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New species and records of Pyxine (Caliciaceae) in China 1
New species and records of Pyxine (Caliciaceae) in
China
Mei-Xia Yang1,2,3, Xin-Yu Wang3, Dong Liu3,4, Yan-Yun Zhang3, Li-Juan Li3,
An-Cheng Yin3, Christoph Scheidegger1,2, Li-Song Wang3
1 Swiss Federal Institute for Forest, Snow and Landscape Research (WSL), Zürcherstrasse 111, CH-8903
Birmensdorf, Zurich, Switzerland 2 University of Bern, Hochschulstrasse 6, 3012 Bern, Switzerland 3 Key
Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy
of Sciences, Heilongtan, Kunming, Yunnan 650201, China 4 Korean Lichen Research Institute (KoLRI), Sun-
chon National University, 255 Jungang-Ro, Suncheon, Korea
Corresponding author: Li-Song Wang (wanglisong@mail.kib.ac.cn)
Academic editor: G. Rambold|Received20 September 2018|Accepted 10 December 2018|Published @@ ######2018
Citation: Yang M-X, Wang X-Y, Liu D, Zhang Y-Y, Li L-J, Yin A-C, Scheidegger C, Wang L-S (2018) New species
and records of Pyxine (Caliciaceae) in China. MycoKeys @: @@–@@. https://doi.org/10.3897/mycokeys.@.29374
Abstract
In this study, the diversity of Pyxine Fr. in China was assessed based on morphological and chemical traits and
molecular data are inferred from ITS and mtSSU sequences. Nineteen species were recognised, including
three that are new to science (i.e. P. avicans M. X. Yang & Li S. Wang, P. hengduanensis M. X. Yang & Li S.
Wang and P. yunnanensis M. X. Yang & Li S. Wang) and three records new to China were found (i.e. P.cog-
nata Stirt., P. himalayensis Awas. and P. minuta Vain.). Pyxine yunnanensis is diagnosed by the small size of the
apothecia, a white medulla of the stipe and the presence of lichexanthone. Pyxine avicans is characterised
by broad lobes, a pale yellow medulla of the stipe and the presence of atranorin. Pyxine hengduanensis can be
distinguished by its pale yellow medulla, marginal labriform soralia and the absence of atranorin. Detailed
descriptions of each new species are presented, along with a key to the known species of Pyxine in China.
Keywords
China, lichenised fungi, new species, phylogeny
Copyright Mei-Xia Yang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC
BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
MycoKeys @: @@–@@ (2018)
doi: 10.3897/mycokeys.@.29374
http://mycokeys.pensoft.net
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RESEARCH ARTICLE
Mei-Xia Yang et al. / MycoKeys @: @@–@@ (2018)
2
Introduction
e lichen genus Pyxine was rst established by Fries (1825). Molecular data support the
placement of Pyxine in a clade of taxa that were previously placed in Physciaceae and the
circumscription of the family has thus changed to Caliciacese (Wedin and Grube 2002;
Crespo et al. 2004; Gaya et al. 2012; Prieto and Wedin 2016). Pyxine is characterised
by an adnate foliose thallus, an internal stipe colour of apothecia, dark brown hypothe-
cium and generally two-celled brown ascospores (Awasthi 1982; Elix 2009; Kalb 1987;
Kalb 2004). e genus Pyxine consists of approximately 70 species. Most species are
pantropical to subtropical and a few species extend into temperate or oceanic regions
(Elix 2009; Mongkolsuk et al. 2012; Kalb 1987; Moberg 1983; Wei and Hur 2007).
Regional studies on this genus have been carried out in Australia (Elix 2009), Bra-
zil (Aptroot et al. 2014), India (Awasthi 1982; Nayaka et al. 2013), ailand (Mong-
kolsuk 2012) and North and Central America (Imshaug 1957; Jungbluth and Marcelli
2011). Prior to this study, 13 species have been reported in China, including Pyxine
berteriana, P. cocoes, P. consocians, P. copelandii, P. coralligera, P. endochrysina, P. limbu-
lata, P. meissnerina, P. microspora, P. petricola, P. philippina, P. sorediata and P. subcinerea
(Hu and Chen 2003; Obermayer and Kalb 2010; Wei 1991).
Although many studies have been conducted, few molecular phylogenetic analyses
have been completed (Gaya et al. 2012; Schmull et al. 2011; Prieto and Wedin 2016).
In this study, morphological, chemical and molecular phylogenetic analyses were com-
bined in order to re-evaluate the species composition and phylogenetic relationship
of this genus in China. In our study, 31 sequences were newly generated from freshly
collected specimens.
