Content uploaded by Alden Dirks
Author content
All content in this area was uploaded by Alden Dirks on Apr 09, 2024
Content may be subject to copyright.
Phytotaxa 644 (1): 049–055
https://www.mapress.com/pt/
Copyright © 2024 Magnolia Press Article PHYTOTAXA
ISSN 1179-3155 (print edition)
ISSN 1179-3163 (online edition)
Accepted by Sajeewa Maharachchikumbura: 21 Mar. 2024; published: 9 Apr. 2024
https://doi.org/10.11646/phytotaxa.644.1.7
49
Licensed under Creative Commons Attribution-N.C. 4.0 International https://creativecommons.org/licenses/by-nc/4.0/
Gyromitra persicula sp. nov. (Discinaceae, Pezizales), a novel discoid Gyromitra
from Northwestern North America
ALDEN C. DIRKS1,5*, MICHAEL W. BEUG2,6, ANDREW S. METHVEN3,7, ANDREW N. MILLER4,8 & TIMOTHY
Y. JAMES1,9
1University of Michigan Herbarium, Research Museums Center, Department of Ecology and Evolutionary Biology, 3600 Varsity Drive,
Ann Arbor, MI 48108, USA
2The Evergreen State College, 2700 Evergreen Pkwy, Olympia, WA 98505, USA
33622 Catherine Lake Cove, Leland, NC 28451, USA
4Illinois Natural History Survey, University of Illinois Urbana-Champaign, 1816 South Oak Street, Champaign, IL 61820, USA
5
�
adirks@umich.edu; https://orcid.org/0000-0002-3997-0122
6
�
beugm@evergreen.edu; https://orcid.org/0000-0001-6767-6134
7
�
asmethven@eiu.edu; https://orcid.org/0000-0002-5675-7438
8
�
amiller7@illinois.edu; https://orcid.org/0000-0001-7300-0069
9
�
tyjames@umich.edu; https://orcid.org/0000-0002-1123-5986
*Corresponding author:
�
adirks@umich.edu
ABSTRACT
Gyromitra persicula (Discinaceae, Pezizales), a new species from Washington state, USA, is described. Like its sister
species G. leucoxantha, G. persicula has reticulate ascospores with blunt apiculi that form concave depressions resembling
a fishtail. Gyromitra persicula is differentiated from G. leucoxantha by its smaller size, peach to buff coloration, and nrDNA
ITS. Further collections are required to better understand the distribution and abundance of this discoid Gyromitra, which is
currently only known from a small geographic area of temperate rainforest.
Key words: false morel, gyromitrin, one new taxon, systematics, Washington
INTRODUCTION
Discinaceae is a morphologically diverse family of mushrooms ranging from discoid or saucer-shaped to stipitate with
an apical hymenophore, as well as hypogeous, truffle-like forms (O’Donnell et al. 1997). Taxa with discoid ascomata
have traditionally been placed in the genus Discina (Fries) Fries (1849: 348) and those with stipitate ascomata in
the genera Gyromitra Fries (1849: 346), Neogyromitra Imai (1932: 174), or Pseudorhizina Jaczewski (1913: 414).
Harmaja (1969) challenged the generic delimitation based on gross morphology and lumped these taxa together under
Gyromitra, arguing that their microscopic characteristics represented a continuum—a taxonomic framework we follow
here. Discinaceae species are difficult to separate with morphological analysis alone; contemporary studies utilize
molecular phylogenetics in an integrative approach to resolve long-standing questions in Discinaceae taxonomy, tease
apart species complexes, and describe new taxa (Miller et al. 2020, 2022a). As part of ongoing studies concerning
the gyromitrin mycotoxin in Discinaceae (Dirks et al. 2023), Dr. Michael W. Beug contributed collections from
Washington, USA, belonging to an unknown but locally abundant species. In Ascomycete Fungi of North America,
Beug called them “mini peach pig’s ears” due to the pinkish buff color of the hymenium (Beug et al. 2014: 145).
While comparable to Gyromitra leucoxantha (Bresadola) Harmaja (1969: 11) in microscopic traits, their coloration
and consistently small size suggested they were an undescribed species. Here, we confirm this suspicion and describe
the mini peach pig’s ears as new to science.
