Mycologia, 106(4), 2014, pp. 856–864. DOI: 10.3852/13-377 # 2014 by The Mycological Society of America, Lawrence, KS 66044-8897

Acidomelania panicicola gen. et sp. nov. from switchgrass roots in acidic New Jersey pine barrens Emily Walsh Jing Luo Ning Zhang1

Abstract: A new genus, Acidomelania, is described based on molecular phylogenetic analyses and ecological and morphological characters. Isolated from switchgrass roots in acidic and oligotrophic New Jersey pine barrens in this study, Acidomelania likely has a global distribution because its internal transcribed spacer (ITS) sequence has high similarity with a number of GenBank sequences resulted from various ecological studies. Apparently these samples all were from roots of plants that lived in acidic, nutrient-poor environments. Phylogenetic analyses based on ITS, LSU and ACT sequence data strongly supported the fact that Acidomelania isolates formed a monophyletic clade in Helotiales, distinct from any known taxa. Phylogenetically Acidomelania is closely related to Loramyces, Mollisia and Phialocephala fortinii, Acephala applanata species complex (PAC), the dark septate endophytes. Acidomelania also can be distinguished from Loramyces and Mollisia by its association with living grass roots. While taxa in PAC also are root endophytes, they have more complex phialid arrangement than Acidomelania. Results from this work will facilitate ecological and evolutionary studies on root-associated fungi. Key words: dark septate endophytes, fungi, grass, Leotiomycetes, phylogeny, root, taxonomy

States is the 1.4 million acre pine barrens of New Jersey, where the soil is highly acidic, sandy and nutrient poor. Much remains unknown about the fungal diversity in the New Jersey pine barrens, even though they play critical roles in litter decomposition, nutrient absorption and cycling (Forman 1998, Tuininga and Dighton 2004). Dark septate endophytes (DSE) are a heterogeneous group of plant root-colonizing ascomycetes that produce melanized, septate hyphae (Stoyke and Currah 1991, Jumpponen and Trappe 1998, Knapp et al. 2012). They inhabit a wide range of hosts including conifers, grasses, terrestrial orchids and ericaceous plants ( Jumpponen and Trappe 1998). The best-studied DSE are the Phialocephala fortiniiAcephala applanata complex (PAC), a group of asexual fungi in Helotiales of Leotiomycetes (Wang et al. 2006). In addition to darkly pigmented hyphae, fungi in PAC are characterized by producing branched conidiophores and hyaline phialides with collarettes. PAC are the dominant root associates of many tree species, especially conifers and Ericaceae plants in the forests of the northern hemisphere (Menkis et al. 2004, Gru¨nig et al. 2008b). Despite the recognized ubiquity of plant rootassociated fungi, their ecological roles, phylogenetic relationships and taxonomy still remain poorly understood (Mandyam and Jumpponen 2005, Knapp et al. 2012). In this study we examined fungi associated with apparently healthy switchgrass roots from New Jersey pine barrens. Based on phylogenetic analyses, ecology and morphology, a new genus, Acidomelania, is proposed in Helotiales. Its phylogenetic relationships with PAC and other allied taxa are discussed.

INTRODUCTION

MATERIALS AND METHODS

Pine barrens (pinelands) is a unique type of ecosystem that is oligotrophic, drought- and fire-prone. Pine barrens occur throughout northeastern USA from New Jersey to Maine (Forman 1998). Pines and oaks are the most common trees in pine barrens, while the understory is composed of grasses (Poaceae), sedges (Cyperaceae), blueberries and other members of heath family (Ericaceae). The largest and most uniform area of pine barrens in the United

Fungal isolation.—Native switchgrass (Panicum virgatum) roots were collected from two locations (N39 26.4738, W74 34.934 and N40 04.084 W74 26.696) in the New Jersey pine barrens in 2011 and 2012. Soil pH of the sampling locations was about 5.2. Root samples were rinsed thoroughly to remove soil from the surface, cut into 10–20 mm segments then surface sterilized with sequential washes of 95% ethanol for 30 s, 0.5% NaOCl for 2 min and 70% ethanol for 2 min. Then samples were rinsed in sterile water and dried. Root samples were cut into 5 mm pieces and plated on acidified malt extract agar (AMEA, 1.5 mL 85% lactic acid per liter of 2% malt extract agar). Plates were incubated at room temperature with 12 h light and 12 h

Department of Plant Biology and Pathology, 201 Foran Hall, 59 Dudley Road, and Department of Biochemistry and Microbiology, 76 Lipman Drive, Rutgers University, New Brunswick, New Jersey 08901

