Morphological characters and molecular data reveal a new species of Hydnocristella (Gomphales, Basidiomycota) from southwestern China

Abstract

Two hydnoid and resupinate specimens were collected from Sichuan Province, southwestern China. They are described and illustrated here as a new species, Hydnocristella latihypha, based on a combination of morphological characters and rDNA sequences data. The new species is characterized by an annual growth habit, resupinate and hydnoid basidiocarps, a monomitic hyphal structure with clamped generative hyphae, wide tramal hyphae, and smooth fusiform basidiospores measured as 10.2–12.2 × 4.2–5.5 μm. In the phylogenetic perspective, H. latihypha is closely related to H. himantia, the generic type, and nested within the Lentariaceae clade, but the latter species has narrow tramal hyphae and smaller basidiospores measured as 8–10 × 4–5 μm.

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Nova Hedwigia Vol. xx (201x) Issue x–x, xxx–xxx
published online xxxxxxxxx x, 201x; published in print xxxxxxx 201x Article
C
Morphological characters and molecular data reveal a new
species of Hydnocristella (Gomphales, Basidiomycota)
from southwestern China
Jia-Jia Chen1, Lu-Lu Shen1 and Bao-Kai Cui*
Institute of Microbiology and Beijing Key Laboratory for Forest Pest Control, P.O. Box 61,
Beijing Forestry University, Beijing 100083, China
With 3 gures and 1 table
Abstract: Two hydnoid and resupinate specimens were collected from Sichuan Province, southwestern
China. They are described and illustrated here as a new species, Hydnocristella latihypha, based on a
combination of morphological characters and rDNA sequences data. The new species is characterized
by an annual growth habit, resupinate and hydnoid basidiocarps, a monomitic hyphal structure with
clamped generative hyphae, wide tramal hyphae, and smooth fusiform basidiospores measured as
10.2–12.2 × 4.2–5.5 µm. In the phylogenetic perspective, H. latihypha is closely related to H. himantia,
the generic type, and nested within the Lentariaceae clade, but the latter species has narrow tramal
hyphae and smaller basidiospores measured as 8–10 × 4–5 µm.
Key words: hydnoid fungi, Lentariaceae, phylogeny, taxonomy, wood-rotting fungi.
Introduction
Kavinia Pilát (1938), typified by K. sajanensis (Pilát) Pilát (=Kavinia alboviridis
(Morgan) Gilb. & Budington), was introduced for fungi with an annual growth habit,
resupinate and hydnoid basidiocarps, a monomitic hyphal structure with clamp
connections, and oblong, subcylindrical or fusiform and non-amyloid basidiospores
bearing cyanophilous warts (Eriksson & Ryvarden 1976, Boidin & Gilles 2000,
Bernicchia & Gorjón 2010). Petersen (1971) indicated that K. himantia (Schwein.)
J.Erikss. differs from other Kavinia species by its smooth basidiospores, and he proposed
another genus Hydnocristella R.H. Petersen to accommodate the hydnoid fungal species
with smooth basidiospores. So far only the type species of Hydnocristella, H. himantia
(Schwein.) R.H.Petersen, was recorded in the genus. Recently, phylogenetic studies
1Jia-Jia Chen and Lu-Lu Shen contributed equally to this work and shared the first author
*Corresponding author: cuibaokai@yahoo.com

