Phytotaxa 403 (1): 025–038
https://www.mapress.com/j/pt/
Copyright © 2019 Magnolia Press
ISSN 1179-3155 (print edition)
Article
PHYTOTAXA
ISSN 1179-3163 (online edition)
https://doi.org/10.11646/phytotaxa.403.1.2
Three new species of Incrucipulum (Lachnaceae, Helotiales, Ascomycota) from
Japan
YUKITO TOCHIHARA1,2* & TSUYOSHI HOSOYA2
1
Department of Biological Sciences, Graduate Scholl of Science, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo, 113-0003,
Japan
2
Department of Botany, National Museum of Nature and Science, Amakubo 4-1-1, Tsukuba, Ibaraki, 305-0005, Japan
* Corresponding author: tochi@kahaku.go.jp
Abstract
Three new species of Incrucipulum were described from Japan: I. foliicola and I. pseudosulphurellum on Myrica gale subsp.
tomentosa and I. hakonechloae-macrae on Hakonechloa macra. Disposition to Incrucipulum was justified by molecular
phylogenetic analysis based on ITS-5.8S, LSU and RPB2 regions, and monophyly of Incrucipulum was also confirmed.
Some apomorphic characters of Incrucipulum were identified. By addition of three new species, the genus Incrucipulum
now contains 13 species.
Key words: Incrucipulum foliicola, Incrucipulum hakonechloae-macrae, Incrucipulum pseudosulphurellum, mycobiota,
taxonomy
Introduction
The genus Incrucipulum Baral (1985: 71) belongs to the family Lachnaceae Raitviir (Helotiales, Leotiomycetes,
Ascomycota). Members of Incrucipulum were separated from wider genus Dasyscyphus Nees ex Gray (1821: 670)
(=currently recognized congeneric with Lachnum Retzius (1769: 255) (Hosoya et al. 2010)) based on their thickwalled (0.5–1.3 μm), cubic ectal excipular cells with densely granulated surface (Baral & Krieglsteiner 1985) as
distinguishing characters. Thin to thick-walled hairs with crystallized and thick-walled apices were also mentioned as
common characters (Baral & Krieglsteiner 1985). The structure of ectal excipulum was noted also by Le Gal (1939)
for I. ciliare (Schrader 1799: 63) Baral (1985: 72) and I. capitatum (Peck 1878: 60) Baral (1985: 72) but had not
been adopted as a genus-level taxonomic character at that time. Now the generic concept of Incrucipulum is widely
accepted.
Four species (I. capitatum, I. ciliare [type species], I. sulphurellum (Peck 1878: 59) Baral (1985: 72) and I.
virtembergense (Matheis 1977: 240) Baral (1985: 73)) were originally included (Baral & Krieglsteiner 1985). Six
species were later added: I. densiseptatum (Raitviir & R. Galán (in Galán & Raitviir 1994: 464)) Raitviir (2006a: 45),
I. longispineum (Hosoya & Issh. Tanaka (in Tanaka & Hosoya 2001: 598)) Sasagawa & Hosoya (in Hosoya et al. 2010:
175), I. radiatum (Issh. Tanaka & Hosoya 2001: 606) Sasagawa & Hosoya (in Hosoya et al. 2010: 175), I. saccardoi
(Raitviir & Sacconi (in Raitviir 1991: 166)) Raitviir (2006a: 47), I. sinegoricum (Raitviir 1985: 3) Raitviir (2006b:
140) and I. uralense (Chlebicki 2002: 84) Chlebicki & Suková (in Chlebicki 2005: 109) (Baral & Krieglsteiner 1985,
Index Fungorum 2018, Raitviir 2006b). Species of Incrucipulum occur on various substrates such as fallen twigs,
culms and leaves and have strong selectivity to host species and their parts.
In the process of elucidating Japanese lachnaceous mycobiota, four undocumented species of Incrucipulum,
including three undescribed species and one new record from Japan, were collected. Molecular phylogenetic analysis
was conducted to confirm the validity of inclusion of the three new species into Incrucipulum and identify apomorphic
characters in Incrucipulum.
Accepted by Ruvishika Jayawardena: 17 Apr. 2019; published: 9 May 2019
Licensed under a Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0
25
Material and methods
Collection and Isolation
Five Incrucipulum specimens of three new species and I. capitatum were collected from four localities in Japan.
Specimens were air-dried naturally for one week in 20 °C and deposited in the mycological herbarium of the National
Museum of Nature and Science (TNS). Multi-spored isolates were obtained by collecting discharged ascospores on
potato dextrose agar (PDA, Nissui, Tokyo) and kept on PDA slants at 4 °C. Isolates were numbered beginning with
‘FC-’ and will be deposited to NITE National Bioresource Center (NBRC).
