences in the relative carbon cost to origin of EcM fungal isolates. By using the data in public plants and efficiency in enzymatic access to organically bound sequence databases, Hynson et al. (2013), Tedersoo et al. nutrients, nutrient uptake and nutrient transfer (Courty et al., (2013a) and Veldre et al. (2013) recently detected additional pu- 2010). Species of EcM fungi differ strongly in their potential tative EcM lineages within the Serendipitaceae (Sebacinales enzymatic capacity, which is a function of both the substrate group B), Pyronemataceae and Ceratobasidiaceae. These and climatic conditions (Courty et al., 2010). Evidence suggests fungal lineages were previously considered root endophytes, that key enzyme functions are highly variable between (and saprotrophs or parasites. The limited number of sequences within) EcM lineages and are partly predictable based on from these lineages suggests that they are uncommon in phylogenetic relationships among the EcM fungi (Tedersoo EcM fungal communities or were considered root contami- et al., 2012b). Not surprisingly, the abundance and nants in the original studies (e.g. Oberwinkler et al., 2013). morphology of the extraradical mycelial system is the single It is important to understand which fungal taxa are EcM most important variable in determining enzymatic capacity and which are not. EcM fungi play fundamentally different (Tedersoo et al., 2012b). The presence and characteristics of roles in forest communities and in ecosystems compared to extraradical hyphae and rhizomorphs serve as proxies for other functional guilds such as fungal root endophytes and foraging strategies referred to as “exploration types” (Agerer, decomposers. For example, EcM fungi are uniquely adapted 2001, 2006). Species of medium-distance and long-distance to facilitate mineral nutrition of plants and to distribute recent exploration types tend to exhibit similar responses to climatic photosynthates into the mycorrhizosphere soil (Buee et al., gradients (Ostonen et al., 2011), N fertilization or pollution 2009). Mycorrhizal fungi have mostly lost the powerful en- (Lilleskov et al., 2011; Kjøller et al., 2012) and carbon influx zymes used for attacking plant cell walls and degrading (Markkola et al., 2004). Most fungi with long-distance explora- organic compounds such as lignin (Eastwood et al., 2011). tion strategies appear specialized in N uptake from organic Furthermore, since EcM fungi are often the most abundant sources and they apparently expend significant carbon re- organisms in forest soils, it is important to understand sources on rhizomorphs so they appear to lose their relative their ecology for the purposes of management and conserva- benefits or competitive abilities in disturbed systems tion. This is particularly relevant for taxa in the Ceratobasidia- (Lilleskov et al., 2011). In contrast, smooth and short- ceae, since this group includes beneficial orchid and distance exploration types are more frequently detected in Lineages of ectomycorrhizal fungi revisited 85

mineral soils and these types are more resilient in response to et al., 2010), and subjected to RaXML Maximum Likelihood an- disturbance, apparently because they can easily regenerate alyses with the GTR þ G þ I evolutionary model and 1000 fast their reduced system of extraradical hyphae. So far, in-depth bootstrap replicates (Stamatakis et al., 2008). We used a series studies of EcM morphology and examination of subterranean of Fisher’s Exact tests to investigate the null hypothesis that foraging strategies cover only the most common genera and sequences derived from EcM root tips are non-randomly species (reviewed in Agerer, 2006). Based on these studies it distributed in the target lineage and its sister groups. We appears that EcM morphology and exploration types are anticipate that a posteriori selection of the ingroup and out- mostly phylogenetically conserved (Eberhardt, 2002; Agerer, group, presence of redundant sequences (i.e. similar se- 2006), but that there are variations in foraging strategy within quences from the same study) and multiple testing may the largest and most well studied genera (e.g. Russula, Lactarius introduce biases to these results. and Tomentella). This information is increasingly referred to in We evaluated all EcM-associated sequences and mycor- fruit body, root tip and soil mycelium-based community ana- rhizal literature published since our previous review lyses (e.g. Deslippe et al., 2011; Ostonen et al., 2011), but most (Tedersoo et al., 2010) in order to determine EcM fungal line- studies do not determine these types in situ. ages that may be present in the database but have not been We have three main purposes in this review. First, we formally recognized. In order to be considered EcM symbionts, gather and interpret information about cryptic EcM lineages a fungal lineage had to meet at least one of the three following from environmental sequences in public databases and we criteria: 1) the EcM-derived sequences form a well-supported give these lineages formal names and designated reference monophyletic clade that includes all or mostly EcM fungal se- sequences. Second, we revise the lineage concept in several quences and does not include sequences from non-EcM habi- groups of EcM fungi based on newly available taxonomic tats (e.g. sequences from roots of non-EcM plants, agricultural and ecological information. Third, we compile existing pub- soil, etc.); 2) EcM root tip vouchers (obtained directly from the lished and unpublished information about the exploration study authors) exhibited typical EcM morphology and the types of all EcM fungal genera by lineages. Information about morphological features were both consistent among different all of the EcM lineages detailed in this paper and by Tedersoo vouchers and also consistent with the phylogenetic position et al. (2010) are provided in Table S1 and regularly updated in of the fungal lineage (cf. Agerer, 2006); and 3) a phylogenetic the UNITE homepage (http://unite.ut.ee/EcMlineages.php). test statistic indicating that there is a greater probability of as- sociation with EcM root tips in the ingroup taxa as compared to outgroup taxa. 2. Methods To determine exploration types of fungi, we relied on an extensive online database about morphological and anatom- We searched the fungal ITS sequence data deposited in Inter- ical descriptions of EcM at www.deemy.de (see also Agerer, national Nucleotide Sequence Database consortium (INSDc) 2006). For groups for which no exploration type data are avail- and UNITE (as of 01.12.2012) using the PlutoF workbench, an able, we studied the publications that included brief EcM de- online tool that allows users to permanently annotate scriptions or directly contacted study authors to obtain sequence quality and metadata (Abarenkov et al., 2010). We additional information on EcM morphology. Lastly, we used updated the metadata on EcM fungi (source of isolation, host light microscopy to re-analyze the EcM morphology of vouch- or substrate, and geographical origin) in PlutoF by searching ered EcM root tips from our own community studies. in the published studies or directly consulting the authors of the studies. We primarily focused on sequences with the term “ectomycorrhiza” listed as the “isolation source” in Plu- 3. Novel lineages of ectomycorrhizal fungi toF. All of the sequences with this designation were assigned to EcM fungal lineages or given a status “non-ectomycorrhi- Based on ITS sequence data, together with updated metadata zal” or “uncertain” based on sequence alignments and phylo- and findings from previous studies, we identified 20 previ- genetic analyses following the protocols of Tedersoo et al. ously unrecognized EcM lineages (11 in Basidiomycota, 7 in (2011a). We also consulted recent publications to evaluate Ascomycota and 2 in Zygomycota) (Table 1). Eight lineages the opinion of other authors on previously unrecognized are derived from separating previously described lineages EcM taxa. We downloaded sequences and associated meta- into two groups in light of accumulated sequence and phylo- data for all these EcM groups and their non-EcM relatives (out- genetic information. We also briefly describe what is known group sequences) as determined by blastN searches against about the EcM exploration types of these new lineages. Infor- INSDc and UNITE. We used multiple outgroup taxa when mation about the exploration types of all EcM lineages and possible, because this improved our ability to evaluate the genera are given in Table S1. EcM status and monophyly of the ingroup taxa. All of the an- alyses used ITS rDNA alignments but in several cases we were Basidiomycota also able to use the flanking LSU alignments in order to enhance the phylogenetic signal. In one group of EcM- The /agaricales1 lineage is known from fruit body collections associated Agaricomycetes (/agaricomycetes1), LSU se- (representative specimen TH9235; INSDc accession quences were used separately to determine their broad place- KC155374) and associated root tips from Dicymbe jenmanii (un- ment among Basidiomycota. Briefly, all downloaded der the same accession; Smith et al., 2013a) and Dicymbe cor- sequences were aligned with MAFFT 7 (Katoh and Strandley, ymbosa (M.E. Smith and T.W. Henkel, unpublished) from 2013), trimmed and manually corrected in SeaView 4 (Gouy Guyana. The ITS region of this group is short compared with 86 L. Tedersoo, M. E. Smith

Table 1 e Information about the newly described EcM lineages Lineage Number of sequences in UNITE Representative sequence P-value from Fisher’s exact (as of 01.05.2013) in INSDc or UNITE test (total n)

/agaricales1 1 KC155374 nd /agaricomycetes1 8 UDB008409 0.003 (16) /atheliales1 10 UDB014265 nd /atheliales2 10 JN168682 0.015 (12) /boletopsis 23 UDB016630 nd /hydropus 4 JN168774 0.071 (8) /serendipita1 66 UDB002608 <0.001 (153) /serendipita2 21 EU909214 <0.001 (43) /sordariales1 41 UDB017246 <0.001 (50) /sordariales2 33 UDB008982 <0.001 (42) /tulasnella1 53 AY192445 nd /tulasnella2 15 UDB002679 nd /xenasmatella 10 JN168733 <0.001 (24) /aleurina 33 KC905032 nd /pustularia 52 EU649088 nd /rhodoscypha 2 FJ236854 nd /pyronemataceae1 11 JN569352 <0.001 (31) /pyronemataceae2 3 AY702741 0.012 (9) /endogone1 1 AY977045 nd /endogone2 2 UDB002714 nd