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proved that Kavinia and Hydnocristella are distantly related and showed that the two
genera belong to Gomphales (Hosaka et al. 2006, Larsson 2007, Giachini et al. 2010).
Taxonomy and phylogeny of hydnoid wood-rotting fungi in China have been carried
out in the last ten years, and some new species have been described from the country
(Dai et al. 2004; Yuan & Dai 2005, 2009a, b; Dai 2010, 2011; Dai & Li 2010). As
a continuation of these surveys, an additional new hydnoid species belonging to
Hydnocristella was found, and its phylogenetic analysis of the internal transcribed
spacer (ITS) regions and the nuclear large subunit (nLSU) ribosomal RNA gene regions
confirmed its affinity within the genus.
Materials and methods
Morphological study: The studied specimens were deposited in the herbarium of the Institute of
Microbiology, Beijing Forestry University (BJFC). The microscopic routines followed Li et al. (2014).
Macro-morphological descriptions were based on field notes. Color terms followed Petersen (1996).
Microscopic measurements and drawings were made from slide preparations of dried specimens
stained with Cotton Blue, KOH and Melzer’s reagent. Sections were studied at ultimate magnification
×1000 using Nikon Eclipse 80i microscopy and phase contrast illumination. Drawings were made with
the aid of drawing tube. Spores were measured in spine sections. In presenting spore size variation, 5%
of measurements were excluded from each end of the range and given in parentheses. The following
abbreviations were used: KOH = 5% potassium hydroxide, CB = Cotton Blue, CB- = acyanophilous,
IKI = Melzer’s reagent, IKI- = both inamyloid and indextrinoid, L = mean spore length (arithmetic
average), W = mean spore width (arithmetic average), Q = L/W ratio for a specimens studied, n (a/b)
= number of spores (a) measured from given number of specimens (b).
Molecular phylogeny: A CTAB rapid plant genome extraction kit (Aidlab Biotechnologies Co., Ltd,
Beijing) was used to obtain total genomic DNA from dried specimens, according to the manufacturer’s
instructions with some modifications (Chen & Cui 2014). The DNA was amplified with the primers:
ITS5 and ITS4 for ITS (White et al. 1990), and LR0R and LR7 for nLSU (http://www.biology.duke.
edu/fungi/mycolab/primers.htm). The PCR procedure for ITS was as follows: initial denaturation at
95°C for 3 min, followed by 35 cycles at 94°C for 40 s, 54°C for 45 s and 72°C for 1 min, and a final
extension of 72°C for 10 min. The PCR procedure for nLSU was as follows: initial denaturation at
94°C for 1 min, followed by 35 cycles at 94°C for 30 s, 50°C for 1 min and 72°C for 1.5 min, and
a final extension of 72°C for 10 min. The PCR products were purified and sequenced in Beijing
Genomics Institute, China, with the same primers.
Sequences generated in this study were aligned with additional sequences downloaded from
GenBank (Table 1) using ClustalX (Thompson et al. 1997) and manually adjusted in BioEdit (Hall
1999). Sequence alignment was deposited at TreeBase (http://treebase.org/treebase-web/home.html;
submission ID 16335).
Maximum parsimony phylogenetic analysis followed Zhao et al. (2014). It was applied to the
combined dataset of ITS and nLSU sequences using PAUP* version 4.0b10 (Swofford 2002).
Sequences of Bondarzewia sp. and Russula violacea Quél. were used as outgroups to root trees
following Giachini et al. (2010). All characters were equally weighted and gaps were treated as
missing data. Trees were inferred using heuristic search option with TBR branch swapping and 1,000
random sequence additions. Max-trees were set to 5,000, branches of zero length were collapsed
and all parsimonious trees were saved. Clade robustness was assessed using bootstrap analysis with
1,000 replicates (Felsenstein 1985). Descriptive tree statistics tree length (TL), consistency index (CI),
retention index (RI), rescaled consistency index (RC), and homoplasy index (HI) were calculated
for each maximum parsimonious tree generated.
MrModeltest2.3 (Nylander 2004) was used to determine the best-fit evolution model for the combined
dataset of ITS and nLSU sequences for estimating Bayesian inference (BI). Bayesian inference

