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Research Article
Pseudolepraria, a new leprose genus revealed in Ramalinaceae (Ascomycota, Lecanoromycetes, Lecanorales) to accommodate Lepraria stephaniana
expand article infoMartin Kukwa‡, Magdalena Kosecka‡, Agnieszka Jabłońska‡, Adam Flakus§, Pamela Rodriguez-Flakus§, Beata Guzow-Krzemińska‡
‡ University of Gdańsk, Gdańsk, Poland
§ W. Szafer Institute of Botany, Polish Academy of Sciences, Kraków, Poland

Corresponding author: Martin Kukwa ( dokmak@ug.edu.pl )

Corresponding author: Beata Guzow-Krzemińska ( beata.guzow@biol.ug.edu.pl )

Academic editor: Thorsten Lumbsch
© 2023 Martin Kukwa, Magdalena Kosecka, Agnieszka Jabłońska, Adam Flakus, Pamela Rodriguez-Flakus, Beata Guzow-Krzemińska.
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: Kukwa M, Kosecka M, Jabłońska A, Flakus A, Rodriguez-Flakus P, Guzow-Krzemińska B (2023) Pseudolepraria, a new leprose genus revealed in Ramalinaceae (Ascomycota, Lecanoromycetes, Lecanorales) to accommodate Lepraria stephaniana. MycoKeys 96: 97-112. https://doi.org/10.3897/mycokeys.96.98029
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Abstract

The new genus Pseudolepraria Kukwa, Jabłońska, Kosecka & Guzow-Krzemińska is introduced to accommodate Lepraria stephaniana 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.

Keywords

Lichenized fungi, morphology, Neotropics, secondary metabolites, sterile lichens, taxonomy

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. (Ascomycota, Lecanoromycetes, Lecanorales, Stereocaulaceae), a well-known genus which up to quite recently comprised only crustose lichens with morphologically simple thalli consisting of soredia-like granules laying directly on substrate or on a layer of hypothalline hyphae (e.g., Ekman and Tønsberg 2002; Kukwa 2002; Sipman 2004; Flakus and Kukwa 2009; Flakus et al. 2011a; Lendemer 2011a, b, 2013a; Lendemer and Hodkinson 2013; Guzow-Krzemińska et al. 2019a). However, Lendemer and Hodkinson (2013) found, based on molecular data, that some fruticose species previously referred to Leprocaulon Nyl. also represented Lepraria s.str. and they were subsequently transferred to the latter genus. In contrast to their simplified morphology, the species produce a vast variety of secondary lichen metabolites, which are an invaluable tool, together with morphological characters that may be sparse, in the recognition of species and their identification (e.g., Laundon 1989, 1992; Tønsberg 1992; Sipman 2004; Kantvilas and Kukwa 2006; Flakus and Kukwa 2007; Saag et al. 2009; Flakus et al. 2011a; Lendemer 2011a, 2013a; Lendemer and Hodkinson 2013; Guzow-Krzemińska et al. 2019a; Kukwa 2019). It is also noteworthy that some species until recently classified as Lepraria have been shown to belong to other genera (e.g., Leprocaulon and Septotrapelia Aptroot & Chaves; Bungartz et al. 2013; Lendemer and Hodkinson 2013) or even new genera were established for some peculiar species, e.g., Andreiomyces Hodkinson & Lendemer within Arthoniomycetes (Hodkinson and Lendemer 2013), Botryolepraria Canals et al., related to Verrucariaceae in Eurotiomycetes (Kukwa and Pérez-Ortega 2010) and Lithocalla Orange in Lecanorales (probably in Ramalinaceae) in Lecanoromycetes (Orange 2020).

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 1989, 1992; Tønsberg 1992; Lendemer 2011a, 2012, 2013a; Lendemer 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.

Materials and methods

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).

DNA extraction, PCR amplification and DNA sequencing

DNA was extracted using a modified CTAB method (Guzow-Krzemińska and Węgrzyn 2000). We analysed four fungal markers: nucITS rDNA, mtSSU rDNA, nucLSU rDNA, and RPB2 gene. For this purpose we used the following primers: ITS1F (Gardes and Bruns 1993) and ITS4A (Kroken and Taylor 2001) for nucITS rDNA; mrSSU1 and mrSSU3R (Zoller et al. 1999) for mtSSU rDNA; ITS4A-5’ (Kroken and Taylor 2001; Nelsen et al. 2011) and LR5 (Vilgalys and Hester 1990) for nucLSU rDNA; fRPB2-5F and fRPB2-7cR (Liu et al. 1999) for RPB2 gene. Additionally, nucITS rDNA region from green algal partner was amplified using Al1500bf (Helms et al. 2001) and ITS4M primers (Guzow-Krzemińska 2006). PCR was performed in a volume of 25 µl using StartWarm HS-PCR Mix (A&A Biotechnology) following the manufacturer’s protocol. 1 µl of genomic DNA was used for amplification. The PCR cycling parameters are available in Suppl. material 1.