Methods
Morphological and chemical analyses
e specimens examined in this study were collected from the Hengduan Mountains re-
gion, Taiwan, Zhejiang, Hainan et al. from 1941 to 2016 and deposited in KUN-L (325
specimens) and in the Institute of Microbiology (HMAS-L, 5). Morphological character-
istics were studied using a dissecting microscope (Nikon SMZ745T) and a light micro-
scope (Nikon Eclipse Ci-S; Nikon Instruments, Tokyo Japan). Sections were made with a
razor blade under a dissecting microscope and anatomical characteristics were examined
and measured using a micrometer under light microscopy. Ten measurements each of the
thallus, apothecia and ascospore dimensions were taken from a single apothecium per
specimen and the ranges of these measurements, from smallest to largest, are presented
in this study. e lichen secondary metabolites were analysed using spot reactions and
thin-layer chromatography in a solvent C system, according to Orange et al. (2001).
New species and records of Pyxine (Caliciaceae) in China 3
DNA extraction and sequencing
Total genomic DNA was extracted from dried herbarium specimens using AxyPrep
Multisource Genomic DNA Miniprep Kit 50-prep (Qiagen) according to the man-
ufacturer’s instructions. ITS (nrDNA ITS1-5.8S-ITS2) and mtSSU (mitochondrial
small subunit rDNA) were amplied by polymerase chain reactions (PCR) using the
primer pairs ITS1F (Gardes and Bruns 1993), ITS4 (White et al. 1990) and mtSSU1/
mtSSU2R (Zoller et al. 1999).
Amplications were performed in a 25 μl volume comprising 12.5 μl of 2×
MasterMix (TapDNA Polymerase, 0.1 units/μl; technologies Co. Ltd), 1.0 μl of
each primer, 8.5 μl ddH2O and 2 μl DNA. Conditions for the PCR were: initial
denaturation at 94°C for 4 min, 34 cycles at 94 °C for 1 min, 54 °C for 1 min and
72 °C for 1.5 min, with a nal extension at 72 °C for 10 min. PCR products were
sequenced in an ABI3730X using amplication primers manufactured by Tsingke
(Kunming, China).
ITS and mtSSU sequences were assembled with Seqman 7.0 (DNAStar) and man-
ually edited using Mega6. DNA sequences were aligned with MAFFT version 7 with
default parameters (Katoh et al. 2005) via the online resource (http://mat.cbrc.jp/
alignment/server/index.html).
Phylogenetic analyses
Maximum likelihood (ML) and Bayesian inference (BI) were conducted based on
the two gene fragments combining ITS and mtSSU. e best-tting substitution
model was determined using MrModeltest 2.3 (Nylander 2005) and PAUP*4b10
(Swoord 2003), where the AIC values were calculated using JModelTest 3.7 (Posa-
da 2008). ML analyses were performed using RAxML7.0.4 (Stamatakis 2006) with
default settings (GTR) and support values were inferred from the 70% majority-
rule tree based on 1000 non-parametric bootstrap pseudo-replicates. e Bayesian
analyses were performed using MrBayes v3.1.2 (Huelsenbeck and Ronquist 2001)
with 2,000,000 generations and four incrementally heated chains. MCMC (Markov
Chain Monte Carlo) analysis started from a random tree that was sampled every
1000th generation, with the rst 10% of trees discarded as burn-in. A majority-
rule consensus tree was constructed from the remaining trees to estimate posterior
probability (PP), with values greater than or equal to 0.95 considered indicative of
strong support. Tracer v1.6 (Rambaut and Drummond 2003) was used to ensure
that stationarity was achieved by checking whether the log-likelihood values of sam-
ple points reached a stable equilibrium. Phylogenetic trees were visualised using the
programme FigTree 1.4.0 (Rambaut 2012). Physcia dubia and Dirinaria applanata
were selected as outgroups.
Mei-Xia Yang et al. / MycoKeys @: @@–@@ (2018)
4
Results
Nineteen species were recognised, including three that are new to science (i.e. Pyxine
avicans M. X. Yang & Li S. Wang, P. hengduanensis M. X. Yang & Li S. Wang and P.
yunnanensis M. X. Yang & Li S. Wang) and three records new to China were found (i.e.