most other species of Agaricales (ITS1: 111 bp; ITS2: 149 bp), and M. Bahram, unpublished), Cameroon, Gabon and which may partly explain why this taxon has no close Madagascar (Tedersoo et al., 2011b). In Indian and African matches in the INSDc and UNITE databases. Nuclear and sites, members of the /atheliales1 EcM lineage were locally mitochondrial LSU sequences and nuclear SSU sequences abundant. Species in this fungal lineage form EcM of a indicate that this fungus has evolutionary affinities with medium-distance mat exploration type characterized by pink-spored or white-spored Agaricales, but it is distinct abundant mycelium, rhizomorphs and binding of soil from any previously recognized EcM lineage. This fungus forms dense clusters of white ectomycorrhizas of the medium-distance fringe exploration type beneath fruit- AB733124 Gloeophyllum sepiarium Unkn a HM536098 Neolentinus lepideus Unkn bodies in sandy soils (see Fig 4 of Smith et al., 2013a). EF644168 EcM Populus tremula Austria 86 FJ524308 Plant root Oryza sativa China The /agaricomycetes1 lineage comprises a monophyletic 100 89 GU256215 EcM Quercus suber Italy group of eight sequences (BS ¼ 70) that were obtained from EF619876 Fungal mycelium USA 100 98 DQ421269 Soil USA 100 JQ247534 Soil USA healthy EcM root tips. This lineage is split into two strongly JQ247539 Soil USA supported clades (BS ¼ 100; Fig 1a), a Southern temperate JQ247538 Soil USA 100 DQ273361 EcM Lithocarpus densiflorus USA AB218075 EcM Fagus crenata Japan group (Horton et al., 2013; Nouhra et al., 2013) and a Northern 70 HQ022288 EcM Angiospermae USA 100 UDB008467 EcM Nothofagus pumilio Argentina temperate group (Bergemann and Garbelotto, 2006; Ishida JF960849 EcM Eucalyptus Australia 100 UDB008409 EcM Nothofagus pumilio Argentina et al., 2007). Both the ingroup and its sister clade form a UDB008445 EcM Nothofagus pumilio Argentina 0.1 deep branch among the Agaricomycetes and cannot be placed UDB008469 EcM Nothofagus pumilio Argentina b /agaricomycetes1 DQ831025 Pseudohydnum gelatinosum Unkn into any of the currently recognized orders (Fig 1b). We have DQ917658 Thanatephorus cucumeris Unkn 100 FJ040363 Soil USA examined four Argentinean EcM root tip vouchers that repre- 84 JX316518 EcM Nothofagus pumilio Argentina 95 JX316510 EcM Nothofagus pumilio Argentina DQ273492 EcM Lithocarpus densiflorus USA sent two distinct species. Both species exhibit short-distance 74 JN649333 Clavulicium macounii Unkn 100 EU669381 Brauniellula albipes USA ycetes1 exploration types with narrow, thin-walled hyphae and an JQ408244 Ramaria sp. USA 100 AY586710 Rickenella fibula Unkn GQ845003 Phanerochaete viticola Unkn EcM mantle anatomy that is intermediate between the pseu- 99 EU118674 Tomentellopsis bresadoliana Sweden AM490944 Amaurodon aquicoeruleus Unkn doparenchymatous and plectenchymatous types. One of the AY586687 Lopharia cinerascens Unkn /agaricom JQ700272 Antrodia primaeva Russia species has long, unicellular, needle-like cystidia, while the 90 AY218409 Sparassis sp. USA EU118664 Radulomyces notabilis Spain 94 HM536077 Neolentinus lepideus Unkn other species lacks cystidia. AF393059 Gloeophyllum sepiarum Unkn GU187581 Jaapia argillacea Unkn The /atheliales1 lineage represents a monophyletic group 83 DQ469289 Piloderma fallax Unkn AF506465 Russula violacea Unkn ¼ 89 JF706317 Tricholoma viridolivaceum Unkn (BS 96) of tropical EcM root tip isolates and unidentified fruit 0.05 GU187605 Tapinella panuoides Unkn body specimens. Phylogenetic analysis was unable to resolve a sister group (Fig 2). An unpublished sequence from a fruit Fig 1 e Unrooted maximum likelihood phylogram demon- body collected in a Cuban Coccoloba uvifera stand and an EcM strating phylogenetic placement of sequences in the /agar- isolate from dry dipterocarp forest in Thailand (Phosri et al., icomycetes1 lineage based on a) ITS sequences; and b) LSU 2012) are relatively similar and they form a monophyletic sequences. Significant bootstrap support (>70) is indicated clade (BP ¼ 100) that is basal to the rest of the lineage. Other above branches. Abbreviations: EcM, ectomycorrhiza; ErM, sequences in this group are derived from EcM roots of Dipter- ericoid mycorrhiza; OrM, orchid mycorrhiza; Unkn, un- ocarpaceae in Malaysia (Peay et al., 2010), India (L. Tedersoo known country. Lineages of ectomycorrhizal fungi revisited 87

UDB013805 Atheliales Zambia associate with well-known EcM fungi in Quercus-Castanopsis EU292347 Soil USA 100 UDB004875 EcM Dipterocarpaceae Thailand forests (Okayama et al., 2012). The /atheliales1 and /athe- 96 UDB016773 Atheliales Cuba GQ268573 EcM Dipterocarpaceae Malaysia liales2 lineages are phylogenetically distinct from the previ- 99 100 UDB014265 Atheliales Vateria indica India UDB014480 EcM Vateria indica India 96 UDB014483 EcM Vateria indica India ously known EcM lineages within Atheliales (e.g. /piloderma, 100 UDB004324 EcM Microberlinia bisulcata Cameroon UDB007555 EcM Uapaca densifolia Madagascar /amphinema-tylospora and /byssocorticium), but the relative UDB004502 EcM Marquesia excelsa Gabon /atheliales1 UDB004400 EcM Caesalpiniaceae Cameroon positions of these different groups within the order remain UDB002469 Tylospora asterophora Germany 100 UDB008296 Atheliaceae Croatia unclear. UDB008272 Amphinema byssoides Austria 88 UDB001722 Amphinema byssoides Sweden The /boletopsis lineage is erected in this study based on se- 100 FJ152491 EcM Tsuga heterophylla Canada 70 UDB002468 Tylospora fibrillosa Germany 100 JN133914 EcM Quercus incana Pakistan quences of Boletopsis fruit-bodies and mycorrhizas that form a

/amphinema- JQ975955 EcM Pinus pinaster Unkn tylospora strongly supported monophyletic group with no close rela- 100 UDB016819 Atheliales Madagascar UDB001725 Byssocorticium pulchrum Finland m ~ 9896 UDB016321 Byssocorticium caeruleum Estonia tives (Cooper and Leonard, 2012;U.Koljalg et al., unpublished). UDB013132 Byssocorticium sp. Castanopsis PNG JF519122 EcM Fagus sylvatica Austria Boletopsis is well-known for its conspicuous fruit-bodies with a UDB000031 Byssocorticium atrovirens Denmark GQ162813 Athelia singularis Norway poroid hymenium but this group has been recovered only GQ162817 Fibulomyces mutabilis Germany GQ268574 EcM Dipterocarpaceae Malaysia twice in EcM community studies (Izzo et al., 2005; UDB016429 Athelia bombacina Estonia /byssocorticiu U85798 Athelia neuhoffii Unkn 97 U85797 Athelia decipiens Unkn Bergemann and Garbelotto, 2006) suggesting that this lineage 97 UDB016352 Athelia decipiens Estonia U85789 Fibulorhizoctonia carotae Unkn is rare on EcM roots. This lineage is distributed in the U85793 Athelia epiphylla Unkn U85792 Athelia arachnoidea Unkn temperate and boreal zone of the Northern hemisphere as UDB004285 Atheliales Angiospermae Ecuador 100 UDB016345 Athelopsis glaucina Estonia well as in Nothofagus forests in New Zealand. It is possible 85 JN168682 EcM Dicymbe sp Guyana 97 100 AB597712 OrM Lecanorchis trachycaula Japan that Boletopsis nothofagi was introduced to New Zealand from 75 AB597711 OrM Lecanorchis trachycaula Japan GQ268572 EcM Dipterocarpaceae Malaysia the Northern Hemisphere, but more data are needed (Cooper 93 AB597636 OrM Lecanorchis flavicans Japan AB597634 OrM Lecanorchis flavicans Japan and Leonard, 2012). AB597635 OrM Lecanorchis flavicans Japan AB597632 OrM Lecanorchis flavicans Japan AB597631 OrM Lecanorchis flavicans Japan /atheliales2 The putative /hydropus lineage represents a small mono- AB597633 OrM Lecanorchis flavicans Japan FJ236851 EcM Tsuga heterophylla Canada phyletic group of sequences derived from EcM root tips in UDB002738 EcM Pomaderris apetala Australia 86 UDB004045 EcM Nothofagus Australia Malaysia (Peay et al., 2010) and Guyana (Smith et al., 2011), JQ711873 EcM Pinus contorta Canada JQ711918 EcM Pinus contorta Canada and from leaf litter in a Dicymbe-dominated forest in Guyana 100 JQ711808 EcM Pinus contorta Canada UDB001740 Piloderma fallax Sweden (K. McGuire, unpublished). This group is nested within the JQ888191 EcM Pinus sylvestris Scotland 100 UDB001615 Piloderma sp. Great Britain genus Hydropus (Agaricales) that comprises putative saprobes UDB001723 Tretomyces lutescens Norway EU645645 EcM Pseudotsuga menziesii Canada /piloderma FM992893 EcM Pinaceae Sweden (Fig 3). Compared to the outgroup taxa, the association with DQ377372 EcM Pinus taeda USA GQ162820 Tretomyces lutescens Sweden EcM roots was not significant in the ingroup sequences 0.1 UDB001724 Tretomyces lutescens Sweden (Fisher’s Exact test: P ¼ 0.071) leaving open the possibility that /hydropus lineage is a parasitic or saprotrophic taxon Fig 2 e Unrooted maximum likelihood phylogram demon- that invades EcM roots. strating phylogenetic placement of sequences in the Athe- The /serendipita lineage as defined in Tedersoo et al. (2010) liales based on combined ITS and LSU sequences. The is re-considered here based on new information. In a series of /piloderma, /amphinema-tylospora and /byssocorticium publications, Warcup (1988, 1990b) suggested that certain lineages are represented by a few divergent sequences for strains of Serendipita vermifera (Sebacinales clade B sensu illustration purposes. Significant bootstrap support (>70) is Weiss et al., 2004) isolated from orchid roots are able to form indicated above branches. Abbreviations: EcM, ectomycor- EcM in axenic conditions. However, subsequent field and rhiza; ErM, ericoid mycorrhiza; OrM, orchid mycorrhiza; experimental studies have failed to confirm these findings in Unkn, unknown country. Australia and elsewhere. Notably, Warcup (1988) reported on EcM formation in arbuscular mycorrhizal herbs and EcM Myr- taceae, but the mantle was usually fragmentary and only one particles. Taxa in this lineage were found to exhibit strong or two cell layers deep. This suggests that he used an overly leucine aminopeptidase enzyme activity, whereas members relaxed definition for ectomycorrhizas that probably included of other EcM lineages from the same site did not (Tedersoo any root endophytes and saprobes that attach to roots et al., 2012b). (Brundrett, 2009). To the best of our knowledge, the isolates The /atheliales2 lineage constitutes a monophyletic group Warcup considered EcM have not been sequenced (M. Weiss (BS ¼ 97) that has been recovered only from EcM root tips and P. McGee, pers. comm March, 2013). More recently, how- and the roots of orchids in tropical and subtropical ecosys- ever, molecular studies have identified species of Serendipita tems. An unidentified Atheliales sp. from Ecuador (BP ¼ 100) from EcM root tips in temperate, boreal, and subarctic com- and the non-EcM taxon Athelopsis glaucina from Estonia munities throughout the Northern hemisphere (Ryberg et al., (BP ¼ 85) form successive sister groups to this EcM lineage 2009; Stefani et al., 2009). In northern boreal and temperate (Fig 2). An isolate from a Dicymbe root tip (Smith et al., 2011) forests, fungi identified as Serendipita spp. are dominant on forms a basal group within the /atheliales2 lineage (BP ¼ 97). roots of Pyrola japonica seedlings, indicating their substantial Another EcM root tip from a Dipterocarpaceae host (Peay role in both carbon and mineral nutrition of the ericoid plant et al., 2010) is nested within uncultured isolates from non- (Hashimoto et al., 2012). Although these fungi also occur on photosynthetic orchids (Lecanorchis spp.) from Shikoku, a sub- adult Pyrola rotundifolia (Vincenot et al., 2009), Hynson et al. tropical island of Japan (Okayama et al., 2012). We consider the (2013) were the first to suggest that several Serendipita spp. orchid association to be strongly indicative of EcM habit since associating with Pyroleae are actually ectomycorrhizal based Lecanorchis spp. are mycoheterotrophic and their sister taxa on their tight clustering in phylogenetic trees. Based on full- 88 L. Tedersoo, M. E. Smith

