Psora altotibetica (Psoraceae, Lecanorales), a new lichen species from the Tibetan part of the Himalayas

In the present study, we describe the new species, Psora altotibetica, from nine localities in China (Tibetan area) and Nepal. The study includes analyses of anatomy, secondary chemistry, and DNA sequence data of P. altotibetica and presumed close relatives. Psora altotibetica resembles P. indigirkae morphologically, but is phylogenetically closer to P. tenuifolia and P. vallesiaca. It differs from P. indigirkae in the colour of the apothecia, the size of the ascospores, and in the secondary chemistry. The species is terricolous and was collected in the alpine zone of the Great Himalayas between 4230 and 5000 m altitude. Psora tenuifolia and P. vallesiaca are here reported as new to China and the Himalayas.


Introduction
The genus Psora consists of c. 30 species growing on soil and rock, mainly in arid areas, from the arctic to the subtropical regions of the world (Timdal 2002).The current concept of the genus was proposed by Schneider (1980) and emended by Timdal (1984).No world monograph exists, but there are two revisions of the genus in North America (Timdal 1986(Timdal , 2002) ) and keys to the species in Europe (Poelt and Vězda 1981) and Asia (Timdal and Zhurbenko 2004).Ekman and Blaalid (2011) published a molecular phylogeny of the Psoraceae, including 18 species of Psora.
On a visit to the herbarium of Institut für Pflanzenwissenschaften, Karl-Franzens-Universität Graz (GZU) in 1992, one of us (ET) discovered an apparently undescribed species of Psora collected near the Khumbu Glacier south of Mt Everest (Nepal) by Josef Poelt in 1962.Two years later, ET was asked by Torstein Engelskjøn to identify some specimens collected by him in 1993 in the Rongbuk Valley north of Mt Everest (Tibet) during a joint Chinese-Norwegian scientific expedition.The material comprised the same apparently undescribed species of Psora.The material from both expeditions was rather poor and put aside pending richer collections.In 2013, during a visit to GZU, MB was made aware of the Psora collection that WO had made in the Himalayas in 1994 and 2000.This collection contained the putative new species, and a renewed study, including DNA analysis, has led us to describe it here as Psora altotibetica.

The specimens
This study is based on 15 collections of Psora made by WO in the Himalayas in 1994 and 2000, the collection of Psora altotibetica by J. Poelt (GZU), the two by T. Engelskjøn (TROM), two by G. & S. Miehe made in the Himalayas in 1993 (GZU), four collections from the Karakoram Range (Pakistan) in GZU which are here identified as P. himalayana and P. vallesiaca (Schaer.)Timdal, and two collections of P. indigirkae Timdal & Zhurb.from Yakutia in O. Voucher data for these 26 specimens are given in the list of examined specimens and in Table 1.The table also contains voucher data for the specimens of which DNA was downloaded from GenBank or obtained through the Norwegian Barcode of Life project (NorBOL).With the exception of one collection (P.himalayana, Zhurbenko 98161), we have examined all collections listed in Table 1 by morphology during this project or previously.

Anatomy
Microscope sections were cut on a freezing microtome and mounted in water, 10% KOH (K), lactophenol cotton blue, a modified Lugol's solution in which water was replaced by 50% lactic acid, and chlor-zinc-iodine.Amyloid reactions were observed in the modified Lugol's solution after pretreatment in K. Chlor-zinc-iodine was used to locate remnants of algae in the cortex, and polarized light was used to locate crystals of secondary metabolites and calcium oxalate.Calcium oxalate was identified by adding 25% sulphuric acid to the section; the oxalate crystals dissolve and needle shaped crystals of calcium sulphate precipitate.Ascospore measurements are given as X ± 1.5×SD, where X is the arithmetic mean and SD -the standard deviation.

Secondary chemistry
Thin-layer chromatography (TLC) was performed in accordance with the methods of Culberson (1972), modified by Menlove (1974) & Culberson and Johnson (1982).All specimens were examined by TLC, with the exception of Miehe & Miehe 9573/23/03 and Obermayer 4502, which were omitted due to being represented by richer material from the same localities.