Submitted 27 Nov 2013; accepted for publication 10 Feb 2014. 1 Corresponding author. E-mail: [email protected]

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WALSH ET AL.: ACIDOMELANIA, dark cycles. Fungal cultures were transferred to fresh AMEA and purified by subculturing from emergent hyphal tips. The color names of colonies follow Ridgway (1912). Culture morphology and growth rate.— Purified fungal isolates were grown on cellophane overlaid with 2% MEA and 2% water agar (WA). Three replicates of each culture were incubated at 20 C in dark. Colony diameter was measured after 20 d. DNA isolation, amplification and sequencing.—Genomic DNA was extracted from fungal mycelium with the UltraClean Soil DNA isolation kit (MoBio, California) following the manufacturer’s instructions. PCR was performed with Taq 23 Master Mix (New England BioLabs, Maine) following the manufacturer’s instructions. PCR cycling conditions for the internal transcribed spacer (ITS) and the large subunit of ribosomal RNA genes (LSU) consisted of an initial denaturation step at 95 C for 2 min, 35 cycles of 95 C for 45 s, 54 C for 45 s, 72 C for 1.5 min and a final extension at 72 C for 5 min. For the actin gene (ACT ) the cycling conditions included an initial denaturation step at 95 C for 2 min, 35 cycles of 95 C for 1 min, 58 C for 1 min, 72 C for 30 s, and a final extension at 72 C for 10 min. Primers used in this study are as follows: ITS1 and ITS4 for the ITS region (White et al. 1990), ITS1 and LR5 for the LSU locus (Rehner and Samuels 1995), and ACT-512F and ACT-783R for the ACT gene (Carbone and Kohn 1999). PCR products were purified with ExoSAP-IT (Affymetrix, California) and sequenced with the PCR primers by Genscript Inc. (Piscataway, New Jersey). Phylogenetic analyses.—Four representative isolates of the new taxon (61R8, 61R41, 61R50B, CM16s1) as well as other reference Leotiomycetes species (TABLE I) were included in phylogenetic analyses. The LSU dataset included four new sequences from this study and 26 reference sequences of Helotiales and Rhytismatales. The ITS dataset included sequences of the four new isolates from this study and 18 reference sequences of Helotiales. The ACT dataset included six sequences. Sequences were aligned with MUSCLE (Edgar 2004). Maximum likelihood (ML) tree was generated with MEGA 5 (Tamura et al. 2011). Models with the lowest BIC scores (Bayesian information criterion) were considered to describe the substitution pattern the best. The best models for LSU, ITS and ACT dataset were Tamura-Nei, Kimura 2 parameter and Kimura 2 parameter respectively. Initial tree(s) for the heuristic search were obtained automatically by applying neighbor-joining and BioNJ algorithms to a matrix of pairwise distances estimated with the maximum composite likelihood approach and then selecting the topology with superior log likelihood value. A discrete gamma distribution was used to model evolutionary rate differences among sites. Bootstrap was computed for 1000 replications. Maximum parsimony (MP) trees were generated with MEGA 5 (Tamura et al. 2011) using the closeneighbor-interchange on random trees, with a search level of 1, and with the addition of 10 random trees. All positions containing gaps and missing data were partially deleted with a site coverage cutoff of 95%. Bootstrap consensus trees were inferred from 1000 replicates.

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RESULTS Culture morphology and growth rate.—Isolates 61R8, 61R41, 61R50B and CM16s1 produced dense, dark mycelium on MEA and light brown sparse aerial mycelium on WA. Colony diameters for isolate 61R8 after 20 d were 33 mm on average on MEA with standard deviation (SD) of 3 and 35 mm on average on WA with SD of 5. Phylogenetic analyses.—A total of 770 characters were in the LSU alignment, 413 in ITS and 197 in ACT (TreeBASE number S15068). Maximum likelihood trees based on LSU, ITS and ACT sequences are illustrated (FIGS. 2–4). The topologies of the MP trees were similar to the ML trees. All three phylogenies (FIGS. 2–4) supported the new isolates as forming a monophyletic clade in Helotiales separated from known taxa. The LSU tree indicated that they were close to Loramyces macrosporus, Mollisia cinerea and PAC. The ITS tree showed that these new isolates formed a well-supported clade with Mollisia melaleuca and M. fusca. Loramyces macrosporus, M. cinerea and Phialocephala scopiformis are also close relatives. Four taxa from GenBank (last four in TABLE I) also grouped with our New Jersey pine barren isolates. In the ITS tree, isolates CM16s1 and 61R50B formed a well-supported group while 61R8 and 61R41 formed another. However, in the ACT tree, isolate 61R41 and 61R50B grouped together. There was no variation among the LSU sequences of the four new isolates. Based on the molecular phylogenetic analyses and morphological characters, a new genus is proposed. TAXONOMY Acidomelania E. Walsh & N. Zhang, gen. nov. MycoBank MB807849 Etymology: ‘‘Acid’’ refers to the acidic soil where the fungus thrives; ‘‘melan’’ refers to the black-pigmented hyphae.

