Squamarina (lichenised fungi) species described from China belong to at least three unrelated genera

Abstract New collections of six Squamarina species from type localities in China were studied. The comparison of morphological characteristics and secondary metabolites with those of the type specimens and phylogenetic analyses suggest that S. callichroa and S. pachyphylla belong to Rhizoplaca, S. semisterilis belongs to Lobothallia and S. chondroderma should be retained in Lecanora temporarily. Only two species, S. kansuensis and S. oleosa, remain in Squamarina. The new combinations Lobothallia semisterilis (H. Magn.) Y. Y. Zhang, Rhizoplaca callichroa (Zahlbr.) Y. Y. Zhang and R. pachyphylla (H. Magn.) Y. Y. Zhang are proposed. Detailed descriptions to aid the identification of these species, distributions and phylogenetic trees, based on multiple collections, are presented. The generic concept of Squamarina is recircumscribed in this study.


introduction
The genus Squamarina Poelt was first erected by Poelt (1958) and is characterised by thick squamules, large apothecia and a "Squamarina-type" thallus, consisting of a wellseparated and more or less equally high upper cortex, algal layer and medulla. Two sections, S. sect. Squamarina and S. sect. Petroplaca, were distinguished by Poelt (1958), based on the former having a larger thallus and larger apothecia, and the latter smaller thallus and apothecia. Hafellner (1984) accommodated the genus in a new family, Squamarinaceae, based on asci with an evenly amyloid tholus without any axial body. However, the circumscription of Squamarina or Squamarinaceae has been disputed for a long time and molecular studies for this genus are largely lacking (Hafellner 1984;Haugan and Timdal 1992;Hertel and Rambold 1988;Poelt 1958). Recent studies showed that the species of the sect. Squamarina have asci with an amyloid tube in the tholus, resembling those of Porpidiaceae and that the ascus structure of sect. Petroplaca resembles that of Protoparmeliopsis muralis (Schreb.) Rabenh. (Haugan and Timdal 1992;Hertel and Rambold 1988). Hence, the detailed circumscription of the genus Squamarina is urgently needed and it was also one of the aims of this study.
Nine species of Squamarina have so far been reported from China (Wei 1991), of which six were originally collected in China by Birger Bohlin and Heinrich Frh.  (Magnusson 1940;Zahlbruckner 1930), no more collections have, however, been reported since then, except for S. chondroderma and molecular data are not available for any of them in GenBank. Therefore, studies on the identification, distribution and phylogeny of these species are necessary. We have undertaken several field trips along the collection routes of Birger Bohlin (1930)(1931)(1932) and Handel-Mazzetti (1914-1915 in the past few years and collected fresh material of the six species from the type localities for the molecular study presented here.

Morphological and chemical studies
Type specimens were loaned from the Museum of Natural History Vienna (W) and the Swedish Museum of Natural History (S). The fresh material collected for this study is deposited in Kunming Institute of Botany, Chinese Academy of Sciences (KUN-L). Morphological features were studied under a dissecting microscope (Nikon SMZ745T). Apothecia and thalli were sectioned with an S-30 microtome with a KS-34 cryostat (Zeiss, Jena) and microscopic traits were observed and measured using a microscope (Leica 020-518.500). Secondary metabolites were analysed by spot reactions and thinlayer chromatography (TLC) in solvents A, B and C (Orange et al. 2001).