Morphological examination
External appearance of apothecia was examined using SZ61 stereoscopic microscope (Olympus, Tokyo, Japan) and
microphotographs were taken using DS-L4 (Nikon, Tokyo, Japan). To examine microscopic characters, dried apothecia
were detached from the substrate and rinsed in 70% ethanol followed by rehydration in ion-exchanged water for six
hours, and then observed in cotton blue dissolved in lactic acid (CB/LA) in squash mount using BX51 microscope
equipped with Nomarski interference contrast device (Olympus). Microphotographs were taken using DS-L3 with the
camera head DS-Fi2 (Nikon). Line drawings were prepared using drawing device U-DA (Olympus) equipped with
BX51. Ascal apex iodine reaction was checked by Melzer’s reagent.
DNA extraction
Two mL of 2% malt extract broth was inoculated with isolates and cultivated at room temperature for two weeks.
The cultivated mycelia were frozen at −80 °C for two hours, dried by a Bulk Tray Dryer (Labconco, Kansas, USA)
and then crushed using TissueLyser (Quiagen, Hilden, Germany). Powdered fungal samples were incubated in
cetyltrimethylammonium bromide buffer (CTAB buffer, 2% CTAB, 100 mM Tris-HCl pH 8.0, 1.4 M NaCl, 20 mM
EDTA) at 65 °C for one hour, and protein was removed using the mixture of chloroform/isoamylalcohol (24:1). The
solution was purified by 6M sodium iodine buffer (6 M NaI, 50 mM Tris-HCl pH 7.4, 10 mM EDTA, 0.1 M Na2SO3)
(Hosaka & Castellano 2008) with GLASSMILK (Funakoshi, Tokyo, Japan) and washed by ethanol/buffer solution (10
mM Tris-HCl pH 7.4, 1 mM EDTA, 100 mM NaCl, 50% EtOH). Purified DNA was eluted and dissolved in Tris-EDTA
buffer (TE buffer, 10 mM Tris-HCl, 1 mM EDTA).
When isolates were not available, DNA was extracted directly from a fresh apothecium using DNA extraction
buffer (SDS 0.35 mM, Proteinase K 0.1mg/ml, Tris-HCl 10 mM, MgCl2 1.5 mM, KCl 50 mM, dH2O to adjust to
total volume 50 mL). Apothecium was put in a 1.5 mL Eppendorf tube and smashed manually using 1.5 mL pellet
pestle (Fisher Scientific, Hampton, USA). 50 μl DNA extraction buffer was added and incubated for 90 min. at 40 °C
followed by 10 min. incubation at 90 °C. After centrifuged, the supernatant was preserved as extracted DNA.
Extracted DNA samples were deposited to Molecular Biodiversity Research Center in the National Museum of
Nature and Science (Tsukuba, Ibaraki, Japan) and available for molecular phylogenetic researches.
Polymerase chain reaction (PCR) and sequencing
For phylogenetic analysis, the internal transcribed spacer region of nuclear ribosomal DNA containing partial ITS15.8S-ITS2 (ITS-5.8S), the partial large subunits nuclear ribosomal RNA gene (LSU) and the section ‘6–7’ of the
second largest subunit of the nuclear RNA polymerase II gene (RPB2) were used, because analysis using only ITS5.8S did not make robust trees in inter- and intra-genus level (Hosoya et al. 2010). Extracted DNA was amplified by
PCR using EmeraldAmp PCR Master Mix (Takara, Kusatsu, Japan). For ITS-5.8S, the forward primer ITS1F (CTT
GGT CAT TTA GAG GAA GTA A) (Gardes & Bruns 1993) and the reverse primer ITS4 (TCC TCC GCT TAT TGA
TAT GC) (White et al. 1990) were used. For LSU, the forward primer LR0R (ACC CGC TGA ACT TAA GC) and
the reverse primer LR5 (TCC TGA GGG AAA CTT CG) (Vilgalys & Hester 1990) were used. For RPB2, the forward
primer RPB2-P6F (TGG GGW YTS GTM TGY CCT GC) and the reverse primer RPB2-P7R (CCC ATS GCY TGY
TTA CCC AT) (Liu et al. 1999) were used.
For ITS-5.8S and LSU, 10 μl PCR reaction were performed in a following protocol: initial denaturation for 3 min
at 95 °C, 30 cycles of 94 °C for 35 s, 51 °C for 30 s and 72 °C for 1 min, and a final extension at 72 °C for 10 min. The
PCR products were purified using ExoProStar Sequencing Clean-up kit (Illumina, San Diego, USA).
For RPB2, 20 μl PCR reaction solution was used in a following protocol: initial denaturation for 3 min at 95 °C,
followed by 25 cycles of 95 °C for 45 s, 52 °C for 40 s and 74 °C for 2 min, then another 25 cycles of 95 °C for 50 s, 52
°C for 45 s and 74 °C for 2 min 5 s, and a final extension at 10 min for 72 °C. Appropriate length of amplified DNA in
26 • Phytotaxa 403 (1) © 2019 Magnolia Press
TOCHIHARA & HOSOYA
the agarose gel were checked using the LED transilluminator (Fujifilm Wako, Tokyo, Japan) and cut out using a spatula
and then purified using MonoFas DNA Purification Kit I (GL Sciences, Tokyo, Japan) following the manufacturer’s
instruction.