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was calculated with MrBayes3.1.2 (Ronquist & Huelsenbeck 2003). Four Markov chains were run
for 2 runs from random starting trees for 2 million generations, and trees were sampled every 100
generations. The first one-fourth generations were discarded as burn-in. Majority rule consensus
tree of all remaining trees was calculated. Branches that received bootstrap support for maximum
parsimony (MP) and Bayesian posterior probabilities (BPP) greater than or equal to 75% (MP) and
0.95 (BPP) respectively were considered as significantly supported.
Results
Molecular phylogeny: The ITS+nLSU dataset included sequences from 22 fungal
specimens representing 18 species. The dataset had an aligned length of 1307 characters,
of which 817 are constant, 109 are variable but parsimony-uninformative, and 381 are
Table 1. A list of species, specimens and GenBank accession number of sequences used in this study.
New sequences are shown in bold.
Species Sample no. GenBank accessions
ITS nLSU
Beenakia fricta Maas Geest. K 2083 – AY574693
Bondarzewia sp. DAOM F-415 DQ200923 DQ234539
Clavariadelphus occidentalis Methven OSC 37018 – AY574648
C. truncates Donk OSC 67280 – AY574649
Gautieria crispa E.L.Stewart & Trappe OSC 61308 – DQ218484
G. otthii Trog REG 636 – AF393058
Gloeocantharellus novae-zelandiae (Segedin)
Giachini ZT 68-657 – AF261547
G. okapaensis (Corner) Corner ZT 7135 – AF261548
Hydnocristella himantia (Schwein.)
R.H.Petersen LL 98 AY463435 AY586682
H. latihypha He 20120911-3 KM489521 KM489523
H. latihypha He 20120914-4 KM489522 KM489524
Kavinia alboviridis (Morgan) Gilb. &
Budington EL 16-98 – AY463434
K. alboviridis O 102140 – AY574692
Lentaria dendroidea (O.R.Fr.) J.H.Petersen SJ 98012 EU118640 EU118641
Phaeoclavulina argentea (R.H.Petersen)
Giachini AGK 042 JQ408234 JQ408234
P. argentea AGK 036 JQ408231 JQ408231
Ramaria araiospora Marr & D.E.Stuntz OSC 81497 EU669297 EU669297
R. araiospora OSC 108292 EU669293 EU669293
R. cyaneigranosa Marr & D.E.Stuntz OSC 65703 EU669298 EU669298
R. foetida R.H.Petersen AGK 058 JQ408239 JQ408239
R. maculatipes Marr & D.E.Stuntz Trappe 23383 EU669348 EU669348
Russula violacea Quél. SJ 93009 AF506465 AF506465

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parsimony-informative. Maximum parsimony analysis yielded 4 equally parsimonious
trees (TL = 1144, CI = 0.670, RI = 0.660, RC = 0.442, HI = 0.330). Best model of
evolution for the combined dataset estimated and applied in the Bayesian analysis was
GTR+I+G. Bayesian analysis resulted in a similar topology as MP analysis, with an
average standard deviation of split frequencies = 0.005381.
The newly sequenced specimens from southwestern China were embedded in the
Lentariaceae clade as a distinct lineage with strong support (100% MP and 0.99
BPPs), and is closely related to H. himantia (100% MP and 1.00 BPPs). The resulting
phylogenetic tree was overally consistent with the one reported by Hosaka et al. (2006)
and it resolved a strongly supported Gomphales clade (100% MP and 1.00 BPPs).
Fig. 1. Maximum parsimony strict consensus tree illustrating the phylogeny of Hydnocristella
latihypha and its related taxa based on ITS+nLSU sequences. Branches are labeled with parsimony
bootstrap proportions (before slanting line) higher than 50% and Bayesian posterior probabilities (after
slanting line) more than 0.95. Bold names = New species. The tree is rooted with Bondarzewia sp.

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taxonoMy: Hydnocristella latihypha Jia J.Chen, L.L.Shen & B.K.Cui, sp. nov. (Figs
2, 3)
MycoBank no.: MB 810133
Differs from other Hydnocristella species by its distinctly wide tramal hyphae with
acyanophilous crystals variable in shape and size, and hyaline, large basidiospores
measuring 10.2–12.2 × 4.2–5.5 µm.
Type: China, Sichuan Province, Jiuzhaigou County, Jiuzhaigou Nature Reserve, on fallen trunk of
Abies, 11 Sep 2012, He 20120911-3 (Holotype in BJFC 14552).
rdna sequence ex holotype: KM489521 (ITS), KM489523 (nLSU).
etyMology: latihypha (Lat.) referring to the distinctly wide tramal hyphae.
Fruiting Body: Basidiocarps annual, resupinate, loosely adnate, without odor or
taste when fresh, becoming corky upon drying, up to 12 cm long, 5 cm wide, 10 mm
thick at centre. Spines cream to pinkish buff when fresh, becoming greyish brown to
clay-buff upon drying, up to 5 mm in length, 4–5 per mm at base. Margin with white
rhizomorphs, cottony, white, fibrillose, up to 1 cm wide. Subiculum cottony, white to
cream, up to 5 mm thick.
hyphal structure: Hyphal system monomitic; generative hyphae with clamp
connections, IKI-, CB-; tissues unchanged in KOH. Ampullate septa sometimes present
in the subiculum.
Fig. 2. A fresh basidiocarp of Hydnocristella latihypha (Holotype). Scale bars = 1 cm.