Phylogenetic analyses
Sequences were assembled and edited using SeqMan 7.1 (DNAstar packages). An nrITS matrix of Lobothallia (Clauzade & Cl. Roux) Hafellner, an nrLSU matrix of Squamarina and a 5-locus (nrITS, nrLSU, RPB1, RPB2 and mtSSU) concatenated matrix of Rhizoplaca Zopf and related genera were generated using Geneious R8. Single-gene analyses were conducted, based on the Maximum Likelihood (ML) method to assess the conflict amongst individual genes and no significant incongruence was detected. Matrices were aligned with MAFFT, using the web service (http://mafft. cbrc.jp/alignment/server/index.html). Ambiguous positions were removed, using the web service of Guidance (http://guidance.tau.ac.il/ver2/). MrModeltest2.3 (Nylander 2004), based on Akaike Information Criterion (AIC), was used to estimate the bestfitting substitution model for each dataset for Maximum Likelihood (ML) and Bayesian Inference (BI). The selected model for nrITS-Lobothallia was HKY+I and, for the other matrices, GTR+I+G. Bayesian reconstructions of phylogenies were performed with MrBayes 3.1.2 (Huelsenbeck and Ronquist 2001), using four Markov chains running for 2 million generations for single locus matrices and 10 million generations for the concatenated dataset. Trees were sampled every 100 generations. ML analyses were performed with RaxmlHPC, using the General Time Reversible model of nucleotide substitution with the gamma model of rate heterogeneity (GTRGAMMA). Support values were inferred from the 70% majority-rule tree of all saved trees obtained from 1000 non-parametric bootstrap replicates. Trees were visualised in FigTree v1.4.0 (Rambaut 2012).

Results and discussions
A total of 84 sequences of the nrITS, nrLSU, RPB1, RPB2 and mtSSU were newly generated for the species Squamarina chondroderma, S. semisterilis, S. callichroa, S. pachyphylla, S. gypsacea (Sm.) Poelt, S. kansuensis and S. oleosa in this study (Table 1). The BLAST results showed that these species belong to at least three unrelated genera,       ) is sister to the genus Rhizoplaca, but differs in growing on moss and meadow and the presence of numerous rhizinose strands that are never present in its related genera. It is also distinct from the genus Squamarina by the Lecanora-type ascus and the strongly gelatinised lower cortex. This species could belong to a genus separate from Lecanora s. str. and closely related to the genera Rhizoplaca and Protoparmeliopsis, but as only one species from this group was included here, further exploration is needed in the future and we prefer to retain this species in Lecanora here. The remaining two species, Squamarina kansuensis and S. oleosa, proved to belong in Squamarina. Squamarina kansuensis is sister to S. lentigera, but differs in the larger thallus and the presence of psoromic and 2'-O-demethylpsoromic acids. Squamarina oleosa is a basal clade of the genus, which is close to the species S. cartilaginea (With.) P. James and S. gypsacea.
We revised the previously reported ascus structure for the two sections of Squamarina (Haugan and Timdal 1992;Hertel and Rambold 1988) and verified that the species in sect. Squamarina display a Porpidia-type ascus and the species in sect. Petroplaca form a Lecanora-type ascus. Our phylogenetic analyses, containing the type species of the two sections, S. callichroa and S. gypsacea, were in accordance with the ascus type: the sect. Squamarina is close to the genus Stereocaulon (Schreb.) Schrad., which also has a Porpidia-type ascus (Högnabba 2006); section Petroplaca is nested within the genus Rhizoplaca having a Lecanora-type ascus. Therefore, we suggest excluding the section Petroplaca from the genus Squamarina and recircumscribe this genus as follows: thallus saxicolous or terricolous, squamulose, placodioid or subfoliose, squamules or lobes dispersed, continuous to irregularly overlapping, very thick, usually with a white, thickened and slightly upturned marginal rim; upper surface white, yellowish-green, grey green to olive green, smooth to strongly cracked and wrinkled; lower surface white, pale brown to blackish-brown, well defined but without cortex; thallus section with well-differentiated upper cortex, algae layer and medulla; upper cortex with pale brown granules, turning colourless in potassium hydroxide (KOH); algal layer continuous; medulla very thick, filled with grey calcium oxalate crystals that become needle shaped after treatment with 25% sulphuric acid (H 2 SO 4 ); apothecia lecanorine type, algal layer usually absent from the margin and only present under hypothecium, rarely biatorine type because of the strong convex disc; disc light yellow, yellow, pale brown to reddishbrown, pruinose or not; ascus narrowly clavate, Porpidia-type, 8-spored; ascospores colourless, ellipsoid to subfusiform, non-septate; pycnidia yellowish-brown, conidia filiform, curved; usnic acid always present and psoromic acid also present in most species. Apothecia rounded, sessile, constricted at the base, up to 2 mm in diam.; disc plane to slightly convex, blackish-brown, non-pruinose; thalline margin entire, concolorous with thallus; hymenium colourless, ca. 60 μm high; subhymenium and hypothecium colourless, I + blue; epihymenium consisting of brown granules, ca. 15 μm high; paraphyses simple, slightly thickened at the apex, ca. 3 μm in diam.; asci Aspicilia-type, 8-spored; ascospores colourless, ellipsoid, 9-13 × 5-9 μm.