UDB016643 Gerronema sp. Gabon 83 sequences 100 EU883594 Gerronema nemorale Korea 80 HQ154277 Root Impatiens noli-tangere Germany HM359000 EcM China UDB015081 Gerronema sp. Gabon 71 AB520600 Soil Japan AB520599 Soil Japan DQ192178 Clitocybula oculus USA EU554931 Soil Populus Canada EU554923 Soil Populus Canada 100 EU669314 Hydropus marginellus USA 100 EU554995 Soil Populus Canada DQ490627 Hydropus marginellus USA EU554997 Soil Populus Canada 86 AY187618 EcM Salix USA GQ268683 EcM Dipterocarpaceae Malaysia EU554697 EcM Populus Canada 79 HQ154328 Root Oxalis acetosella Germany GQ268691 EcM Dipterocarpaceae Malaysia UDB008509 EcM Betula Estonia 100 EU554893 Soil Populus Canada JN889949 Plant litter Guyana EU554927 Soil Populus Canada EU554682 EcM Populus Canada JN168774 EcM Dicymbe altsonii Guyana /hydropus 97 EU554869 Soil Populus Canada EU554899 Soil Populus Canada 94 JF908050 Hydropus atramentosus France JX030285 EcM Castanea dentata USA 0.1 JQ917844 EcM Pinus radiata Mexico JF691233 OrM Orchidaceae Reunion FJ626934 Soil Populus Canada EU554901 Soil Populus Canada 100 JQ824883 EcM Populus canescens France EU910898 Root Calamagrostis Germany Fig 3 e Unrooted maximum likelihood phylogram demon- AJ875375 Root Phragmites australis Germany UDB002608 EcM Betula pendula Estonia 88 JX844771 EcM Fagus sylvatica Europe strating phylogenetic placement of sequences in the /hy- JX844774 EcM Fagus sylvatica Europe JX844773 EcM Fagus sylvatica Europe dropus lineage based on ITS sequences. Significant JX844772 EcM Fagus sylvatica Europe 95 JX844775 EcM Fagus sylvatica Europe DQ068978 EcM Pinus sylvestris Lithuania bootstrap support (>70) is indicated above branches. Ab- EF611157 EcM Tilia Finland EU554714 EcM Populus Canada breviations: EcM, ectomycorrhiza; ErM, ericoid mycorrhiza; 71 AB669683 EcM Pyrola asarifolia Japan AB669684 EcM Pyrola asarifolia Japan DQ054559 Root Italy OrM, orchid mycorrhiza; Unkn, unknown country. AB669634 EcM Pyrola asarifolia Japan AB669688 EcM Pyrola asarifolia Japan JX316811 EcM Populus tremuloides USA AB669686 EcM Pyrola asarifolia Japan AB669685 EcM Pyrola asarifolia Japan

AB244045 EcM Betula Japan /serendipita1 AB669673 EcM Pyrola asarifolia Japan AB669691 EcM Pyrola asarifolia Japan AB669674 EcM Pyrola asarifolia Japan length ITS sequences of the Sebacinales clade B, it appears AB669675 EcM Pyrola asarifolia Japan AB669689 EcM Pyrola asarifolia Japan that EcM taxa fall into two major monophyletic clades (Figs AB669679 EcM Pyrola asarifolia Japan AB669676 EcM Pyrola asarifolia Japan AB669678 EcM Pyrola asarifolia Japan 4, S1), hereafter referred to as the /serendipita1 and /serendip- AB669677 EcM Pyrola asarifolia Japan AB669687 EcM Pyrola asarifolia Japan AB669672 EcM Pyrola asarifolia Japan ita2 EcM lineages. In both of these groups, the sebacinalean AB669680 EcM Pyrola asarifolia Japan AB669667 EcM Pyrola asarifolia Japan taxa were also recovered from non-EcM roots by specifically AB669666 EcM Pyrola asarifolia Japan AB669636 EcM Pyrola asarifolia Japan AB669681 EcM Pyrola asarifolia Japan targeting these taxa using lineage-specific primers and some- AB669671 EcM Pyrola asarifolia Japan HM136664 Root France times cloning (Weiss et al., 2011; Garnica et al., 2013). Use of HQ212339 Soil USA HQ212320 Soil USA 100 HQ212299 Soil USA specific primers may result in detection of species that are FJ553298 Soil Canada HQ212269 Soil USA not necessarily prevalent; we suggest that this incidental HQ211712 Soil USA HQ212204 Soil USA HQ212319 Soil USA detection of these taxa from non-EcM root systems does not HQ212184 Soil USA HQ212355 Soil USA exclude them as EcM symbionts. DQ421202 Soil USA FJ556808 Root Medicago Italy GQ240918 EcM Pinus China The /serendipita1 lineage forms a monophyletic group HQ180295 Root Trifolium Unkn 71 HQ154382 Root Polygala Germany ¼ JX135065 EcM Populus Europe (BS 97). Species of this group associate with Salicaceae, FJ556805 Root Medicago lItaly GU189719 Root Elymus Unkn Fagaceae, Betulaceae, Pinaceae, Tiliaceae and Pyroleae in HQ154347 Root Trifolium Germany GU189704 Root Elymus Unkn GU189693 Root Poaceae Unkn temperate, boreal, subalpine and subarctic habitats of the GU189689 Root Potentilla Unkn GU189699 Root Poaceae Unkn Northern Hemisphere (Fig 4). We have been able to study the HQ154381 Root Polygala Germany HQ154361 Root Trifolium Germany HQ154365 Root Trifolium Germany anatomy of only a single root tip isolate, UDB002608, that is EU910924 Root Trifolium Germany HQ154388 Root Trifolium Germany characterized by a plectenchymatous mantle and smooth sur- DQ421201 Soil USA HQ154280 Root Campanula Germany GU189680 Root Poaceae Unkn face with no hyphae. In another isolate, JX316811, sparse FJ237152 Soil Austria FM992970 EcM Pinaceae Sweden EU909214 Plant leaf Riccardia Germany extraradical hyphae were observed (C. Andrew, pers. comm. EU668270 Root Fagus sylvatica Germany GU184073 EcM USA March, 2013). Thus, the examined species of the /serendipita1 GU452533 EcM Pseudotsuga Canada 98 FJ197211 EcM Pinus muricata USA AM231795 EcM Pseudotsuga menziesii USA lineage belong to either contact or short-distance exploration DQ273405 EcM Lithocarpus densiflorus USA EU668934 EcM Pyrola rotundifolia Estonia types. JX561233 EcM Pyrola rotundifolia Germany JQ711784 EcM Pinus contorta Canada AY587753 EcM Pinus jeffreyi USA The /serendipita2 lineage is phylogenetically distinct and it JQ711843 EcM Pinus contorta Canada ¼ UDB007423 EcM Pinus ponderosa USA forms a monophyletic group (BS 98; Fig 4). Members of this DQ377407 EcM Pinus taeda USA /serendipita2 JQ616802 Root Pinus taeda USA JN704824 EcM Pinus montezumae Mexico lineage associate predominantly with Pinaceae, but to a lesser HQ271379 EcM Pinus Mexico EF619757 Soil Pinus taeda USA extent with Fagaceae and Pyroleae in subtropical and AB669633 EcM Pyrola asarifolia Japan AB669632 EcM Pyrola asarifolia Japan temperate forest ecosystems. Although no root tips were 291 sequences 0.1 available to us for study, species of this group probably fall into the contact or short-distance exploration types described Fig 4 e Cut-out of an unrooted maximum likelihood phylo- for other Sebacinales (Urban et al., 2003; Agerer, 2006). gram demonstrating phylogenetic placement of EcM line- The /tulasnella lineage (sensu Tedersoo et al., 2010) is simi- ages in the Serendipitaceae family (Sebacinales clade B) larly split into two groups. These two lineages are monophy- based on ITS sequences. The inclusive tree (in the rectangle) letic, but distantly related to one another within the is given in Fig S1. Significant bootstrap support (>70) is Tulasnellaceae phylogeny (J. Oja, unpublished). indicated above branches. Abbreviations: EcM, ectomycor- The /tulasnella1 lineage comprises a deeply diverging rhiza; ErM, ericoid mycorrhiza; OrM, orchid mycorrhiza; monophyletic clade of closely related fungal species that colo- Unkn, unknown country. nize both the rhizoids of hepatics (Bidartondo et al., 2003; Bidartondo and Duckett, 2010) and EcM root tips of Betula pen- (Deslippe et al., 2011) fall into this group, but the sequences dula and Pinus sylvestris (Bidartondo et al., 2003; I. Ostonen were recently withdrawn from INSDc. The EcM formed by et al., unpublished) in temperate forests of Europe. In addition, Tulasnella are smooth with no extraradical mycelium and sequences from Betula nana-dominated tundra soil in Alaska correspond to the contact exploration type. Lineages of ectomycorrhizal fungi revisited 89

