﻿Pseudolepraria, a new leprose genus revealed in Ramalinaceae (Ascomycota, Lecanoromycetes, Lecanorales) to accommodate Leprariastephaniana

﻿Abstract The new genus Pseudolepraria Kukwa, Jabłońska, Kosecka & Guzow-Krzemińska is introduced to accommodate Leprariastephaniana Elix, Flakus & Kukwa. Phylogenetic analyses of nucITS, nucLSU, mtSSU and RPB2 markers recovered the new genus in the family Ramalinaceae with strong support. The genus is characterised by its thick, unstratified thallus composed entirely of soredia-like granules, the presence of 4-O-methylleprolomin, salazinic acid, zeorin and unknown terpenoid, and its phylogenetic position. The new combination, P.stephaniana (Elix, Flakus & Kukwa) Kukwa, Jabłońska, Kosecka & Guzow-Krzemińska, is proposed.


Introduction
During the evolution in some groups of lichenized fungi the ability to reproduce sexually has been apparently lost completely and some phylogenetic lineages are known to develop exclusively asexual lichenized propagules. This includes Lepraria Ach.
Lepraria includes at present c. 75 species (Wijayawardene et al. 2017;Guzow-Krzemińska et al. 2019a;Barcenas-Peña et al. 2021), most of which were described based on chemical (secondary metabolites) and morphological features and some also by molecular markers (e.g., Laundon 1989Laundon , 1992Tønsberg 1992;Lendemer 2011aLendemer , 2012Lendemer , 2013aLendemer and Hodkinson 2013;Guzow-Krzemińska et al. 2019a;Barcenas-Peña et al. 2021). One of the species that was placed in Lepraria based solely on morphological similarity to other members of the genus was L. stephaniana Elix, Flakus & Kukwa (Flakus et al. 2011a). This species is characterised by the thick, unstratified and non-lobed thallus composed of coarse soredia-like granules with soft appearance, and the production of 4-O-methylleprolomin, salazinic acid and terpenoids. 4-O-methylleprolomin was known only in a single Pannaria species before its discovery in L. stephaniana (Flakus et al. 2011a). Lepraria stephaniana has been known until recently only from the type locality, however during field studies in 2017 in Bolivia we found two new localities of the species (one close to the type locality) (Guzow-Krzemińska et al. 2019b). Sequencing of molecular markers of those two recently collected specimens revealed that L. stephaniana is unrelated to other species of Lepraria s.str., but instead it appeared to be nested within Ramalinaceae as a previously unsequenced lineage close to Cliostomum Fr., Ramalina Ach. and allied genera. In this paper we introduce the new genus Pseudolepraria for this peculiar lineage within Ramalinaceae.

Taxon sampling
The studied specimens are deposited in B, BG, KRAM, LPB, NY and UGDA herbaria. Morphology was examined by using Nikon SMZ 800N stereomicroscope. The secondary chemistry of all samples was studied by thin layer chromatography (TLC) following methods by Culberson and Kristinsson (1970) and Orange et al. (2001a).
The efficiency of the PCR was checked by visualising the reaction products on a 1% agarose gels stained with SimplySafe (Eurx) dye in order to determine DNA fragment lengths. Afterwards, PCR products were purified using Clean-Up Concentrator (A&A Biotechnology). The sequencing was performed in Macrogen Europe (The Netherlands), using amplification primers. The newly obtained sequences were deposited in GenBank database and their accession numbers are listed in Table 1.