Ecology and distribution. Growing on soil in very dry habitats at elevations of 1760-3151 m. This species was previously only known from Gansu Prov. and is reported here as new to Qinghai Prov., China.
Notes. The holotype consists of numerous fragments on soil, without apothecia but numerous pycnidia. This species was originally described as a Lecanora by Magnusson (1940) and transferred to Squamarina by Wei (1991). We initially treated our materials as "S. semisterilis" since their morphology was identical with the holotype, which is characterised by the pruinose and lobate thallus containing norstictic acid, terricolous habit, pycnidia resembling apothecia and bacilliform conidia. We transfer this species to the genus Lobothallia, based on the phylogenetic reconstruction. Its position within this genus is supported by the lobate and slightly convex thallus, the Aspicilia-type ascus, the bacilliform conidia and the absence of usnic acid.
Ecology and distribution. Growing on rock in arid environments at elevations of 984-2100 m. Previously only known from Yunnan Prov., here reported as new to Sichuan Prov., China.
Notes. The isotype grows on quartzitic rock ca. 2 cm across, containing several intact apothecia. The spore size of "Squamarina callichroa", given in the protologue, is 15-20 × 8-9 μm (Zahlbruckner 1930); however, Poelt (1958) measured the spore size of the type material as 11-12 × 8-9 μm. Our measurements of the freshly collected materials, 9.5-13.5 × 6-9 μm, are in accordance with Poelt's results and the other characteristics, elevation and locality of our collections are more or less identical with the isotype. We did not find any specimens around the type locality having those long ascospores as in the description of the protologue. Therefore, we treat our specimens as "Squamarina callichroa".This species was originally described as a Lecanora by Zahbruckner (1930) and transferred to Squamarina as the type species of the section Petroplaca by Poelt (1958). We transfer this species to the genus Rhizoplaca, primarily based on its nested position within the R. chrysoleuca group in the phylogeny (Fig. 4) and also based on the orange apothecia, the Lecanora-type ascus and the presence of usnic and placodiolic acids. The genus Rhizoplaca is a small genus containing eleven species (Lücking 2017). We added nine of these species as intergroups to assess the phylogenetic position of R. callichroa in the genus. The results show that R. callichroa is sister to R. chrysoleuca and R. huashanensis J.C. Wei, which differ by the umbilicate thallus, narrower ascospores, (7)8.5-12 × 3.5-6 μm and the monophyllus thallus and black apothecia, respectively (Nash et al. 2002;Wei 1984). Rhizoplaca subdiscrepans (Nyl.) R. Sant. is similar to R. callichroa in the squamulose thallus and orange apothecia, but differs in the very convex and smaller (0.3-1 mm) squamules and the narrower ascospores 7-12 × 3.5-5 μm.    Apothecia common, usually densely grouped, irregular in shape, up to 5 mm in diam.; disc black, pruinose at the centre, plane when young, strongly concave with age; thalline margin thin and crenate, strongly bending towards inside with age; hymenium colourless, I+ blue, ca. 50 μm high; epihymenium containing yellowish-brown granules, ca. 9.5 μm high; subhymenium and hypothecium colourless; paraphyses evenly septate, simple, 2-3 μm in diam., apex more or less swollen and bluish-green, ca. 4.5 μm in diam.; ascus Lecanora-type, 8-spored; ascospores regular in shape, ellipsoid, colourless, 5.8-8 × 3-4.5 μm.
Ecology and distribution. Growing on rock at elevations of 3291-3909 m. Only known from Gansu Prov., China.
