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Two new species of Phaeohelotium (Leotiomycetes: Helotiaceae) from Chile and their putative ectomycorrhizal status.

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Affiliations

  • 1. Department of Ecology and Evolutionary Biology, University of Colorado Boulder, Boulder, CO 80309, USA.
      Authors
    • Grupe Ii AC 1
    • Weier A 1
    • Quandt CA 1
    (3 authors)
  • 2. Department of Plant Pathology, University of Florida, Gainesville, FL 32611, USA.
      Authors
    • Smith ME 2
    • Healy R 2
    (2 authors)
  • 3. Department of Microbiology and Plant Pathology, University of California Riverside, Riverside, CA 92521, USA.
      Authors
    • Caiafa MV 3
    (1 author)
  • 4. Department of Organismic and Evolutionary Biology & Farlow Reference Library and Herbarium of Cryptogamic Botany, Harvard University, Cambridge MA 20138, USA.
      Authors
    • Pfister DH 4
    (1 author)
  • 5. Faculty of Science, University of South Bohemia, 37005 České Budějovice, Czech Republic.
      Authors
    • Haelewaters D 5
    (1 author)

ORCIDs linked to this article

Fungal Systematics and Evolution, 15 Dec 2022, 10:231-249
https://doi.org/10.3114/fuse.2022.10.10 PMID: 36741556 PMCID: PMC9875694

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Abstract 

Species of the genus Phaeohelotium (Leotiomycetes: Helotiaceae) are cup fungi that grow on decaying wood, leaves, litter, and directly on soil. Northern Hemisphere species are primarily found on litter and wood, whereas in the Southern Hemisphere the genus includes a mix of saprotrophs as well as taxa that grow on soil in association with ectomycorrhizal trees. The diversity of this genus has not been fully explored in southern South America. Here we describe two species from Chile, Phaeohelotium maiusaurantium sp. nov. and Ph. pallidum sp. nov., found on soil in Patagonian Nothofagaceae-dominated forests. We present macro- and micromorphological descriptions, illustrations, and molecular phylogenetic analyses. The two new species are placed in Phaeohelotium with high support in our 15-locus phylogeny as well as phylogenetic reconstructions based on the internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene. Our ITS phylogeny places both Ph. maiusaurantium and Ph. pallidum in a well-supported subclade that includes ectomycorrhizal root tip samples from Australasia. Similar species can be separated from these new taxa based on morphological characteristics, biogeography, substrate, and sequence data. In addition, two unnamed species from Chilean Nothofagaceae forests (Phaeohelotium sp. 1 and Phaeohelotium sp. 2) are documented from scant collections and sequence data and await description until more material becomes available. Citation: Grupe II AC, Smith ME, Weier A, Healy R, Caiafa MV, Pfister DH, Haelewaters D, Quandt CA (2022). Two new species of Phaeohelotium (Leotiomycetes: Helotiaceae) from Chile and their putative ectomycorrhizal status. Fungal Systematics and Evolution 10: 231-249. doi: 10.3114/fuse.2022.10.10.

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Fungal Syst Evol. 2022 Dec; 10: 231–249.
Published online 2022 Dec 15. https://doi.org/10.3114/fuse.2022.10.10
PMCID: PMC9875694
PMID: 36741556

Two new species of Phaeohelotium (Leotiomycetes: Helotiaceae) from Chile and their putative ectomycorrhizal status

A.C. Grupe II, 1 M.E. Smith, 2 A. Weier, 1 R. Healy, 2 M.V. Caiafa, 3 D.H. Pfister, 4 D. Haelewaters, 5 , 6 , * and C.A. Quandt 1 , *
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Abstract

Species of the genus Phaeohelotium (Leotiomycetes: Helotiaceae) are cup fungi that grow on decaying wood, leaves, litter, and directly on soil. Northern Hemisphere species are primarily found on litter and wood, whereas in the Southern Hemisphere the genus includes a mix of saprotrophs as well as taxa that grow on soil in association with ectomycorrhizal trees. The diversity of this genus has not been fully explored in southern South America. Here we describe two species from Chile, Phaeohelotium maiusaurantium sp. nov. and Ph. pallidum sp. nov., found on soil in Patagonian Nothofagaceae-dominated forests. We present macro- and micromorphological descriptions, illustrations, and molecular phylogenetic analyses. The two new species are placed in Phaeohelotium with high support in our 15-locus phylogeny as well as phylogenetic reconstructions based on the internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene. Our ITS phylogeny places both Ph. maiusaurantium and Ph. pallidum in a well-supported subclade that includes ectomycorrhizal root tip samples from Australasia. Similar species can be separated from these new taxa based on morphological characteristics, biogeography, substrate, and sequence data. In addition, two unnamed species from Chilean Nothofagaceae forests (Phaeohelotium sp. 1 and Phaeohelotium sp. 2) are documented from scant collections and sequence data and await description until more material becomes available.

Citation: Grupe II AC, Smith ME, Weier A, Healy R, Caiafa MV, Pfister DH, Haelewaters D, Quandt CA (2022). Two new species of Phaeohelotium (Leotiomycetes: Helotiaceae) from Chile and their putative ectomycorrhizal status. Fungal Systematics and Evolution 10: 231–249. 10.3114/fuse.2022.10.10

Keywords: Ascomycota, four new taxa, multilocus phylogeny, Nothofagaceae, Patagonia, taxonomy

INTRODUCTION

Leotiomycetes (Ascomycota: Pezizomycotina) is a diverse group of fungi that are taxonomically and systematically challenging. This class contains approximately 12 orders and roughly 60 families of which multiple lineages remain unassigned (Quandt & Haelewaters 2021). The genus Phaeohelotium (Helotiales: Helotiaceae) was erected to accommodate species that did not fit well with the generic concept of Helotium based on the anatomy of the ectal excipulum and color of ascospores (Kanouse 1935, Dennis 1981, Gamundí & Messuti 2006). Helotium was replaced by Hymenoscyphus on nomenclatural grounds (Dennis 1964). Phaeohelotium currently comprises taxa that have been transferred from as many as 16 different genera, including Discinella, Helotium, Peziza, and others (Kanouse 1935, Dennis 1971, Svrček & Matheis 1979, Baral et al. 2013, Wijayawardene et al. 2022). The type species is Ph. flavum (= Ph. monticola), which was first found on a decaying log in Harbor Springs, Michigan, USA (Kanouse 1935, Dennis 1964, Dumont 1981, Baral et al. 2013).

The trophic mode for species in this genus has yet to be determined, but ascomata of many species are found on rotten wood, twigs, and leaves suggesting a saprotrophic ecology. However, close matches to ectomycorrhizal (ECM) root tip sequences along with observations of ascoma-production on bare soil suggest that some species may be associates of Eucalyptus (Myrtaceae) and Nothofagus (Nothofagaceae) (Baral et al. 2013, Tedersoo & Smith 2013).

Five species of Phaeohelotium have thus far been reported in South America. These are Ph. castaneum, Ph. flavum, Ph. luteum, Ph. nothofagi, and Ph. recurvum from Argentina, Brazil, and Chile (Gamundí & Messuti 2006). All of these have been recorded from plant substrates. New collections of Phaeohelotium species were made from 2012 to 2019 as part of a larger project to survey fungal biodiversity in Patagonian Nothofagaceae-dominated forests, with a special focus on ECM fungi (Truong et al. 2017a). Sequence data analysis identified four putatively ECM Phaeohelotium species that were collected directly on soil. The objective of this paper is to describe two of these species, Phaeohelotium maiusaurantium and Ph. pallidum spp. nov., to compare them to all previously documented South American Phaeohelotium species, and to provide ITS-based and multi-locus molecular phylogenetic analyses to determine their placement within Helotiaceae.