DIRKS ET AL.
50 • Phytotaxa 644 (1) © 2024 Magnolia Press
MATERIALS AND METHODS
Specimens were collected by Dr. Michael W. Beug from Gifford Pinchot National Forest and Mt. Baker-Snoqualmie
National Forest in Washington, USA, between the years 2005 and 2022. Ascomata were dried at 35 °C in a food
dehydrator and later deposited at the University of Michigan fungarium (MICH). Micromorphological features were
studied by shaving off thin sections from dried ascomata and rehydrating the tissue in 5 % KOH stained with phloxine
B. Sections were examined with an LW Scientific i4 Infinity compound microscope (Lawrenceville, GA) at × 400
and × 1000 magnification. Photographs and measurements were taken with an AmScope MU500 microscope camera
(AmScope, Irvine, CA). When present, 25 mature, freely liberated ascospores, 25 paraphyses, and 10 asci were
measured. Sizes are presented as the range consisting of the mean ± one standard deviation. The mean length (Lm),
mean width (Wm), length-width ratio (Q), and mean length-width ratio (Qm) are also reported. Ascospore measurements
include the perispore and apiculi consistent with Miller et al. (2022a). Images were processed with Adobe Illustrator
2024 and Adobe Photoshop 2024 (Adobe Systems Inc., Mountain View, California).
DNA extractions and PCR amplifications of the nuclear rDNA internal transcribed spacer region ITS1-5.8S-ITS2
(ITS barcode) and the D1–D2 domains of nuclear 28S large subunit rDNA (LSU) were carried out according to Dirks et
al. (2023). PCR products were purified with ExoSAP-IT (Applied Biosystems, Waltham, Massachusetts) and submitted
to Azenta Life Sciences for Sanger sequencing (Azenta Life Sciences, South Plainfield, New Jersey). Forward and
reverse Sanger sequences were assembled with Geneious Prime (Dotmatics, Boston, Massachusetts) and submitted
to GenBank (PP051281–PP051297) (Table 1). ITS and LSU sequences were aligned separately with MUSCLE as
implemented in SeaView 5.0.5 (Edgar 2004; Gouy et al. 2021). Ambiguous regions of the alignments were trimmed
with trimAl using the “automated1” option (Capella-Gutiérrez et al. 2009) and concatenated with catfasta2phyml 1.1.0
(github.com/nylander/catfasta2phyml). ModelFinder was used to determine the optimal model of sequence evolution
according to the BIC (Kalyaanamoorthy et al. 2017). Maximum likelihood phylogenetic analyses of the individual
and partitioned concatenated alignments were conducted with 1000 ultrafast and SH-aLRT bootstrap replicates using
IQ-TREE 1.6.12 (Guindon et al. 2010; Hoang et al. 2018; Nguyen et al. 2015). The most likely tree was visualized in
FigTree 1.4.4 (github.com/rambaut/figtree) and further edited in Adobe Illustrator 2024.
TABLE 1. Taxa used in the molecular phylogenetic analysis with their corresponding fungarium accession number, voucher
or collection number, location, GenBank accession numbers, and source.
Species Fungarium ID Location ITS LSU Reference
Gyromitra leucoxantha MICH352087 ACD0326 USA-NY ON693641 ON693593 Dirks et al. 2023
Gyromitra leucoxantha ILLS00121418 MP 2018-136 Andorra MW078428 - Miller et al. 2020
Gyromitra leucoxantha MICH25407 NSW4536 USA-MI ON693659 ON693613 Dirks et al. 2023
Gyromitra leucoxantha HMAS279665 NV2017.06.10 Spain MG846991 MG847020 Wang & Zhuang 2019
Gyromitra leucoxantha HOLOTYPE S-F11771 NA Italy OP265175 - Miller et al. 2022b
Gyromitra melaleucoides MICH352039 ACD0308 USA-OR ON693638 ON693590 Dirks et al. 2023
Gyromitra melaleucoides NA iNat48465814 USA-WA OQ701114 - Bouchillon et al.