Colonies on MEA and WA darkly pigmented. Conidiophores simple or occasionally branched. Conidiogenous cells hyaline, straight. Collarettes cylindrical. Conidia aggregated in slimy heads, globose to subellipsoidal, aseptate, hyaline, smooth. Chlamydospores catenulate, intercalary, subglobose. Type species: Acidomelania panicicola Habitat: Endophytic in roots of Panicoideae Known distribution: New Jersey, United States. Acidomelania panicicola E. Walsh & N. Zhang, sp. nov. FIG. 1A–E MycoBank MB807850 Etymology: ‘‘panici’’ refers to the host genus or subfamilial name and ‘‘cola’’ means loving.

Species

CBS468.94 CBS119280

CBS477.97 CBS122030 CBS300.62 CBS109309 CBS443.86 CBS109302

AFTOL49 CBS811.85 AFTOL1 CBS235.53 AFTOL1292 CBS122029 ICMP18083 CBS486.48 CBS589.84 CBS122031

AFTOL166 CBS161.38

CBS118.31

CBS109321 CBS109318 CBS123555 61R8 61R41 61R50B CM16s1 CBS251.80

Isolate No.a Location

log with moss Malus sp. slime in pulp mill forest soil Pinus sylvestris, root forest soil Picea abies Picea abies, bark Picea abies, root Picea abies, root Tsuga Canadensis

fallen log Actinidia deliciosa cv. Hayward Rhododendron sp. Pinea abies, needle

Alnus sp., cones Aster ageratoides var. ovata Chrysolepis chrysophyla Equisetum limosum

Regensburg, Germany Bo¨dmeren, Switzerland Czech Republic Tennessee, USA Svalbard, Norway

Zernez, Switzerland Suonenjoki, Finland Oberschlatt, Switzerland

Finland New York, USA Oregon, USA

Portland, Oregon, USA Kaarina, Finland Oregon, USA Honshu, Japan Oregon, USA UK Tennessee, USA Alsea Falls, Oregon, USA New Zealand Netherlands Germany

Bu¨dmerenwald, Switzerland Knesebeck, Germany Hubertusstock, Germany New Jersey Pine Barrens, USA New Jersey Pine Barrens, USA New Jersey Pine Barrens, USA New Jersey Pine Barrens, USA New Haven, Connecticut, USA Oregon, USA Germany Oregon, USA New Zealand foam in stream Nova Ves, Czech Republic Tsuga sp., cones and small sticks Oregon, USA Acer rubrum Bear Island, Ontario, Canada

Picea abies, root Pinus sylvestris, root Pinus sylvestris, root Panicum virgatum, root Panicum virgatum, root Panicum virgatum, root Schizachyrium scoparium, root

Host

Species name, isolate number, host, location and GenBank accession numbers of the fungi used in this study

Acephala applanata Acephala applanata Acephala macrosclerotium Acidomelania panicicola Acidomelania panicicola Acidomelania panicicola Acidomelania panicicola Ascocoryne cylichnium Botryotinia fuckeliana Bulgaria inquinas Chloroscypha chloromela Chlorovibressea sp. Collembolispora aristata Cudoniella clavus Dermea acerina Fabrella tsugae Gelatinodiscus flavidus Hyaloscypha vitreola Lachnum virgineum Lambertella subsubrenispora Leotia lubrica Loramyces macrosporus Microglossum rufum Mollisia cinerea Mollisia dextrinospora Mollisia fusca Mollisia melaleuca Monilinia laxa Neobulgaria lilacina Neobulgaria pura Neofabrea malicorticis Phialocephala dimorphospora Phialocephala europea Phialocephala fortinii Phialocephala helevetica Phialocephala letzii Phialocephala scopiformis Phialocephala subalpina Phialocephala turiciensis Spathularia velutipes Varicosporium elodeae

TABLE I.

