Research Article |
Corresponding author: Li-Song Wang ( wanglisong@mail.kib.ac.cn ) Academic editor: Gerhard Rambold
© 2019 Mei-Xia Yang, Xin-Yu Wang, Dong Liu, Yan-Yun Zhang, Li-Juan Li, An-Cheng Yin, Christoph Scheidegger, Li-Song Wang.
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.
Citation:
Yang M-X, Wang X-Y, Liu D, Zhang Y-Y, Li L-J, Yin A-C, Scheidegger C, Wang L-S (2019) New species and records of Pyxine (Caliciaceae) in China. MycoKeys 45: 93-109. https://doi.org/10.3897/mycokeys.45.29374
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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. flavicans 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.). Pyxine yunnanensis is diagnosed by the small size of the apothecia, a white medulla of the stipe and the presence of lichexanthone. Pyxine flavicans 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.
China, lichenised fungi, new species, phylogeny
The lichen genus Pyxine was first 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 (
Regional studies on this genus have been carried out in Australia (
Although many studies have been conducted, few molecular phylogenetic analyses have been completed (
The specimens examined in this study were collected from the Hengduan Mountains region, 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 characteristics were studied using a dissecting microscope (Nikon SMZ745T) and a light microscope (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. The lichen secondary metabolites were analysed using spot reactions and thin-layer chromatography in a solvent C system, according to
Total genomic DNA was extracted from dried herbarium specimens using AxyPrep Multisource Genomic DNA Miniprep Kit 50-prep (Qiagen) according to the manufacturer’s instructions. ITS (nrDNA ITS1-5.8S-ITS2) and mtSSU (mitochondrial small subunit rDNA) were amplified by polymerase chain reactions (PCR) using the primer pairs ITS1F (
Amplifications 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 final extension at 72 °C for 10 min. PCR products were sequenced in an ABI3730X using amplification primers manufactured by Tsingke (Kunming, China).
ITS and mtSSU sequences were assembled with Seqman 7.0 (DNAStar) and manually edited using Mega6. DNA sequences were aligned with MAFFT version 7 with default parameters (
Maximum likelihood (ML) and Bayesian inference (BI) were conducted based on the two gene fragments combining ITS and mtSSU. The best-fitting substitution model was determined using MrModeltest 2.3 (
Nineteen species were recognised, including three that are new to science (i.e. Pyxine flavicans 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
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 1 | China: Yunnan | KUN 12-36993 | KY611891 | KY751398 |
P. sorediata 2 | China: Yunnan | KUN 15-48546 | KY611892 | KY751399 |
P. sorediata 3 | Sweden | Wetmore 91254 (UPS) | JX000111 | – |
– | KX512973 | |||
P. hengduanensis 1 | China: Yunnan | KUN 15-48082 | KY611889 | KY751396 |
P. hengduanensis 2 | China: Yunnan | KUN14-43258 | KY611890 | KY751397 |
P. endochrysina 1 | China: Xizang | KUN 14-46462 | KY611887 | KY751394 |
P. endochrysina 2 | China: Xizang | KUN 14-46439 | KY611888 | KY751395 |
P. limbulata 1 | China: Taiwan | KUN 15-49117 | KY611885 | KY751392 |
P. limbulata 2 | China: Taiwan | KUN 15-49153 | KY611886 | KY751393 |
P. himalayensis 1 | China: Yunnan | KUN 12-36055 | KY611881 | KY751388 |
P. himalayensis 2 | China: Xizang | KUN 14-46410 | KY611882 | KY751389 |
P. flavicans 1 | China: Yunnan | KUN 14-43995 | KY611883 | KY751390 |
P. flavicans 2 | China: Yunnan | KUN 15-48196 | KY611884 | KY751391 |
P. meissnerina 1 | China: Yunnan | KUN 12-34386 | KY611877 | KY751384 |
P. meissnerina 2 | China: Yunnan | KUN 12-34377 | KY611878 | KY751385 |
P. consocians 1 | China: Yunnan | KUN 15-49942 | KY611879 | KY751386 |
P. consocians 2 | China: Yunnan | KUN 15-47417 | KY611880 | KY751387 |
P. petricola 1 | China: Yunnan | KUN 13-40715 | KY611875 | KY751382 |
P. petricola 2 | China: Sichuan | KUN 13-39419 | KY611876 | KY751383 |
P. cocoes 1 | China: Taiwan | KUN 15-49457 | KY611874 | KY751381 |
P. minuta 1 | China: Yunnan | KUN 13-40695 | KY611872 | KY751379 |
P. minuta 2 | China: Yunnan | KUN 13-40630 | KY611873 | KY751380 |
P. yunnanensis 1 | China: Yunnan | KUN 13-41372 | KY611870 | KY751377 |
P. yunnanensis 2 | China: Yunnan | KUN 13-40596 | KY611871 | KY751378 |
P. berteriana 1 | China: Yunnan | KUN 15-47921 | KY611868 | KY751375 |
P. berteriana 2 | China: Yunnan | KUN 14-43730 | KY611869 | KY751376 |
P. subcinerea 1 | China: Taiwan | KUN 15-48998 | KY611866 | KY751373 |
P. subcinerea 2 | China: Taiwan | KUN 15-49012 | KY611867 | KY751374 |
P. subcinerea | USA | NC 27708 | HQ650705 | – |
Spain | MAF9852 | – | AY464080 | |
P. cognata 1 | China: Yunnan | KUN 14-43569 | KY611864 | KY751371 |
P. cognata 2 | China: Yunnan | KUN 13-40767 | KY611865 | KY751372 |
P. berteriana var. himalaica 1 | China: Yunnan | KUN 14-43571 | KY611862 | KY751369 |
P. berteriana var. himalaica 2 | China: Yunnan | KUN 13-40706 | KY611863 | KY751370 |
Dirinaria applanata | India | – | EU722342 | – |
Spain | MAF 9854 | – | AY464079 | |
Physcia dubia | Finland | T. Ahti 69359 | JQ301695 | – |
– | JQ301536 |
The ITS and mtSSU datasets were analysed separately and concatenated; both parsimony and Bayesian trees of ITS vs. mtSSU were congruent. A maximum likelihood phylogenetic tree was inferred from the combined dataset of ITS and mtSSU (Fig.