P. cognata Stirt., P. himalayensis Awas. and P. minuta Vain.). Of the 39 sequences that
were included in the phylogenetic analyses, 31 were newly generated (Table 1). A phy-
logenetic analysis using ITS and mtSSU sequences revealed 15 species. We were unable
to obtain sequences from P. copelandii, P. coralligera, P. microspora and P. philippina, but
the Chinese specimens agreed morphologically and chemically with the current circum-
scription of these species (Hu and Chen 2003; Obermayer and Kalb 2010; Wei 1991).
e ITS and mtSSU datasets were analysed separately and concatenated; both
parsimony and Bayesian trees of ITS vs. mtSSU were congruent. A maximum likeli-
hood phylogenetic tree was inferred from the combined dataset of ITS and mtSSU
(Fig. 1). e monophyly of each species and the phylogenetic relationships between
species were well supported (Fig. 1; MLBS > 90%, PP > 0.95). Specically, the three
new species were all monophyletic with a high support value: Pyxine yunnanensis
(MLBS = 97%, PP = 1.00), P. avicans (MLBS = 99%, PP = 0.99) and P. hengduan-
ensis (MLBS = 98%, PP = 1.00).
Species of Pyxine were separated into two main clades, as inferred from the phylo-
genetic tree with strong support (Fig. 1). e ten species in Clade 1 are all characterised
by the presence of soralia or isidia on the thallus, whereas the ve species in Clade 2
contain lichexanthone and lack soralia and isidia. e two species P. petricola and P.
cocoes are characterised by the presence of both lichexanthone and soralia.
Taxonomic treatment
Nineteen Pyxine species were conrmed in China, including three species new to science
and three species hereby newly reported for the country, based on the following charac-
teristics: presence of isidia and soredia, colour of the medulla, main compounds, reaction
of K on the internal stipe of apothecia, nature of the substrate and colour of the thallus.
New species
Pyxine avicans M. X. Yang & Li S. Wang, sp. nov.
MycoBank No.: MB 819956
Figure 2
Holotype. CHINA, YUNNAN PROVINCE, Nujiang Perf., Chide Vil., 1916 m el-
evation, 27°42'32"N, 98°43'19"E, on Juglans, 4 Aug 2015, L. S. Wang et al. KUN-
L15-48196. GenBank accession No.: ITS = KY611884, mtSSU = KY751391.
New species and records of Pyxine (Caliciaceae) in China 5
Table 1. Specimen information and GenBank accession numbers for taxa used in this study. Newly
obtained sequences are shown in bold.
Taxa Locality Voucher specimens Accession Number
ITS mtSSU
Pyxine sorediata 1China: Yunnan KUN 12-36993 KY611891 KY751398
P. sorediata 2China: Yunnan KUN 15-48546 KY611892 KY751399
P. sorediata 3Sweden Wetmore 91254
(UPS)
JX000111 –
– KX512973
P. hengduanensis 1China: Yunnan KUN 15-48082 KY611889 KY751396
P. hengduanensis 2China: Yunnan KUN14-43258 KY611890 KY751397
P. endochrysina 1China: Xizang KUN 14-46462 KY611887 KY751394
P. endochrysina 2China: Xizang KUN 14-46439 KY611888 KY751395
P. limbulata 1China: Taiwan KUN 15-49117 KY611885 KY751392
P. limbulata 2China: Taiwan KUN 15-49153 KY611886 KY751393
P. himalayensis 1 China: Yunnan KUN 12-36055 KY611881 KY751388
P. himalayensis 2China: Xizang KUN 14-46410 KY611882 KY751389
P. avicans 1China: Yunnan KUN 14-43995 KY611883 KY751390
P. avicans 2China: Yunnan KUN 15-48196 KY611884 KY751391
P. meissnerina 1China: Yunnan KUN 12-34386 KY611877 KY751384
P. meissnerina 2China: Yunnan KUN 12-34377 KY611878 KY751385
P. consocians 1China: Yunnan KUN 15-49942 KY611879 KY751386
P. consocians 2China: Yunnan KUN 15-47417 KY611880 KY751387
P. petricola 1China: Yunnan KUN 13-40715 KY611875 KY751382
P. petricola 2China: Sichuan KUN 13-39419 KY611876 KY751383
P. cocoes 1China: Taiwan KUN 15-49457 KY611874 KY751381
P. minuta 1China: Yunnan KUN 13-40695 KY611872 KY751379
P. minuta 2China: Yunnan KUN 13-40630 KY611873 KY751380
P. yunnanensis 1China: Yunnan KUN 13-41372 KY611870 KY751377
P. yunnanensis 2China: Yunnan KUN 13-40596 KY611871 KY751378
P. berteriana 1China: Yunnan KUN 15-47921 KY611868 KY751375
P. berteriana 2China: Yunnan KUN 14-43730 KY611869 KY751376
P. subcinerea 1China: Taiwan KUN 15-48998 KY611866 KY751373
P. subcinerea 2China: Taiwan KUN 15-49012 KY611867 KY751374
P. subcinerea USA NC 27708 HQ650705 –
Spain MAF9852 – AY464080
P. cognata 1China: Yunnan KUN 14-43569 KY611864 KY751371
P. cognata 2China: Yunnan KUN 13-40767 KY611865 KY751372
P. berteriana var.
himalaica 1China: Yunnan KUN 14-43571 KY611862 KY751369
P.berteriana var.