MATERIALS AND METHODS

Morphological study

Fresh specimens were collected during expeditions to Nothofagaceae forests across Chile between 2012 and 2019 (Truong et al. 2017a, Nouhra et al. 2021). We searched for cup fungi exposed on the soil or fruiting below the leaf litter by gently removing the litter with rakes or by hand (Kraisitudomsook et al. 2020). Specimens were photographed in situ and in the field laboratory. Macroscopic features were described from fresh specimens using terminology for ascomata outlined in Weber et al. (1997). Collections of fresh ascomata were air-dried or dried in a forced air dryer for 48 h at 40 °C. Micromorphological features of dried specimens were examined with an Olympus BX43 (Olympus, Center Valley, Pennsylvania) compound microscope. Separate mounts of fungal tissue were made in deionized (DI) water, tap water, 3 % KOH, and Melzer’s solution. At least 20 individual ascospores and other structures were measured at 400–1 000× magnification for each collection after having been rehydrated in 3 % KOH for 20 min. Outlying measurements observed in less than 5 % of the measured population are indicated in parentheses. Histological preparation was done as follows: dried samples were initially rehydrated in 3 % KOH for 20 minutes before being placed in a Formal-Fixx Concentrate (Epredia, Kalamazoo, Michigan) at a dilution of 2 mL Formal-Fixx concentrate to 8 mL dH2O for 24–48 h before proceeding with the fixative steps. Samples were formalin fixed and paraffin embedded using a Sakura Tissue-Tek VIP5 automated tissue processor (Sakura Finetek, Torance, California) and a Tissue-Tek TEC II embedding center (Sakura Finetek, Torance, California) as follows: samples were placed in a formalin solution for 5 min, a 70 % alcohol solution for 15 min, a 95 % alcohol solution for 40 min, a 100 % alcohol for 40 min, a xylene solution for 30 min, and a paraffin solution for 50 min. All steps were done while agitating, a pressure vacuum engaged, and at 40 °C, except for the paraffin stage which was at 58 °C. A Leica ST5010 Autostainer XL (Leica Biosystems, Wetzlar, Germany) was used for hematoxylin & eosin staining of samples before sectioning on a Jung BioCut 2030 Rotary Microtome (Leica Biosystems, Wetzlar, Germany) at a thickness of 5 μm at the University of Colorado Cancer Center Research Histology Services. Fungal collections are deposited in the following herbaria: FH (Farlow Herbarium at Harvard University), FLAS (Florida Museum of Natural History Fungarium), NY (New York Botanical Garden), and SGO (Museo Nacional de Historia Natural de Chile) (Thiers 2022).

Molecular protocols

DNA was extracted from ascomata using the Extract-N-Amp Plant kit (Sigma-Aldrich, St. Louis, Missouri) or using a modified CTAB extraction protocol utilizing an initial phenol/chloroform step (Gardes & Bruns 1993). We amplified the following loci: nuclear small and large nuclear ribosomal subunits (SSU and LSU), internal transcribed spacer region of the ribosomal DNA (ITS), the mitochondrial locus MS456 containing the gene MCM7, and the RNA polymerase II largest and second-largest subunits (rpb1 and rpb2). Primer combinations were as follows: NS1/NS4 for SSU (White et al. 1990), ITS1F/ITS4 for ITS (White et al. 1990, Gardes & Bruns 1993), LR0R/LR5 for LSU (Vilgalys & Hester 1990, Hopple 1994), Mcm7-709for/Mcm7-1348rev for MCM7 (Schmitt et al. 2009), RPB1-Af/RPB1-Cr for rpb1 (Stiller & Hall 1997, Matheny et al. 2002), and RPB2-f5F/RPB2-7cR for rpb2 (Liu et al. 1999). All 25-μL PCR reactions were conducted on a C1000 Touch Thermal Cycler (Bio Rad, Hercules, California) and consisted of 2.5 μL of 10× Takara Ex Taq buffer (Takara Bio, Shiga, Japan), 1.25 μL of each 10 μM primer, 0.2 μL of Takara Ex Taq, up to 18.8 μL of sterile distilled water, and 1–2 μL of DNA extract. PCR protocols followed those outlined in the original publications. PCR products were visualized on 1.5 % agarose gels stained with ethidium bromide. Amplicons were sequenced by GENEWIZ (South Plainfield, New Jersey). Raw sequence reads were assembled and then edited using FinchTV 1.4 (Geospiza Inc., Seattle, Washington). Newly generated sequences (Table 1) were used as queries for BLAST searches against NCBI GenBank’s standard nr/nt nucleotide database (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to identify closest relatives.

Table 1.

Fungarium and GenBank accession numbers for studied collections.

Species Collector’s label Herbarium accession SSU ITS LSU MCM7 rpb1 rpb2
Phaeohelotium maiusaurantium DHP-CH-74FH 00284861– OP868959 ––––
DHP-CH-80FH 00284867– OP868958 ––––
DHP-CH-93FH 00284876– OP868960 ––––
MES-2905FLAS-F-65460 OP951346 OP868961 OP962215 –––
MES-2976TFLAS-F-65526 OP951346 OP868953 OP962216 –––
MES-2925FLAS-F-65476– OP868951 – OP957112 OP957114 OP957116
Phaeohelotium pallidum MES-1068TFLAS-F-63072–KY462415 OP962212 –––
MES-2850FLAS-F-65411– OP868956 OP962213 –––
MES-2852FLAS-F-65413– OP868955 OP962211 –––
MES-3308Specimen destroyed– OP868954 ––––
MES-3573FLAS-F-66003– OP868950 ––––
MES-3574FLAS-F-66004– OP868952 – OP957113 OP957115 OP957117
Phaeohelotium sp. 1MES-2358FLAS-F-65016–MH930302––––
Phaeohelotium sp. 2MES-2650FLAS-F-65244–MH930289––––
MES-2851FLAS-F-65412– OP868957 OP962214 –––

TStands for holotype specimen. Accession numbers of sequences generated during this study are in boldface.

Phylogenetic analyses

First, we used T-BAS v. 2.1 (Carbone et al. 2019) and the “Place Unknowns” tool to place newly generated Phaeohelotium sequences onto the 15-locus Leotiomycetes-wide tree from Haelewaters et al. (2021), named “Leotiomycetes v1.” Seven FASTA files with unaligned SSU, LSU, ITS, MCM7, rpb1, and rpb2 sequences of Phaeohelotium were uploaded to the T-BAS interface. We selected the de novo option for the RAxML phylogenetic analysis, with GTRGAMMA as substitution model, 500 bootstrap replicates, and Neurospora crassa and Xylaria hypoxylon (Sordariomycetes) as outgroup taxa.

Next, we constructed two ITS datasets: one to place the genus Phaeohelotium among related genera and the second to place the newly generated Phaeohelotium sequences among other species in the genus. The first ITS dataset included 106 taxa belonging to Helotiaceae and Tricladiaceae (outgroup) (Table 2). We also included Bulgariella pulla (currently classified as Leotiomycetes incertae sedis, Iturriaga et al. 2017), Connersia rilstonii and Pleuroascus nicholsonii (Pleuroascaceae), and Roesleria subterranea (Roesleriaceae, Baral 2016) because these taxa were found to be most closely related to Helotiaceae and Tricladiaceae by Haelewaters et al. (2021). Sequences were aligned using MUSCLE v. 3.7 (Edgar 2004), available on the Cipres Science Gateway v. 3.3 (Miller et al. 2010). Sequences were trimmed at the conserved motifs 5’–CATTA–3’ (3’ end of the SSU) and 5’–GACCT(CAAA…)–3’ (5’ end of the LSU) (Dentinger et al. 2011). Because the two spacers (ITS1, ITS2) and 5.8S have different rates of evolution, we treated them as individual partitions in the phylogenetic analysis. Maximum likelihood (ML) inference was performed using IQ-TREE v. 1.6.7 from the command line (Nguyen et al. 2015) under partitioned models (Chernomor et al. 2016). Nucleotide substitution models were selected under the Akaike information criterion (AIC) with the help of the built-in program ModelFinder (Kalyaanamoorthy et al. 2017). Ultrafast bootstrap analysis was implemented with 1 000 replicates (Hoang et al. 2017).

Table 2.

Species included in the ITS-based phylogenetic analyses to place the genus Phaeohelotium among related genera (placement) and to place the newly described species among other species of Phaeohelotium (genus). For each species, the following information is shown: GenBank accession number, DNA isolate/voucher information, type status (type species, ex-type specimen), dataset (placement or genus), and source reference. Accession numbers of sequences generated during this study are in boldface.