unpublished
Gyromitra melaleucoides MICH1455 NSW4520 USA-CO ON693658 - Dirks et al. 2023
Gyromitra persicula MICH346505 ACD0508 USA-WA PP051290 PP051281 This study
Gyromitra persicula MICH346493 ACD0515 USA-WA PP051291 PP051282 This study
Gyromitra persicula MICH346500 ACD0521 USA-WA PP051292 PP051283 This study
Gyromitra persicula MICH346515 ACD0524 USA-WA - PP051284 This study
Gyromitra persicula MICH346519 ACD0525 USA-WA PP051293 PP051285 This study
Gyromitra persicula MICH346499 ACD0526 USA-WA PP051294 PP051286 This study
Gyromitra persicula MICH346523 ACD0547 USA-WA PP051295 PP051287 This study
Gyromitra persicula HOLOTYPE MICH346504 ACD0548 USA-WA PP051296 PP051288 This study
Gyromitra persicula MICH346513 ACD0552 USA-WA PP051297 PP051289 This study
Gyromitra persicula FLAS-F-61956 04mwb050917 USA-WA MN653042 - Healy unpublished
Gyromitra sp. NA 420526MF0212 China ON554780 ON527645 Wang et al. 2023
A NOVEL DISCOID GYROMITRA FROM NORTH AMERICA Phytotaxa 644 (1) © 2024 Magnolia Press • 51
RESULTS
The ITS was > 99.7 % identical (no more than two bp differences) across Gyromitra persicula specimens, excluding
MICH346519 (Table 1). Gyromitra persicula MICH346519 had 7–10 bp differences in the ITS (98.9–99.0 % identical
to other specimens) and one bp difference in the LSU, which was otherwise identical across all specimens. All G.
persicula specimens were collected in Washington. For comparison, specimens of G. leucoxantha came from different
continents (Table 1), and their ITS had no more than three bp differences (> 99.3 % identical). Between G. persicula
and G. leucoxantha, the ITS was 95–97 % identical, except for G. persicula MICH346519 and G. leucoxantha
MICH25407, which exhibited 97.8 % similarity. The individual ITS and LSU phylograms were congruent, thus we
present just the phylogram inferred from the concatenated dataset (Fig. 1). The concatenated alignment contained 19
sequences with 1799 nucleotide sites, 334 of which were parsimony-informative. The best-fit substitution models
were HKY+F+G4 for ITS and TNe+I for LSU. The most likely tree strongly supports the delimitation of Gyromitra
leucoxantha and Gyromitra persicula as distinct species.
FIGURE 1. Phylogram of Gyromitra persicula and related taxa obtained from IQ-TREE maximum likelihood phylogenetic analysis of a
concatenated ITS and LSU dataset. The tree is rooted with Gyromitra melaleucoides (Seaver) Pfister (1980: 615). The numbers above the
branches are SH-aLRT and UFBoot support values. Only SH-aLRT values ≥ 80% and UFBoot values ≥ 95% are shown. Sequences from
holotype specimens are in bold. The bar indicates the number of nucleotide substitutions per site.
DIRKS ET AL.
52 • Phytotaxa 644 (1) © 2024 Magnolia Press
FIGURE 2. Ascomata and microscopic features of Gyromitra persicula (A–G) and ascospores of Gyromitra leucoxantha (H–I). A.
Ascomata of the holotype collection (MICH346504). B. Peach-colored ascoma (MICH346523). C. Hymenium and excipulum
(MICH346493). D. Asci and paraphyses with brown contents in KOH (MICH346493). E. Medullary excipulum of textura epidermoidea
(MICH346493). F. Ascospores at various stages of maturity; note the blunt apiculi with a concave depression, large central guttule,
and minute ornamentation (MICH346519). G. Close up of ascospores showing the pronounced “fishtail” apiculi and fine reticulation
(MICH346519). H–I. Ascospores of Gyromitra leucoxantha (MICH25407); similar to those of G. persicula but with a more pronounced
reticulation. Mounting medium: 5% KOH (C–D), 5% KOH stained with phloxine B (E–F, leftmost ascospore in G, H), Melzer’s reagent
(two rightmost ascospores in G, I). Scale bars: 2 cm (A), 0.5 cm (B), 100 µm (C), 50 µm (D–F, H), 10 µm (G, I).