himalaica 2China: Yunnan KUN 13-40706 KY611863 KY751370
Dirinaria
applanata
India – EU722342 –
Spain MAF 9854 – AY464079
Physcia dubia Finland T. Ahti 69359 JQ301695 –
– JQ301536
Description. allus 5–9 cm wide, attached to closely adnate. Lobes radiating,
plane to convex, but often slightly concave towards the tips, (0.5) 1–3 (4) mm wide,
subround at the apices. Upper surface white-grey to celadon, sparsely pruinose at the
lobe tips or epruinose, isidia and soredia absent. Medulla pale yellow above, white
below. Lower surface black in the centre, paler towards the margin; rhizines dense,
Mei-Xia Yang et al. / MycoKeys @: @@–@@ (2018)
6
Figure 1. Phylogenetic relationship of Pyxine species occurring in China inferred from ITS and
mtSSU sequences using maximum likelihood (values refer to ML bootstrap frequencies and Bayesian
posterior probabilities).
furcate. Apothecia common, (0.5) 0.8–1.5 (2) mm wide, constricted at base, plane
to possibly convex; margin black. Hymenium height 80–120 μm; hypothecium light
brown to brown, internal stipe K– pale yellow to yellow; spores brown, two-celled,
18–20 × 6–8 μm. Upper cortex K+ yellowish, UV–; medulla K–, C–; containing atra-
norin, chloroatranorin (minor), zeorin and unknown terpenes.
Habitat and distribution. Growing on bark of Quercus and Picea spp. and on
rocks around 1916–4000 m elevation in semi-arid environments; only known from
south-western China.
Etymology. e epithet avicans refers to the yellow medulla and internal stipe of
the apothecia.
New species and records of Pyxine (Caliciaceae) in China 7
Figure 2. Pyxine avicans (KUN-L 15-48196) photographed by Li-Song Wang and Meixia Yang.
Ahabit B yellow internal stipe of apothecia C hymenium D ascospores from GAW (glycerine:alcohol:w
ater=1:1:1). Scale bars: 5 cm (A); 0.5 cm (B); 100 μm (C); 20 μm (D).
Notes. Pyxine avicans is characterised by at corticated yellowish-grey to brown-
ish-grey thalli, a constricted base, a pale yellow medulla and the presence of atranorin.
is species resembles P. berteriana in terms of lobe size, saxicolous habitat and
internal stipe, but the latter has a yellow to yellowish-orange medulla and produces li-
chexanthone (Hu and Chen 2003). Pyxine avicans is similar to P. australiensis Kalb re-
garding the absence of soredia and isidia and both species are frequently lignicolous but
occasionally grow on rocks. However, P. avicans diers from P. australiensis in having
marginal and laminal pseudocyphellae, lichexanthone and a white medulla in the stipe
(Elix 2009). Pyxine avicans is similar to P. himalayensis in terms of the type of apothe-
cia and lack of lichexanthone. However, P. himalayensis has a colourless internal stipe.
Selected specimens examined (KUN). CHINA: SICHUAN PROVINCE: Muli
Co., 2850 m elev., on Pinus yunnanensis, 23 Aug 1983, L. S. Wang 83-1869(A); XI-
ZANG PROVINCE: Chayu Co., along the road from Muruo Vil. to Bingzhongluo,
3833 m elev., 28°35.781'N, 98°06.404'E, on Pinus armandii, 26 Sep 2014, L. S. Wang
et al. 14-46763; YUNNAN PROVINCE: Jianchuan Co., Shibao Mt., 2620 m elev.,
26°22.920'N, 99°49.811'E, on bark, 24 Jun 2014, L. S. Wang et al. 14-43995; Nuji-
ang Co., Chide Vil., 1916 m elev., 27°42'32.40"N, 98°43'18.59"E, on Juglans, 4 Aug
2015, L. S. Wang et al. 15-48196.
Mei-Xia Yang et al. / MycoKeys @: @@–@@ (2018)
8
Pyxine hengduanensis M. X. Yang & Li S. Wang, sp. nov.
MycoBank No.: MB 819957
Figure 3
Holotype. CHINA, YUNNAN PROVINCE, Nujiang Pref., Dizhengdang Vil., 1858
m elevation, 28°05'00.86"N, 98°19'39.97"E, on bark, 2 Aug 2015, L. S. Wang et al.
KUN-L 15-48082. GenBank accession No.: ITS = KY611889, mtSSU = KY751396.