DNA extraction, PCR and sequencing
We performed DNA extraction, PCR amplification, PCR purification, and cycle sequencing as described by Bendiksby and Timdal (2013).DNA was extracted from apothecia of 13 specimens (Table 1; GenBank Accession Numbers KU863631-KU863656).All DNA isolates produced for the present study are deposited in the DNA collection at Natural History Museum, University of Oslo.We amplified and sequenced the nuclear ribosomal internal transcribed spacer (ITS) and the mitochondrial ribosomal small subunit (mtSSU) using the primer pairs ITS5/ITS4 (White et al. 1990) and mtSSU1/mtSSU3R (Zoller et al. 1999), respectively.

Data analyses
Sequences were assembled and edited using SEQUENCHER v.4.1.4(Gene Codes Corporation, Ann Arbor, Michigan, U.S.A.).Alignments were established in BIO-EDIT 7.2.3 (Hall 1999) using the "ClustalW/Multiple alignment" option with subsequent manual adjustments.We analysed and summarized the data with parsimony and Bayesian phylogenetic methods, including model testing, as described in Bendiksby et al. (2015).The nuclear and mitochondrial datasets were analysed separately and in combination (concatenated) with indels treated as missing data.

Anatomy
The following key characters for including P. altotibetica in Psora were observed in the new species: the upper cortex contained remnants of algae throughout both the lower stainable layer and the upper epinecral layer ('Scheinrindentyp' of Poelt 1958); the hypothecium contained calcium oxalate crystals; the epihymenium contained orange crystals which dissolved in K with a purple diffusion (assumed to be anthraquinones); and the ascus contained a well-developed, amyloid tholus with a central, deeper amyloid tube structure (Porpidia-type).
The following species level characters were observed in P. altotibetica: Upper cortex composed of thin-walled hyphae with rounded lumina; lower cortex composed of mainly periclinally oriented hyphae; crystals of calcium oxalate and assumedly gyrophoric acid (dissolving in K) present both in upper cortex and medulla; no crystals in lower cortex; ascospores 9-14 × 5-7 µm.

Secondary Chemistry
The results of the TLC examinations are given in Table 1.All examined specimens of P. altotibetica contained gyrophoric acid; no traces of fatty acids were detected.

Molecular data
Altogether 26 DNA sequences were generated from 13 specimens for the present study (13 ITS and 13 mtSSU), including two specimens of P. indigirkae from Yakutia in O.In addition, seven unpublished ITS sequences of P. globifera (Ach.)A.Massal. and P. vallesiaca from Norway were generated by the lichen DNA barcode project, OLICH, at the Norwegian Barcode of Life (NorBOL).Moreover, 24 ITS and mtSSU sequences from 13 Psora specimens were downloaded from GenBank.GenBank accession numbers of all 57 sequences are given in Table 1.

Alignments and phylogenetic analyses
The ITS matrix of 32 accessions was 676 basepairs long and contained 181 parsimonyinformative characters.The basepairs and parsimony-informative characters for the mtSSU matrix of 25 accessions were 881 and 28, respectively.The estimated best fit model of evolution for ITS was SYM+G and for mtSSU it was HKY+I+G.Both parsimony jackknife and Bayesian trees of ITS vs mtSSU were congruent but resolved to various extents (not shown).Therefore, for the final analyses, a concatenated dataset of 1557 bp was used.In the Bayesian analysis of the concatenated dataset, the average standard deviation of split frequencies had fallen to 0.0045 at termination (four million generations) and the first 1000 saved trees (i.e.25%) were discarded as burn-in.The Bayesian 50% majority rule consensus tree, rooted with P. testacea, is presented with both Bayesian and parsimony branch support superimposed (Fig. 1).The molecular data group, with high support, multiple accessions of each species according to species determination based on morphology.The single exception is one accession of P. himalayana, which falls out nested within a P. vallesiaca clade.The latter consists of strongly supported subclades.Psora tenuifolia is strongly supported as phylogenetic sister species to P. altotibetica.The P. tenuifolia -P.altotibetica clade is in turn sister to the P. vallesiaca clade.A clade consisting of P. hyporubescens Timdal and P. pacifica Timdal is also strongly supported.Apart from this, the molecular data does not support any further inter-species relationships.