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suBiculuM: Generative hyphae hyaline, thin-walled, occasionally branched, interwoven,
2.5–5 µm in diameter, encrusted with fine, hyaline, variable and acyanophilous crystals.
Big rhombic and acyanophilous crystals occasionally present.
spines: Generative hyphae hyaline, thin- to thick-walled, frequently branched,
interwoven; thick-walled hyphae dominant, smooth, 5.5–10 µm in diameter; thin-walled
hyphae rare, encrusted with tiny, hyaline and acyanophilous crystals, 3–4 µm in diameter.
HyMeniuM: Cystidia and cystidioles absent; basidia clavate with capitate tips, bearing
four sterigmata and a basal clamp connection, 22–27 × 8–10 µm; basidioles similar
to basidia in shape, but smaller.
Spores: Basidiospores more or less fusiform, hyaline, thin-walled, smooth, IKI-, CB-,
(9.8–)10.2–12.2(–12.5) × (4–)4.2–5.5(–5.8) µm, L = 11.45 µm, W = 5.08 µm, Q =
2.18–2.32 (n = 60/2).
additional speciMen exaMined: China, Sichuan Province, Songpan County, Huanglong Nature
Reserve, on fallen trunk of Abies, 14 Sep 2012, He 20120914-4 (Paratype in BJFC 14575).
Fig. 3. Microscopic structures of Hydnocristella latihypha (Holotype). a: Basidiospores. b: A vertical
section through a spine. c: Hyphae from subiculum. d: Generative hyphae bearing irregular and big
crystals in subiculum. e: Generative hyphae bearing tiny crystals in subiculum and spines. f: Rhombic
crystals in subiculum. Scale bars: a–f = 10 µm.

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Discussion
Morphologically, Hydnocristella latihypha is characterized by an annual growth habit,
resupinate and hydnoid basidiocarps, a monomitic hyphal structure with clamped
generative hyphae, presence of distinctly wide and smooth tramal hyphae, presence
of variable crystals, and smooth fusiform basidiospores. Both morphology and rDNA
sequences data confirmed that H. latihypha is a new species in Hydnocristella.
Phylogenetically, Hydnocristella latihypha is closely related to H. himantia (Fig. 1),
both species sharing an annual growth habit, resupinate and hydnoid basidiocarps,
a monomitic hyphal structure with clamp connections, and smooth and inamyloid
basidiospores. However, H. himantia has narrower and smooth tramal hyphae
measuring as 3–4 µm in width, cyanophilous crystals on hyphae, and shorter
basidiospores measured as 8–10 × 4–5 µm (Bernicchia & Gorjón 2010).
In the rDNA-based phylogeny (Fig. 1), Hydnocristella latihypha is nested in the Hydno-
cristella clade and distant from Beenakia D.A. Reid, Kavinia and Lentaria Corner.
Morphologically, Lentaria can be readily distinguished from Hydnocristella mainly
by the coralloid basidiocarps (Petersen 2000); while Kavinia and Beenakia produce
basidiospores bearing small warts, which can be separated them from Hydnocristella
(Núñez & Ryvarden 1994; Bernicchia & Gorjón 2010).
Acknowledgements
The authors are grateful to Prof. Yu-Cheng Dai (BJFC, China) for improving the text. Special thanks
are due to Dr. Shuang-Hui He (BJFC, China) for collecting specimens. The research was financed
by Beijing Higher Education Young Elite Teacher Project (No. YETP0774).
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Manuscript submitted September 10, 2014; accepted November 15, 2014.