Description. allus corticolous, 4–9 cm wide, rmly to loosely adnate to sub-
strate. Lobes linear, compact, imbricate to discrete, (0.5) 1–2.5 mm wide, upper cortex
plane but often slightly concave towards the tips; pseudocyphellae linear, marginal; up-
per surface grey to greyish-green, lower-side black; rhizines dense, squarrosely branched.
Soralia marginal, labriform; soredia grey to bluish-grey, powdery to granular. Medulla
pale yellow. Dactyls and isidia absent. Apothecia absent. Upper cortex K+ yellowish,
UV–; medulla K–, C–; containing chloroatranorin (minor) and unknown terpenes.
Habitat and distribution. Growing on bark of Quercus and Alnus spp. Range
1700 –3060 m elevation in semi-arid environments; known only from Yunnan, Si-
chuan and Xizang in China.
Etymology. e epithet hengduanensis refers to the type locality of the species, the
Hengduan Mountains region.
Figure 3. Pyxine hengduanensis: A (KUN-L 09-30247) photographed by Li-Song Wang, in situ at the
type locality B–D (KUN-L 15-48082), photographed by Mei-xia Yang B allus C upper surface of thal-
lus D marginal labriform soralia. Scale bars: 2 cm (B); 5 mm (C); 0.5 mm (D).
New species and records of Pyxine (Caliciaceae) in China 9
Notes. Pyxine hengduanensis is characterised by a corticolous habit, yellowish-grey
to greyish-green thallus, marginal labriform soralia, pale yellow medulla and the ab-
sence of atranorin. Pyxine hengduanensis is most closely related to P. sorediata, as in-
ferred from the phylogenetic tree (Fig. 1); P. sorediata is also corticolous but has a
yellow or yellow-orange medulla and soralia that develop marginally from ssures and
then become laminal and orbicular (Elix 2009), while P. hengduanensis has marginal
labriform soralia developing from the centre of the pseudocyphellae, grey to bluish-
grey soredia and a pale yellow medulla. Pyxine hengduanensis also resembles P. retiru-
gella Nyl. (Elix 2009) in the marginal and laminal pseudocyphellae, but it diers in
having white or creamy and K+ yellow turning red medulla and norstictic acid as the
main compound (Mongkolsuk et al. 2012).
Selected specimens examined (KUN). CHINA: SICHUAN PROVINCE: Dukou
Co., Yanbian Vil., Shibao Mt., 2900 m elev., 29 Jun 1983, L. S. Wang 83-635; Nanping
Co., Jiuzhaigou, 2000 m elev., on Pinus, 23 Sep 1986, L. S. Wang 86-2591. XIZANG
PROVINCE: Linzhi Co., 3060 m elev., 2°50'249"N, 94°44'728"E, on Quercus spp. 20
Aug 2007, L. S. Wang et al. 07-28389; YUNNAN PROVINCE: Luquan Co., 30 km
from Sapanying Co. to Zehei Co., 2540 m elev., 26°04'24.53"N, 102°36'19.15"E, on
moss, 19 Apr 2014, L. S. Wang et al. 14-43258; Lufeng Co., Heijin Vil., 1800 m elev.,
25°20'146"N, 102 05'835"E, on bark, 1 May 2009, L. S. Wang 09-30247.
Pyxine yunnanensis M. X. Yang & Li S. Wang, sp. nov.
MycoBank No.: MB 819958
Figure 4
Holotype. CHINA, YUNNAN PROVINCE, Yongren Co., Lagu Vil., 1050 m eleva-
tion, 26°23.239'N, 101°25.120'E, on rock, 4 Dec 2013, L. S. Wang et al. KUN-L 13-
41372. GenBank accession No.: ITS = KY611870, mtSSU = KY751377.
Description. allus saxicolous, up to 7 cm in diam., closely appressed to the
substrate. Lobes radiating, irregularly branched, plane to slightly concave, 0.2–1.0mm
wide, subround to truncate at the apices. Upper surface pale grey to yellowish-grey,
sparsely pruinose at the lobe tips or epruinose. Lower surface brownish-black, rarely
pale brown, rhizines indistinct, sparse to moderately abundant, brownish-black to
black. Isidia and soredia absent. Medulla pale yellow in the upper part, white in the
lower part. Apothecia abundant, 0.2–0.8 mm wide, constricted at base, plane to pos-
sibly convex; margin black. Hymenium height 80–120 μm; hypothecium light brown
to brown, internal stipe white; spore brown with two cells, 10–15 × 4–7 μm. Upper
cortex K+ yellowish, UV+ yellow; medulla K–, C–; containing lichexanthone, chloro-
atranorin (minor), zeorin and unknown terpenes (detected by TLC).
Habitat and distribution. Growing on rocks around 1050–1650 m elevation in
secondary forests in a dry to semi-arid environment; known only from Yunnan.
