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Short communication
Coltriciella minuscula sp. nov., a new species of poroid fungus on Pinus
merkusii from an Indonesian tropical forest
Dewi Susan
a
, Atik Retnowati
b
, Nampiah Sukarno
c
,
*
a
Graduate School of Microbiology, Department of Biology, Faculty of Mathematics and Natural Sciences, Bogor Agricultural University, Kampus IPB
Darmaga, Bogor 16680, Indonesia
b
Herbarium Bogoriense, Botany Division, Research Center for Biology, Indonesian Institute of Sciences, Jl. Raya Jakarta-Bogor KM 46 Cibinong, Bogor 16911,
Indonesia
c
Department of Biology, Faculty of Mathematics and Natural Sciences, Bogor Agricultural University, Kampus IPB Darmaga, Bogor 16680, Indonesia
article info
Article history:
Received 27 September 2016
Received in revised form
5 August 2017
Accepted 9 August 2017
Available online 7 December 2017
Keywords:
Basidiomycota
Hymenochaetales
Phylogeny
Taxonomy
abstract
Coltriciella minuscula sp. nov. is described and illustrated from a specimen collected on Pinus merkusii
from Bogor, Indonesia. This species is characterized by the size of its basidiocarp up to 4 mm in diam,
with long hair on the stipe and with ornamented spores. Both morphological distinctiveness and
phylogenetic separation based upon analyses of nrDNA ITS sequences support the establishment of this
new species. Morphological dissimilarities between C. minuscula and closely related species are
discussed.
©2017 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
The genus Coltriciella was erected by Murrill (1904) as a
monotypic genus typified by C. dependens (Berk. &M.A. Curtis)
Murrill to accommodate species similar to Coltricia Gray but which
are epixylous and have a vertically-attached pileus. The generic
concept was broadened by the discovery of species which form
basidiocarps varying from resupinate, effuse-reflexed, pendent or
stipitate with a monomitic hyphal system, blackish in KOH, colored
and ornamented basidiospores (Corner, 1991; Ryvarden, 2004).
Based on morphological characteristics, Coltriciella was formerly
accommodated in the family Hymenochaetaceae (Dai, 2010;
Ryvarden, 2004; Ryvarden &Johansen, 1980). However, recent
molecular phylogenetic evidence indicates that the genus is distinct
from Hymenochaetaceae, as is the genus Coltricia. Together, these
two genera form a joint Coltricia subclade (Larsson et al., 20 06;
Wagner &Fischer, 2002). Coltriciella comprises 13 species from
tropical and temperate areas (Aime, Henkel, &Ryvarden, 2003;
Valenzuela et al., 2012), Asia (Bian &Dai, 2015; Corner, 1991; Dai,
2010; Dai &Li, 2012; Dai, Cui, He, &Schigel, 2014; Dai &Niemel€
a,
2006), and Australia (Reid, 1963). Species of Coltriciella are
commonly found on wood (Aime et al., 2003; Corner, 1991;
Ryvarden, 2004). Formation of ectomycorrhizal associations is
also known (Tedersoo, Suvi, Beaver, &K~
oljalg, 2007a; Tedersoo,
Suvi, Beaver, &Saar, 2007b).
Coltriciella and Coltricia share similar morphological features,
but the latter differs in having smooth basidiospores (Dai, 2010;
Ryvarden, 1991, 2004). Phylogenetically, Coltriciella and Coltricia
comprise a monophyletic clade (Tedersoo et al., 2007b; Wagner &
Fischer, 2002), but Larsson et al. (2006) has contended that
phylogenetic analysis does not support a separation of the two
genera.
No species of Coltriciella has previously been reported from
Indonesia, but during a recent study of poroid fungi from West Java,
a specimen of Coltriciella was collected from Pinus merkusii Jungh. &
de Vriese. Due to its morphological distinction from other members
of the genus, we describe this as a new species. This is also sup-
ported by phylogenetic analysis inferred from the nrDNA Internal
Transcribed Spacer (ITS) region.
Macroscopic and microscopic features of basidiocarps collected
from Haurbentes Experimental Forest in Bogor, West Java, were
observed in fresh and dried conditions. All the materials were
examined under a Nikon 80i microscope (Nikon Corporation,
Tokyo, Japan). Line drawings were made with the aid of a Nikon Y-
IDN drawing tube (Nikon Corporation). Slides were prepared from
*Corresponding author.
E-mail address: nampiahsukarno@gmail.com (N. Sukarno).
Contents lists available at ScienceDirect
Mycoscience
journal homepage: www.elsevier.com/locate/myc
https://doi.org/10.1016/j.myc.2017.08.005
1340-3540/©2017 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
Mycoscience 59 (2018) 49e53
dried specimens mounted in 5% (w/v) KOH solution, 1% (w/v)
phloxine solution, Melzer's reagent (0.5 g iodine, 1.5 g potassium
iodide, 22 g chloral hydrate, 20 g water) and aniline blue solution
(0.1 mg aniline blue dissolved in 60 g pure lactic acid) (Ryvarden &
Johansen, 1980). Abbreviations used in the morphological
description were as follows: IKI (Melzer's reagent; with
IKI¼inamyloid), CB (cotton blue; CBþ¼cyanophylous;
CB¼acyanophylous), L(mean spore length), W(mean spore
width), Q(the L/W ratio) and n (number of spores measured). Color
terminology followed that of Kornerup and Wanscher (1967). The
examined specimens were afterwards deposited in Herbarium
Bogoriense (BO), Botany Division, Research Center for Biology,
Indonesian Institute of Sciences.