Cycle sequence reaction was conducted with BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems;
Thermo Fisher Scientific, Waltham, MA, USA) using the same PCR primers mentioned above. Sequencing was carried
out using Applied Biosystems 3500xL Genetic Analyzer. Sequences obtained in the present study were assembled using
ATGC Ver. 7 (Genetyx, Tokyo, Japan), and then registered to DNA Data Bank of Japan (DDBJ) database synchronized
with the GenBank.
For TNS-F-81441, DNA was also extracted directly from an apothecium in addition to DNA extraction from an
isolate (FC-6879), because the isolate turned out to be contaminated by bacteria. Sequences derived from the isolate
and the apothecia were examined separately.
Phylogenetic analysis
For species of Incrucipulum, all available sequences obtained by Hosoya et al. (2010) and newly obtained sequences
were assorted, and 12 OTUs composed of seven species were obtained (Table 1).
For other lachnaceous members, one or two species were selected from five major lachnaceous genera (Albotricha
Raitviir (1970: 40), Brunnipila Baral (1985: 49), Erioscyphella Kirschstein (1938: 384), Lachnellula P. Karsten (1884:
138), Lachnum (sensu stricto)), since monophyly of these genera were already suggested (Hosoya et al. 2010, Perić
& Baral 2014). Species of Capitotricha (Raitviir 1970: 88) Baral (1985: 60), Neodasyscypha Suková & Spooner (in
Suková 2005: 163) and Proliferodiscus J.H. Haines & Dumont (in Haines 1983: 536) not included in Hosoya et al.
(2010) were also added. Since Lachnum varians (Rehm 1900: 94) M.P. Sharma (1986: 411) was presumed as a separate
lineage from Lachnum (sensu stricto) (Hosoya et al. 2010, Guatimosim et al. 2016), L. varians was also included.
Two species of Dasyscyphella Tranzschel (1899: 11), whose generic affiliation is still unresolved, were incorporated.
Species of Lasiobelonium Ellis & Everhart (1897: 136), Trichopeziza Fuckel (1870: 295) and Trichopezizella Dennis
ex Raitviir (in Raitviir 1969: 68) were not included because they were indicated to be phylogenetically apart from
other lachnaceous genera (Hosoya et al. 2010). Hyaloscypha spiralis (Velenovský 1934) J.G. Han, Hosoya & H.D.
Shin (in Han et al. 2014: 161), Urceolella carestiana (Rabenhorst 1866: 189) Dennis (1963: 335) and Hymenoscyphus
varicosporoides Tubaki (1966: 346) were selected as outgroup taxa based on molecular phylogenetic assessment
of Hyaloscyphaceae (Han et al. 2014). In total, 27 lachnaceous OTUs of 22 species and three outgroup OTUs were
gathered (Table 1).
Sequences of 30 OTUs were initially aligned by MAFFT 7 (Katoh 2013) and adjusted manually for each region.
Molecular phylogenetic analysis of 30 OTUs was executed based on Maximum likelihood (ML; Felsenstein 1981)
and Bayesian inference (BI) analyses. The combined sequence datasets were divided into five data partitions (ITS5.8S, LSU and each codon position of RPB2). The optimal model was estimated using Kakusan 4 (Tanabe 2011) with
default settings. In the ML analysis, the separate model was selected based on Akaike’s information criterion (AIC).
ML tree search and ML bootstrap analysis (Felsenstein 1985) with 1,000 bootstrap replications were performed using
RAxML v.8.2.X (Stamatakis 2016). In the BI analysis, proportional model was selected based on Bayesian information
criterion (BIC). Four chains of Metropolis Coupled Markov chain Monte Carlo (MCMCMC) analysis resulted in 5
million generations and sampled every 100 generations using MrBayes v.3.2 (Ronquist et al. 2012). The average
standard deviation of split frequencies (ASDSF) was verified to become <0.01 and each run was checked to become
convergent using Tracer v1.7.1 (Rambaut 2018a). Bayesian Posterior probabilities (BI PPs) were used to evaluate
reliability and 50% majority rule consensus tree was adopted.
Trees were illustrated using FigTree v1.4.4 (Rambaut 2018b) based on ML analysis and ML bootstrap value
(BS) and BI PPs were plotted on each branch. The data matrix was deposited in TreeBASE with accession ID S24265
(available from http://purl.org/phylo/treebase/phylows/study/TB2:S24265).
THREE NEW SPECIES OF INCRUCIPULUM FROM JAPAN
Phytotaxa 403 (1) © 2019 Magnolia Press • 27
28 • Phytotaxa 403 (1) © 2019 Magnolia Press
TABLE 1. Taxa investigated in the molecular phylogenetic analysis. ‘-’ appears when DNA was extracted not from an isolate but directly from an apothecium. Specimens and sequences of
Incrucipulum newly obtained in this study were shown in bold.
Taxon
Specimen no. Locality
Substrate
Collecting
Isolate /
GenBank/DDBJ accession no.
ITS
LSU
RPB2
date
Culture no.
TNS-F-16740 Japan, Nagano, Sugadaira
Culm of bamboo
6/17/2006
NBRC
AB481234 LC438571 AB481354
Albotricha acutipila (P. Karst.) Raitv.