Species Accession number Isolate/voucher Country Note Dataset Reference
Brunaudia phormigena KF727423PDD:75309New Zealandtype speciesplacementP.R. Johnston & D. Park unpubl. data
Bulgariella pulla KU845537TL 2011Norwaytype speciesplacement Iturriaga et al. (2017)
MH578505PDD:111518 / ICMP:22812New Zealandtype speciesplacementP.R. Johnston & D. Park unpubl. data
KJ704848DHP-06-607USAtype speciesplacement Iturriaga et al. (2017)
KU845540DHP 15-215Chiletype speciesplacement Iturriaga et al. (2017)
Connersia rilstonii KJ755499CBS:537.74Canadaex-typeplacement Malloch et al. (2016)
Cudoniella acicularis DQ202512CBS:100273DenmarkplacementN. Boonyuen et al. unpubl. data
Cudoniella indica DQ202513CBS:430.94Indiaex-typeplacementN. Boonyuen et al. unpubl. data
DQ202505SS 708N/AplacementN. Boonyuen et al. unpubl. data
Dicephalospora albolutea MK425601HMAS 279693Chinaex-typegenus, placement Zheng & Zhuang (2019)
Dicephalospora rufocornea AB926055TNS:F-40024Japantype speciesgenus, placement Zhao et al. (2016)
KU668565Zhuang 10106Chinatype speciesgenus, placement Zhuang et al. (2016)
Dicephalospora sessilis MK584947MFLU:18-1823Chinaex-typegenus, placement Ekanayaka et al. (2019)
Endoscypha perforans KF727424PDD:102231New Zealandtype speciesplacementP.R. Johnston & D. Park unpubl. data
Graddonia coracina JQ256423ILLS:60491USAtype speciesplacement Hustad et al. (2011)
Helicodendron tubulosum EF029195ICMP:14615New ZealandplacementJ.A. Cooper unpubl. data
Helicodendron westerdijkae EF029196ICMP:14616New ZealandplacementJ.A. Cooper unpubl. data
Helotiaceae sp.MH682236ICMP:22540New ZealandplacementP.R. Johnston & D. Park unpubl. data
Hymenoscyphus albidoides KF188722HMAS 264140Chinaex-typeplacement Zheng & Zhuang (2014)
Hymenoscyphus aurantiacus NR_154907HMAS 264143Chinaex-typeplacement Zheng & Zhuang (2015)
Hymenoscyphus caudatus AY348578HMAS 82063Chinaplacement Zhang & Zhuang (2004)
KF188730HMAS 264150Chinaplacement Zheng & Zhuang (2014)
AB926065TNS:F-40056Japanplacement Zhao et al. (2016)
Hymenoscyphus equiseti UDB038358TAAM:194261Russiaex-typeplacement Lebeuf et al. (2021)
OL679972D. Haelew. F-1493cSwedenplacement Lebeuf et al. (2021)
Hymenoscyphus fraxineus PRJNA297734 (NCBI genome)CBS:133217LuxembourgplacementC. Sambles et al. unpubl. data
Hymenoscyphus fraxineus [as H. pseudoalbidus]GU586904ZT:Myc 2022Switzerlandex-typeplacement Queloz et al. (2011)
GU586933CBS:650.92Germanytype speciesplacement Queloz et al. (2011)
AB926057TNS:F-44644Japantype speciesplacement Zhao et al. (2016)
Hymenoscyphus ginkgonis NR_119669KUS F51352South Koreaex-typeplacement Han & Shin (2008)
Hymenoscyphus haasticus NR_137108PDD:57577 / ICMP:19598New Zealandex-typeplacement Johnston & Park (2013)
“Hymenoscyphus” imberbisKC411993H.B. 6797Germanyplacement Baral et al. (2013)
“Hymenoscyphus” cf. imberbisOL679974D. Haelew. F-262Germanyplacement Lebeuf et al. (2021)
Hymenoscyphus infarciens PRJNA297733 (NCBI genome)CBS:122016FranceplacementC. Sambles et al. unpubl. data
Hymenoscyphus kiko NR_137110PDD:82964 / ICMP:19613New Zealandex-typeplacement Johnston & Park (2013)
Hymenoscyphus koreanus KP068057KUS F52847_01South Koreaex-typeplacement Gross & Han (2015)
Hymenoscyphus macrodiscus KJ472296HMAS:264158Chinaex-typeplacement Zheng & Zhuang (2015)
Hymenoscyphus macroguttatus DQ431179H.B. 7034Spainplacement Baral et al. (2007)
Hymenoscyphus as “menthae”AB926063TNS:F-40052Japanplacement Zhao et al. (2016)
Hymenoscyphus menthae KM114537H.B. 5846Liechtensteinplacement Gross et al. (2015)
Hymenoscyphus occultus KP068058CBS:139469South Koreaex-typeplacement Gross & Han (2015)
Hymenoscyphus ohakune NR_137109ICMP:19601New Zealandex-typeplacement Johnston & Park (2013)
Hymenoscyphus pusillus MH476516HMC 21525Polandex-typeplacement Kowalski & Bilañski (2019)
Hymenoscyphus qinghaiensis KJ472297HMAS:264175Chinaex-typeplacement Zheng & Zhuang (2015)
Hymenoscyphus repandus PRJNA297738 (NCBI genome)CBS:341.76GermanyplacementC. Sambles et al. unpubl. data
Hymenoscyphus salicellus PRJNA297737 (NCBI genome)CBS:111550The NetherlandsplacementC. Sambles et al. unpubl. data
Hymenoscyphus scutula KC481695CBS:480.97USAplacement Hamelin et al. (2013)
AB926114TNS:F-17507Japanplacement Zhao et al. (2016)
Hymenoscyphus tamaricis DQ431167br020Spainplacement Baral et al. (2007)
Hymenoscyphus tetrasporus KJ472302HMAS:266592Chinaex-typeplacement Zheng & Zhuang (2015)
“Hymenoscyphus” varicosporoides(JGI genome)Hymvar1USAplacement Grigoriev et al. (2014)
AB481291FC-2038Japanplacement Hosoya et al. (2010)
Hymenoscyphus waikaia KC164666PDD:66379New Zealandplacement Johnston & Park (2013)
KC164667PDD:102886New Zealandex-typeplacement Johnston & Park (2013)
Hymenoscyphus yui KJ472303HMAS:266595Chinaex-typeplacement Zheng & Zhuang (2015)
Hymenotorrendiella dingleyae MH578484ICMP:22793New ZealandplacementP.R. Johnston & D. Park unpubl. data
Hymenotorrendiella eucalypti MH578483ICMP:22792New Zealandtype speciesplacementP.R. Johnston & D. Park unpubl. data
Mycofalcella calcarata KC834065CCM F-10289UKex-typeplacement Baschien et al. (2013)
Phaeohelotium confusum MH921873PDD:112244New Zealandgenus, placementP.R. Johnston & D. Park unpubl. data
MH578507PDD:111536New Zealandgenus, placementP.R. Johnston & D. Park unpubl. data
MH578519PDD:111537New Zealandgenus, placementP.R. Johnston & D. Park unpubl. data
Phaeohelotium confusum [as Discinella confusa]OL653012PDD:112680New Zealandgenus, placementJ.A. Cooper & P.R. Johnston unpubl. data
Phaeohelotium confusum [as Discinella terrestris]GU222294PDD:89062New Zealandgenus, placementP.R. Johnston & D. Park unpubl. data
“Phaeohelotium” epiphyllumAB926061TNS:F-40042Japanplacement Zhao et al. (2016)
KT876976H.B. 9911GermanyplacementH.-O. Baral & G. Marson unpubl. data
Phaeohelotium geogenum MF908475UBC:F33074CanadagenusM.L. Berbee et al. unpubl. data
KC411992H.B. 7222AGermanygenus, placement Baral et al. (2013)
Phaeohelotium flavum [as monticola]KC411991H.B. 8612Germanytype speciesgenus, placement Baral et al. (2013)
Phaeohelotium maiusaurantium OP868960 FH 00284876Chilegenus, placementThis paper
KY462393FH 00284882Chilegenus Truong et al. (2017a)
OP868951 FLAS-F-65476Chilegenus, placementThis paper
OP868961 FLAS-F-65460Chilegenus, placementThis paper
OP868953 FLAS-F-65526Chileex-typegenus, placementThis paper
OP868958 FH 00284867Chilegenus, placementThis paper
OP868959 FH 00284861ChileplacementThis paper
Phaeohelotium pallidum OP868956 FLAS-F-65411Chilegenus, placementThis paper
KY462415FLAS-F-63072Chileex-typegenus, placementThis paper
OP868955 FLAS-F-65413Chilegenus, placementThis paper
OP868954 MES-3308Chilegenus, placementThis paper
OP868950 FLAS-F-66003Chilegenus, placementThis paper
OP868952 FLAS-F-66004Chilegenus, placementThis paper
Phaeohelotium pateriforme complexMK932825PDD:112176 / ICMP:23087New Zealandgenus, placementP.R. Johnston & D. Park unpubl. data
MK932826PDD:94748 / ICMP:23370New Zealandgenus, placementP.R. Johnston & D. Park unpubl. data
MK932832PDD:116634 / ICMP:23365New ZealandplacementP.R. Johnston & D. Park unpubl. data
Phaeohelotium sp.MK932818ICMP:23361New ZealandplacementP.R. Johnston & D. Park unpubl. data
Phaeohelotium sp. 1 OP868962 FLAS-F-65016Chilegenus, placementThis paper
Phaeohelotium sp. 2MH930289FLAS-F-65244Chilegenus, placementThis paper
OP868957 FLAS-F-65412Chilegenus, placementThis paper
Phaeohelotium succineoguttulatum KC411989AH7643Spainplacement Baral et al. (2013)
Phaeohelotium tasmanicum complexMK932819PDD:111541 / ICMP:23082New Zealandgenus, placementP.R. Johnston & D. Park unpubl. data
MK932821PDD:116635 / ICMP:23372New Zealandgenus, placementP.R. Johnston & D. Park unpubl. data
MK932823PDD:111926 / ICMP:23083New Zealandgenus, placementP.R. Johnston & D. Park unpubl. data
Phaeohelotium undulatum KC411988AH7337Spainplacement Baral et al. (2013)
Phaeohelotium cf. undulatumMK432759PDD:105499New Zealandgenus, placementJ.A. Cooper et al. unpubl. data
Pleuroascus nicholsonii KJ755519CBS:345.73USAex-type, type speciesplacement Malloch et al. (2016)
Roesleria subterranea EF060298CBS:407.51Italytype speciesplacement Kirchmair et al. (2008)
AB628057TNS:F-38701Japantype speciesplacementY. Degawa et al. unpubl. data
Spirosphaera floriformis HQ696658CBS:402.52The Netherlandsex-typeplacement Voglmayr et al. (2011)
Tricladium obesum KC834068CCM F-14598Czech Republicex-typeplacement Baschien et al. (2013)
Tricladium splendens AY204635CCM F-16599Czech Republictype speciesplacement Baschien et al. (2006)
Uncultured Ascomycota ECM root tip ex Eucalyptus delegatensisJF960619clone BH2125RAustraliagenus Horton (2011)
Uncultured fungus ECM root tip ex Eucalyptus delegatensisJF960772clone BH1255RAustraliagenus Horton (2011)
JF960769clone BH3140FAustraliagenus Horton (2011)
Uncultured fungus ECM root tip ex Eucalyptus regnansFN298678H004x_L3399XafAustraliagenus, placement Tedersoo et al. (2009)
Uncultured fungus ECM root tip ex Nothofagus cunninghamiiFN298750H71_L3609sAustraliagenus, placement Tedersoo et al. (2009)
FN298679H004y_L3677Australiagenus, placement Tedersoo et al. (2009)
FN298677H004z_L3620Australiagenus Tedersoo et al. (2009)
Uncultured fungus ECM root tip ex Pomaderris apetalaFN298689H010x_L3161bAustraliagenus, placement Tedersoo et al. (2009)
Uncultured Helotiales ECM root tip ex Eucalyptus delegatensisJF960780clone BH3615RAustraliagenus Horton (2011)
Zalerion varium AF169303ATCC:28788USAtype speciesplacementBill et al. (1999)