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.

Table 1.
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Species used in this study with their GenBank accession numbers. New sequences are marked in bold.

Species nucITS rDNA nucLSU rDNA mtSSU RPB2 Algal nucITS rDNA
Aciculopsora salmonea MG925948 – MG925842 –
Aciculopsora srilankensis MK400258 – MK400211 –
Bacidia arceutina AF282083 MG926041 MG925846 MG926230
Bacidia rosella AF282086 AY300829 AY300877 AM292755
Bacidina arnoldiana AF282093 MG926048 MG925854 MG926238
Bacidina phacodes AF282100 MG926049 AY567725 MG926240
Badimia dimidiata MG925956 MG926052 AY567774 –
Bellicidia incompta AF282092 MG926043 MG925849 MG926233
Biatora globulosa AF282073 MG926055 KF662414 KF662450
Biatora vacciniicola MG925960 MG926060 MG925861 MG926245
Biatora vernalis AF282070 DQ838752 DQ838753 –
Bibbya albomarginata MG926024 MG926115 MG925927 MG926286
Bibbya vermifera AF282109 MG926047 MG925852 MG926237
Bilimbia sabuletorum AM292670 AY756346 AY567721 AM292761
Boreoplaca ultrafrigida HM161512 DQ986797 DQ986813 DQ992421
Catillaria scotinodes AM292673 MG926064 AM292720 AM292763
Catinaria atropurpurea MG925965 MG926065 MG925865 MG926246
Catolechia wahlenbergii HQ650649 DQ986794 DQ986811 DQ992424
Cenozosia inanis – MG926066 MG925866 –
Cliomegalaria symmictoides MW622003 MW621867 MW622006 –
Cliostomum corrugatum MG925966 MG926067 AY567722 KF662436
Cliostomum haematommatis MK446224 – MK446223 –
Eschatogonia prolifera MG925969 MG926070 MG925870 MG926249
Kiliasia athallina MG926023 MG926114 – MG926284
Kiliasia sculpturata MG926034 MG926122 MG925938 MG926295
Krogia coralloides MG925977 MG926072 MG925875 MG926251
Lecania aipospila MG925978 MG926073 MG925876 MG926252
Lecania erysibe AM292682 MG926074 AM292733 AM292769
Lecania fuscella AM292684 MG926075 MG925877 –
Lecidea albohyalina KF650950 MG926079 KF662398 KF662438
Lithocalla ecorticata KT962179 – KT962184 –
Lithocalla malouina KT962178 – MT857015 –
Lueckingia polyspora MG925984 MG926082 MG925882 –
Megalaria grossa AF282074 MG926083 MG925883 MG926257
Megalaria versicolor – AY584651 AY584622 DQ912401
Mycobilimbia pilularis KF650954 – KF662402 KF662442
Mycobilimbia tetramera – KJ766600 KJ766439 KJ766957
Niebla homalea MG925987 – MG925888 –
Namibialina melanothrix MG926038 MG926128 MG925945 MG926303
Parallopsora brakoae MG925989 – MG925891 –
Parallopsora leucophyllina MG925994 – MG925897 MG926265
Phyllopsora breviuscula MG925990 MG926087 MG925892 MG926262
Phyllopsora gossypina MG925967 MG926068 MG925867 MG926247
Phyllopsora parvifoliella MG925999 MG926092 MG925902 MG926267
Physcidia wrightii MN334233 – MN334227 –
Pseudolepraria stephaniana Kukwa 19740 OQ172237 OQ172242 OQ172251 – OQ303855
Pseudolepraria stephaniana Kukwa 19267 OQ172236 OQ172243 OQ172250 OQ160272 OQ303854
Ramalina dilacerata MG926013 MG926104 MG925917 –
Ramalina fraxinea MG926014 MG926105 MG925918 MG926277
Ramalina mannii MG926019 MG926111 – MG926280
Ramalina pollinaria MG926017 MG926108 AM292752 MG926278
Ramalina sinensis MG926018 MG926110 MG925921 –
Rolfidium bumammum MG926027 MG926117 MG925930 MG926288
Ropalospora lugubris MG926020 – MG925922 –
Scutula circumspecta – – MG925848 –
Sporacestra pertexta MG926000 MG926093 MG925903 MG926268
Stirtoniella kelica MG926021 – MG925923 –
Thalloidima candidum AF282117 MG926118 MG925932 MG926290
Thalloidima toninianum MG926036 MG926124 MG925942 MG926298
Thamnolecania brialmontii AF282066 MG926112 MG925925 MG926283
Toninia cinereovirens AF282104 AY756365 AY567724 AM292781
Toninia populorum MG925950 MG926039 MG925843 MG926228
Toniniopsis aromatica AF282126 MG926113 MG925926 MG926284
Toniniopsis subincompta AF282125 MG926046 MG925851 MG926236
Tylocliostomum viridifarinosum NR_174049 – – –
Tylothallia biformigera AF282077 MG926129 MG925946 MG926304
Waynea californica – MG926130 MG925947 MG926305
Vermilacinia breviloba MN811352 MN811548 – MN757330