104380
5/18/2005
NBRC
AB481235 LC424943 AB481340
Albotricha albotestacea (Desm.) Raitv. TNS-F-16497 Japan, Nagano, Sugadaira
Culm of Miscanthus sinensis
101346
TNS-F-16635 Japan, Gunma, Agatsuma
Unidentified leaf
4/27/2006
NBRC
AB481255 LC424945 AB481348
Brunnipila fuscescens (Pers.) Baral
104365
TNS-F-65670 Switzerland, Graubünden, Filisur Twig of Prunus spinosa
6/6/2016
FC-6101
LC424834 LC424942 LC425011
Capitotricha bicolor (Bull.) Baral
TNS-F-65752 Switzerland, Verwaltungskreis,
6/4/2016
FC-6075
LC438560 LC438573 LC440395
Capitotricha rubi (Bres.) Baral
Twig of Rubus idaeus
Saicourt
TNS-F-16439 Japan, Kanagawa, Yamakita
4/17/2005
NBRC
AB481239 LC424947 AB481331
Dasyscyphella longistipitata Hosoya
Cupule of Fagus crenata
101335
TNS-F-16527 Japan, Gunma, Agatsuma
Unidentified wood
5/21/2005
NBRC
AB481242 LC438577 AB481336
Dasyscyphella montana Raitv.
102336
TNS-F-80478 Japan, Shizuoka, Oyama
Unidentified wood
6/26/2017
NBRC
LC424837 LC424949 LC425009
Erioscyphella abnormis (Mont.) Baral
113934
Incrucipulum capitatum (Peck) Baral TNS-F-81420 Japan, Hokkaido, Sapporo
LC424838 LC424954 LC438592
6/17/2018
NBRC
Leaf of Quercus crispula
TOCHIHARA & HOSOYA
7/26/2018
113938
NBRC
113940
NBRC
113941
NBRC
113942
-
LC438569 LC438586 LC438599
8/12/2018
-
LC438564 LC438581 LC438594
7/29/2006
NBRC
102347
FC-6822
Incrucipulum ciliare (Schrad.) Baral
Incrucipulum ciliare (Schrad.) Baral
TNS-F-81514 Japan, Saitama, Ogano
TNS-F-81516 Japan, Saitama, Ogano
Leaf of Quercus crispula
Leaf of Castanea crenata
8/12/2018
8/12/2018
Incrucipulum ciliare (Schrad.) Baral
Leaf of Quercus crispula
8/18/2018
Incrucipulum foliicola Tochihara
TNS-F-81520 Japan, Shizuoka, Shizuoka,
Umegashima
TNS-F-81508 Japan, Hokkaido, Nemuro
7/20/2018
Incrucipulum foliicola Tochihara
TNS-F-81526 Japan, Hokkaido, Oshamambe
Incrucipulum hakonechloae-macrae
Tochihara
Incrucipulum longispineum (Hosoya &
Issh. Tanaka) Sasagawa & Hosoya
Incrucipulum pseudosulphurellum
Tochihara
TNS-F-81512 Japan, Saitama, Ogano
TNS-F-17632 Japan, Miyagi, Sendai
Leaf of Myrica gale var.
tomentosa
Leaf of Myrica gale var.
tomentosa
Leaf and culm of Hakonechloa
macra
Leaf of Lyonia ovalifolia
TNS-F-81441 Japan, Hokkaido, Oshamambe
Twig of Myrica gale var.
7/26/2018
LC438568
LC438565
LC438585
LC438582
LC438598
LC438595
LC438566
LC438583
LC438596
LC438567 LC438584 LC438597
AB481256
LC438579 AB481362
LC438563 LC438588 LC438601
tomentosa
...Continued on next page
THREE NEW SPECIES OF INCRUCIPULUM FROM JAPAN
TABLE 1. (Continued)
Taxon
Incrucipulum pseudosulphurellum
Tochihara
Incrucipulum radiatum (Issh. Tanaka &
Hosoya) Sasagawa
Incrucipulum radiatum (Issh. Tanaka &
Hosoya) Sasagawa & Hosoya
Lachnellula calyciformis (Batsch) Dharne
Specimen no.
Locality
TNS-F-81441
Japan, Hokkaido, Oshamambe Twig of Myrica gale
var. tomentosa
Japan, Nagano, Sugadaira
Leaf of Fagus
crenata
Japan, Niigata, Tokamachi
Leaf of Fagus
crenata
Japan, Hokkaido, Engaru
Twig of Abies
TNS-F-16769
TNS-F-36248
TNS-F-81248
Substrate
sachalinensis
Twig of Larix
kaempfer
Unidentified leaf
Phytotaxa 403 (1) © 2019 Magnolia Press • 29
Lachnellula suecica (de Bary ex Fuckel)
Nannf.
Lachnum soppittii (Massee) Raitv.
TNS-F-16529
Japan, Nagano, Sugadaira
TNS-F-16551
Japan, Ibaraki, Mt. Tsukuba
Lachnum varians (Rehm) Spooner
TNS-F-17631
Lachnum virgineum (Batsch) P. Karst.