JN164946 Trametes membranacea Puerto Rico JX043116 Soil USA EU118657 Phlebia unica Sweden JF908719 Ramsbottomia asperior Italy FJ820500 Air Germany HM123685 Lichen USA 99 UDB016396 Xenasmatella tulasnelloidea Estonia UDB014228 Pyronemataceae Fagaceae Vietnam UDB016408 Xenasmatella ardosiaca Estonia 99 EU118658 Xenasmatella aff. ardosiaca Costa Rica 100 AB218200 EcM Betula grossa Japan 99 99 FJ820639 Air Germany FN298731 EcM Nothofagus Australia DQ008219 Xenasmatella vaga Sweden JF960624 Pyronemataceae Australia EU118659 Xenasmatella christiansenii Finland KC905033 Aleurina echinata Nothofagus Chile EF619631 Fungal mycelium USA UDB008458 EcM Nothofagus pumilio Argentina 100 100 JN593374 Leaf litter USA 100 UDB008420 EcM Nothofagus pumilio Argentina UDB000519 Xenasmatella vaga Denmark 77 JX860488 Dead wood USA 100 UDB008406 EcM Nothofagus pumilio Argentina FR682218 Dust Finland UDB007063 EcM Nothofagus dombeyi Argentina FJ475671 Soil Sweden 100UDB007006 EcM Nothofagus dombeyi Argentina AY781267 Xenasmatella vaga Sweden UDB007184 EcM Nothofagus alpina Argentina 99 AM901937 Dust Finland UDB007032 EcM Nothofagus dombeyi Argentina FJ475670 Soil Sweden 100 UDB007026 EcM Nothofagus dombeyi Argentina FJ475672 Soil Sweden UDB007133 EcM Nothofagus obliqua Argentina EU118660 Xenasmatella vaga Sweden UDB007160 EcM Nothofagus alpina Argentina 100 EF619673 Fungal mycelium USA UDB013201 Polyporales PNG UDB007075 EcM Nothofagus obliqua Argentina UDB007176 EcM Nothofagus alpina Argentina UDB013341 Polyporales Malaysia 100 79 GQ268630 EcM Dipterocarpaceae Malaysia UDB007029 EcM Nothofagus dombeyi Argentina JN168733 EcM Dicymbe corymbosa Guyana UDB007190 EcM Nothofagus alpina Argentina /aleurina 72 AY394673 Root Graffenrieda emarginata Ecuador 99 100 UDB007095 EcM Nothofagus obliqua Argentina JN168734 EcM Aldina insignis Guyana atella UDB007076 EcM Nothofagus obliqua Argentina 69 KC155368 EcM Dicymbe jenmanii Guyana JQ272394 Living culture Rhododendron USA UDB007007 EcM Nothofagus dombeyi Argentina GQ268629 EcM Dipterocarpaceae Malaysia UDB007185 EcM Nothofagus alpina Argentina GQ268625 EcM Dipterocarpaceae Malaysia UDB007134 EcM Nothofagus obliqua Argentina GQ268627 EcM Dipterocarpaceae Malaysia /xenasm UDB007115 EcM Nothofagus obliqua Argentina 99 0.1 JX860490 Dead wood USA UDB008460 EcM Nothofagus pumilio Argentina UDB007202 EcM Nothofagus alpina Argentina e UDB007051 EcM Nothofagus dombeyi Argentina Fig 5 Unrooted maximum likelihood phylogram demon- UDB007146 EcM Nothofagus alpina Argentina strating phylogenetic placement of sequences in the /xe- UDB007089 EcM Nothofagus obliqua Argentina UDB008424 EcM Nothofagus pumilio Argentina nasmatella lineage based on combined ITS and LSU UDB017001 Pyronemataceae Nothofagus dombeyi Argentina 0.1 KC905032 Aleurina argentina Nothofagus Chile sequences. Significant bootstrap support (>70) is indicated above branches. Abbreviations: EcM, ectomycorrhiza; ErM, Fig 6 e Unrooted maximum likelihood phylogram demon- ericoid mycorrhiza; OrM, orchid mycorrhiza; Unkn, un- strating phylogenetic placement of sequences in the known country. /aleurina lineage based on ITS sequences. Significant boot- strap support (>70) is indicated above branches. Abbrevia- tions: EcM, ectomycorrhiza; ErM, ericoid mycorrhiza; OrM, The /tulasnella2 lineage is restricted to southern temperate orchid mycorrhiza; Unkn, unknown country. habitats. The isolates have been found from Nothofagus spp. in Argentina (Nouhra et al., 2013) and Nothofagus cunninghamii, Eucalyptus regnans, and Pomaderris apetala in Tasmania (Tedersoo et al., 2008a, 2009a). Individual species of the /tulas- Several other Basidiomycota species have been suggested nella2 lineage share a gelatinous mantle; some species as EcM symbionts since 2010. For example, Walker et al. possess long, thick-walled cystidia, whereas others do not. (2012) consistently recovered Alloclavaria purpurea and a Extraradical mycelium is absent or scarce. This group belongs related species from EcM root tips of Pseudotsuga menziesii in to the contact exploration type. Canada and considered these to be EcM fungi. However, the The putative /xenasmatella lineage forms a strongly sup- 13C and 15N stable isotope signatures were equivocal and could ported monophyletic group that is nested within the genus not definitively determine the likely source of nutrients for Xenasmatella (Fig 5). The EcM-associated groups originate these fungi. Because the root tips had contrasting from roots of Dipterocarpaceae and Caesalpinioideae in morphology, ranging from cream and hairy (Amphinema- Malaysia and Guyana (Peay et al., 2010; Smith et al., 2011, type) to black and rough (Tomentella-type; J.K.M. Walker, 2013). Although taxa in this group have been regularly pers. comm. January, 2013), Alloclavaria probably functions encountered on EcM root tips and form apparently normal as an endophyte or saprobe and should not be considered EcM morphology (as /polyporales1 in Smith et al., 2011), the ectomycorrhizal. Nouhra et al. (2013) similarly found that a phylogeny provides conflicting evidence about the trophic Rickenella sp. and a Tulasnella sp. (closely related to Cypripedium mode of this group. EcM-forming isolates are phylogenetically symbionts) frequently colonized root tips of Nothofagus spp. at placed alongside sequences from dead wood, a non-EcM tree several study sites in Argentina. Based on the high variation in (contrary to the assertions of Haug et al. (2004) we consider EcM mantle anatomy, both groups were considered saprotro- Graffenrieda to be non-EcM), and cultures from ericoid mycor- phic or endophytic. Besides these groups, EcM root tips are rhizal plants. Considering these features and the conflicting commonly colonized by basidiomycetous yeasts such as data, we suggest that the /xenasmatella lineage may repre- Atractiellales and Cryptococcus based on INSDc records and sent a non-mycorrhizal, opportunistic, facultative root associ- our own results. These are common soil fungi and their func- ation that colonizes mycorrhizas of other species. Another tion on the surface and inside roots is not clear. However, possibility is that fungi in this group form extensive rhizo- certain tropical orchids have evolved mutualistic associations morphs similar to those in temperate Xenasmatella spp. and with Atractiellales (Kottke et al., 2010) and some species in this they are tightly bound to roots and preferentially amplified group are frequently isolated as root endophytes from EcM during PCR. Further work is definitely needed to clarify the trees. Among Agaricomycetes, species of Gymnopus, Rhodocol- nutritional mode and ecology of the putative /xenasmatella lybia, Trechispora, Bjerkandera, Mycena and Pleurotus have all lineage. been occasionally recovered from EcM root tips based on the 90 L. Tedersoo, M. E. Smith

EU669386 Pseudaleuria quinaultiana USA GU222313 Aleuria sp New Zealand GU366703 Soil USA JX489801 Soil China 100 HM123451 Culture Lecidea tessellata USA AF351582 "Trichophaea hybrida" Canada HM123025 Culture Flavoparmelia praesignis USA 99 100 AJ893249 EcM Betula pendula Estonia EF434053 Soil USA JQ975970 EcM Pinus pinaster Unkn 100 AM181380 EcM Orthilia secunda Estonia FJ660486 EcM Pinus USA 100 100 JN129415 EcM Keteleeria davidiana China AB506091 EcM Pinus densiflora South Korea