Etymology. e epithet yunnanensis refers to the province of the type locality of
the species.
Mei-Xia Yang et al. / MycoKeys @: @@–@@ (2018)
10
Figure 4. Pyxine yunnanensis: A (KUN-L 09-30247) photographed by Li-Song Wang, in situ at the
type locality B–D (KUN-L 13-41372) photographed by Mei-xia Yang B white internal stipe of apothecia
Chymenium D ascospores from GAW (glycerine:alcohol:water=1:1:1). Scale bars: 1 cm (A); 1 mm (B);
50μm (C); 10 μm (D).
Notes. Pyxine yunnanensis is characterised by small and saxicolous thalli, rather
small narrow apothecia (up to 0.8 mm in diam.), a white internal stipe and the pres-
ence of lichexanthone. Pyxine minuta Vain. (up to 3 cm in diam.) resembles P. yun-
nanensis (up to 7 cm in diam.) in its small thalli and the presence of lichexanthone,
but diers in that its internal stipe is absent or indistinct and it has a white medulla
(Awasthi 1982). Pyxine pyxinoides (Müll. Arg.) Kalb and P. elixii Kalb also grow on
rocks, but P. pyxinoides diers from P. yunnanensis in that it has a white medulla, an
indistinct internal stipe of the apothecia and smaller ascospores (10–15 × 4–7 μm)
than those of P. pyxinoides (10–16 × 4.5–8.0 μm). Pyxine elixii can be distinguished by
its orange medulla and lack of lichexanthone (Elix 2009).
Pyxine yunnanensis is closely related to P. berteriana in that they have a similar type
and size of apothecia and lichexanthone is present, but Pyxine berteriana diers in that it
occurs in incorticolous habitat and has a yellow medulla and a yellow medulla of the stipe.
Selected specimens examined (KUN). CHINA: YUNNAN PROVNCE:
Yongsheng Co., Dongjiang of Renhe Town, 1130 m elev., 26°20.448'N, 101°06.908'E,
New species and records of Pyxine (Caliciaceae) in China 11
on rock, 7 Dec 2013, L. S. Wang et al. 13-41413; Shawan Village of Renhe Town,
1160 m elev., 26°19.449'N, 101°05.200'E, 7 Dec 2013, L. S. Wang et al. 13-40643,
13-40694, 13-40686, 13-40641, 13-40684; Lijiang City, east of Jinan Bridge, 1310 m
elev., 26°47.725'N, 100°25.640'E, on rock, 8 Dec 2013, L. S. Wang et al. 13-40596.
New records
Pyxine cognata Stirt
= Pyxine berteriana var. himalaica D.D. Awasthi
Description. Upper surface white to whitish-grey or grey-brown; isidia and soredia ab-
sent; medulla orange-yellow to orange; lower surface black in the centre, paler towards
the margin; apothecia common, (0.3) 0.5–1.0 (1.5) mm wide; internal stipe upper part
orange, K+ purple, P–; lower part yellow or much paler than upper part, K–, P–. Upper
cortex K–, UV+ yellow, medulla K– or K+ pale red, C–, P– or P+ orange; containing
lichexanthone (major), triterpenes, unknown pigment (minor) (detected by TLC).
Habitat and distribution. Growing on bark of Quercus and Juglans spp. Range
1090–2230 m elevation in semi-arid environments. Worldwide distribution: India
(Awasthi 1982), Brazil (Aptroot 2014), ailand (Mongkolsuk et al. 2012) and Aus-
tralia (Elix 2009); newly recorded in China.
Notes. Pyxine berteriana var. himalaica was described by Awasthi (1982) as a va-
riety based on the pale yellow to yellow medulla and a narrow distribution from the
Himalayan region and central India. Pyxine cognata is very similar to P. berteriana
var. himalaica in the presence of lichexanthone, the pigmented medulla and the lack
of isidia and soredia. However, Pyxine cognata is distinguished by a faint pruina on
the lobe tips, deep yellow to rust coloured medulla and slightly larger spores, as well
as for being widely distributed in tropical regions. erefore, the morphological and
ecological dierences between these two species are minor. In this study, we collected
specimens of both species and found that they have a similar ecology and distribution
pattern. Phylogenetic analysis inferred that Pyxine berteriana var. himalaica is clus-
tered with P. cognata with a high support value (MLBS = 100%, PP = 1.00). Based on
the combination of molecular, morphological and ecological information, we propose
P.berteriana var. himalaica as a synonym for P. cognata.