TNS-F-16583
Japan, Kagoshima, Yakushima Stem of unidentified
fern
Japan, Kanagawa, Yamakita
Unidentified wood
Neodasyscypha cerina (Pers.) Spooner
TNS-F-65625
Proliferodiscus alboviridis (Sacc.) Spooner TNS-F-17436
Switzerland, Verwaltungskreis,
Saicourt
Japan, Ibaraki, Tsukuba
Botanical Garden
Japan, Kumamoto, Kikuchi
TNS-F-17909
Hyaloscypha spiralis (Velen.) J.G. Han,
Hosoya & H.D. Shin (outgroup)
TNS-F-16472
Japan, Ibaraki, Kasumigaura
Hymenoscyphus varicosporoides Tubaki
(outgroup)
TNS-F-18014
Japan, Iwate, Hanamaki
Urceolella carestiana (Rabenh.) Dennis
(outgroup)
Newly described species as well as specimen number and sequences are shown in bold.
a) The duplicate sequence is registered to DDBJ as ‘AB705235’.
Collecting
date
Isolate /
Culture no.
7/26/2018
-
LC438570
LC438587
LC438600
9/24/2006
NBRC
104385
NBRC
113933
NBRC
AB481261
LC438572
AB481359
LC438559
LC438580
LC438593
LC438561
LC438574
LC438590
AB481248
LC424944
AB481341
AB481266
LC438578
AB481344
AB481267
LC438576
AB481330
AB481268a) AB926119
AB481343
LC424836
LC424948
LC425013
LC438558
LC424950
LC438589
LC438602
LC438604
LC438606
AB926052
LC424952
AB481329
LC438603
LC438605
LC438607
9/20/2010
7/12/2017
5/21/2005
5/28/2005
10/23/2005
7/2/2005
Twig of Crataegus
sp.
Unidentified wood
6/8/2016
Unidentified wood
10/10/2005
Unidentified wood
5/5/2005
Stem of Thelypteris
nipponica
5/23/2006
7/8/2006
113935
NBRC
101348
NBRC
104361
NBRC
102332
NBRC
104358
FC-6068
NBRC
108594
NBRC
108585
NBRC
104355
NBRC
108588
GenBank/DDBJ accession no.
ITS
LSU
RPB2
Taxonomy
Incrucipulum foliicola Tochihara, sp. nov. (Figs. 1, 2)
MycoBank no.:—MB829358
Ecology:—Saprotrophic on dead leaves of Myrica gale subsp. tomentosa (C.DC.) E. Murray. Abundant on the fallen
leaves accumulated on the wetlands.
Holotype:—JAPAN. Hokkaido: Yamakoshi-gun, Oshamanbe-cho, Shizukari wetland, 5 m, 42.573644° N,
140.430907° E, 26 June 2018, on dead leaves of Myrica gale subsp. tomentosa, Y. Tochihara (TNS-F-81526!).
FIGURE 1. Incrucipulum foliicola. A: Apothecia scattered on the substrate. B: Fresh apothecium. C: Dried apothecium. D: Ascus. E:
Ascal bases with croziers. F: Paraphysis. G: Hairs with cruciate crystals like ‘cross shuriken’. H: Ascospores. I: Ectal excipular cells with
granulate surface. TNS-F-81508 (A, C–I), TNS-F-81526 (B). Scale bars = 1 cm (A), 0.5 mm (B, C), 10 μm (D–I).
Paratype:—JAPAN. Hokkaido: Nemuro-city, Cape Ochiishi wetland, 47 m, 43.166311° N, 145.512608° E, 20
July 2018, on dead leaves of Myrica gale subsp. tomentosa, Y. Tochihara (TNS-F-81508!).
Etymology:—Referring to the part of substrates.
Description:—Apothecia scattered on the substrate, superficial, cup-shaped, 0.1–0.4 mm in diameter when fresh
and dry, having long and slender stipes, up to 0.6 mm high, externally covered with hairs, lemon yellow throughout
when fresh and dry. Disc concave, concolourous with external receptacle. Ectal excipulum textura prismatica
composed of hyaline cubic cells with granulated surface arranged in parallel rows, thick-walled; wall up to 2 μm thick.
Medullary excipulum textura intricata of hyaline hyphae up to 2 μm wide. Hairs straight, cylindrical, up to 90 × 3–5
μm, densely septate, hyaline, granulated throughout, thick-walled; wall up to 2 μm thick; individual cells 2.5–10 μm
long; apex blunt or a little bit swollen, equipped with one cross-shaped crystal like “cross shuriken” ca. 10 μm across
or one tetrahedral crystal detached easily in squash mounts. Asci 27–37 × 2.5–4 μm, 8-spored, cylindrical-clavate with
slightly protruding apices; apical pore blue in Melzer’s reagent without 3% KOH pretreatment; croziers present at the
basal septa. Ascospores 5–8 × 1.2–1.5 μm, long ellipsoid to narrowly fusiform, aseptate, containing some small lipid
bodies. Paraphyses straight, lanceolate, septate, up to 4 μm wide, exceeding the asci up to 25 μm.