Our second ITS dataset included 43 taxa, all within the genus Phaeohelotium, and with Dicephalospora spp. as outgroup (Table 2). In this analysis, we included uncultured ectomycorrhizal root tip sequences as well as the type species Ph. flavum (as Ph. monticola), for which a single ITS sequence is available in the National Center for Biotechnology Information (NCBI) GenBank database (acc. no. KC411991, Baral et al. 2013). As above, sequences were aligned using MUSCLE v. 3.7 (Edgar 2004) and trimmed at the conserved motifs sensu Dentinger et al. (2011). The selection of nucleotide substitution models with ModelFinder v. 1.6.7 (Kalyaanamoorthy et al. 2017) was done under the Akaike information criterion corrected for small sample size (AICc). Maximum likelihood (ML) was performed under partitioned models using IQ-TREE (Nguyen et al. 2015, Chernomor et al. 2016, Hoang et al. 2017). Visualization of phylogenetic reconstructions with bootstrap (BS) values was done in FigTree v. 1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/). A link to the final alignments and unedited trees is available here: https://github.com/Quandt-Mycology-Lab/Lab_Codes_and_workflows/tree/master/Phaeohelotium_paper.

RESULTS

Nucleotide alignment datasets and phylogenetic analyses

Placement onto the Leotiomycetes v1 tree in T-BAS reveals the position of three of our samples in Phaeohelotium in family Helotiaceae, in a well-supported clade with Phaeohelotium geogenum (Fig. 1). Phaeohelotium is retrieved (ML bootstrap = 81 %) among Pleuroascaceae, Bulgariella pulla, Roesleriaceae, and Helotiaceae. The relationship of these five clades to one another is unresolved. Our first ITS dataset (Fig. 2) includes 106 isolates and 628 characters, of which 287 are constant and 270 are parsimony-informative. The following models were selected by ModelFinder (AIC): TIM2+F+G4 for ITS1, TIM3e+I+G4 for 5.8S, and SYM+I+G4 for ITS2. The phylogenetic reconstruction of this ITS dataset results in the placement of Phaeohelotium pallidum as sister to Phaeohelotium sp. 2 with maximum BS support. The clade (Ph. pallidum, Ph. sp. 2) is retrieved as sister to Ph. maiusaurantium (BS = 91 %) (Fig. 2). Connersia and Pleuroascus are resolved in a clade with maximum support, placed sister to a subclade of Phaeohelotium with Ph. flavum (type species), Ph. geogenum, Ph. pateriforme complex, and Ph. tasmanicum complex. The Connersia–Pleuroascus–Phaeohelotium clade is sister to a clade with Hymenoscyphus isolates belonging the “Hymenoscyphus” 1 clade sensu Lebeuf et al. (2021: fig. 22), although with low support (BS = 69 %).

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Excerpt of the best-scoring ML tree (-lnL = 685940.513110), showing the placement of Phaeohelotium species in the 15-locus Leotiomycetes v1 tree in T-BAS. The topology is the result of a RAxML analysis with 500 bootstrap replicates. For each node, the ML bootstrap support (≥ 70 %) is presented above or below the branch leading to that node. Phaeohelotium highlighted in gray, new species highlighted in yellow and green, T indicating ex-type sequences.

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Phylogeny of Helotiaceae and Tricladiaceae (outgroup), reconstructed from an ITS dataset. The consensus tree topology (-lnL = 8626.682) is the result of ML inference performed with IQ-TREE. For each node, the ML bootstrap support (≥ 70 %) is presented above or in front of the branch leading to that node. “Hymenoscyphus” 1 and 2 annotations sensu Lebeuf et al. (2021: fig. 22), Phaeohelotium highlighted in gray, new species highlighted in yellow and green, T indicating ex-type sequences.

Our second, genus-level ITS (ITS1 + 5.8S + ITS2) dataset includes 43 isolates and 226 + 158 + 162 characters, of which 113 + 143 + 49 are constant and 77 + 9 + 85 are parsimony-informative (Fig. 3). The following models were selected by ModelFinder (AICc): SYM+G4 for ITS1, TIM3e+I for 5.8S, and TIM2e+G4 for ITS2. The phylogenetic reconstruction of this dataset recovers two intrageneric subclades of Phaeohelotium (Fig. 3): the first with saprotrophic taxa Ph. geogenum, Ph. monticola, Ph. pateriforme, and Ph. tasmanicum; and the second with Phaeohelotium spp. 1 and 2, Ph. confusum, Ph. cf. undulatum, the two newly described species from Chile Ph. maiusaurantium and Ph. pallidum, and ECM root tip sequences associated with multiple ECM tree host tree genera across three families (Eucalyptus in Myrtaceae, Nothofagus in Nothofagaceae, Pomaderris in Rhamnaceae). Support for these subclades is moderate to high (BS = 95 % for the putatively saprotrophic subclade, BS = 76 % for the putatively ECM clade). Phaeohelotium maiusaurantium is placed as sister to an ECM root tip sequence associated with Nothofagus cunninghamii, whereas Ph. pallidum is sister to an undescribed Chilean species, Phaeohelotium sp. 2.

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Phylogeny of Phaeohelotium and Dicephalospora (outgroup), reconstructed from an ITS dataset. The consensus tree topology (-lnL = -3437.109) is the result of ML inference performed with IQ-TREE. For each node, the ML bootstrap support (≥ 70 %) is presented above or in front of the branch leading to that node. T indicating ex-type sequences, Phaeohelotium maiusaurantium sp. nov. highlighted in yellow, Ph. pallidum sp. nov. highlighted in green.

Taxonomy

Leotiomycetes O.E. Erikss. & Winka, Myconet 1: 7. 1997.