Sequence alignment and phylogenetic analysis
The newly generated sequences were compared to the sequences available in the Gen-Bank database (http://www.ncbi.nlm.nih.gov/BLAST/) using BLASTn search (Altschul et al. 1990). For the phylogenetic analyses we used representatives of Ramalinaceae and Boreoplaca ultrafrigida Timdal and Ropalospora lugubris (Sommerf.) Poelt were used as outgroup taxa according to previous studies (Kistenich et al. 2018;Orange 2020; van den Boom and Magain 2020). The independent alignments for each marker were generated in MAFFT using auto option and default parameters (Katoh and Standley 2013). The datasets were then subjected to Guidance2 server (Landan and Graur 2008;Penn et al. 2010;Sela et al. 2015; http://guidance.tau.ac.il/) for further analysis. The MSA algorithm was set to MAFFT and 100 bootstrap replicates were used.
The Guidance confidence scores were calculated and columns with a score < 0.93 were excluded from the alignments. The terminal ends were trimmed. Single-locus matrices consisted of 61 sequences for nucITS, 62 sequences for mtSSU, 51 sequences for nu-cLSU, and 44 sequences for RPB2. The best ML tree was inferred for each locus using IQ-TREE with 1000 ultrafast bootstrap replicates as implemented in the IQ-TREE web server (Nguyen et al. 2015;Chernomor et al. 2016;Kalyaanamoorthy et al. 2017;Hoang et al. 2018). Congruence was examined by eye and no significant conflict between loci was observed.
For the final analysis, we concatenated four markers which resulted in a dataset of 66 terminals and 3766 positions. The concatenated dataset was subjected to IQ-TREE analysis to find best-fitting nucleotide substitution models (Nguyen et al. 2015;Chernomor et al. 2016;Kalyaanamoorthy et al. 2017;Hoang et al. 2018). The model selection was restricted to models implemented in MrBayes and the following nucleotide substitution models for the four predefined subsets were selected: GTR+F+I+G for mtSSU rDNA, K2P+I+G for nucITS, and SYM+I+G for both nucLSU rDNA and RPB2 markers. The search for maximum likelihood tree was performed in IQ-TREE and followed with 1000 standard bootstrap replicates (Nguyen et al. 2015;Chernomor et al. 2016;Kalyaanamoorthy et al. 2017;Hoang et al. 2018).
Bayesian analysis was carried out using a Markov Chain Monte Carlo (MCMC) method, in MrBayes v. 3.2.6 (Huelsenbeck and Ronquist 2001;Ronquist and Huelsenbeck 2003) on the CIPRES Web Portal (Miller et al. 2010) using previously selected models (see above). Two parallel MCMC runs were performed, each using four independent chains and ten million generations, sampling every 1000 th tree. The resulting log files were analysed using Tracer 1.7.2 (Rambaut et al. 2018). Posterior probabilities (PP) were determined by calculating a majority-rule consensus tree after discarding the initial 25% trees of each chain as the burn-in. The convergence of the chains was confirmed by the convergent diagnostic of the Potential Scale Reduction Factor (PSRF), which approached 1 and the 'average standard deviation of split frequencies' was < 0.01). Phylogenetic trees were visualised using FigTree v. 1.4.3 (Rambaut 2009) and modified in Inkscape (https://inkscape.org/). Bootstrap support (BS values ≥ 70) and PP values (values ≥ 0.95) are given near the branches on the phylogenetic tree. The data were deposited at TreeBASE (Submission ID: 30149).