An attempt was made to culture the fungi on Modified Merlin
Norkrans agar medium, however, fungal growth did not occur.
Dried material of the fungus (ca. 10 mg) was ground to powder
with sterile sand. A Genomic DNA Mini Kit (Plant) (Geneaid Biotech
Ltd., New Taipei City, Taiwan) was used to extract DNA from the
dried specimen following the manufacturers' instructions. ITS re-
gion sequences were obtained using primer sets ITS1F/ITS4B
(Gardes &Bruns, 1993). Reactions were performed with the
following cycling parameters: initial denaturation at 94
C for
1 min, then 35 cycles at 94
C for 30 s, annealing at 51
C for 1 min
and extension at 72
C for 1 min, with a final extension at 72
C for
10 min. The amplicons were sequenced by 1st BASE (Selangor,
Malaysia) using primer sets ITS1F/ITS4B.
The ITS sequences obtained in this study were deposited under
GenBank accession Nos. KX086684 for Coltriciella minuscula and
KX159769 for C. aff. subglobosa. Each sequence was compared to the
reference sequences in GenBank, NCBI using BLAST search (http://
blast.ncbi.nlm.nih.gov/Blast.cgi). The fungal taxa used in the
phylogenetic analysis are listed in Table 1. The sequences were then
aligned to those sequences downloaded from GenBank using
MEGA. Phylogenetic analyses were performed using maximum
parsimony and Bayesian inference. Maximum Parsimony analysis
was conducted with PAUP* v.4.0b10 (Swofford, 2003). All charac-
ters were equally weighted and gaps were treated as missing data.
Trees were inferred using the heuristic search option with TBR
branch brach swapping. Clade robustness was assessed using
bootstrap analysis with 1000 replicates (Felsenstein, 1985).
Bayesian analysis was performed with MrBayes 3.2.1 (Ronquist &
Huelsenbeck, 2003). The best-fit model for the dataset was
selected by jModelTest 2.1.5 (Darriba, Taboada, Doallo, &Posada,
2012). Two independent runs with 1,000,000 generations were
performed, with sampling of every 100th generation. The first 25%
of trees were discarded while the last 75% of the trees were used to
construct consensus trees.
1. Taxonomy
Coltriciella minuscula Susan, Retnowati &Sukarno, sp. nov.
Figs. 1 and 2.
MycoBank no.: MB 817889.
Basidiocarps annual, pendent, 1e4 mm diam, stipe short, fibrillose,
hirsute. Hyphal system monomitic. Basidiospores ellipsoid, golden
brown, finely verruculose.
Type: INDONESIA, West Java, Bogor, Haurbentes Experimental
Forest, on trunk base of Pinus merkusii, 4 Jan 2014, leg. D. Susan
DWS1192 (holotype, BO22806).
Gene sequences ex-holotype: KX086684 (ITS).
Etymology: minuscula, referring to the size of the basidiocarp.
Basidiocarps pendent, light in weight. Stipe dorsally attached or
Table 1
eList of GenBank accession numbers of sequences used in the phylogenetic analysis.
Species name Specimen reference Origin GenBank accession no.
Coltriciella baoshanensis Dai 13075 China, Yunnan, Gaogling Mts. KC857266
C. baoshanensis Dai13072 China KU360700
C. dependens TU103078 Seychelles, Mah
e, Casse Dent AM412254
C. dependens TAA195099 Seychelles, Mah
e, L'Abondance AM412253
C. dependens TU103078 China, Fujian, Fuzhou AM412252
C. globosa L.S. Bian &Y.C. Dai Cui 7545 China, Guangdong, Ruyang KJ540930
C. navispora T.W. Henkel, Aime &Ryvarden TH9529 Guyana KT339262
C. navispora MCA3921 Guyana KC155386
C. oblectabilis (Lloyd) Kotl., Pouzar &Ryvarden TH9187 Guyana KC155387
C. pseudodependens Cui 8138 China, Yunnan, Baoshan KJ540931
C. pusilla (Imazeki &Kobayashi) Corner Dai15168 China KU360701
C. subglobosa Y.C. Dai Dai15158 China KU360702
Coltricia confluens P.-J. Keizer TAA181460 Estonia AM412241
C. confluens GO-2009-444 Mexico, Puebla, Chalchicomula De Sesma KC152085
C. confluens GO-2009-008 Mexico, Mexico State, Temascaltepec KC152083
C. confluens GO-2009-483 Mexico, Tlaxcla, Huamantla KC152084
C. perennis (L.) Murrill AFTOL-ID 447 clone 2 DQ234560
C. perennis AFTOL-ID 447 clone 1 DQ234559
C. perennis AFTOL-ID 447 clone 3 DQ234561
Inonotus zonatus Y.C. Dai &X.M. Tian Cui 6631 China JQ860305
Trametes elegans (Spreng.) Fr. USA AY684178
Phellinus fragrans M.J. Larsen &Lombard CBS 202.90 USA AY558619
Fig. 1. Basidiocarps of Coltriciella minuscula (BO22806). Bar: 0.5 cm.