Discussion
Psora tenuifolia is the sister species of P. altotibetica in our phylogeny (Fig. 1).It differs in having thinner, ascending, less pruinose, more white-edged squamules containing zeorin and usually norstictic acid, and in having a well-developed lower cortex composed of mainly anticlinally oriented hyphae which are densely covered by calcium oxalate crystals (Timdal 1986).Psora tenuifolia was previously known from Alaska and arctic Canada (Timdal 1986) and from Yakutia (Zhurbenko 2003).In two specimens (Obermayer 4525 and 5236) norstictic acid was not detected by TLC; these specimens Figure 1.The Bayesian 50% majority rule consensus tree based on a concatenated alignment of ITS and mtSSU sequences of 33 accessions of 14 Psora species (see Table 1).Parsimony jackknife support values above 50% are shown below branches and Bayesian posterior probabilities above.The curly branch leading to P. testacea has been shortened to reduce the size of a broad figure .represent a previously unknown chemotype of P. tenuifolia.The species is here reported as new to China (Sichuan and Xizang) and the Himalayas.One collection (Obermayer 4487) is a mixture of P. altotibetica and P. tenuifolia.Psora vallesiaca is the phylogenetic sister species of the P. altotibetica -P.tenuifolia clade (Fig. 1).It differs from P. altotibetica in having less pruinose squamules with a more up-turned and white-edged margin, and in containing norstictic acid.It is morphologically more similar to P. tenuifolia than to P. altotibetica; see Timdal (1986) for discussion on the differences between P. tenuifolia and P. vallesiaca.Two specimens here identified as P. vallesiaca (Obermayer 4482 and 5279) do not contain lichen substances and were first thought to represent P. himalayana.We obtained sequences from the latter, and it clusters with P. vallesiaca in our phylogeny (Fig. 1, specimen P. vallesiaca 3).The sequence of P. himalayana downloaded from GenBank (specimen not examined by us, chemistry unknown) also clusters with those of P. vallesiaca in our phylogeny.Timdal (1986) remarked that P. himalayana and P. vallesiaca are sometimes difficult to distinguish morphologically, but that the chemistry is diagnostic.It now seems that there is a norstictic acid deficient chemotype of P. vallesiaca, making the taxonomic status of P. himalayana in need of revision.Psora vallesiaca is here reported as new to China (Sichuan and Xizang) and the Himalayas.
The other species of Psora known from the Himalayas, P. decipiens, differs in having orange to red or rose, more regularly rounded squamules with a usually more upturned and crenulate margin, in having strictly marginal apothecia, and in lacking lichen substances or more rarely containing norstictic acid or very rarely hyposalazinic and hypostictic acids (see, e.g., Timdal 2002).
Habitat and distribution.The species is terricolous and known from nine localities in China (Tibet) and Nepal at altitudes between 4230 and 5000 m (Fig. 3).
Etymology.The name refers to its occurrence at high altitude in the Tibetan part of the Himalayas.cially supported by the Austrian Science Fund in 1994 (project number P09663-BIO) and in 2000 (project number P13676-BIO).The seven Norwegian ITS sequences were provided by the Norwegian Barcode of Life project, funded by the Norwegian Taxonomy Initiative (Norske Artsprosjektet) administered by the Norwegian Biodiversity Information Centre (ArtsDatabanken) (project number 70184216).

Table 1 .
Psora specimens used in this study with voucher information, major lichen substances, and GenBank accession numbers.New sequences are indicated by accession numbers in bold.

Taxon, Specimen Voucher Information Major Lichen Substances GenBank Accession Number ITS mtSSU
P. altotibetica1 China, Xizang, Obermayer