99 AB636444 EcM China ilcoxina AJ968678 EcM Picea abies Estonia 97 UDB002634 EcM Estonia /w FJ158081 EcM Pinus sylvestris Poland DQ200834 Trichophaea cf. hybrida USA EU292476 Soil USA 99 JX545195 Soil USA 99 AY220836 Scutellinia colensoi Unkn FJ235141 Scutellinia scutellata USA AY220819 Scutellinia superba Unkn AY971745 Plant leaf Picea mariana Canada 100 HM123093 Culture Peltigera canina USA 99 EF159464 Plant litter USA EF159435 Plant litter USA 100 HM069461 Soil Finland AY546004 Plant leaf Pinus tabulaeformis China FJ025247 Plant leaf Unkn 91 JQ761457 Culture Lecanora oreinoides USA HM123462 Culture Dermatocarpon tenue USA EU715596 Trichophaea abundans Mexico HM123322 Culture Physcia caesia USA GQ240938 EcM Castanopsis fargesii China 72 UDB000994 Sphaerosporella brunnea Finland UDB007916 EcM Betula pendula Estonia GU301279 EcM Pinus contorta USA 84 HM164581 EcM Populus tremuloides USA JQ758662 Culture Cassiope tetragona USA 100 JN569355 EcM Carya illinoinensis USA 88 JN704836 EcM Pinus montezumae Mexico GQ240937 EcM Castanopsis fargesii China FJ626917 Soil Canada GQ205368 EcM Pinus pinaster Portugal JN704819 EcM Pinus montezumae Mexico GU553372 EcM Castanea dentata USA JF419498 EcM Fagus sylvatica Poland 89 EF484935 EcM Pinus Spain EU847259 EcM Betula pendula Finland JX844781 EcM Fagus sylvatica Unkn 90 UDB013625 EcM Tilia Estonia 100 UDB005694 EcM Betula Iran UDB005824 EcM Fagaceae Iran EU334893 EcM Quercus USA DQ974751 EcM Quercus USA 86 EF411124 EcM Quercus USA AY634164 OrM Epipactis helleborine Germany /sphaerosporella EU819507 EcM Castanea dentata USA 100 GQ281482 EcM Pinus tabuliformis China JF748089 EcM Quercus wutaishanica China UDB007975 EcM Tilia Estonia UDB005828 EcM Fagaceae Iran HM370456 EcM Ostrya carpinifolia Italy 100 DQ200835 Trichophaea woolhopeia Denmark UDB005716 EcM Betula Iran UDB008881 EcM Salix caprea Estonia AJ968676 EcM Betula pendula Estonia UDB005780 EcM Fagaceae Iran UDB008859 EcM Salix caprea Estonia UDB013540 EcM Tilia Estonia HM123700 Culture Usnea hirta USA HM122819 Culture Xanthoparmelia viriduloumbrina USA a- 89 GU327420 OrM Epipactis atrorubens Czech Republic 100 JQ917866 EcM Pinus radiata USA AY702741 EcM Abies USA EU649078 EcM Pinus USA FJ788782 OrM Pterygodium volucris RSA

/pyronem 100 JQ836559 Tricharina praecox var praecox Unkn taceae2 HQ602672 Culture Pinus monticola USA 100 AF387652 Picoa lefebvrei Spain 91 FR694203 Geopora cooperi Spain 100 JQ393126 EcM USA JQ836556 Tricharina striispora Unkn 96 FJ788768 OrM Pterygodium volucris RSA

GQ153175 Plant leaf Cupressus arizonica Unkn /geopora HM123159 Culture Xanthoparmelia viriduloumbrina USA JQ836557 Ascorhizoctonia BCS1 Unkn JQ836558 Tricharina ochroleuca Unkn 100 JQ976017 EcM Pinus pinaster Spain JQ824884 EcM Populus canescens France JQ409293 Root Populus France HM036637 EcM Pinus sylvestris Lithuania HM545741 EcM Pinus pinaster Italy FJ013060 EcM Pinus pinaster Spain EF484931 EcM Pinus Spain EU557319 EcM Pinaceae Argentina GU181851 EcM Larix decidua Italy HM044534 EcM Larix decidua Italy AJ410863 EcM Pinus halepensis France GU181861 EcM Larix decidua Italy HM044538 EcM Larix decidua Italy GU181904 EcM Pinus cembra Italy GU181871 EcM Larix decidua Italy EU562601 EcM Pinus contorta Argentina UDB005745 EcM Fagaceae Iran FR852318 EcM Fagaceae Iran DQ069008 EcM Pinus sylvestris Lithuania DQ320132 EcM Picea abies Lithuania DQ508802 EcM Picea abies Poland FJ210746 EcM Pinus Italy JQ409292 Root Populus France JQ409294 Root Populus France JN704828 EcM Pinus montezumae Mexico

GQ985430 Unkn Pinus tabulaeformis China /pustularia DQ069010 EcM Pinus sylvestris Lithuania UDB013715 EcM Tilia Estonia DQ069013 EcM Picea abies Lithuania EU726302 Fungal mycelium Pinus sabiniana USA JQ393125 EcM USA EU649088 EcM Pinus sabiniana USA JX198543 EcM Betulaceae USA GU184036 EcM USA EU726303 Fungal mycelium Pinus ponderosa USA DQ822806 EcM Pinus muricata USA JQ393124 EcM USA JN704826 EcM Pinus montezumae Mexico JQ917867 EcM Pinus radiata USA JQ917849 EcM Pinus radiata USA JX316808 EcM USA HQ445326 Root Dryas octopetala Norway HQ445327 Root Dryas octopetala Svalbard GU452518 EcM Pseudotsuga menziesii Canada JF792511 EcM Pseudotsuga menziesii Canada 0.1 AY587740 EcM Abies concolor Pinus jeffreyi USA JX003300 EcM Pseudotsuga menziesii Canada

Fig 7 e Unrooted maximum likelihood phylogram demonstrating phylogenetic placement of EcM lineages in the Scutellinia- Trichophaea clade of Pyronemataceae (sensu Hansen et al., 2013) based on combined ITS and LSU sequences. The /wilcoxina, /geopora and /sphaerosporella lineages are represented by a few divergent sequences for illustration purposes. Significant bootstrap support (>70) is indicated above branches. Abbreviations: EcM, ectomycorrhiza; ErM, ericoid mycorrhiza; OrM, orchid mycorrhiza; Unkn, unknown country.

records in INSDc. However, these are not concentrated in spe- unpublished; Perry et al., 2007; Tedersoo et al., 2013a). Based cific clades nor do they form monophyletic groups of EcM iso- on LSU, the /aleurina lineage is nested within the Pyronema- lates, leading us to treat them as parasitic, endophytic or taceae family and it has Southern Hemisphere distribution saprotrophic root colonizers rather than true EcM symbionts. (Tedersoo et al., 2013a). However, the more inclusive ITS data- set of this EcM lineage suggests that the distribution involves Ascomycota both the Southern Hemisphere and Eastern Asia. In particular, this group has been found in natural forests in Japan (Ishida The /aleurina lineage is comprised of species of Aleurina, Geli- et al., 2007) and Vietnam (L. Tedersoo and co-workers, unpub- nipes nom. prov. and Unicava nom. prov. (J.M. Trappe et al., lished). In Argentina, the /aleurina lineage is divided into five Lineages of ectomycorrhizal fungi revisited 91

AJ875384 Root Phragmites australis Germany JN689974 Podospora Lithuania HQ115722 Air Austria 100 AY587913 Cercophora sulphurella Unkn 100 FJ553261 Soil Canada JN673050 Lasiosphaeria ovina USA FN397309 Soil France AY587915 Lasiosphaeria glabrata Unkn 72 EU826918 Soil Korea 91 EF029210 Cordana ellipsoidea New Zealand 97 HQ829414 Air China JN689975 Porosphaerella cordanophora Lithuania JX171178 Heydenia alpina Unkn FJ903316adicoides bina Latvia JX489837 Soil China JF340260adicoides bina Latvia 100 EU784396 Paurocotylis pila New Zealand 82 100 EF159493 Leaf litter USA EU784395 Paurocotylis pila Great Britain HM123351 Plant leaf USA 100 100 HM123210 Culture Pseudevernia intensa USA HE820817 Plant leaf Unkn HQ641122 Plant litter China JX847756 Fimetariella rabenhorstii Great Britain 100 Z96991 Geopyxis rehmii Norway 77 JQ761957 Lichen USA JN048890 Hydnocystis piligera Unkn 100 UDB004428 EcM Eucalyptus grandis Cameroon HM123458 Culture Xanthoparmelia USA UDB004580 EcM Eucalyptus grandis Zambia 98 GQ153227 Plant leaf Cupressus arizonica Unkn JX630429 EcM Salix arctica Greenland JQ759163 Culture Masonhalea richardsonii USA 94 EF635832 Soil Austria 99 Z96990 Geopyxis carbonaria Norway EF635801 Soil Austria JQ759358 Culture Flavocetraria cucullata USA EU516932 Soil Austria 100 HM123534 Culture Juniperus deppeana USA 100 AB244053 EcM Salix Japan JQ759854 Culture Parmelia omphalodes USA AY187597 EcM Salix USA JQ759645 Culture Peltigera aphthosa USA 100 AY187611 EcM Salix USA UDB008146 EcM Isoberlinia doka Benin AY187613 EcM Salix USA 75 FJ008037 EcM Quercus prinus USA AB089817 EcM Japan 100 FJ008038 EcM Quercus palustris USA AY187596 EcM Salix USA 100 JN569353 EcM Carya illinoensis USA AY187598 EcM Salix USA 100 EU202707 EcM Quercus sp. USA AY187612 EcM Salix USA GQ240939 EcM Castanopsis fargesii China AB244052 EcM Salix Japan 100 100 UDB007008 EcM Nothofagus dombeyi Argentina 100 AY916067 EcM Salix caprea Slovenia UDB007164 EcM Nothofagus alpina Argentina UDB008926 EcM Salix triandra Estonia JN569352 EcM Carya illinoensis USA 99100 UDB008987 EcM Salix cinerea Estonia 97 JN569359 EcM Carya illinoensis USA UDB008951 EcM Salix pentandra Estonia 92 UDB008916 EcM Salix pentandra Estonia FJ008039 EcM Quercus palustris USA 95 /sordariales2 FJ008040 EcM Quercus palustris USA /pyronemataceae1 UDB008982 EcM Salix fragilis Estonia 100 GQ153115 Plant leaf Juniperus deppeana Unkn 98 UDB008925 EcM Salix triandra Estonia HM123560 Culture Lecidea tessellata USA AY916066 EcM Salix caprea Slovenia 100 EU480240 Soil USA EF635676 Soil Austria EU144522 Plant root Bouteloua gracilis USA EF635830 Soil Austria 91 FN397181 Soil France EU195342 OrM Epipactis atrorubens Estonia 100 DQ421286 Soil USA GU817139 Plant root Bistorta vivipara Norway JX042935 Soil USA GU817153 Plant root Bistorta vivipara Norway FN397376 Soil France GU817149 Plant root Bistorta vivipara Norway AY916068 EcM Salix caprea Slovenia 100 UDB008880 EcM Salix triandra Estonia UDB008862 EcM Salix caprea Estonia GU817145 Plant root Bistorta vivipara Norway UDB000987 Pulvinula convexella Estonia DQ447981 EcM Salix Slovenia 99 FM992943 EcM Pinus sylvestris Sweden JX630928 EcM Salix arctica Canada EU668294 EcM Germany JN890112 Soil Guyana DQ508805 EcM Picea abies Poland 100 JN936997 Conlarium duplumascospora Unkn FJ210748 EcM Italy 96 JX489782 Soil China JN704812 EcM Pinus montezumae Mexico 98 UDB007828 EcM Asteropeia micraster Madagascar JN704838 EcM Pinus montezumae Mexico 96 AY627787 Ericoid mycorrhiza Epacris pulchella Australia JQ917851 EcM Pinus radiata USA AY230783 Ericoid mycorrhiza Woollsia pungens Australia GU817103 Plant root Bistorta vivipara Norway /pulvinula 99 EU041813 Rhodoveronaea varioseptata Germany AJ877201 Plant stem China 100 JN704831 EcM Pinus montezumae Mexico UDB008883 EcM Salix caprea Estonia JQ760162 Lichen USA UDB008860 EcM Salix caprea Estonia 99 UDB013022 EcM Gnetum gnemon PNG 0.1 AF289074 Pulvinula constellatio Unkn UDB004751 EcM Cryptosepalum exfoliatum Zambia 90 UDB004039 EcM Nothofagus cunninghamii Australia 99 UDB004030 EcM Nothofagus cunninghamii Australia UDB004088 EcM Nothofagus cunninghamii Australia Fig 8 e Unrooted maximum likelihood phylogram demon- UDB004070 EcM Nothofagus cunninghamii Australia UDB004004 EcM Nothofagus cunninghamii Australia 70 AB218173 EcM Fagus crenata Japan strating phylogenetic placement of the /pulvinula and /py- AB218074 EcM Carpinus japonica Japan 82 HE814159 EcM China ronemataceae1 lineages based on combined ITS and LSU EF040884 Soil Castanea Italy HE814150 EcM China sequences. Significant bootstrap support (>70) is indicated 100 HE814078 EcM China UDB005281 EcM Carpinus Iran GQ900524 EcM Castanopsis China above branches. Abbreviations: EcM, ectomycorrhiza; ErM, 69 GQ268554 EcM Dipterocarpaceae Malaysia HE814116 EcM China ericoid mycorrhiza; OrM, orchid mycorrhiza; Unkn, un- 96 GQ268561 EcM Dipterocarpaceae Malaysia GQ268564 EcM Dipterocarpaceae Malaysia known country. UDB017246 EcM Vateriopsis sechellarum Seychelles UDB004848 EcM Dipterocarpaceae Thailand UDB004377 EcM Uapaca staudtii Cameroon JF273543 EcM China