D. Susan et al. / Mycoscience 59 (2018) 49e5350
more contracted vertex, 1e2 mm long, fibrillose, hirsute. Pilei
1e4 mm diam, conico-campanulate when young, becoming more
or less peltate, fused laterally when mature, brown (6F8); margin
encircling, entire, thin, light brown (6D7), 1 mm wide; surface
fibrillose tomentose, soft and cottony when dry, no radial zones.
Pore surface light brown (6D7), angular, 2e3/mm, dissepiments
thin, entire. Context rust brown (6E8), soft, spongy, fibrillose. Tubes
dark brown (6F7), up to 2 mm deep. Mycelial strands easily seen on
substrates.
Hyphal system monomitic, generative hyphae with simple
septa, IKI,CB; tissue darkening in KOH. Contextual hyphae hy-
aline to yellowish-brown, slightly thick-walled, moderately
branched, interwoven, 2e4
m
m diam, some finely verruculose, up to
1 mm thick, black in KOH. Tramal hyphae yellowish brown to
brown, moderately thick-walled, moderately branched, more or
less parallel along the tubes or loosely interwoven, 6e8
m
m diam.
Cystidia absent. Basidia shortly clavate to shortly cylindrical, thin-
walled, 2-sterigmate with basal simple septum. Basidiospores
ellipsoid, golden brown, thick walled, finely verruculose, IKI,CB,
(5.7e)5.8e7.2(e7.3) (3.7e)3.8e4.8(e5.1)
m
m. L¼6.6
m
m.
W¼4.3
m
m. Q¼1.53 (n ¼30/1).
Habit, habitat and distribution: Congregated on trunk base of
living Pinus merkusii. Known from type locality.
Phylogenetic tree constructed by Maximum Parsimony and
Bayesian Inference analyses has yielded well resolved clades with
high bootstrap support. Maximum Parsimony analysis yielded the
most parsimonious trees (TL ¼1648, CI ¼0.677, RI ¼0.761,
RC ¼0.515, HI ¼0.323). All clades were supported with a bootstrap
Fig. 2. Line drawing of Coltriciella minuscula (BO22806). A: Basidiocarps. B: Basidiospores. C: Basidia. D: Hyphae from trama. E: Hyphae from context. Bars: A 1 mm; B 2.5
m
m; C
5
m
m; D, E 10
m
m.
D. Susan et al. / Mycoscience 59 (2018) 49e53 51
value >50%. All accessions were well separated from outgroups
(bootstrap value 100%). The new sequence for the Indonesian ma-
terials was embedded in the Coltriciella clade (Fig. 3). The tree was
submitted to TreeBASE (TB2:S20266).
Morphological and molecular evidence show that C. minuscula is
distinct from known species of Coltriciella.Coltriciella minuscula is
characterized by the small and pendent basidiocarps, lack of a
distinct concentric zone on pileus surface, presence of distinctly
hirsute stipe, 2 sterigmate basidia, and basidiospores measuring
5.8e7.2 3.8e4.8
m
m. This species is morphologically similar to
C. dependens (Berk. &M.A. Curtis) Murrill and C. pseudodependens
L.S. Bian &Y.C. Dai by having pendant basidiocarps. The former
species, however, has a larger pileus up to 20 mm wide with
distinct zones, a stipe up to 1 cm long, 4 sterigmate basidia, and
larger basidiospores measuring 7e10 4e6
m
m(Ryvarden &
Johansen, 1980). The latter differs from C. minuscula by the pileus
up to 6 mm wide, 4 sterigmate basidia, and basidiospores
9e11.8 5e6.2
m
m(Bian &Dai, 2015).
Disclosure
The authors declare no conflicts of interest. All the experiments
undertaken in this study comply with the current laws of Indonesia.
Acknowledgments
This study was supported in part by a Ministry of Research and
Technology-Higher Level Study Directory scholarship 2013, and by
the JST-JICA SATREPS Project FY 2015. We thank Dr. Iman Hidayat,
Microbiology Division, Research Center for Biology, Indonesian
Institute of Sciences for constructive suggestions and to the Center
for Forest Productivity Improvement Research and Development,
Fig. 3. Strict consensus tree inferred from Maximum Parsimony and Bayesian inference analyses of ITS region. Parsimony bootstrap values (before the slash markers) higher than
50% and Bayesian posterior probabilities (after the slash markers) more than 0.80 were indicated along branches.
D. Susan et al. / Mycoscience 59 (2018) 49e5352
Forestry Research and Development Agency (FORDA) for giving
permission to collect research material from Haurbentes Experi-
mental Forest. We also thank Mr. Liam Trethowan from Manchester
Metropolitan University (MMU), United Kingdom and Dr. Graham
E. Eagleton (New South Wales, Australia for English language
editing.
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