Notes. The holotype grows on rock with Lecidea tessellata Flörke, Lecanora asiatica H. Magn. and Xanthoria elegans (Link) Th. Fr. and contains numerous apothecia. This species was originally described as a Lecanora by Magnusson (1940) and transferred to Squamarina by Wei (1991). It is characterised by the yellowish, areolate and very thick thallus, the black lecanorine apothecia and the very small ascospores. We transfer this species to Rhizoplaca, primarily based on the phylogenetic results (Fig.  4) and also based on the yellow thallus, the large, concave apothecia with margins bending towards the inside and the Lecanora-type ascus. Rhizoplaca pachyphylla is phylogenetically closely related to R. callichroa, R. chrysoleuca and R. huashanensis, but differs in the very thick and areolate thallus without lobate margin and the very small ascospores, 5.8-8 × 3-4.5 μm. Rhizoplaca subdiscrepans is similar to R. pachyphylla in the squamulose thallus, but differs in the orange apothecia, longer ascospores, 7-12 × 3.5-4.5 μm, and the presence of pseudoplacodiolic or placodiolic acids. Rhizoplaca melanophthalma (DC.) Leuckert is also similar to the species in having black apothecia, but differs in the umbilicate thallus and the larger ascospores, 6.5-12 × 4-7 μm. Description. Thallus to 6 cm across, squamulose or lobate, growing on moss over rock or on the meadow; squamules 0.5-2 mm across, convex, continuous to slightly overlapped; marginal lobes branched, convex, 0.5-2 mm wide, 2-4 mm long; the apex of squamules and lobes rounded, bent downwards; upper surface smooth, pale green to straw, covered by white pruina; lower surface pale to dark brown in the centre and white to pale brown at the margin; rhizinose strands blackish-brown. Upper cortex very thin, ca. 16 μm, filled with yellowish-brown granules dissolving in KOH; algal layer continuous, 48-60 μm thick, medulla filled with grey to pale brown granules, 129-161 μm high, medullary hyphae very loose, more or less hollow in centre; lower cortex well separated from medulla, evenly thick with strongly gelatinised and anticlinally arranged hyphae, ca. 80 μm thick, colourless, hyphae at lower part brown. Apothecia lecanorine, sessile, with constricted base, rounded, scattered or in small groups, up to 3 mm in diam.; disc pruinose, reddish to dark brown, slightly concave when young, slightly convex with age; thalline margin concolorous with thallus, entire to flexuose, forming a well-delimited cortex consisting of strongly gelatinised and anticlinally arranged hyphae; hymenium colourless, 58-80 μm; epihymenium filled with yellowish-brown granules, 10-15 μm; paraphyses simple, evenly septate; ascus Lecanora-type, 8-spored; ascospores colourless, ellipsoid to slightly ovoid, 7-13 × 6.5-9 μm. Chemistry. Upper cortex K-, C-, P + yellow, medulla K+ yellow, C-, P-; usnic acid and zeorin present in each sample, placodiolic and isousnic acids also present in most samples.
Ecology and distribution. Growing on moss over rock or in meadow at 3600-4968 m elevation in the alpine zone. Worldwide distribution: China, India and Nepal. China: Sichuan Prov., reported here as new to Yunnan and Xizang provinces.
Notes. The holotype of Lecanora chondroderma consists of several fragments, containing numerous apothecia.
Lecanora chondroderma was originally described by Zahbruckner (1930) and transferred to Squamarina by Wei (1991). We transfer this species back to Lecanora temporarily because of its Lecanora-type ascus and phylogenetic position being closely related to the genera Rhizoplaca and Protoparmeliopsis (Fig. 4). Although Lecanora chondroderma is highly supported as a basal clade of the genus Rhizoplaca in our topology, it differs in dwelling on moss and meadow and having numerous rhizinose strands. Given that there are still many taxa of Lecanora which have not been included in our analyses and the phylogenetic relationships between Rhizoplaca and its related genera have still not been thoroughly resolved, we prefer to retain this species in Lecanora temporarily, rather than treat it as Rhizoplaca. Lecanora chondroderma is only known from the Himalayan region at elevations between 3600-4968 m. The morphology of the species varies amongst localities, with samples growing on moss over rock in Yunnan and Sichuan provinces, having broad (1-2 mm) and pale green lobes and samples from meadows at higher altitudes in Xizang Prov. developing narrower (0.5-1 mm) and more branched lobes with a yellowish appearance. These populations, however, share a pruinose thallus, convex lobes with rounded and downwards bent apices, a loose medulla, a well-delimited cortex of the thalline margin and lower cortex and the presence of usnic acid and zeorin.