GQ268556 EcM Dipterocarpaceae Malaysia /sordariales1 99 HE814196 EcM China HE814195 EcM China 87 91 GQ268552 EcM Dipterocarpaceae Malaysia JN889970 Soil Guyana species based on ITS sequences from EcM roots (Nouhra et al., UDB017247 EcM Vateriopsis sechellarum Seychelles UDB004850 EcM Dipterocarpaceae Thailand 2013) and at least two of these taxa correspond to named UDB004847 EcM Dipterocarpaceae Thailand 97 UDB004555 EcM Marquesia excelsa Gabon species (Aleurina echinata KC905033 and Aleurina argentina 89 UDB004750 EcM Brachystegiaiciformis Zambia UDB007822 EcM Sarcolaena multiflora Madagascar 100 UDB007821 EcM Asteropeia micraster Madagascar KC905032; Pfister & Smith, unpublished). In Australia, this UDB007827 EcM Sarcolaena multiflora Madagascar UDB007826 EcM Sarcolaena multiflora Madagascar group is thus far represented by a single species (Tedersoo UDB007825 EcM Sarcolaena multiflora Madagascar UDB007823 EcM Intsia bijuga Madagascar et al., 2008a; Horton et al., 2013; Fig 6). We studied several UDB007824 EcM Asteropeia Madagascar 005 UDB007820 EcM Intsia bijuga Madagascar EcM root tips from this group and all had a typical coarse pseu- doparenchymatous mantle and either thick-walled hyphae or Fig 9 e Unrooted maximum likelihood phylogram demon- no hyphae. Thus, species of this lineage belong to either con- strating phylogenetic placement of EcM lineages within tact or short-distance exploration types. Sordariales based on combined ITS and LSU sequences. The /pustularia lineage was recovered based on an LSU Significant bootstrap support (>70) is indicated above match between a Pustularia patavina fruit body and se- branches. Abbreviations: EcM, ectomycorrhiza; ErM, ericoid quences of EcM root tips (Tedersoo et al., 2013a). Based on mycorrhiza; OrM, orchid mycorrhiza; Unkn, unknown ITSsequences,thisisamonophyleticgroup(BS¼ 100) of country. closely related isolates that have been found throughout the boreal and temperate forests of the Northern Hemi- sphere. This lineage is most commonly recovered from EcM roots of Pinaceae, but they are occasionally identified transferred to Tarzetta;thereforethegenusnameofP. pata- from Fagaceae, Tiliaceae, Salicaceae and Rosaceae (Fig 7). vina may change. Members of this group exhibit mantle structure similar to The /rhodoscypha lineage comprises a small monophyletic that of Geopora (Tedersoo et al., 2006) and sparse extraradi- group of sequences that was discerned based on LSU cal hyphae that correspond to the short-distance explora- (Tedersoo et al., 2013a). There are only two EcM-derived ITS tion type. The genus name Pustularia had a very wide sequences available in INSDc and both originated from Pina- useanditstypespecies,Pustularia cupularis, has been ceae in SW Canada (Jones et al., 2008; S. Lim, unpublished). 92 L. Tedersoo, M. E. Smith

The monotypic genus Rhodoscypha is distributed in the boreal common root endophytes that are frequently detected by mo- and temperate forests of the Northern Hemisphere and is rare lecular (Tedersoo et al., 2009a,c) and culture-based surveys throughout its range. Unfortunately no root tips were avail- (Vralstad et al., 2002; Kernaghan and Patriquin, 2011; Menkis able from the authors for microscopy. and Vasaitis, 2011) of EcM tissues. Phylogenetic analyses of The /pyronemataceae1 lineage comprises a heterogeneous subgroups of Helotiales indicate that endophytic, ericoid group of EcM root-derived sequences that have no fruit body mycorrhizal, soil-derived and EcM-associated isolates occur representatives. This group displays affinities to the Pulvi- throughout the phylogeny (Vralstad et al., 2002; Hambleton nula-Lazuardia group, but is distinct from the /pulvinula EcM and Sigler, 2005). Without statistical approaches or informa- fungal lineage (Tedersoo et al., 2013a; Fig 8). ITS sequences tion about EcM morphology and behavior in culture, it is diffi- of this group were derived from EcM roots of Fagaceae and cult to assign a nutritional mode to an individual species in Juglandaceae in the USA (Cavender-Bares et al., 2009; Bonito this group. An additional problem is the thin mantle and et al., 2012), Nothofagaceae in Argentina (Nouhra et al., 2013) sometimes poorly developed Hartig net in these helotialean and from Caesalpinioideae in Benin (L. Tedersoo and N.S. EcM (Yu et al., 2001; Tedersoo et al., 2008b; Munzenberger€ Yorou, unpublished). The Argentinean root tip vouchers et al., 2009). These anatomical features have led to many have sparse hyphae and have a mantle structure that is inter- ambiguous reports on EcM formation between Helotiales mediate between the pseudoparenchymatous and plecten- and various plants (Kohn et al., 1986; Haug et al., 2004; chymatous types. We believe that this lineage is best Peterson et al., 2008; Comandini et al., 2012). In Pezizales, characterized by short-distance exploration type. where root endophytes have rarely been recovered in molecu- The /pyronemataceae2 lineage is a monophyletic group lar and culture-based surveys, assignment of nutritional (BS ¼ 100) of three sequences recovered exclusively from mode to particular taxa is more straightforward (Tedersoo EcM root tips of Pinaceae in California (Izzo et al., 2005; et al., 2013a). In only one of the pezizalean families, the Pyro- Smith et al., 2009) and Northern Mexico (Hoeksema et al., nemataceae, we documented five novel lineages. Previous 2012; Fig 7). This small group is closely related to but clearly recognition of these groups was hampered by the lack of ITS distinct from the /geopora lineage. Based on 97 % ITS similar- sequences from sporocarps, because molecular taxonomic ity, each sequence could be recognized as a distinct taxon. No work has mostly utilized LSU and protein-encoding genes root tips were available for microscopy. (Hansen et al., 2013). However, recent ecological studies The /sordariales lineage of Tedersoo et al. (2010) is divided have started to routinely sequence both ITS and partial LSU into two groups based on additional sequence data and to improve biogeographic and taxonomic resolution (e.g. phylogenetic analyses of combined ITS and LSU sequences Smith et al., 2007). Matching LSU genes from fruit-bodies (Fig 9). and EcM root tips can prove useful to assign nutritional The /sordariales1 lineage encompasses sequences from mode to pezizalean groups (Tedersoo et al., 2006, 2013a; various angiosperm hosts in tropical (Tedersoo et al., Smith et al., 2007). 2007, 2011b; Peay et al., 2010; Phosri et al., 2012), southern temperate (Tedersoo et al., 2009a) and warm northern Zygomycota temperate (Ishida et al., 2007; Bahram et al., 2012) biomes, and forms a well-supported monophyletic group (BS ¼ 99; There is only limited phylogenetic information available for Fig 9). Based on descriptions of EcM roots from the Seychelles EcM fungal lineages in Zygomycota (Desiro et al., 2013). The (Tedersoo et al., 2007) as well as material from Australia and /endogone lineage has been separated into two parts based Africa, members of the /sordariales1 lineage form short- on phylogenetic information from SSU, ITS and LSU genes distance exploration type. (L. Tedersoo et al., unpublished data). Of species studied by The /sordariales2 lineage represents a monophyletic group Warcup (1990a), the Pinaceae-associated Endogone flammicor- of sequences (BS ¼ 94; Fig 9). While the bulk of the sequences ona and Endogone lactiflua constitute the /endogone1 lineage, originates from arctic, alpine and temperate habitats in asso- whereas the Australian Endogone aggregata, Endogone tubercu- ciation with Salix (Trowbridge and Jumpponen, 2004; Nara, losa and Sclerogone eucalypti form the /endogone2 lineage. 2006b; Timling et al., 2012; Tedersoo et al., 2013b) and Bistorta Probably numerous sequenced EcM root tips from the Austra- vivipara (Brevik et al., 2010), species in the basal clade of this lian Myrtaceae, Nothofagaceae and Rhamnaceae (Tedersoo lineage are EcM symbionts of Eucalyptus introduced to Africa. et al., 2008a, 2009a) also fall into the latter group. Notably, This group was common on eucalypts but absent on indige- the type species of Endogone, Endogone pisiformis, is non-EcM nous trees, suggesting their co-introduction to Africa (Jairus (e.g. Berch, 1983). Sequences from old collections of Densospora et al., 2011). Root tip vouchers from Estonia and Africa have spp., associated with Myrtaceae (McGee 1996), are phylogenet- a thin mantle and abundant hyphae, pointing to a short- ically distinct from the /endogone1 and /endogone2 lineages distance exploration type. (L. Tedersoo et al., unpublished data). In Ascomycota, there are many EcM fungal lineages that are comprised of only a few species (e.g. /cenococcum and Doubtful groups several lineages in the Helotiales). Many of these species- poor lineages apparently are either limited in their distribu- Several EcM lineages such as /catathelasma, /sowerbyella, tion or are infrequently detected in EcM community studies, /endogone1 and /densospora have never been recovered in perhaps due to their specific ecological requirements. The lin- EcM community studies. Root tips of the /catathelasma line- eages within Helotiales have remained challenging to accu- age have been documented only once, when specifically rately detect and identify, because they are closely related to searching for them under a fruit body of Catathelasma Lineages of ectomycorrhizal fungi revisited 93

