New species and records of Pyxine (Caliciaceae) in China

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.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.). Pyxineyunnanensis is diagnosed by the small size of the apothecia, a white medulla of the stipe and the presence of lichexanthone. Pyxineflavicans is characterised by broad lobes, a pale yellow medulla of the stipe and the presence of atranorin. Pyxinehengduanensis 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.


Introduction
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 (Wedin and Grube 2002;Crespo et al. 2004;Gaya et al. 2012;Prieto and Wedin 2017). Pyxine is characterised by an adnate foliose thallus, an internal stipe colour of apothecia, dark brown hypothecium and generally two-celled brown ascospores (Awasthi 1982;Elix 2009;Kalb 1987;Kalb 2004). The 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).
Although many studies have been conducted, few molecular phylogenetic analyses have been completed (Gaya et al. 2012;Schmull et al. 2011;Prieto and Wedin 2017). In this study, morphological, chemical and molecular phylogenetic analyses were combined 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.

Morphological and chemical analyses
The specimens examined in this study were collected from the Hengduan Mountains region, Taiwan, Zhejiang, Hainan et al. from 1941 to 2016 anddeposited 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 Orange et al. (2001).
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 ddH 2 O 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).

Phylogenetic analyses
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 (Nylander 2005) and PAUP*4b10 (Swofford 2003), where the AIC values were calculated using JModelTest 3.7 (Posada 2008). ML analyses were performed using RAxML7.0.4 (Stamatakis 2006) with default settings (GTR) and support values were inferred from the 70% majorityrule tree based on 1000 non-parametric bootstrap pseudo-replicates. The 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 1000 th generation, with the first 10% of trees discarded as burn-in. A majorityrule 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 sample 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.

Results
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 1). A phylogenetic 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 circumscription of these species (Hu and Chen 2003;Obermayer and Kalb 2010;Wei 1991).
Species of Pyxine were separated into two main clades, as inferred from the phylogenetic tree with strong support (Fig. 1). The ten species in Clade 1 are all characterised by the presence of soralia or isidia on the thallus, whereas the five species in Clade 2 contain lichexanthone and lack soralia and isidia. The two species P. petricola and P. cocoes are characterised by the presence of both lichexanthone and soralia.

Taxonomic treatment
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.   Description. 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.
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. The epithet flavicans refers to the yellow medulla and internal stipe of the apothecia. Notes. 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 (Hu and Chen 2003). Pyxine flavicans is similar to P. australiensis Kalb regarding the absence of soredia and isidia and both species are frequently lignicolous but occasionally grow on rocks. However, P. flavicans differs from P. australiensis in having marginal and laminal pseudocyphellae, lichexanthone and a white medulla in the stipe (Elix 2009). Pyxine flavicans is similar to P. himalayensis in terms of the type of apothecia and lack of lichexanthone. However, P. himalayensis has a colourless internal stipe. Description. 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.
Habitat and distribution. 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.
Etymology. The epithet hengduanensis refers to the type locality of the species, the Hengduan Mountains region. Notes. 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. 1); P. sorediata is also corticolous but has a yellow or yellow-orange medulla and soralia that develop marginally from fissures and then become laminal and orbicular (Elix 2009), while P. hengduanensis has marginal labriform soralia developing from the centre of the pseudocyphellae, grey to bluishgrey soredia and a pale yellow medulla. Pyxine hengduanensis also resembles P. retirugella Nyl. (Elix 2009) in the marginal and laminal pseudocyphellae, but it differs in having white or creamy and K+ yellow turning red medulla and norstictic acid as the main compound (Mongkolsuk et al. 2012).  Description. 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).
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. The epithet yunnanensis refers to the province of the type locality of the species. 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 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 (Awasthi 1982). Pyxine pyxinoides (Müll. Arg.) Kalb and P. elixii Kalb also grow on rocks, but P. pyxinoides differs 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 differs in that it occurs in incorticolous habitat and has a yellow medulla and a yellow medulla of the stipe.
Notes. Pyxine berteriana var. himalaica was described by Awasthi (1982) as a variety 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. Therefore, the morphological and ecological differences 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 clustered 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 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. (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 differs in that it has marginal soralia and obscurascens-type apothecia (Elix 2009). (Fig. 1).
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 flora 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 first described by Awasthi (1982) and it is characterised by an orange medulla and colourless internal stipe of apothecia. The closely related Pyxine limbulata is described as having a yellow medulla and a brown internal stipe (Hu and Chen 2003). There are 24 specimens of this species in the KUN-L. The phylogenetic analysis of ITS and mtSSU sequences confirm that these are independent species.
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. 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 (Aptroot et al. 2014;Kalb 1987;Huneck et al. 1987), the characteristics of Pyxine pyxinoides are: Thallus without isidia, pustules or soredia, usually 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 without apothecia showed traces of a substance running like norstictic acid (Obermayer and Kalb 2010); neotropical. We did not find any specimens of P. pyxinoides in our collections.