AF486121 AY078137 AB671499 AY078136 AY078130 AF486126 EF093161 EF093162

AY259137 AY259136

DQ491498

DQ471005

DQ491485

EU652349

AF141164

U92311

AY078145 AY078151 HM189719 KF874619 KF874617 KF874618 KF874620 HM152544

ITS

FJ99786 JN941371

AB671466

AY544670 EU940141 FJ176865 AY544662 AB671465

FJ477058 AY544646 DQ470978 AY544644 DQ470957 DQ470981 DQ470942 HM116757

DQ257352 KC005811 DQ470944 DQ247801 AF356694

KF943828

KF943825 KF943823 KF943824 KF943826

KF874622 KF874623 KF874624 KF874621 AY544651 DQ470960

KF943827

ACT

KF951051b

LSU

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Phialocephala sp.c Uncultured Leotiomycetes clonec

a AFTOL 5 Assembling the Fungal Tree of Life project; ATCC 5 American Type Culture Collection, Manassas, Virginia, USA; CBS 5 Centraalbureau voor Schimmelcultures, Utrecht, the Netherlands; ICMP 5 International Collection of Microorganisms from Plants, Lincoln, New Zealand. b Numbers in boldface indicating new sequences from this study. c Taxon name was copied from GenBank. Phylogenetic analysis in this study indicated that they belong to Acidomelania panicicola.

HM230888 Ecuador Cavendishia bracteata

Wales

JN995647

FJ176874 EU434854 JX481974 JN655572 EU880589 Ontario, Canada Luxembourg Sumava Mountains, Czech Republic Mufu Forest Park, China CBS258.91 CBS 132843

Populus, submerged root xeric bark of Salix sp. Pseudorchis albida, roots Rhododendron fortunei, root hairs

ITS Species

Vibressea truncorum Xerombrophila crystallifera Helotiales sp.c Mycorrhizal fungal sp.c

TABLE I.

Continued

Isolate No.a

Host

Location

LSU

ACT

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Colonies on MEA 33 mm diam after 20 d in the dark at 20 C. Vandyke brown, surface velvety, aerial hyphae sparse and light brown, reverse pigmented, oil green. Colonies on WA reaching 35 mm diam after 20 d in the dark at 20 C. Buckthorn brown, aerial hyphae sparse, reverse pigmented, tawny brown. Conidia formed on WA after 20 d at 20–25 C in dark or light. Conidiophores simple or occasionally branched. Conidiogenous cells hyaline, straight, 6– 15 3 1.6–3.5 mm (n 5 50). Collarettes cylindrical, 2.8– 6.2 3 1.6–3.5 mm, 1.6–3.1 mm wide at base (n 5 50). Conidia aggregated in slimy heads, globose to subellipsoidal, aseptate, hyaline, smooth, 1.1–2.7 3 1.1–2.6 mm (n 5 50). Chlamydospores present on WA, catenulate, intercalary, dark brown, subglobose, 4.1– 14 3 4–13.9 mm (n 5 20). Specimens examined: UNITED STATES. NEW JERSEY: pine barrens, N39 26.4738, W74 34.934, 3 m. Roots of Panicum virgatum, 1 Jun 2011, E. Walsh & N. Zhang 61R8 (HOLOTYPE, RUTPP-61R8; ex-holotype culture CBS 137156). UNITED STATES. NEW JERSEY: pine barrens, N39 26.4738, W74 34.934, 3 m. Roots of Panicum virgatum, 1 Jun 2011, E. Walsh & N. Zhang 61R41, 61R50B (RUTPP61R41 and RUTPP-61R50B). UNITED STATES. NEW JERSEY: pine barrens, Colliers Mills, N40 04.084 W74 26.696, 5 m. Roots of Schizachyrium scoparium, 30 Aug 2012, E. Walsh & N. Zhang CM16s1 (RUTPP-CM16s1).

Notes: Acidomelania differs from Loramyces in its phialidic conidia and association with living plant roots, while Loramyces species are associated with submerged dead plants and apparently do not produce conidia (Ingold and Chapman 1952, Weston 1929, Digby and Goos 1987). Cadophora, the anamorph of some Mollisia species, and Phialocephala produce phialidic conidia, but the arrangement of the phialides is more complex than Acidomelania (Day et al. 2012). In addition, some Phialocephala cultures took a year to sporulate at 4 C, while Acidomelania sporulated at room temperature within 3 wk. Acidomelania panicicola also exhibited slower growth than P. fortinii in dark at 20 C on both WA and MEA (Gru¨nig and Sieber 2005). Moreover, A. panicicola has 92% or less ITS sequence identities to the above-mentioned close relatives or any other described species with accessible ITS sequences. DISCUSSION Phylogenetic relationships among taxa in Leotiomycetes are poorly understood due to inadequate taxon sampling and lack of multilocus sequence data (Wang et al. 2006). Based on the ITS and LSU trees, Acidomelania described in this study belongs to Helotiales, a heterogeneous and the largest order in Leotiomycetes that includes endophytes, plant pathogens and saprobes. Acidomelania is phylogenetically