Pyxine cognata is most similar to P. berteriana in that it contains lichexanthone,
lacks isidia and soredia and has a pigmented medulla; however, P. cognata can be dis-
tinguished by the presence of lichexanthone in the cortex, an orange medulla and an
orange-yellow internal stipe of apothecia with K+ purple. In comparison, P. berteriana
has a pale yellow to yellow medulla and the internal stipe is pale yellow to yellow. (Kalb
1987). Despite the broad similarities, these species are not closely related; P. cognata
seems to share a unique ancestor with P. subcinerea. Pyxine subcinerea diers in that it
has marginal soralia and obscurascens-type apothecia (Elix 2009). (Fig. 1).
Mei-Xia Yang et al. / MycoKeys @: @@–@@ (2018)
12
Selected specimens examined (KUN). CHINA: SICHUAN PROVINCE: Miyi Co.,
Malong north slope, 2100 m elev., on Carya spp., 5 Jul 1983, L. S. Wang 83-698; Dukou
Co., Dabaoding, 1900 m elev., 21 Jun 1983, L. S. Wang 83-212. YUNNAN PROVINCE:
Yuanmou Co. Langbapu Forest Soil, 1612 m elev., 25°41'01.76"N, 101°41'25.78"E, on
branch, 21 Apr 2014, c14-43569, 14-43539; Yongren Co., from Menghu to Wanma,
1543 m elev., 26°13'45.15"N, 101°25'56.86"E, 3 Dec 2013, L.S. Wang et al. 13-40767.
Pyxine himalayensis Awas
Description. allus grey-white, soredia and isidia absent; medulla yellow to orange-
yellow; apothecia common, laminal, constricted at base, up to 2 mm in diam.; internal
stipe colourless, K–, hypothecium 50–80 μm thick, spores brown, 15–25 × 6–9 μm.
Upper cortex K+ yellow, UV–, medulla K–, C–, P–; containing atranorin (major),
+/– zeroin, triterpense.
Habitat and distribution. Growing on bark of Rhododendron, Quercus, Alnus,
Juglans, Sophora, Lonicera and Lyonia spp. and rarely on rocks, at elevations of 1330–
3600 m in semi-arid environments. Worldwide distribution: India (Awasthi 1982) and
added here to the ora of China.
Notes. Pyxine himalayensis is distinctive for having lobes 1.5–3.0 mm wide, an
orange medulla and a lack of isidia and soredia, lichexanthone and norstictic acid.
Pyxine himalayensis was rst described by Awasthi (1982) and it is characterised by an
orange medulla and colourless internal stipe of apothecia. e closely related Pyxine
limbulata is described as having a yellow medulla and a brown internal stipe (Hu and
Chen 2003). ere are 24 specimens of this species in the KUN-L. e phylogenetic
analysis of ITS and mtSSU sequences conrm that these are independent species.
Selected specimens examined (KUN). CHINA: SICHUAN PROVINCE: Dukou
City, Shibao Mt., 2800 m elev., 29 Jun 1983, L. S. Wang 83-628; Yuanyang Co., Bailing
commune, 3100 m elev., on Quercus, 11 Aug 1983, L. S. Wang 83-1508; 3250 m elev., on
stone, 10 Aug 1983, L. S. Wang 83-1483; Muli Co., Yazui forest farm, on Quercus, 3000
m elev., 20 Aug 1983, L. S. Wang 83-1589, 83-1596; Donglang, 3000 m elev., on bark,
10 Sep 1983, L. S. Wang 83-2220. XIZANG PROVINCE: Bomi Co., Gang vil., 2688
m elev., 29°52.983'N, 095°33.593'E, on branch of Populus yunnanensis, 20 Sep 2014, L.
S. Wang et al. 14-46203, 14-46162. YUNNAN PROVINCE: Luquan Co., 30 km from
Sapanying Co. to Zehei Co., 2540 m elev., 26°04'24.53"N, 102°36'19.15"E, on Quercus,
19 Apr 2014, L. S. Wang et al. 14-43218, 14-43204; Luquan Co., Zhongcun Vil., 2350 m
elev., 25°20'146"N, 102 05'835"E, on bark of Quercus, 1 May 2009, L. S. Wang 09-30279.
Pyxine minuta Vain
Description. Pyxine minuta is characterised by narrow lobes, centrally subcrustaceous,
saxicolous thalli, a whitish-grey or grey-brown upper surface; brownish-black lower
New species and records of Pyxine (Caliciaceae) in China 13
surface with black and simple rhizines, a lack of isidia and soredia and a white or whit-
ish stramineous medulla. Apothecia common, 0.5–1.5 mm wide; internal stipe absent
or not distinct. Upper cortex K+ yellowish, UV+ yellow, medulla K–, C–; containing
lichexanthone (major) and terpenoids (detected by TLC).