Ecology and distribution. Growing on soil at 1310-4730 m of elevation. Previously only known from Gansu Prov. and reported here as new to Neimenggu, Ningxia, Sichuan, Xizang, Xinjiang and Yunnan provinces, China.
Notes. The holotype consists of several small fragments on soil, bearing a single small apothecium. This species was originally described as a Lecanora by Magnusson (1940) and transferred to Squamarina by Poelt (1958). It is characterised by the pruinose, greenish-to straw-coloured thallus, lobes with white, thickened and slightly upturned edges, exposing a milk-white to pale lower surface, without rhizines and the presence of psoromic and 2'-O-demethypsoromic acids. This species is very common in the deserts and alpine zones of China. In desert regions, the thallus is usually irregular in outline with wider lobes and becomes rosette-like with narrower lobes when growing in the alpine zone.
The genus Squamarina (= S. sect. Squamarina) includes eleven species (Poelt 1958) and there are three species with sequences in GenBank. We integrated the data from GenBank with the newly-produced data here to reconstruct the phylogeny of the genus Squamarina to assess the phylogenetic position of the species S. kansuensis (Fig.  6). The results show that S. kansuensis is a sister species to S. lentigera which, in turn, is also very similar in morphology, but differs in the larger thallus and by containing psoromic and 2'-O-demethypsoromic acids. Squamarina nivalis Frey & Poelt and S. provincialis Clauzade & Poelt are similar to S. kansuensis in having a strongly white pruinose thallus; however, S. nivalis differs in the smaller thallus, ca. 2 cm, not cracked upper surface, the apices of lobes bent downwards and the absence of psoromic acid; S. provincialis differs in the continuous but never overlapped lobes, the absence of the white thickened edges of lobes and the presence of atranorin. So far, the two species, S. nivalis and S. provincialis, are only known from very restricted places from Europe. Description. Thallus placodioid to subfoliose, rather closely attached to calcareous rocks, olive-green turning to yellowish-brown in the herbarium, up to 8 cm across and 5 mm high in the centre; lobes 2-4 mm long, 1.5-2.5 mm wide, ca. 1 mm thick, apices usually detached from the substrate with a white thickened edge; upper surface pruinose at least on the margins, matt to somewhat shiny, centrally cracked and faveolate-wrinkled, strongly convex, giving the thallus centre a bullate appearance, the base of the bullae carbonised, black; lower surface covered with pale brown to blackish-brown pulvinate hyphae, with sparse to numerous rhizinose strands; rhizinose strands brown to black, irregularly branched, up to 5 mm long. Upper cortex filled with yellowish-brown granules, turning colourless in KOH, 62-75 μm high, without epinecral; algal layer continuous, 65-70 μm thick; medulla filled with grey crystals of calcium oxalate and brick-red hyphae in lower part; lower cortex lacking. Apothecia common but not abundant, laminal, scattered to slightly grouped, up to 4 mm in diam.; disc concave, plane to convex, light yellow, covered by yellowish pruina; thalline margin pruinose or not, darker than thallus, shiny, entire and distinctive when young, excluded with age. Hymenium 75-85 μm high, hyaline, I+ blue; epihymenium filled with yellowish-brown granules, not disperse into hymenium, turning colourless in KOH, 5-12.5 μm high; thalline margin without algae in the upper part, cortex filled with yellowish-brown granules, 112-125 μm thick; paraphyses septate, tips not swollen; hypothecium colourless, 100-162 μm thick, with pale