imperiale (L. Tedersoo, unpublished). Although Sowerbyella is suggest that it should be treated as Geopora clausa (Tul. and C. suggested to form EcM based on its habitat and the 15N iso- Tul.) Burds. (Burdsall 1968; M.E. Smith, unpublished). topic signature (Hobbie et al., 2001), there are still no direct The /leucangium lineage is appended with the recently observations on EcM in this group. Of novel lineages, /hydro- erected, monophyletic genus Kalapuya that is nested within pus and /xenasmatella require further support for their EcM this group (Trappe et al., 2010). While the genus Fischerula is status (see above). distantly placed in some phylogenetic studies (Healy et al., 2013), most studies with more focused taxonomic sampling or inclusive ingroup sampling support placement of Fischerula 4. New EcM fungal genera within the /leucangium lineage (Trappe et al., 2010; Alvarado et al., 2011). Since taxonomists continually lump, split, and create new Several recently sequenced and/or newly described genera genera, the list of EcM groups is subject to change. There are accommodated in the /marcelleina-peziza gerardii line- is still substantial controversy as to whether monophyletic age. Delastria rosea, a rare sequestrate species was recently lineages that contain different fruit body types should be placed as a sister group to species of Hydnobolites (Alvarado treated as monogeneric or multigeneric groups. In partic- et al., 2011; Healy et al., 2013). D. rosea isolate JN102449 exhibits ular, sequestrate fungi have traditionally been named in 99.5 % ITS sequence similarity with an EcM root tip isolate separate genera, but since these truffle-like taxa are often FJ013057 from Portugal (Rincon and Pueyo, 2010) and slightly nested within groups of epigeous species, the nomenclature less to many other root tip isolates, indicating that Delastria has become problematic. For example, the polyphyletic spp. are ectomycorrhizal. Kovacs et al. (2011) described the sequestrate fungi within the /cortinarius lineage were previ- new sequestrate fungal genera Temperantia and Stouffera that ously treated within the genus Thaxterogaster, but have now are nested within the /marcelleina-peziza gerardii lineage all been synonymized within the genus Cortinarius.In (Kovacs et al., 2011; Healy et al., 2013), suggesting that these contrast, the sequestrate fungi within the /boletus and /rus- genera are ectomycorrhizal. sula-lactarius lineage have mostly been retained as separate The /tuber-helvella lineage includes the Southern Hemi- genera (but see Desjardin (2003) and Lebel and Tonkin (2007) sphere genera Gymnohydnotrya and Nothojafnea in addition to for exceptions). Within the /boletus lineage, many new previously reported taxa (Bonito et al., 2013). The genus Under- genera of both epigeous and hypogeous taxa have been woodia appears to be split into two distinct groups that likely erected in recent years. Peziza sensu lato represents the constitute one genus for the Northern Hemisphere and one opposite scenario and this genus is still treated as a large, for the Southern Hemisphere (Bonito et al., 2013, M.E. Smith, paraphyletic group even though it includes several well- unpublished). Loculotuber and Paradoxa also belong to this defined linages that include sequestrate and epigeous spe- group, but these taxa will probably be synonymized with Tuber cies as well as both EcM and non-EcM taxa. (Kinoshita et al., 2011; Alvarado et al., 2012; Bonito et al., 2013). For 14 previously recognized lineages, we provide updated In contrast to most Tuber species, the basal Southern Hemi- information about the genera that are currently included. In sphere species in Gymnohydnotrya and Underwoodia sensu lato total, we add 47 genera to the list of EcM fungi, most of which exhibit a contact exploration type that is similar to species have been described very recently. We also remove three in the Helvellaceae. genus-level taxa (Hydnocystis piligera, Tricharina ochroleuca, A recently sequenced isolate of Discinella terrestris from Rubinoboletus rubinus; see below). The updated list of EcM New Zealand (GU222294) has 96e97 % ITS match to multiple fungal genera is found in Table S1. species within the /helotiales4 lineage that has so far only From matching fruit body and EcM root tip LSU sequences, documented from Australia (Tedersoo et al., 2008a; Horton there is good evidence that the Pezizalean genus Parascutellinia et al., 2013). The core group of Discinella sensu stricto is probably is ectomycorrhizal with Salix spp. in various habitats in non-ectomycorrhizal, because the type species Discinella bou- Estonia (Tedersoo et al., 2013b). Unfortunately, no ITS se- dieri has a Northern Hemisphere distribution and it has not quences of Parascutellinia fruit-bodies are available in INSDc. been sequenced thus far. Although Parascutellinia was considered a saprotrophic sister The /hebeloma-alnicola lineage is widened by the addition group to the /genea-humaria lineage in Tedersoo et al. of the genus Psathyloma (nom. prov.; P.B. Matheny, pers. (2010), recent phylogenetic analyses support its placement comm. January 2013) that has been found mostly in New Zea- within this group (Hansen et al., 2013; Tedersoo et al., 2013b). land as fruit-bodies, but also as EcM root tips in Tasmania In previous reports by Tedersoo et al. (2010) and Comandini (Tedersoo et al., 2009a; Horton et al., 2013) and Argentina et al. (2012), the fungus T. ochroleuca was reported as an EcM- (Nouhra et al., 2013). In contrast to other members of this line- forming member of the /geopora lineage. However, all reports age, EcM of Psathyloma spp. exhibit a short-distance explora- of EcM formation by T. ochroleuca are apparently based on par- tion type with abundant dark brown hyphae but no tial ITS matches to sequences from cultures that are phyloge- rhizomorphs. At least one species from the genus Wakefieldia netically placed outside the /geopora lineage (Fig 7; see also (Wakefieldia macrospora) also belongs to the /hebeloma-alni- Stielow et al., 2013). We find no credible evidence that any Tri- cola lineage, because it is phylogenetically placed among spe- charina species form EcM. Similarly, H. piligera (type species) cies of Alnicola, Hebeloma and Hymenogaster (Kaounas et al., forms a sister group to the non-EcM genus Stephensia and 2011). Sequences from EcM root tips (isolates HQ204662, therefore we consider the genus Hydnocystis to be non-EcM. HQ204659) of Quercus ilex in France (Richard et al., 2011) are However, at least one species of Hydnocystis s. lato, Hydnocystis 99.8 % similar to W. macrospora. However, the type species, clausa, is an EcM member of the /geopora lineage. However, we Wakefieldia striaespora was described from SE Asian 94 L. Tedersoo, M. E. Smith