30 • Phytotaxa 403 (1) © 2019 Magnolia Press
TOCHIHARA & HOSOYA
FIGURE 2. Incrucipulum foliicola (TNS-F-81508). A: Apothecium. B: Ascospores. C: Hair with a crystal. D: Ectal excipular cells. E:
Paraphyses. F: Asci.
Notes:—Incrucipulum foliicola, I. pseudosulphurellum and I. sulphurellum occur on Myrica gale L. (including M.
gale. subsp. tomentosa) and share tetrahedral or cross-shaped crystals of hairs (Figs. 1G, 2C) as a common character.
Incrucipulum foliicola much resembles I. sulphurellum which differs in the absence of croziers and parts of host,
based on the original description (Peck 1878) and redescription of Haines (1989). Incrucipulum foliicola differs from
I. pseudosulphurellum in short stipes, the presence of croziers, hyaline ectal excipular cells and parts of host.
Considering that all Incrucipulum species and other lachnaceous species have strong selectivity to the parts of
host and that foliicolous species never occur on twigs and other parts, I. foliicola should be treated as a new species.
Incrucipulum foliicola is currently known from two wetlands in Hokkaido.
Incrucipulum hakonechloae-macrae Tochihara, sp. nov. (Figs. 3, 4)
MycoBank no.:—MB829308
Ecology:—Saprotrophic on fallen and damp leaves, culms and other leaf-like parts of Hakonechloa macra (Munro)
Honda stuck to the ground.
Holotype:—JAPAN. Saitama Pref.: Chichibu-gun, Ogano-machi, Mt. Futagoyama, 1008 m, 36.068342° N,
138.867364° E, 12 August 2018, on dead leaves and culms of Hakonechloa macra, Y. Tochihara (TNS-F-81512!).
Etymology:—Referring to the host plant Hakonechloa macra.
Description:—Apothecia scattered on the substrate, superficial, cup-shaped, 0.2–0.4(–0.5) mm in diameter,
externally covered with short, white, capitate hairs bearing drops of dew when moist, stipitate, up to 0.2–0.5 mm high
when fresh and dry; stipe white, sometimes brown in the lower part. Disc concave, lemon yellow to orange when fresh
and dry. Ectal excipulum textura prismatica composed of hyaline cubic cells like stone pavings, 2.5–15 × 5–13 μm,
containing yellow oil drops, thick-walled; wall up to 3 μm thick with granulate surface; cells arranged in parallel rows.
Medullary excipulum textura intricata of hyaline hyphae up to 2 μm wide. Hairs straight, cylindrical, sometimes
with rounded apices, 38–90 × 3–8 μm, 2–5-septate, hyaline, completely covered by granules, thin to relatively thickwalled; wall up to 1.5 μm thick; apices blunt or a little bit swelled, bearing amber crystal caps detached easily in squash
mounts. Asci 46–63(–75) × 6–10 μm, 8-spored, cylindrical-clavate; apical pore blue in Melzer’s reagent without 3%
KOH pretreatment; croziers present at the basal septa. Ascospores 8–14 × 2.5–5 μm, fusiform, aseptate or rarely
1-septate, usually packed by gelatinous sheath, containing some large lipid bodies conspicuous in CB/LA mount.
Paraphyses straight, cylindrical to narrowly lanceolate, rarely swollen at the apices, sometimes branched near the
bases, septate, up to 3 μm wide, containing hyaline to orange oil drops, exceeding the asci up to 7.5 μm.
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FIGURE 3. Incrucipulum hakonechloae-macrae (TNS-F-81512). A: The host Hakonechloa macra and its litter in the collection site of
I. hakonechloae-macrae. B: Scattered apothecia of I. hakonechloae-macrae occurring on the host leaf. C, D: Fresh apothecia. E: Ascus.
F: Ascus arising from a crozier. G, H: Paraphysis. I: Hair arising from ectal excipular cells with granulate surface. J: Ascospores packed
by gelatinous sheath. K: Ascospore with a septum. L: Ascospores containing large lipid bodies. M: Thick-walled ectal excipular cells
containing orange oil drops. Scale bars = 1 cm (B), 0.5 mm (C, D), 10 μm (E–M).
FIGURE 4. Incrucipulum hakonechloae-macrae (TNS-F-81512). A: Apothecium. B: Ascospores. C: Ectal excipular cells. D: Paraphyses.
E: Hairs with crystals. F: Asci.
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TOCHIHARA & HOSOYA
Notes:—Incrucipulum hakonechloae-macrae is distinguishable from other species of Incrucipulum in yellow
to orange disc (Fig. 3C) and ascospores containing very large oil drops and packed by gel sheath (Figs. 3J–3L).
Ascospores did not germinate in PDA at all. Some spores germinated in water agar or corn meal agar, but no further
extension of hyphae was observed.
Incrucipulum hakonechloae-macrae was collected only from Hakonechloa macra, an endemic grass in the central
Honshu, Japan (Katoh & Ebihara 2011). Incrucipulum hakonechloae-macrae is possibly also endemic to the Honshu
Island corresponding to the host distribution, because most of lachnaceous species have strong host selectivity.