Helotiales Nannf. ex Korf & Lizoň, Mycotaxon 75: 501. 2000.

Helotiaceae Rehm, Rabenhorst’s Kryptogamen-Flora, Pilze - Ascomyceten 1: 647. 1886.

Phaeohelotium Kanouse, Pap. Mich. Acad. Sci. 20: 75. 1935.

Type species: Phaeohelotium flavum Kanouse, Pap. Michigan Acad. Sci. 20: 75. 1935.

Synonyms: Helotium monticola Berk., Grevillea 4: 1. 1875.

Phaeohelotium monticola (Berk.) Dennis, Persoonia 3: 54. 1964.

Hymenoscyphus monticola (Berk.) Baral, Fl. Medit. 15: 67. 2005.

Phaeohelotium maiusaurantium A. Grupe, A. Weier, C.A. Quandt & M.E. Sm., sp. nov. Index Fungorum IF 553521. Fig. 4.

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Morphological characteristics of Phaeohelotium maiusaurantium. A. Field habit in Vicente Perez Rosales National Park, Chile (FLAS-F-65526, isotype). B. Close-up of apothecia (FLAS-F-65526, isotype). C. Microtome section, stained with hematoxylin & eosin, of an apothecium showing the mixture of textura intricata and loosely interwoven hyphae of the ectal excipulum and the medullary excipulum (FLAS-F-65476). D, E. Developing asci arising from a series of repeating croziers (FLAS-F-65460). F. Amyloid ascus apical ring and eight fusiform ascospores (FLAS-F-65460). G. Close up of an ascus and the Hymenoscyphus-type amyloid ascus apical ring (FLAS-F-65460). H. Smooth, fusiform ascospores, each with two large, oil drops that fused in the dead state (FLAS-F-65460). I. Multiseptate, filiform paraphysis (FLAS-F-65476). Scale bars: A, B = 5 mm; C = 100 μm; D = 45 μm; E–H = 10 μm, I = 5 μm.

Etymology: Referring to the relatively large (maius) orange (aurantium) apothecia.

Diagnosis: Differs from other southern South American species by the color (light orange) and size (3–6 mm diam.) of its apothecia, and the size of its asci (171–208 × 8–10 μm) and ascospores (14–20 × 6–8 μm). Also differs from Ph. nothofagi in the amyloidity of its ascus apex, and from Ph. castaneum and Ph. nothofagi in its ECM lifestyle.

Typus: Chile, Los Lagos Region, Vicente Perez Rosales National Park, 930 m a.s.l, in Nothofagus dombeyi forests, on soil, 18 Apr. 2017, M.E. Smith & R. Healy MES-2976 (holotype SGO), (isotype FLAS-F-65526). GenBank accession nos. OP951346 (SSU), OP868953 (ITS), OP962216 (LSU).

Description: Apothecia scattered or gregarious in small groups, never fasciculate from a common stipe, 3–6 mm diam. When fresh, initially concave, becoming slightly convex with age, slightly gelatinous consistency, light orange verging on pale orange externally, margin thin, smooth to minutely downy; stipe distinct but buried in substrate, concolorous with the flanks of the apothecia, 1–3 × 1.0 mm. Asci 8-spored with blunt apex, gradually tapering to the base, arising from a series of repeating croziers; (151–)171–208(–223) × 8–10(–12) μm; hyaline in DI water and KOH, amyloid apical ring in Melzer’s reagent without KOH pretreatment, of the Hymenoscyphus-type, inner part of the ring staining strongly, commonly extending laterally, remaining wall inamyloid. Ascospores fusoid to fusiform with obtuse to acute poles, without ornamentation, containing two large guttules that fuse in the dead state; (13–)14–20(–21) × (5–) 6–8(–9) μm; hyaline in DI water and KOH, uncolored in Melzer’s reagent. Paraphyses filiform reaching a slightly enlarged rounded apex, generally non-branching, multiseptate, lacking vacuolar bodies in the rehydrated condition; 2–3 μm diam. at apex; hyaline in DI water and KOH, inamyloid in Melzer’s reagent. Ectal excipulum of textura prismatica at the margins of the hymenium, 74–85 μm thick, a mixture of textura intricata and loosely interwoven hyphae below the margins of the disc to the stipe, 97–220 μm thick, cells (6–)8–24(–35) × (3–)6–10(–12) μm, slightly gelatinized. Medullary excipulum of textura intricata, 51–416 μm thick, cells 5–11(–19) × (2–)4–8 μm, slightly gelatinized. Subhymenium of textura intricata, 38–97 μm thick, cells (4–)6–8(–27) × 3–9 μm, non-gelatinized.

Habitat and distribution: Growing in the Andes mountains of south-central Chile, during the fall. On exposed soil within native Nothofagus dombeyi forests.

Additional specimens examined: Chile, Los Lagos Region, Vicente Perez Rosales National Park, 930 m a.s.l., in Nothofagus dombeyi forests, on soil, 24 Mar. 2008, D.H. Pfister & M.E. Smith DHP-CH-74 (FH 00284861); ibid., 22 Mar. 2008, D.H. Pfister & M.E. Smith DHP-CH-80 (FH 00284867); ibid., 27 Mar. 2008, D.H. Pfister & M.E. Smith DHP-CH-93 (FH 00284876); ibid., 27 Mar. 2008, D.H. Pfister & M.E. Smith DHP-CH-99 (FH 00284882); ibid., 17 Apr. 2017, R. Healy MES-2905 (FLAS-F-65460); ibid., 17 Apr. 2017, R. Healy & M.E. Smith MES-2925 (FLAS-F-65476).

Notes: Phaeohelotium maiusaurantium is morphologically similar to other southern South American species of Phaeohelotium (Ph. castaneum, Ph. nothofagi, Ph. pallidum sp. nov. and Ph. recurvum). Phaeohelotium maiusaurantium can be distinguished morphologically from Ph. pallidum by the color of the apothecia (light orange in Ph. maiusaurantium vs light gray in Ph. pallidum), longer asci (171–208 μm vs 160–175 μm), and longer ascospores (14–20 μm vs 12–15 μm) (Table 3). Both Ph. maiusaurantium and Ph. pallidum putatively share the same ECM tree host genus (Nothofagus). Phaeohelotium maiusaurantium can be separated from the other three species (Ph. castaneum, Ph. nothofagi, Ph. recurvum) by substratum and morphological characteristics. Phaeohelotium castaneum, Ph. nothofagi, and Ph. recurvum are saprotrophs and grow directly on fallen leaves, twigs, and rotten wood. Morphologically, Ph. maiusaurantium can be differentiated from P. castaneum by its larger apothecia (3–6 mm diam. in Ph. maiusaurantium vs 0.5–1.5 mm diam in P. castaneum), the color of the apothecia (light orange vs dirty chestnut), its substantially larger asci (171–208 × 8–10 μm vs 63–96 × 4.8–6.4 μm), and larger ascospores (14–20 × 6–8 μm vs 8–12.8 × 3.2–4.8 μm) (Table 3). In comparison to Ph. nothofagi, Ph. maiusaurantium has light orange-colored apothecia (vs cream or light yellow), and longer and amyloid asci (171–208 μm vs 116–145 μm and inamyloid) (Table 3). Finally, compared to the holotype of Ph. recurvum, Ph. maiusaurantium has longer asci (171–208 μm vs 115 μm in Ph. recurvum) and larger ascospores (14–20 × 6–8 μm vs 10–12.5 × 4–4.5 μm in Ph. recurvum) (Table 3). The collections of Ph. recurvum from Tierra del Fuego (Gamundí & Romero 1998) have even smaller asci (75–77 × 4.5–5.4 μm) and slightly shorter ascospores (9–10 × 3.6–4.5 μm) compared to the holotype as described by Dennis (1958). Sequence data are needed to discern whether the Argentinian and Tasmanian material of Ph. recurvum represents the same species.

Table 3.