Results and discussion
For this work we successfully sequenced nucITS, mtSSUand nucLSU from two specimens and additionally RPB2 from one specimen of Lepraria stephaniana collected in Bolivia (Table 1). BLAST searches of the nucITS, nucLSU, mtSSU and RPB2 markers surprisingly showed the highest similarities to representatives of the family Ramalinaceae, i.e., the genera Cenozosia A. Massal., Cliostomum and Ramalina. Phylogenetic analysis of the concatenated dataset shows that L. stephaniana is nested inside Ramalinaceae. The newly sequenced specimens of the species were resolved in a distinct and highly supported clade sister to a clade consisting of Cliostomum s.str. represented by the type species C. corrugatum (Ach.) Fr., Cenozosia inanis (Mont.) A. Massal. and a subclade of several species of Ramalina, Namibialina melanothrix (Laurer) Spjut & Sérus., Niebla (Ach.) Rundel & Bowler and Vermilacinia breviloba Spjut & Sérus. (Fig. 1). A new monotypic genus, Pseudolepraria, is introduced for this lineage of Lepraria stephaniana and is characterised by a thick, unstratified thallus composed of soredia-like granules, and the presence of 4-O-methylleprolomin, salazinic acid, zeorin and unknown terpenoid.
Pseudolepraria is the first genus forming leprose and sterile thalli that can be placed with high support within Ramalinaceae. Orange (2020) described the genus Lithocalla and placed it with uncertainty in Ramalinaceae. In our phylogeny, Lithocalla forms the sister group to Ramalinaceae sensu Kistenich et al. (2018), but this may be an artifact of the taxon sampling. The particular placement of the genus was beyond the scope of this study. Lithocalla was introduced for two species, which were originally placed, due to morphological similarities, in Lepraria, i.e., L. ecorticata (J. R. Laundon) Kukwa and L. malouina Øvstedal (Kukwa 2006;Fryday and Øvstedal 2012). Both, Lithocalla ecorticata (J. R. Laundon) Orange known from Great Britain and Norway, and L. malouina (Øvstedal) Fryday & Orange found in the Falkland Islands (Fryday and Øvstedal 2012;Orange 2020), differ from Pseudolepraria stephaniana, in their distribution in colder climates, by the production of usnic and fatty acids, the absence of zeorin and the exclusively saxicolous habitat (Orange 2020).
Species resembling Pseudolepraria in the Ramalinaceae have up until recently been included in Crocynia (Ach.) A. Massal. This genus was established for lichens with a non-corticate, byssoid, felt-like thallus and historically included several species now placed mostly in Lepraria (e.g., Laundon 1989Laundon , 1992Kistenich et al. 2018). According to Lücking et al. (2017) Crocynia comprised three species and two of them were included in the phylogeny of Ramalinaceae by Kistenich   formed a clade nested inside Phyllopsora Müll. Arg. Consequently, Crocynia was synonymised with Phyllopsora, also because of morphological similarities (Kistenich et al. 2018). The status of the third species, C. microphyllina Aptroot (Lumbsch et al. 2011), and three species discussed by Sipman (2018) remains uncertain. The species historically placed in Crocynia differ from Pseudolepraria in the byssoid thalli not producing 4-O-methylleprolomin and in sometimes producing apothecia (Cáceres 2007;Lumbsch et al. 2011;Aptroot and Cáceres 2014;Sipman 2018).
Pseudolepraria is very similar to Lepraria s.str. in sharing the same thallus morphology and, to a certain extent, secondary chemistry (presence of salazinic acid and terpenoids) (e.g., Aptroot 2002;Sipman 2004;Saag et al. 2009;Flakus et al. 2011a;Lendemer 2011a, b). They differ, apart from the phylogenetic position, only in the presence of the very rarely reported 4-O-methylleprolomin, a diphenyl ether previously found only in one Pannaria species (Flakus et al. 2011a). Pseudolepraria differs also in the habitat preferences. It was found only in tropical forests at low elevations (300-470 m a.s.l.), whereas Lepraria in tropical South America, including Bolivian ecosystems, are found mostly above 1000 m above sea level (only one locality of L. finkii (B. de Lesd.) R.C. Harris found at the elevation of 890 m), in montane forests and open high Andean vegetation (Flakus and Kukwa 2007;Flakus et al. 2011aFlakus et al. , b, 2012Flakus et al. , 2015Guzow-Krzemińska et al. 2019a). This is in agreement with the statement presented by Orange et al (2001b), who considered Lepraria to be restricted to montane habitats in the tropics. Poelt (1987) considered the genus Lepraria as a 'box of analogous groups of lichens of completely different origin, held together by the same highly specialized thallus type'. Poelt (1987) also stated that the leprarioid thallus type and the loss of generative reproduction developed in evolution through the reduction of the thallus structures as an adaptation for growing in bark crevices and on over-hanging rocks in ecologically specialised group of lichenized fungi, which includes Lepraria, but also, as Poelt (1987) mentioned, Leproplaca (Nyl.) Nyl. and some species of the genus Chrysothrix Mont. (Poelt 1987). However, this is only partly true, as some lichen groups with this type of thallus (e.g., species of Lepraria neglecta group) can grow also in other habitats (e.g., Laundon 1992;Lendemer 2013b). Nevertheless, the statement of Poelt (1987) was true and innovative at this time and it was later shown that the leprarioid thallus indeed originated in several unrelated lichen lineages (e.g., Ekman and Tønsberg 2002;Kukwa and Pérez-Ortega 2010;Hodkinson and Lendemer 2013;Malíček et al. 2018;Guzow-Krzemińska et al. 2017;Orange 2020). Furthermore, some leprarioid genera are known exclusively in sterile state, like Andreiomyces (Arthoniales, Arthoniomycetes), Botryolepraria (Verrucariales, Eurotiomycetes), Lepraria and Lithocalla (both in Lecanorales, Lecanoromycetes) (Ekman and Tønsberg 2002;Kukwa and Pérez-Ortega 2010;Hodkinson and Lendemer 2013;Orange 2020). Pseudolepraria is another addition to this group, however, as only a few collections are available, it may eventually be found with ascomata. Buschbom and Mueller (2006) suggested that the asexual way of reproduction is advantageous because the symbiosis with the optimal photobiont for a given environment allows the rapid dissemination of both partners. Therefore, it is more important for the mycobiont to keep the relationship with suitable algal species; however this does not mean that the symbiosis in asexually reproducing species cannot be broken. Kosecka et al. (2021) showed for some Lepraria species that the mycobiont can form thalli with different, locally adapted algal strains. We partially sequenced the nucITS region of the photobiont of Pseudolepraria stephaniana (Table 1) and found that both thalli associate with the same green algal partner (100% of identity). BLAST hits were closest to Symbiochloris, Dictyochloropsis, Massjukichlorella and Watanabea spp., all of which were quite dissimilar to the photobiont sequences of Pseudolepraria stephaniana. . Thallus crustose, thick, usually not delimited nor lobed, green-grey to creamy-white, not stratified, but sometimes with a poorly differentiated, pseudo-medullary layer of decaying granules. Hypothallus indistinct. Granules coarse with soft appearance, irregularly rounded, up to 100(-200) µm in diam., composed of very lax hyphae mixed with algal cells, usually with projecting hyphae up to c. 30(-50) µm long. Photobiont green, cells globose, up to 12 µm in diam.
Distribution and habitat. The species is known only from three localities in Bolivia. It was found on bark of trees in transition Chaqueño-Amazon or preandean Amazon forests at elevation between c. 300 to 470 m a.s.l. (Flakus et al. 2011a;Guzow-Krzemińska et al. 2019b).