Species of Pyxine were separated into two main clades, as inferred from the phylogenetic tree with strong support (Fig.
Nineteen Pyxine species were confirmed in China, including three species new to science and three species hereby newly reported for the country, based on the following characteristics: 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.
Thallus 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, 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 atranorin, chloroatranorin (minor), zeorin and unknown terpenes.
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.
The epithet flavicans refers to the yellow medulla and internal stipe of the apothecia.
Pyxine flavicans is characterised by flat corticated yellowish-grey to brownish-grey thalli, a constricted base, a pale yellow medulla and the presence of atranorin.
This 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 lichexanthone (
CHINA: SICHUAN PROVINCE: Muli Co., 2850 m elev., on Pinus yunnanensis, 23 Aug 1983, L. S. Wang 83-1869(A); XIZANG 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; Nujiang 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.
Thallus corticolous, 4–9 cm wide, firmly to loosely adnate to substrate. 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; upper 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.
Growing on bark of Quercus and Alnus spp. Range 1700 –3060 m elevation in semi-arid environments; known only from Yunnan, Sichuan and Xizang in China.
The epithet hengduanensis refers to the type locality of the species, the Hengduan Mountains region.
Pyxine hengduanensis is characterised by a corticolous habit, yellowish-grey to greyish-green thallus, marginal labriform soralia, pale yellow medulla and the absence of atranorin. Pyxine hengduanensis is most closely related to P. sorediata, as inferred from the phylogenetic tree (Fig.
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., 29°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.
Thallus saxicolous, up to 7 cm in diam., closely appressed to the substrate. Lobes radiating, irregularly branched, plane to slightly concave, 0.2–1.0 mm 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 possibly 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, chloroatranorin (minor), zeorin and unknown terpenes (detected by TLC).
Growing on rocks around 1050–1650 m elevation in secondary forests in a dry to semi-arid environment; known only from Yunnan.
The epithet yunnanensis refers to the province of the type locality of the species.
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 presence of lichexanthone. Pyxine minuta Vain. (up to 3 cm in diam.) resembles P. yunnanensis (up to 7 cm in diam.) in its small thalli and the presence of lichexanthone, but differs in that its internal stipe is absent or indistinct and it has a white medulla (
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 differs in that it occurs in incorticolous habitat and has a yellow medulla and a yellow medulla of the stipe.
CHINA: YUNNAN PROVNCE: Yongsheng Co., Dongjiang of Renhe Town, 1130 m elev., 26°20.448'N, 101°06.908'E, 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.
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 C hymenium D ascospores from GAW (glycerine:alcohol:water=1:1:1). Scale bars: 1 cm (A); 1 mm (B); 50 μm (C); 10 μm (D).
= Pyxine berteriana var. himalaica D.D. Awasthi
Upper surface white to whitish-grey or grey-brown; isidia and soredia absent; 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).
Growing on bark of Quercus and Juglans spp. Range 1090–2230 m elevation in semi-arid environments. Worldwide distribution: India (
Pyxine berteriana var. himalaica was described by
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 distinguished 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. (
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.
Thallus 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.
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 (
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 first described by
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 is characterised by narrow lobes, centrally subcrustaceous, saxicolous thalli, a whitish-grey or grey-brown upper surface; brownish-black lower surface with black and simple rhizines, a lack of isidia and soredia and a white or whitish 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).
Growing on bark of Quercus spp. or rock around 1090–2230 m elevation in semi-arid environments. Worldwide distribution: India (
There is some confusion in the classification of Pyxine minuta and P. pyxinoides. 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 (
CHINA: SICHUAN PROVINCE: Dukou Co., Dabaoding, 1950 m elev., 21 Jun 1983, L. S. Wang 83-206. YUNNAN 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.
1 | Thallus UV+, lichexanthone present | 2 |
– | Thallus UV–, lichexanthone absent | 12 |
2 | Thallus with vegetative propagules | 3 |
– | Thallus lacking vegetative propagules | 7 |
3 | Thallus with soredia | 4 |
– | Thallus 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 |
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 | Thallus with soralia | 13 |
– | Thallus 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. flavicans |
The authors are grateful to Dr. Melissa Dawes for the scientific editing, as well as Prof. Bernard Goffinet 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. This study was supported by grants from the National Natural Science Foundation of China (No. 31370069, 31400022, 31670028, 31750001), the Second Tibetan Plateau Scientific Expedition and the China Scholarship Council.