Comparative morphology of species in the genus Phaeohelotium

Species Ascus length × width (μm) Ascospore length × width (μm) Amyloid apex Substrate and location Reference Sequences in GenBank
asiaticum 90–100 × 10–1318–24(–26) × 5–6AmyloidOn decorticated branchlet, Japan Korf (1959) No
baileyanum 1 (140–)160–210(–225) × (8–)9–10(–12)(12–)14–20(–23) × (5.5–)6–8(–9)AmyloidUnder Eucalyptus, Australia (Tasmania) Baral et al. (2013) No
carneum 80–90 × 6–712–15 × 3.5–5AmyloidOn twigs and wood debris submerged in water, Britain and Ireland and Switzerland Miśkiewicz (2000) No
carpinicola 85–105 × 10–1212–16 × 4–5AmyloidWith Carpinus betulus and Vaccinium myrtillus, Germany Arendholz 1979 No
castaneum 63–96 × 4.8–6.48–12.8 × 3.2–4.8AmyloidOn a submerged twig, Argentina Gamundí (1962) No
confusum (140–)160–210(–225) × 8–9.5(–10)(11.5–)13–17(–19) × (5.5–)6–7(–7.5)AmyloidOn Nothofagus litter or on soil under Nothofagus, New Zealand Baral et al. (2013) Yes
conicola 85–90 × 12–1515–19 × 4–6.5AmyloidOn wood of a spruce cone, Czech Republic Svrček (1984) No
flavum 2 100–125 × 8–914–16 × 4–5AmyloidOn a decaying log, USA Kanouse (1935) No
fluviatile 80–100 × 6–97.5–10 × 3.5–4.5AmyloidOn fallen log of Abies sp. in a stream, India Raitviir & Sharma (1984) No
fulvidulum 120–140 × 10–1214–20 × 3–4AmyloidRotten leaves of grasses or sedges, France Boudier (1907) No
geogenum 125–150 × 10–1225–30 × 4.5–5AmyloidOn moist, heavy soil, thick fallen oak branches, decorticated wood, Switzerland Svrček & Matheis (1979) Yes
hylocomii 50–60 × 6–6.59–11 × 3.5–4AmyloidHylocomium splendens stems, Estonia Leenurm et al. (2000) No
lilacinum 125 × 10–1214–20 × 5–8AmyloidOn wet rotten wood, UK Dennis (1981) No
luteum 120–140 × 9–11(16–)18–22(–24) × (4–)5–6(–8)N/AOn unknown substrate in subtropical rainforests, Brazil Dennis (1960) No
maiusaurantium (151–)171–208(–223) × 8–10(–12)(13–)14–20(–21) × (5–)6–8(–9)AmyloidOn soil in Nothofagus dombeyi forests, ChileThis paperYes
melleoflavum 50–55 × 4.5–5.57–9 × 1.5–2AmyloidOn decorticated branch of Populus nigra, Czech Republic Svrček (1992) No
monticola 2 95–100 × 7–1012–18 × 4–5AmyloidOn decorticated wood, USA Dennis (1964) Yes
nobile 120–150 × 8–128–15InamyloidOn wet rotting branches of Oak, Czech Republic Velenovský (1934) No
nothofagi 116–145 × 8.5–10(14.5–)18.5–20.5(–21) × (4–)5–6InamyloidOn fallen leaves of Nothofagus dombeyi, Argentina Gamundí & Messuti (2006) No
pallidelilacinum 95–125 × 8–12(7.5–)8.5–12.5 × 4–5AmyloidOn rotten wood of Pinus mugo, Slovakia Svrček (1992) No
pallidum (153–)160–175(–185) × 9–11(–12)12–15 × 6–7(–8)AmyloidOn soil with Nothofagus dombeyi, ChileThis paperYes
pani 95–100 × 6–78–12 × 3–3.5InamyloidOn a decaying Populus sp. twig, Czech Republic Svrček (1984) No
pateriforme 170–180 × 9–1023–28 × 4–5AmyloidOn decorticated wood and dead sticks, Australia (Tasmania) Dennis (1958) Yes
purpureum 40–50 × 4.5–55–8 × 1InamyloidOn dried up branches of Pinus sylvestris, UK Dennis (1974) No
readeri 135–150 × (8–)9–11(–12)(11–)13–16(–18) × 6.8–8.3InamyloidN/A Baral et al. (2013) No
recurvum 115 × 910–12.5 × 4–4.5AmyloidOn moss and rotten wood, Australia (Tasmania) Dennis (1958) No
rubropurpurascens 50–60 × 4–67–10 × 2.5–4AmyloidOn leaves, Czech Republic Svrček (1976) No
samaricola 80–100 × 7–98–13 × 3–4AmyloidOn fallen Acer pseudoplatanus leaves, Czech Republic Svrček (1983) No
subcarneum 80–90 × 6–712–15 × 3.5–5AmyloidOn twigs and wood debris submerged in water, Britain and Ireland and Switzerland Miśkiewicz (2000) No
succineoguttulatum 160–250 × (9–)10–11(–12)(12–)14–20(–24) × (6.2–)7–9.5(–10)InamyloidOn soil under Eucalyptus globulus, Spain Baral et al. (2013) Yes
tasmanicum N/A11–18 × 3–4.5N/AOn dead wood, Australia (Tasmania) Rodway (1925) Yes
terrestre 100–110 × 9–1212–15.5 × 4–5InamyloidOn loamy damp ground, Czech Republic Svrček (1987) No
undulatum 160–250 × (9–)10–11(–12)(12–)14–20(–24) × (6.2–)7–9.5(–10)AmyloidOn soil under Eucalyptus globulus, Spain Baral et al. (2013) Yes

1Phaeohelotium baileyanum was introduced by Baral et al. (2013) as a new name for Discinella terrestris.

2Phaeohelotium monticola was synonymized with the type species Ph. flavum (Dennis 1964, Dumont 1981: 354). Nevertheless, both names are presented here, with their characteristics as reported by Dennis (1964) and Kanouse (1935), respectively.

Phaeohelotium pallidum A. Grupe, A. Weier, C.A. Quandt & M.E. Sm., sp. nov. Index Fungorum IF 553527. Fig. 5.

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Morphological characteristics of Phaeohelotium pallidum. A. Collection of apothecia from Puyehue National Park, Chile (FLAS-F-66004). B. Close-up of apothecia (FLAS-F-66004). C. Microtome section, stained with hematoxylin & eosin, of an apothecium showing organization of the subhymenium and medullary excipulum layers (FLAS-F-63072, isotype). D. Developing ascus arising from a series of repeating croziers (FLAS-F-63072, isotype). E. Close-up of the series of repeating croziers (FLAS-F-63072, isotype). F. A single ascus with eight fusiform ascospores (FLAS-F-63072, isotype). G, H. Close-up of the amyloid apical ascus ring (FLAS-F-63072, isotype). I. Two multiseptated, filiform paraphyses (FLAS-F-63072, isotype). J. Smooth, fusiform ascospores, each with two large, oil drops that fused in the dead state (FLAS-F-63072, isotype). Scale bars: B = 5 mm; C = 50 μm; D = 20 μm; E–H = 10 μm; I–J = 5 μm.

Etymology: Referring to the pallid color of fresh apothecia.

Diagnosis: Differs from other southern South American species by the color (light gray) and size (2–4 mm diam.) of its apothecia, and the size of its asci (160–175 × 9–11 μm) and ascospores (12–15 × 6–7 μm). Also differs from Ph. nothofagi in the amyloidity of its ascus apex, and from Ph. castaneum and Ph. nothofagi in its ECM lifestyle.

Typus: Chile, Los Lagos Region, Puyehue National Park, ~1 000 m a.s.l., in deep bamboo patches with Nothofagus dombeyi, 5 May 2015, R. Healy MES-1068 (holotype SGO), (isotype FLAS-F-63072). GenBank accession nos. OP951345 (SSU), KY462415 (ITS), OP962212 (LSU).

Description: Apothecia scattered or gregarious in small groups, never fasciculate from a common stipe, 2–4 mm diam when fresh, initially concave, becoming convex with age, slightly gelatinous consistency, light gray, margin thin, smooth to minutely downy; stipe distinct but buried in substrate, concolorous with the flanks of the apothecia, 1–2 × 0.6 mm. Asci 8-spored with blunt apex, gradually tapering to the base, arising from a series of repeating croziers; (153–)160–175(–185) × 9–11(–12) μm; hyaline in DI water and KOH, amyloid apical ring in Melzer’s reagent without KOH pretreatment, of the Hymenoscyphus-type, inner part of the ring staining strongly, commonly extending laterally, remaining wall inamyloid. Ascospores fusiform with obtuse to acute poles, unornamented, containing two large guttules that fuse in the dead state; 12–15 × 6–7(–8) μm; hyaline in DI water and KOH, uncolored in Melzer’s reagent. Paraphyses filiform reaching a slightly enlarged rounded apex, generally non-branching, septate, lacking vacuolar bodies in rehydrated condition; 2–3 μm diam at apex; hyaline in DI water and KOH, inamyloid in Melzer’s reagent. Ectal excipulum a mixture of textura prismatica at the margins of the disc, 24–67 μm thick, cells 5–23(–31) × 4–9 μm, a mixture of textura intricata and loosely interwoven hyphae below the flanks of the hymenium to the stipe, 65–180 μm thick, slightly gelatinized. Medullary excipulum of textura intricata, 87–275 μm thick, cells 6–24 × 5–9(–16) μm, slightly gelatinized. Subhymenium of textura intricata, 51–73 μm thick, cells (4–)6–23 × (3–)5–9(–14) μm, non-gelatinized.