dipterocarp forests and it may belong to a different group (Sutara, 2008). Australopilus and Harrya are Australian genera based on the combination of morphology and habitat (M.E. that have been erected as segregates of the Northern Hemi- Smith, unpublished). sphere genus Tylopilus (Halling et al., 2012b). The genus Sutor- In the /inocybe lineage, Tubariomyces has been erected from ius has been erected from Boletus (Halling et al., 2012a). For Inocybe (Alvarado et al., 2010). Tubariomyces is inferred to asso- some reason, no ITS sequences exist for Australopilus, Gym- ciate with Cistaceae. However, since relatively few studies nogaster, Harrya, Phylloboletellus, Royoungia and Sutorius, and have focused on EcM communities of Cistaceae there are therefore root tip matches to these genera cannot be evalu- currently no available root-derived sequences that correspond ated. The type species of Rubinoboletus, R. rubinus is nested to Tubariomyces). within the non-EcM Chalciporus, but a few other species are The /piloderma lineage includes a recently erected genus nested among EcM taxa within the /boletus lineage (Nuhn Tretomyces that has been recorded only from boreal and et al., 2013). Nuhn et al. (2013) listed a number of genera that temperate Pinaceae forests (Kotiranta et al., 2011; Fig 2). belong to the Boletaceae (Boletochaete, Gastroleccinum, Paxil- Destuntzia fusca was considered putatively EcM (Tedersoo logaster, Sinoboletus) or Paxillaceae (Austrogaster, Hoehne- et al., 2010) and is now a confirmed member of the /ramaria- logaster, Meiorganum) based on morphological characters, but gautieria lineage based on ITS sequence data. The isolate they lack ITS or LSU sequence data to evaluate their phyloge- EU697269 matches a group of ectomycorrhizal Ramaria species netic position and their EcM status. (not shown). Rhopalogaster belongs to the /suillus-rhizopogon lineage DNA sequences of ITS and LSU from the genus Fevansia based on LSU sequence data (Hosaka et al., 2006). Rhopalogaster indicate that this genus belongs to the EcM /albatrellus lineage transversarium isolate DQ218599 has 96.6 % LSU sequence (Smith et al., 2013b). Although Fevansia does not match closely identity with Suillus hirtellus fruit body AY612828. ITS se- with any EcM root tip sequences, this species is consistently quences of this genus are lacking from sequence databases. found among EcM roots of Pinaceae and all of its closest rela- tives form EcM. 5. Confirmed non-EcM genera The /boletus lineage has been enriched with several recently described genera or genera with newly generated mo- The genus Amogaster that produces sequestrate fruit-bodies lecular data (Nuhn et al., 2013). The LSU sequences of the was originally considered a member of Boletales. However, sequestrate Gymnogaster boletoides falls into the /boletus line- this species is nested within the saprotrophic genus Lepiota age (Halling et al., 2012b). The monotypic Heliogaster was and does not form EcM (Ge and Smith, 2012). Similarly, Amer- segregated from Octaviania and it represents a sequestrate ican species of Gigasperma are nested within Lepiota and are Xerocomus species (Orihara et al., 2010). No EcM root tips are currently treated in the sequestrate, non-EcM genus Cryptole- matched to Heliogaster columellifera, but the phylogenetic posi- piota (Kropp et al., 2012). Sequestrate forms have evolved mul- tion indicates that it is ectomycorrhizal. Tubosaeta belongs to tiple times in Lepiotaceae (Ge and Smith, 2012), which is a the /boletus lineage based on ITS sequences (Brock et al., common phenomenon in Basidiomycota. The type species of 2009). However, none of the sequenced specimens match Gigasperma, Gigasperma cryptica is found exclusively in New ITS sequences from EcM root tips. Rossbeevera was erected to Zealand and is nested within the /cortinarius EcM lineage accommodate a monophyletic group of sequestrate fungi (Kropp et al., 2012). The fruit body sequence generated by from Australia and Japan (Lebel et al., 2012). Sequences corre- Kropp et al. (2012) has 93.7 % ITS sequence match with Corti- sponding to this group have been found from EcM root tips in narius elaiops (JX000369). Neopaxillus forms a monophyletic sis- Australia (Tedersoo et al., 2009a; Horton et al., 2013). Turmali- ter group to Crepidotus and Simocybe and no EcM-derived ITS nea is a recently described sequestrate genus that is sister to sequences fall into this group so Neopaxillus is therefore Rossbeevera; this group is phylogenetically related to other considered non-ectomycorrhizal (Vizzini et al., 2012). EcM-forming members of the /boletus lineage and Turmalinea species are consistently found with EcM trees (Orihara et al., 2013). Spongiforma represents a recently described sequestrate 6. Biodiversity and biogeography genus that is related to Porphyrellus (Desjardin et al., 2009). The pileate genus Borofutus has been described to accommodate a The lack of randomly obtained sequences from certain line- sister species to Spongiforma (Hosen et al., 2013). No EcM iso- ages suggests that, despite our good overall understanding lates correspond to these two genera but both groups are of EcM fungal communities, several EcM groups still await dis- inferred as EcM. Zangia has been erected from Tylopilus and covery due to their natural rarity. Based on fruit body records, this group is currently represented only by Chinese species we previously suggested that tropical-endemic EcM lineages (Li et al., 2011). No EcM root tip sequences correspond to Zangia were either rare or absent (Tedersoo et al., 2010). However, roseola, the only species for which an ITS sequence is avail- here we report four putatively EcM groups that are hitherto able. The recently described sequestrate genus Solioccasus is known only from tropical habitats (/agaricales1, atheliales1, phylogenetically allied with Bothia and is inferred as EcM as /hydropus, /xenasmatella) as well as one lineage that is found it is always encountered among ECM Myrtaceae and Alloca- in both tropical and subtropical ecosystems (/atheliales2). All suarina in Northern Queensland and Papua New Guinea these lineages are relatively rare and species-poor except for (Trappe et al., 2013). Corneroboletus has been described to the /atheliales1 lineage (see above). Given that four of these accommodate Boletus indecorus (Zeng et al., 2012). No EcM groups are distributed on multiple continents, it is reasonable root isolate corresponds to Corneroboletus. Hemileccinum was to assume that these lineages are either relatively old or have described to accomodate Boletus impolitus and Boletus depilatus excellent capacity for dispersal. However, given the rarity of Lineages of ectomycorrhizal fungi revisited 95

these taxa, we hypothesize that vicariance is probably more Nevertheless, we believe that our summary of current knowl- important than long-distance dispersal in explaining the dis- edge on exploration types will be useful for analysis and inter- tribution of these EcM groups. Nonetheless, we acknowledge pretation of results from molecular studies that rely on fungal that the present data are too scanty to generate any realistic DNA from hyphal mesh bags and soil. biogeographic scenarios to explain their origins. In addition, several common groups of EcM fungi such as the /inocybe and /clavulina lineages may have evolved in tropical regions based on molecular data (Matheny et al., 2009; Smith et al., Acknowledgments 2011; Kennedy et al., 2012). These groups have effectively spread to both temperate and arctic ecosystems, suggesting We thank C. Andrew, M. Bahram, M. Bidartondo, G.M. Bonito, ~ that long-term migration from tropical to temperate climates T.W. Henkel, U. Koljalg, K.-H. Larsson, T. Leski, S. Lim, P. € is possible for some EcM groups with tropical origins. We McGee, A. Morte, E. Nouhra, J. Oja, M. Opik, D. Pfister, D. South- b deduce that the rare tropical lineages described here may worth, J.M. Trappe, J.K.M. Walker and M. Wei for discussing not have been able to expand beyond the tropics because of fungal taxonomy or EcM morphology of their root tip collec- inefficient dispersal capacities or due to an inability to with- tions. L.T. receives financial support from Estonian Science stand cold temperatures. Based on large-scale biogeographic Foundation grants 9286, PUT171, FIBIR, and EMP265). M.E.S. distribution patterns of EcM taxa (Geml et al., 2012; Tedersoo acknowledges the University of Florida’s Institute of Food et al., 2012a; Timling et al., 2012; Bahram et al., 2013), we sug- and Agricultural Sciences (IFAS) for continued financial sup- gest that intolerance of low temperature may have limited port. M.E.S. also received funding for DNA sequencing and ex- migration of many lineages to subarctic and arctic peditions in South America from the Farlow Herbarium and ecosystems. the David Rockefeller Center for Latin American Studies at Harvard University (with D.H. Pfister).

7. Concluding remarks Appendix A. Supplementary data

Based on accumulated ITS sequences and associated meta- Supplementary data related to this article can be found at data, we describe 20 new lineages of EcM fungi. Thus, the http://dx.doi.org/10.1016/j.fbr.2013.09.001. number of distinct EcM lineages is elevated to 78e82 and the number of EcM fungal genus-level taxa is elevated to 251e256. However, several putative lineages require further references morphological or ultrastructural proof, because saprotrophic Ascomycota, Basidiomycota and Zygomycota are all commonly detected as saprotrophs or endophytes with EcM Abarenkov, K., Tedersoo, L., Nilsson, R.H., Vellak, K., Saar, I., root tips (Morris et al., 2008; Lindner and Banik, 2009; Veldre, V., Parmasto, E., Prous, M., Aan, A., Ots, M., Kurina, O., ~ ~ Tedersoo et al., 2009c; Nouhra et al., 2013). Given the rarity Ostonen, I., Jogeva, J., Halapuu, S., Poldmaa, K., Toots, M., ~ e of many lineages in EcM community studies, we suggest Truu, J., Larsson, K.-H., Koljalg, U., 2010. PlutoF a web based workbench for ecological and taxonomic research with an that continued research will reveal new EcM lineages, espe- online implementation for fungal ITS sequences. Evol. Bio- cially in tropical and Southern Hemisphere ecosystems where inform. 6, 189e196. fewer EcM community studies have been conducted. This syn- Agerer, R., 2001. Exploration types of ectomycorrhizae. Mycor- thesis of new data from our own studies and from the INSDc rhiza 11, 107e114. indicates that several uncommon, species-poor lineages Agerer, R., 2006. Fungal relationships and structural identity of e may indeed be limited to tropical and subtropical ecosystems. their ectomycorrhizae. Mycol. Progr. 5, 67 107. Alvarado, P., Manjon, J.L., Matheny, P.B., Estve-Raventos, F., 2010. Information about EcM morphology allowed us to deter- Tubariomyces, a new genus of Inocybaceae from the Mediter- mine the main exploration types for both the novel groups ranean region. Mycologia 102, 1389e1397. and poorly studied taxa that have not been addressed in pre- Alvarado, P., Moreno, G., Manjon, J.L., 2012. A new Tuber without vious in-depth studies. Although most EcM fungal lineages spore ornamentation. Tuber melosporum comb. nov. Bol. Soc. (and genera therein) possess a single exploration type, the Micol. Madrid 36, 23e28. most common and species-rich genera exhibit multiple explo- Alvarado, P., Moreno, G., Manjon, J.L., Gelpi, C., Kaounas, V., ration types (Agerer, 2006). Based on these findings, we Konstantinidis, G., Barseghyan, G.S., Venturella, G., 2011. First molecular data on Delastria rosea, Fischerula macrospora and caution that exploration type cannot be consistently extrapo- Hydnocystis piligera. Bol. Soc. Micol. Madrid 35, 31e37. lated from a few species to the entire genus or lineage. There- Bahram, M., Polme,~ S., Koljalg,~ U., Zarre, S., Tedersoo, L., 2012. fore, we strongly encourage researchers to determine foraging Regional and local patterns of ectomycorrhizal fungal diver- strategies based on original experimental material. To be able sity and community structure along an altitudinal gradient in to seek further molecular or morphological proof and to study the Hyrcanian forests of northern Iran. New Phytol. 193, e the morphology in more detail in future, researchers should 465 473. Endogone pisiformis keep voucher root tips. A large number of in-depth morpho- Berch, S.M., 1983. : axenic culture and associ- ations with Sphagnum, Pinus sylvestris, Allium cepa and Allium logical and anatomical descriptions of unidentified EcM is porrum. Can. J. Bot. 61, 899e905. also available online (www.deemy.de). Molecular identifica- Bergemann, S.E., Garbelotto, M., 2006. High diversity of fungi tion of these well-described EcM samples would further our recovered from the roots of mature tanoak (Lithocarpus densi- understanding of EcM fungal community ecology. florus) in northern California. Can. J. Bot. 84, 1380e1394. 96 L. Tedersoo, M. E. Smith

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