A NOVEL DISCOID GYROMITRA FROM NORTH AMERICA Phytotaxa 644 (1) © 2024 Magnolia Press • 53
TAXONOMY
Gyromitra persicula Dirks & Beug, sp. nov. (Fig. 2)
MycoBank MB852786
Holotype:—USA. Washington: Klickitat Co., Mount Adams Horse Camp, 46.0515 -121.5357, growing from the
ground, 30 May 2022, leg. Michael W. Beug, ACD0548, iNaturalist #119742009 (HOLOTYPE MICH346504;
ISOTYPE ILLS00122612). Sequences: PP051296 (ITS), PP051288 (LSU).
Diagnosis:—Gyromitra persicula and G. leucoxantha are readily distinguished from other Discinaceae species by
the scooped out “fishtail” apiculi of the mature ascospores. These two closely related taxa are separable by their size
and coloration: G. persicula is small (up to 4.5 cm wide) and has a pinkish buff color, whereas G. leucoxantha is larger
(up to 10 cm wide) and has an egg yolk yellow to yellow ochre color.
Description:—Ascomata consisting of a discoid hymenophore and pseudostipe, width 1.5–4.5 cm; hymenium
buff to peach, smooth, becoming wrinkled; sterile underside off-white, drying tan, minutely pubescent; pseudostipe if
present 1 × 1 cm. Medullary excipulum a textura epidermoidea. Paraphyses cylindric, apices subclavate, apical width
6.3–8.5 µm (Wm = 7.4 µm), thin-walled, septate, unbranched, contents golden-brown in KOH. Asci 380–450 µm ×
22–25 µm, cylindric, thin-walled, hyaline, eight-spored. Ascospores 36–44 µm × 16–19 (Lm = 40 µm, Wm = 17 µm,
Q = 2.1–2.5, Qm = 2.3), ellipsoid, with a large central guttule and a varying number of smaller polar guttules, hyaline
in KOH, inamyloid. Perispore present, expanded up to 2 µm in KOH, less noticeable in water, appearing minutely
roughened due to a fine reticulation when mature (best observed in water or Melzer’s reagent), forming blunt apiculi
with concave depressions enlarged in KOH, 3.2–4.9 µm × 6.7–8.9 (Lm = 4.0 µm, Wm = 7.8 µm).
Etymology:—From the Latin persica meaning peach along with the diminutive suffix -ula, referring to the peach
coloration and small size, hence the common name “mini peach pig’s ears”.
Habitat and distribution:—Growing from soil in association with conifers in Washington, 250–1300 m
elevation.
Additional specimens examined:—USA. Washington: King Co., Middle Fork Snoqualmie Natural Area, 274 m,
47.4922 -121.6411, in a mixed conifer forest, 3 June 2011, leg. Michael W. Beug (MWB), ACD0515 (MICH346493);
Klickitat Co., Gotchen Creek Trail, 46.0880 -121.4929, growing from the ground in a mixed conifer forest, 9 June
2011, leg. MWB, ACD0526 (MICH346499) (immature); ibid., 46.1069 -121.5075, in a mixed P. menziesii forest, 21
June 2011, leg. MWB, ACD0521 (MICH346500) (immature); Klickitat Co., Mount Adams Horse Camp, 46.0499
-121.5327, 8 May 2022, leg. MWB, ACD0508, Mushroom Observer #491382 (MICH346505) (immature); Klickitat
Co., Trout Lake, 46.0093 -121.5553, growing from the ground in a mixed conifer forest, 1 May 2005, leg. MWB,
ACD0524 (MICH346515); ibid., 46.0212 -121.5295, on soil, 30 May 2022, leg. MWB, ACD0552, iNaturalist
#119741787 (MICH346513) (immature); Pierce Co., Mt. Rainier National Park, 46.7586 -121.5569, growing from
the ground in an old growth forest, 17 May 2014, leg. MWB, ACD0525 (MICH346519); Skamania Co., Trout
Lake, 45.9688 -121.6573, growing from the ground, 13 June 2022, leg. MWB, ACD0547, iNaturalist #121904369
(MICH346523).