Incrucipulum pseudosulphurellum Tochihara, sp. nov. (Figs. 5, 6)
MycoBank no.:—MB829362
Ecology:—Saprotrophic on fallen twigs of Myrica gale subsp. tomentosa.
Holotype:—JAPAN. Hokkaido: Yamakoshi-gun, Oshamanbe-cho, Shizukari wetland, 5 m, 42.573644° N,
140.430907° E, 26 June 2018, on dead twigs of Myrica gale subsp. tomentosa, Y. Tochihara (TNS-F-81441!).
Etymology:—Referring to the similarity to I. sulphurellum.
FIGURE 5. Incrucipulum pseudosulphurellum (TNS-F-81441). A, B: Fresh apothecia. C, D: Dried apothecium. E: Ascus. F: Ascus
with an irregularly branched base. G: Immature ascus with a crozier-like structure (arrow head). H: Paraphysis. I: Hair with a tetrahedral
crystal. J: Hair with an irregular-shaped crystal. K: Ascospores. L: Hyaline ectal excipular cells. M: Subhyaline ectal excipular cells. N:
Surface of ectal excipular cells with granules (arrow heads). Scale bars = 0.5 cm (A–D), 10 μm (E–N).
Description:—Apothecia scattered, superficial, 0.4–0.8 mm in diameter, having short and dark brown stipes,
up to 0.5 mm high, white to somewhat dull yellow on the upper part and thin to dark brown in the lower part, densely
covered by white, capitate hairs. Disc concave, pure white to orange when fresh and deep orange when dry. Ectal
excipulum textura prismatica to textura angularis, 4–21 × 3–15 μm, hyaline in the upper part and subhyaline to brown
in the lower part, slightly thick-walled; wall up to 2 μm thick; surface smooth or rarely equipped with sparse granules.
Medullary excipulum textura intricata of hyaline hyphae up to 2 μm wide. Hairs straight, cylindrical with slightly
rounded apices, up to 130 × 4.5–7.5 μm, completely granulate, multiseptate, arising from rounded basal cells; hair
cells 5–25 μm long, thin to slightly thick-walled; wall up to 1.5 μm thick; apical cells thick-walled; wall up to 2 μm
thick; apices bearing various shapes of crystals easily detached by squash mounts, i.e. tetrahedral ones, cross-shaped
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ones like ‘cross shuriken’ or amber crystal caps. Asci 33–44(–52) × 3.5–7.5 μm, 8-spored, cylindrical-clavate; pore
blue in Melzer’s reagent without 3% KOH pretreatment; croziers absent, but base sometimes irregularly branched
and forming ‘fake-croziers’. Ascospores 6.3–10 × 1.5–2.2 μm, oblong to narrowly fusiform, aseptate. Paraphyses
straight, lanceolate, up to 5.5 μm wide, exceeding the asci by 25 μm.
Notes:—Incrucipulum pseudosulphurellum resembles I. foliicola and I. sulphurellum, but differs in short and dark
brown stipes (Figs. 5A–5D), brownish ectal excipulum rarely equipped with granules (Figs. 5L–5M). Little granules
on ectal excipular cells is an atypical character of Incrucipulum, but other characters agrees with generic definition.
Incrucipulum pseudosulphurellum is currently known only from the type locality, but probably widely distributed
corresponding to the distribution of host plants.
Wooly appearance and short stipes of this species are similar to Capitotricha bicolor (Bulliard 1789: 410) Baral
(1985: 60) macroscopically but differs in smaller asci and lacking orange oil drops in paraphyses.
FIGURE 6. Incrucipulum pseudosulphurellum (TNS-F-81441). A: Apothecium. B: Ascospores. C: Ectal excipular cells. D: Paraphyses.
E: Hairs with and without crystals. F: Asci.
Results and Discussion
In the molecular phylogenetic analysis, we obtained sequence matrices composed of 350 bases of ITS-5.8S, 808
bases of LSU and 665 bases of RPB2. The optimal model was selected as follow; GTR model with a discrete gamma
distribution (GTR+G) for ML analysis, and K80+G for ITS, GTR+G for LSU, SYM+G for RPB2 first and third codon
position, and HKY85+G for RPB2 second codon position for BI analysis. In the BI analysis, the first 10% of the
generated trees in the cold chain were discarded as the burn-in, because ASDSF became <0.01 and model parameters
converged when 500,000 sample trees generated. ML tree with BI PPs calculated by the remaining trees was shown
(Fig. 7).
All seven species of Incrucipulum formed a strongly supported clade and robust phylogenetic inner relationship
of Incrucipulum was revealed. Inclusion of the three new species into Incrucipulum was justified and monophyly of
Incrucipulum was also confirmed.
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TOCHIHARA & HOSOYA
FIGURE 7. Phylogenetic tree of Incrucipulum and other lachnaceous species constructed by RAxML based on combined sequences
of ITS (350 bases), LSU (808 bases) and RPB2 (665 bases). ML bootstrap values (ML BS; before the slash marks) and BI posterior
probabilities (BI PPs; after the slash marks) were shown in each branch. ‘-’ represents ML BS are under 50% or BI PPs are under 0.90, or a
branch did not appear in BI analysis. ‘䠆’ represents BI PPs are 1.00. Bolded branches show ML BS are 90% or more and BI PPs are 0.95
or more in Incrucipulum clade. Sequences of Incrucipulum species newly described in this study were shown in bold.