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FIG. 1. Morphological characters of Acidomelania panicicola holotype isolate 61R8. A–D. Conidia and conidiogenous cells. E. Chlamydospores. Bar 5 10 mm.

close to Mollisia, Loramyces and PAC but morphologically and ecologically distinguishable to these allies (see TAXONOMY). The phylogenetic affinity of Mollisia, Loramyces and PAC also was supported by Zijlstra et al. (2005) and Wang et al. (2006). The family placement of Acidomelania is not determined. Currently 13 families are placed in Helotiales (Lumbsch and Huhndorf 2010). Phylogenetic analyses in this study indicated that Dermateaceae, Vibrisseaceae and Helotiaceae are likely polyphyletic, which corroborates Wang et al. (2006). The dark, septate hyphal morphology of Acidomelania panicicola and its root-colonizing habit and phylogenetic closeness to PAC indicate that it is likely a DSE. PAC are the most frequently isolated DSE associated with alpine forests (Gru¨nig et al. 2008b). Other reported DSE belong in Pleosporales, Hypocreales and Pezizales of Ascomycota ( Jumpponen and Trappe 1998, Mandyam et al. 2010). The ecological functions of PAC and other DSE in nature still are enigmatic. Host-fungus interaction experiments often yielded different results under different experimental conditions (Mandyam and Jumpponen 2005). Further investigation is needed to address such questions as why A. panicicola is associated with plant roots in acidic and nutrient-poor environments and whether it promotes the growth of the host plants.

Based on our ongoing survey (unpubl), Acidomelania panicicola is one of the dominant fungi associated with grass roots in New Jersey pine barrens. Moreover, BLAST results in GenBank and phylogenetic analysis indicated that A. panicicola may have a global distribution. Seventeen ITS sequences in GenBank had 97–99% identities with that of A. panicicola isolate 61R8 and a few examples are listed here: EU880589, mycorrhizal fungal sp. shylmf10, Rhododendron fortunei root, China; JN655572, Helotiales sp. 1 MV-2011 strain PA 072, Pseudorchis albida root, Czech Republic; and HM230888, uncultured Leotiomycetes clone K627, Cavendishia bracteata root, Ecuador. The host plants of the matched sequences are either Ericaceae or terrestrial orchids, which usually are found in acidic and infertile growing conditions (Keddy 2007). Genetic diversity was observed among the Acidomelania isolates. The ITS gene tree placed the four New Jersey isolates into two well-supported clades, which had a 97% ITS sequence identity. However, the ACT gene tree showed incongruity among the four isolates. Therefore, we recognize the four New Jersey pine barren isolates as a single species based on genealogical concordance phylogenetic species recognition, which measures reproductive isolation (Taylor et al. 2000).

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FIG. 2. Maximum likelihood phylogenetic tree inferred from the large subunit of rRNA gene sequences. Bootstrap values higher than 75% are labeled on branches.

Traditional fungal taxonomy uses morphology of reproductive structures to define taxa. However, some species, including many root-associated fungi, do not sporulate or lack distinguishable morphology when sporulation occurs (Gru¨nig et al. 2008a). Therefore, taxonomy of these fungi has fallen behind even though their molecular data may be available. Recent advances in next-generation metagenomic sequencing enabled production of large amount of DNA sequence data from environmental samples. Generally BLAST queries in GenBank or other databases are performed to identify the environmental sequences, which often yield unnamed species or erroneous taxonomic names due to errors and incomplete taxonomic sampling in the

sequence databases (Bidartondo et al. 2008). Lack of taxonomic information hampers effective scientific communication. Hibbett and Taylor (2013) encouraged taxonomists to name environmental sequences. The fungal barcoding initiative (Schoch et al. 2012) and the development of other well-documented sequence databases will provide a solution to more accurate identification of the environmental sequences. In this study we named an apparently common root-colonizing fungus associated with plants living in acidic, oligotrophic environments. Both culture-based fungal isolates and environmental sequences are included in the new taxon. This taxonomic work will aid future ecological and evolutionary studies.

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FIG. 3. Maximum likelihood phylogenetic tree inferred from the internal transcribed spacer sequences of the rRNA genes. Bootstrap values higher than 75% are labeled on the branches.

FIG. 4. Maximum likelihood phylogenetic tree inferred from the ACT gene sequences. Bootstrap values higher than 75% are labeled on the corresponding branches.

WALSH ET AL.: ACIDOMELANIA, ACKNOWLEDGMENTS The research was financially supported by the National Science Foundation (grant number DEB 1145174), Rutgers Center for Turfgrass Science and US Golf Association to Zhang.

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