Notes:—While separable by macromorphology, other distinguishing features of Gyromitra persicula and G.
leucoxantha may be the degree of ascospore reticulation and prominence of the scooped-out apiculi in water. Whereas
G. persicula shows a notable but somewhat faint ascospore reticulation, McKnight (1969) described the ascospores
of G. leucoxantha as “very distinctly and coarsely reticulate”. We also observed a somewhat more pronounced
reticulation in the one specimen of G. leucoxantha with mature ascospores that we studied (Fig. 2). The ascospores of
both taxa differ in appearance according to the mounting medium (McKnight 1968). In water, the scooped-out apiculi
of G. persicula could be overlooked but are prominent in KOH due to the expansion of the perispore, resulting in an
overall increase in size of 10–15 %. In contrast, the scooped-out apiculi of G. leucoxantha are more obvious in water
and are less likely to be overlooked. Discinaceae species mature over an extended period and are often inadvertently
collected in an immature state when their ascospores have not yet attained their full size, ornamentation, and apiculi.
More observations of ascospores from fresh deposits (i.e., living ascospores) are required to delineate these finer
microscopic details and to establish a uniform comparison across the discoid Gyromitra (Karakehian et al. 2021).
DIRKS ET AL.
54 • Phytotaxa 644 (1) © 2024 Magnolia Press
DISCUSSION
We describe Gyromitra persicula as new to science based on phylogenetic and morphological analyses. A sufficient
barcode gap exists between G. persicula and G. leucoxantha for reliable identification with ITS; however, the ITS
divergence exhibited by G. persicula MICH346519 suggests further collections are needed to document the full
genetic diversity of G. persicula. More collections are also required to better understand the geographic range of the
mini peach pig’s ears mushroom, which so far is limited to temperate rainforests in Washington state. There is clearly
undocumented diversity within the G. leucoxantha group, as indicated by the closely related and potentially novel
species Gyromitra sp. 420526MF0212 sequenced by Wang et al. (2023). Gyromitra persicula should not be consumed
because, like its sister species G. leucoxantha, it produces gyromitrin (Dirks et al. 2023, unpublished data).
ACKNOWLEDGEMENTS
We thank collection manager Dr. Alison Harrington (MICH) for accessioning new specimens and loaning specimens
for study.
REFERENCES
Beug, M., Bessette, A.E. & Bessette, A.R. (2014) Ascomycete fungi of North America: A mushroom reference guide. University of Texas
Press, Austin, TX, 502 pp.
Capella-Gutiérrez, S., Silla-Martínez, J.M. & Gabaldón, T. (2009) trimAl: A tool for automated alignment trimming in large-scale
phylogenetic analyses. Bioinformatics 25: 1972–1973.
https://doi.org/10.1093/bioinformatics/btp348
Dirks, A.C., Mohamed, O.G., Schultz, P.J., Miller, A.N., Tripathi, A. & James, T.Y. (2023) Not all bad: Gyromitrin has a limited distribution
in the false morels as determined by a new ultra high-performance liquid chromatography method. Mycologia 115: 1–15.
https://doi.org/10.1080/00275514.2022.2146473
Edgar, R.C. (2004) MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research 32: 1792–
1797.
https://doi.org/10.1093/nar/gkh340
Fries, E. (1849) Summa vegetabilium Scandinaviae. A. Bonnier, 572 pp.
Gouy, M., Tannier, E., Comte, N. & Parsons, D.P. (2021) Seaview version 5: A multiplatform software for multiple sequence alignment,
molecular phylogenetic analyses, and tree reconciliation. In: Katoh, K. (Ed.) Multiple sequence alignment: Methods and protocols.
Springer US, New York, NY, pp. 241–260.
https://doi.org/10.1007/978-1-0716-1036-7_15
Guindon, S., Dufayard, J.-F., Lefort, V., Anisimova, M., Hordijk, W. & Gascuel, O. (2010) New algorithms and methods to estimate
maximum-likelihood phylogenies: Assessing the performance of PhyML 3.0. Systematic Biology 59: 307–321.
https://doi.org/10.1093/sysbio/syq010
Harmaja, H. (1969) A wider and more natural concept of the genus Gyromitra Fr. Karstenia 2: 9–12.
https://doi.org/10.29203/ka.1969.53
Hoang, D.T., Chernomor, O., von Haeseler, A., Minh, B.Q. & Vinh, L.S. (2018) UFBoot2: Improving the ultrafast bootstrap approximation.