Within Incrucipulum, a strongly supported clade excluding I. foliicola and I. pseudosulphurellum was recognized.
Within this clade another strongly supported clade (Clade A), composed of I. ciliare (type species) and other three
species of Incrucipulum, was recognized. The species in Clade A all had cylindrical to narrowly lanceolate paraphyses
while others had lanceolate paraphyses. Since lanceolate paraphyses are shared by most of lachnaceous genera and
Clade A is terminal in Incrucipulum, cylindrical to narrowly lanceolate paraphyses are presumed to be apomorphic and
evolved multiple times.
Species of Clade A were also characterized by large (>10 μm long) and conspicuously fusiform ascospores and
longer asci (mainly > 40 μm long), while others have small (<10 μm long) and oblong to narrowly fusiform ascospores
and smaller asci (mainly < 40 μm long). Fusiform ascospores and larger asci are also presumed to be apomorphic in
Incrucipulum, as has been suggested in the morphological data of Tanaka & Hosoya (2001).
In the present research, I. capitatum was newly collected from Japan. Eight species of Incrucipulum are currently
known from Japan. Five species, I. foliicola, I. hakonechloae-macrae, I. longispineum, I. pseudosulphurellum and I.
radiatum, have been known only from Japan (Katsumoto 2010, Otani 1967, Raitviir 1977, Tanaka & Hosoya 2001,
this study). Considering that lachnaceous species tend to show strong host specificity, it is presumed that species of
Incrucipulum were diversified in the Far East corresponding to characteristic vascular plant flora, such as tall herb
grasslands and bamboo grove (Raitviir 1979).
With the addition of three new species, Incrucipulum now contains 13 species. In addition to the currently
known members, Lachnum radovii Svrček (1984: 201), L. roseum (Rehm 1881: 41) Rehm (1893: 882) and L. soppitii
(Massee 1895: 330) Raitviir (1986: 2) were mentioned as potential members of Incrucipulum (Baral & Krieglsteiner
1985). Lachnum soppitii is phylogenetically apart from the genus Incrucipulum. (Fig. 7). Based on the description
and the illustration of Raitviir (1977) Lachnum lespedezae (Raitviir 1977: 691) Raitviir (1986: 2) is also a potential
member due to cylindrical paraphyses, thick-walled hairs and thick-walled ectal excipulum like stone pavings, but
the presence or absence of granules on ectal excipulum of the type was not documented. We hesitate to propose their
new combinations in this study because type studies and phylogenetic analyses (excluding L. soppitii) have not been
done.
THREE NEW SPECIES OF INCRUCIPULUM FROM JAPAN
Phytotaxa 403 (1) © 2019 Magnolia Press • 35
Key to species of Incrucipulum
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
-
Having obvious lanceolate paraphyses ...............................................................................................................................................2
Having cylindrical to narrowly lanceolate paraphyses .......................................................................................................................6
Having hairs with >6 septa .................................................................................................................................................................3
Having hairs with ≤6 septa ............................................................................................................................................... I. capitatum
Occurring on Myrica gale ..................................................................................................................................................................4
Occurring on dead culms of grasses .......................................................................................................................... I. densiseptatum
Occurring on dead leaves .................................................................................................................................................... I. foliicola
Occurring on dead twigs .....................................................................................................................................................................5
Having hyaline ectal excipulum .................................................................................................................................. I. sulphurellum
Having brownish ectal excipulum .................................................................................................................... I. pseudosulphurellum
Occurring on dead leaves of ericaceous plants...................................................................................................................................7
Occurring on plants except for Ericaceae ...........................................................................................................................................8
Croziers present at the basal septa of asci ...................................................................................................................I. longispineum
Croziers absent at the basal septa of asci..................................................................................................................I. virtembergense
Occurring on dead stems of tall herbaceous plants .......................................................................................................I. sinegoricum
Occurring on other plants ...................................................................................................................................................................9
Croziers present at the basal septa of asci ........................................................................................................................................10
Croziers absent at the basal septa of asci..........................................................................................................................................11
Occurring on dead leaves of Fagus ....................................................................................................................................I. radiatum
Occurring on dead leaves or culms of grasses...............................................................................................I. hakonechloae-macrae
Spores <12 μm long ..........................................................................................................................................................I. saccardoi
Spores ≥12 μm long .........................................................................................................................................................................12
Occurring mainly on dead leaves of fagaceous plants ........................................................................................................... I. ciliare
Occurring on dead leaves of Dryas octopetala ...................................................................................................................I. uralense
Acknowledgments
We are very thankful to Dr. Hidehisa Koba at Obirin University for identifying a host grass, Hakonechloa macra and
Dr. Kentaro Hosaka & Dr. Yasuhide Nakamura at National Museum of Nature and Science for helpful discussion.
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