Habitat and distribution. Growing on bark of Quercus spp. or rock around 1090–
2230 m elevation in semi-arid environments. Worldwide distribution: India (Awasthi
1982), Australia (Rogers 1986) and newly recorded in China.
Notes. ere is some confusion in the classication of Pyxine minuta and P. pyxi-
noides. Pyxine minuta is characterised by narrow lobes, an absent or indistinct internal
stipe, small spores (11–16 (18) × 5–7 μm) and a white medulla. Based on the world key to
Pyxine species with lichexanthone (Aptroot et al. 2014; Kalb 1987; Huneck et al. 1987),
the characteristics of Pyxine pyxinoides are: allus without isidia, pustules or soredia, usu-
ally with apothecia; Medulla yellow, ochraceous or salmon; apothecium margin black, not
thalline; apothecium without a clear stipe; one TLC run of a portion of the thallus with-
out apothecia showed traces of a substance running like norstictic acid (Obermayer and
Kalb 2010); neotropical. We did not nd any specimens of P. pyxinoides in our collections.
Selected specimens examined (KUN). CHINA: SICHUAN PROVINCE:
Dukou Co., Dabaoding, 1950 m elev., 21 Jun 1983, L. S. Wang 83-206. YUN-
NAN PROVINCE: Yongsheng Co., Shawan village of Renhe town, 1160 m elev.,
26°19.449'N, 101°05.200'E, on rock, 7 Dec 2013, L. S. Wang et al. 13-40630, 13-
40695; Yongren Co., Lagu village, 1050 m elev., 26°23.239'N, 101°25.120'E, on rock,
4 Dec 2013, L. S. Wang et al. 13-41380; Jinggu Co., on the way to Zhenyuan, 1800 m
elev., 21 Aug 1994, L. S. Wang et al. 94-14247.
Key to the species of the genus Pyxine in China
1 allus UV+, lichexanthone present ...........................................................2
– allus UV–, lichexanthone absent ...........................................................12
2 allus with vegetative propagules ..............................................................3
– allus lacking vegetative propagules ..........................................................7
3 allus with soredia ....................................................................................4
– allus with isidia .......................................................................................5
4 Medulla yellow ........................................................................P. subcinerea
– Medulla white ..................................................................................P. cocoes
5 Medulla yellow; isidia dactyliform .......................................P. endochrysina
– Medulla white; isidia cylindrical ..................................................................6
6 Norstictic acid present ............................................................. P. consocians
– Norstictic acid absent ...............................................................P. coralligera
7 Atranorin present ..........................................................................P. cognata
– Atranorin absent .........................................................................................8
8 Medulla pale yellow to yellow .................................................. P. berteriana
– Medulla white .............................................................................................9
Mei-Xia Yang et al. / MycoKeys @: @@–@@ (2018)
14
9 Internal stipe of apothecia absent or indistinct ..........................................10
– Internal stipe of apothecia well developed .................................................11
10 Norstictic acid present, as well as other triterpenes .................. P. microspora
– Norstictic acid absent .................................................................... P. minuta
11 Internal stipe of apothecia brown, K+ red violet ..........................P. petricola
– Internal stipe of apothecia white, K–..................................... P. yunnanensis
12 allus with soralia ................................................................................... 13
– allus lacking vegetative propagules ........................................................16
13 Medulla white, soralia laminal; norstictic acid present.............. P. copelandii
Medulla yellow; soralia marginal; norstictic acid absent ............................14
14 Atranorin absent; soralia labriform .....................................P. hengduanensis
– Atranorin present; soralia granular, laminal or orbicular ............................15
15 Lobe margin without pseudocyphellae; soredia yellow ...........P. meissnerina
– Lobe margin with intermittent pseudocyphellae; soredia grey to bluish-grey .
.................................................................................................. P. sorediata
16 Medulla yellow .........................................................................................17
– Medulla white ..........................................................................P. philippina
17 Internal stipe of apothecia colourless .................................... P. himalayensis
– Internal stipe of apothecia brown or yellow ...............................................18
18 Internal stipe brown, K+ red violet............................................ P. limbulata
– Internal stipe of apothecia pale yellow; upper medulla yellow, lower medulla
white ..........................................................................................P. avicans
Acknowledgements
e authors are grateful to Dr. Melissa Dawes for the scientic editing, as well as Prof.
Bernard Gonet from the University of Connecticut and Prof. James R. Shevock from
the California Academy of Sciences for guidance and help with writing in English.
Sincere thanks are extended to Institute of Microbiology, Chinese Academy of Sciences
for the loan of specimens. is study was supported by grants from the National Natu-
ral Science Foundation of China (No. 31370069, 31400022, 31670028, 31750001),
the Second Tibetan Plateau Scientic Expedition and the China Scholarship Council.
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