Molecular Biology and Evolution 35: 518–522.
https://doi.org/10.1093/molbev/msx281
Imai, S. (1932) Contribution to the knowledge of the classification of Helvellaceae. Shokubutsugaku Zasshi 46: 172–175.
https://doi.org/10.15281/jplantres1887.46.172
Jaczewski, A.L.A. (1913) Opredelitel’ Gribov. Sovershennye Griby (Diploidnye Stadii). I. Fikomitsety. Moscow, 934 pp.
Kalyaanamoorthy, S., Minh, B.Q., Wong, T.K.F., von Haeseler, A. & Jermiin, L.S. (2017) ModelFinder: Fast model selection for accurate
phylogenetic estimates. Nature Methods 14: 587–589.
https://doi.org/10.1038/nmeth.4285
Karakehian, J., Quijada, L., Pfister, D., Tocci, G. & Miller, A. (2021) Methods for observing, culturing, and studying living ascospores.
Asian Journal of Mycology 4: 1–18.
A NOVEL DISCOID GYROMITRA FROM NORTH AMERICA Phytotaxa 644 (1) © 2024 Magnolia Press • 55
https://doi.org/10.5943/ajom/4/2/1
McKnight, K.H. (1968) Artifacts on spores of Discineae induced by common reagents. Mycologia 60: 723–727.
https://doi.org/10.2307/3757447
McKnight, K.H. (1969) A note on Discina. Mycologia 61: 614–630.
https://doi.org/10.1080/00275514.1969.12018775
Miller, A.N., Dirks, A.C., Filippova, N., Popov, E. & Methven, A.S. (2022a) Studies in Gyromitra II: cryptic speciation in the Gyromitra
gigas species complex; rediscovery of G. ussuriensis and G. americanigigas sp. nov. Mycological Progress 21: 1–13.
https://doi.org/10.1007/s11557-022-01832-x
Miller, A.N., Karakehian, J. & Raudabaugh, D.B. (2022b) Next-generation sequencing of ancient and recent fungarium specimens. Journal
of Fungi 8: 932.
https://doi.org/10.3390/jof8090932
Miller, A.N., Yoon, A., Gulden, G., Stensholt, Ø., Van Vooren, N., Ohenoja, E. & Methven, A.S. (2020) Studies in Gyromitra I: the
Gyromitra gigas species complex. Mycological Progress 19: 1459–1473.
https://doi.org/10.1007/s11557-020-01639-8
Nguyen, L.-T., Schmidt, H.A., von Haeseler, A. & Minh, B.Q. (2015) IQ-TREE: A fast and effective stochastic algorithm for estimating
maximum-likelihood phylogenies. Molecular Biology and Evolution 32: 268–274.
https://doi.org/10.1093/molbev/msu300
O’Donnell, K., Cigelnik, E., Weber, N.S. & Trappe, J.M. (1997) Phylogenetic relationships among ascomycetous truffles and the true and
false morels inferred from 18S and 28S ribosomal DNA sequence analysis. Mycologia 89: 48–65.
https://doi.org/10.1080/00275514.1997.12026754
Pfister, D.H. (1980) On “Peziza” melaleucoides—A species of Gyromitra from the Western United States. Mycologia 72: 614–619.
Wang, X.-C., Yang, Z.-L., Chen, S.-L., Bau, T., Li, T.-H., Li, L., Fan, L. & Zhuang, W.-Y. (2023) Phylogeny and taxonomic revision of the
family Discinaceae (Pezizales, Ascomycota). Microbiology Spectrum 11: e00207-23.
https://doi.org/10.1128/spectrum.00207-23
Wang, X.-C. & Zhuang, W.-Y. (2019) A three-locus phylogeny of Gyromitra (Discinaceae, Pezizales) and discovery of two cryptic species.
Mycologia 111: 69–77.
https://doi.org/10.1080/00275514.2018.1515456