Habitat and distribution: Growing in the coastal forests and Andes mountains of south-central Chile in the fall and spring seasons. On exposed soil, typically under leaf litter in mature Nothofagus dombeyi forests.

Additional specimens examined: Chile, Los Lagos Region, Puyehue National Park, 930 m a.s.l., on soil beneath Nothofagus dombeyi, 14 Apr. 2017, C. Truong MES-2850 (FLAS-F-65411); ibid., 14 Apr. 2017, C. Truong MES-2852 (FLAS-F-65413); Los Rios, Parque Nacional Alerce Costero, on soil beneath Nothofagus alpina and N. dombeyi, 13 Oct. 2019, M. Caiafa & M.E. Smith MES-3573 (FLAS-F-66003); ibid., 13 Oct. 2019, P. Sandoval MES-3574 (FLAS-F-66004); Los Rios, Villarica National Park, 10 May 2019, M. Caiafa & M.E. Smith MES-3308 (specimen destroyed during fieldwork, only photos and sequences available).

Notes: Like Ph. maiusaurantium, Ph. pallidum can be distinguished from previously recorded southern South American species of Phaeohelotium (Ph. castaneum, Ph. nothofagi, Ph. recurvum) based on substratum (ECM vs saprotrophic) and morphological characteristics. Phaeohelotium pallidum differs morphologically from Ph. castaneum in apothecial color (light gray in Ph. pallidum vs dirty chestnut in Ph. castaneum), ascus size (160–175 × 9–11 μm in Ph. pallidum vs 63–96 × 4.8–6.4 μm in Ph. castaneum), and ascospore size (12–15 × 6–7 μm in Ph. pallidum vs 8–12.8 × 3.2–4.8 μm in Ph. castaneum). Compared to Ph. nothofagi, Ph. pallidum has light gray apothecia (vs cream or pale yellow in Ph. nothofagi), and larger and amyloid asci (160–175 × 9–11 μm in Ph. pallidum vs 116–145 × 8.5–10 μm and inamyloid in Ph. nothofagi) (Table 3). Finally, compared to the holotype of Ph. recurvum, Ph. pallidum has longer asci (160–175 μm vs 115 μm in Ph. recurvum) and larger ascospores (12–15 × 6–7 μm vs 10–12.5 × 4–4.5 μm in Ph. recurvum) (Table 3).

Phaeohelotium pallidum is phylogenetically most closely related to an undescribed taxon, Phaeohelotium sp. 2 (Fig. 2). The phylogenetically most closely related described species to both Ph. pallidum and Ph. maiusaurantium is Ph. confusum (Fig. 2, ​,3).3). However, Ph. pallidum and Ph. confusum only share 92.11–95.64 % identity, and Ph. maiusaurantium and Ph. confusum share 91.83–95.02 % identity. In addition, Ph. confusum is exclusively found under Nothofagus in New Zealand (Baral et al. 2013). For Ph. baileyanum (synonym Discinella terrestris), which is morphologically similar to Ph. confusum, no sequence data are currently available. Phaeohelotium maiusaurantium and Ph. pallidum differ from Ph. baileyanum in their always hyaline ascospores (vs often brown in Ph. baileyanum). In addition, Ph. baileyanum occurs under Eucalyptus with records in Australia and Tasmania (Baral et al. 2013).

Phaeohelotium pateriforme (Cooke) P.R. Johnst., comb. nov. Index Fungorum IF 553544.

Basionym: Helotium pateriforme Cooke [as “pateraeforme”], Grevillea 11: 102. 1883.

Synonyms: Peziza pateriformis Berk., in Hooker, Bot. Antarct. Voy., III, Fl. Tasmania 2: 276. 1859. Nom. illegit., Art. 53.1, non Peziza pateriformis Durieu & Lév., Ann. Sci. Nat. Bot. (sér. 3) 9: 140. 1848.]

Calycina pateriformis (Berk.) Kuntze, Revis. gen. pl. (Leipzig) 3: 448. 1898.

Phaeohelotium tasmanicum (Rodway) P.R. Johnst., comb. nov. Index Fungorum IF 553546.

Basionym: Helotium tasmanicum Rodway, Pap. & Proc. Roy. Soc. Tasmania 1920: 155. 1921.

Notes: These two new combinations are made based on phylogenetic evidence. Johnston (2019) presented an ITS phylogeny incorporating Phaeohelotium isolates from Australia and New Zealand. Ten isolates of Helotium pateriforme and five isolates of Helotium tasmanicum were placed within the genus Phaeohelotium, with maximum support. Our ITS phylogenies, incorporating additional Phaeohelotium isolates and species compared to Johnston (2019), found the same result: Helotium pateriforme and H. tasmanicum are retrieved within Phaeohelotium with maximum support, positioned closely to the type species, Ph. flavum, as part of the saprotrophic subclade (Fig. 2, ​,3).3). These species are also collected on wood and both taxa are to be referred to as species complexes as per Johnston (2019).

Additional materials examined

Phaeohelotium sp. 1

Specimens examined: Chile, Magellanes, Magellanes National Park, Nothofagus pumilio forest, on soil under moss layer, 3 Apr. 2017, A. Mujic MES-2358 (FLAS-F-65016).

Phaeohelotium sp. 2

Specimen examined: Chile, Los Lagos, Puyehue National Park, Podocarpus nuvigena forest with Nothofagus dombeyi, on bare soil and heavily decayed wood, 3 Apr. 2017, C. Truong MES-2851 (FLAS-F-65412); ibid., 10 Apr. 2017, C. Truong MES-2650 (FLAS-F-65244).

DISCUSSION

Our phylogenetic analyses show that Phaeohelotium in the sense of Baral et al. (2013) is a polyphyletic genus (Fig. 1, ​,2).2). Phaeohelotium epiphyllum and Ph. tamaricis are consistently phylogenetically distinct from the other members of the genus including the type species Ph. flavum (Dennis 1958, 1964, Baral et al. 2013). In addition, in our 15-locus phylogeny, Ph. flavum is phylogenetically distinct from all other Phaeohelotium sequences (unedited tree available through GitHub) but this is likely due to lack of sequence data; only a single ITS sequence is available for Ph. flavum whereas for the other Phaeohelotium isolates sequences of three to five loci are included in this phylogeny.

Here we reveal for the first time the likely divergence in ecological strategy (ECM vs saprotrophic) within the genus Phaeohelotium. Our ITS phylogenies (Fig. 2, ​,3)3) show two supported subclades. One of these lineages is comprised of taxa that are only collected from decaying wood and litter, while the second one includes species that are collected from the soil in forests with ECM plants as well as sequences directly from ECM root tips. This suggests genetic divergence between the two guilds. It might be that the ECM subclade represents a different genus altogether, although currently we do not have enough data to make such a taxonomic decision. To resolve this, more species need to be incorporated in phylogenetic analyses to confirm or reject this hypothesis. In addition, sequences of more loci need to be generated to decipher the position of Phaeohelotium with regard to the closely related Connersia and Pleuroascus (Fig. 2, Johnston 2019, Lebeuf et al. 2021: fig. 22), and careful morphological study of original materials may unlock more taxonomically valuable characteristics. We advocate for further molecular investigation of Phaeohelotium sensu lato by sampling more taxa and sequencing more loci to test the monophyly of this genus.

The type species of the genus, Ph. flavum, was collected from decorticated wood in Michigan, USA (Kanouse 1935, Dennis 1964). Baral et al. (2013) used an ITS sequence from a German specimen of Ph. flavum [as Ph. monticola], collected from a Fagus sylvatica trunk. Phaeohelotium maiusaurantium and Ph. pallidum from Chile, Ph. succineoguttulatum and Ph. undulatum from Spain, and Ph. confusum (= Discinella confusa) from New Zealand all grow directly on soil, and the ITS sequences from isolates form a well-supported subclade with ECM root tip sequences (Fig. 2, ​,3).3). The ectomycorrhizal guild can be assigned to closely related species at low taxonomic levels (such as genus) (Tedersoo et al. 2010, Zanne et al. 2020) and ECM clades rarely or never revert to saprotrophy because they typically lose important carbohydrate-degrading enzymes upon the transition to the ECM lifestyle (Tedersoo & Smith 2013, Kohler et al. 2015). These patterns further support the inference that the taxa in this subclade form ECM associations. The other Phaeohelotium subclade (Ph. flavum, Ph. geogenum, Ph. pateriforme species complex, Ph. tasmanicum species complex) exhibits high to maximum support, and these species have an apparent saprotrophic lifestyle having been collected from fallen decomposing wood, rotting litter, and seeds and cupules (Dennis 1958, 1964, Baral et al. 2013, Johnston 2019). In addition to their trophic differences, species in these subclades also differ in their ascus apex morphology and excipular anatomy. As noted by Johnston (2019), species in the subclade of saprotrophs possess weakly amyloid pores, with two lines restricted to the inner half of the ascus wall. This characteristic differentiates them from species in the putatively ECM subclade, where the amyloid reaction of the pore is present throughout the ascus wall or restricted to the outer portion of the wall. These two subclades are also morphologically separated based on the excipuluar cell walls, which are gelatinized in the ECM subclade and non-gelatinized among the saprotrophic taxa.

Whether these subclades differ in ultimate ascospore coloration is uncertain based on our current sampling. Most species in the genus have ascospores that are hyaline, darkening with age (Kanouse 1954, Dennis 1981, Johnston 2019). Dennis (1981) suggested that this is “probably not an essential character.” Indeed, species seem to vary in the timing of darkening as well as in whether darkening occurs at all (Gamundí & Messuti 2006, Johnston 2019, this paper). In Ph. confusum, Ph. luteum, Ph. maiusaurantium, Ph. pallidum, and Ph. recurvum, brown ascospores have never been observed (Gamundí & Messuti 2006, Baral et al. 2013, this paper). Phaeohelotium baileyanum, on the other hand, has brown ascospores prior to release from the ascus (Johnston 2019). Based on formal descriptions of taxa and their inferred trophic modes, we hypothesize that species with permanently hyaline ascospores and those with ascospores browning with age occur in both the ECM (Baral et al. 2013, this paper) and the saprotrophic subclades (Dennis 1981, Gamundí et al. 2004, Gamundí & Messuti 2006).

The recognition of Ph. maiusaurantium and Ph. pallidum as two new species from Chile and the two new combinations (Ph. pateriforme, Ph. tasmanicum) brings the total number of formally described species in the genus to 32 (Index Fungorum 2022, Table 3). However, sequence data are available for only nine described species within the genus. Note that Hymenoscyphus epiphyllus (synonym Ph. epiphyllum) and Hymenoscyphus tamaricis (synonym Ph. tamaricis) are not considered members of the genus based on molecular phylogenetic data (Fig. 1, ​,2).2). We also generated sequences for two undescribed species of Phaeohelotium, but not enough material was available to warrant formal description as per the guidelines by Aime et al. (2021). The ecological strategy of Ph. maiusaurantium and Ph. pallidum may be ECM; both species grow directly on soil, they are closely related to ECM root tip sequences from New Zealand and Tasmania, and preliminary data revealed Phaeohelotium-like sequences from South American ECM root tip amplicon data (A. Mujic, pers. comm.). If confirmed, this would be another example of independent evolution of the ECM lifestyle from non-mycorrhizal ancestors (Tedersoo & Smith 2013). Fieldwork should be directed to collect ascomata of Phaeohelotium and root tip samples under likely candidate tree associates in both Patagonia and Australasia. Given the significant ecological and genetic changes involved in switching from a free-living saprotrophic state to an ECM symbiont, this may warrant the erection of a new genus to accommodate the Phaeohelotium ECM subclade.

With regard to the biogeography of the ECM subclade, that ECM root tip samples collected in Australasia are retrieved in a clade with Chilean and Australasian collections is a phylogenetic signal of their shared sympatric ancestry from part of the Gondwana supercontinent. Similarly, Truong et al. (2017b) found that Amanita species in southern South America were grouped with relatives in Australia in a 34.5-M-yr-old clade, corresponding to the fragmentation of South American, Australian, and Antarctic Plates. The discovery of Ph. succineoguttulatum and Ph. undulatum in Spain associated with Eucalyptus is certainly due to the exportation of Eucalyptus species to the Northern Hemisphere for silviculture. The introduction of non-native ECM fungi has been documented in other lineages (Giachini et al. 2000, Díez 2005, Hynson et al. 2013, Hayward et al. 2015, Kraisitudomsook et al. 2019). If the Phaeohelotium ECM subclade indeed evolved from a saprotrophic lineage, then the presence of the ECM species Ph. maiusaurantium and Ph. pallidum in Chile and the saprotrophic species Ph. castaneum and Ph. nothofagi in Argentina hints at travel of the ECM lineage potentially migrating along the southern edge of Gondwanaland. This would be possible given the fossil record of Nothofagus trees in Antarctica and the extant members of Nothofagus in Chile, Australia, and New Zealand (Linder & Crisp 1995, Heads 2006). In other Helotiales lineages with a similar geographic pattern, such as Cyttaria, it is theorized that co-migration with host plants, long-distance dispersal events, or vicariance are responsible for the current observed distribution (Johnston 2006, Peterson et al. 2010). Whether the geographic pattern we observe in the putatively ECM subclade is due to a combination of these factors remains an outstanding question for these species of Phaeohelotium. We note that there is 100 % shared identity between an ITS sequence of Phaeohelotium sp. from New Zealand (ICMP:23361) included in our tree and that of a collection from Chile (DHP-CH-11B, GenBank acc. no. KY462387), perhaps providing further evidence for the Chilean–Australasian link.

From what we know about Phaeohelotium thus far, saprotrophic taxa occur in both the Northern and Southern Hemispheres. We do not know, however, where they are most species-rich. It does appear that all ECM species are restricted to the Southern Hemisphere, except for the Spanish species that are documented with introduced Eucalyptus (Baral et al. 2013), as pointed out above. This suggests that the ECM lifestyle in Phaeohelotium may have evolved in the Southern Hemisphere. This phenomenon of unique taxa that evolved the ECM symbiosis with plants in the Southern Hemisphere is known in two genera of Basidiomycota, Austropaxillus (Skrede et al. 2011) and Descolea (Kuhar et al. 2017). However, the ECM lineage within Phaeohelotium may be the first known group of Ascomycota to have evolved the ECM symbiosis in the Southern Hemisphere (Tedersoo & Smith 2013).

With the formal description of Ph. maiusaurantium and Ph. pallidum, we add to the growing recognition of high undescribed diversity in Leotiomycetes. As previously acknowledged (e.g., Truong et al. 2017a, Cazabonne et al. 2022), continued fieldwork will prove important to accumulate fresh Phaeohelotium collections for sequencing and deposition in fungaria. This will undoubtedly result in the discovery of more diversity in the genus and help to determine whether a new genus should be recognized to accommodate species in the ECM subclade. In addition, we hope that future work will elucidate the origins of the switch to the ECM lifestyle within Phaeohelotium through utilizing comparative genomics between species with different trophic modes.

Acknowledgments

We thank Adriana Calle, Maria Jose Diban, Francisco Kuhar, Yeyetsi Maldonado, Alija Mujic, Eduardo Nouhra, Pablo Sandoval, and Camille Truong for sharing their collections and photos used in this work; Candice Perrotta for nomenclatural advice; Jessica Arnold, Allison Quador, and E. Erin Smith for their assistance at the University of Colorado Center Pathology Shared Resource; and Hans-Otto Baral and Peter R. Johnston for their thoughtful suggestions to the manuscript. The Chilean Corporación Nacional Forestal (Gerencia de Áreas Silvestres Protegidas) provided permission to collect fungi in Chile under permit numbers 014/2014 (to M.E.S.) and 013/2019 (to M.V.C. and M.E.S.).

Footnotes

Funding: The work of A.C.G., A.W., and C.A.Q. was partially supported by the National Cancer Institute grant P30CA046934, “University of Colorado Cancer Center Core Support Grant”. This research received support from the U.S. National Science Foundation (DEB-1354802 to M.E.S., DEB-2018098 to D.H., DEB-2018215 to C.A.Q.), the National Geographic Society (Explorer Grant 59603R-19 to M.E.S. and M.V.C.), and the Research Foundation—Flanders (Junior Postdoctoral Fellowship 1206620N to D.H.).

Conflict of interest: The authors declare that there is no conflict of interest.

Data availability statement: All final alignments and unedited trees are available through GitHub: https://github.com/Quandt-Mycology-Lab/Lab_Codes_and_workflows/tree/master/Phaeohelotium_paper.

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