Sequence alignment and phylogenetic analysis

The newly generated sequences were compared to the sequences available in the GenBank 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 nucLSU, 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 1000th 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.

Figure 1. 

Majority-rule consensus tree resulting from MrBayes analysis of the concatenated mtSSU, nucITS, nucLSU and RPB2 markers with Bayesian PP (values ≥ 0.95) and IQ-TREE bootstrap support values (BS values ≥ 70) given near the branches. Pseudolepraria is marked in blue.

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 1989, 1992; Kistenich 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 et al. (2018), where they 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. 2011a, b, 2012, 2015; Guzow-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.

Taxonomy

Pseudolepraria Kukwa, Jabłońska, Kosecka & Guzow-Krzemińska, gen. nov.

MycoBank No: 847408

Diagnosis

Characterised by thick, unstratified thallus composed of soredia-like granules, the presence of 4-O-methylleprolomin, salazinic acid, zeorin, and unknown terpenoid, and the phylogenetic position within Ramalinaceae.

Generic type

Pseudolepraria stephaniana (Elix, Flakus & Kukwa) Kukwa, Jabłońska, Kosecka & Guzow-Krzemińska

Description

As this is a monotypic genus the description below constitutes the generic description.

Etymology

The new name refers to the similarity to the genus Lepraria, in which this particular species was originally placed.

Pseudolepraria stephaniana (Elix, Flakus & Kukwa) Kukwa, Jabłońska, Kosecka & Guzow-Krzemińska, comb. nov.

MycoBank No: 847409
Fig. 2

Lepraria stephaniana Elix, Flakus & Kukwa, in Flakus et al., Lichenologist 43: 64, 2011 (2010). Basionym.

Type

Bolivia. Dept. La Paz: Prov. Iturralde, between Ixiamas and Santa Rosa de Maravillas villages, elev. 305 m, 13°49'16"S, 68°07'18"W, preandean Amazon forest, on bark of tree, 28 July 2008, M. Kukwa 6828 (holotype: UGDA L!; isotypes: B!, BG!, KRAM!, LPB!, NY!).

Description

(adopted from Flakus et al. 2011a). 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.

Chemistry

Substances detected: 4-O-methylleprolomin (major), salazinic acid (minor), zeorin (minor) and an unknown terpenoid (minor) with Rf class values A6, B6, C6. Thallus reactions: K+ yellow turning brownish to red, P+ yellow, C–, KC– (see also Flakus et al. 2011a).

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).

Figure 2. 

Morphology of Pseudolepraria stephaniana (type). Scale bar: 0.5 mm.

Specimens used for DNA extraction

(Table 1). Bolivia. Dept. La Paz: Prov. Abel Iturralde, between Santa Rosa de Maravillas and Tumupasa, 13°58'43"S, 67°58'14"W, elev. 300 m, natural Preandean Amazon forest, corticolous, 25 May 2017, M. Kukwa 19740 (LPB, UGDA). Dept. Santa Cruz: Prov. Ichilo, Parque Nacional y Área Natural de Manejo Integrado Amboró, Sendero a la Cascada, near Villa Amboró, 17°44'02"S, 63°35'05"W, elev. 470 m, transition Chaqueño-Amazon forest, in the valley, corticolous, 11 May 2017, M. Kukwa 19267 (LPB, UGDA).

Acknowledgements

We are very grateful to James C. Lendemer (New York Botanical Garden), Tor Tønsberg (University of Bergen) and an anonymous reviewer for invaluable comments, and the members of Herbario Nacional de Bolivia, Instituto de Ecología, Universidad Mayor de San Andrés, La Paz, for their generous cooperation. This research received funding from the National Science Centre (project no 2015/17/B/NZ8/02441).

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Supplementary material

Supplementary material 1 

The PCR parameters

Beata Guzow-Krzemińska, Magdalena Kosecka

Data type: PCR parameters (word document)

Explanation note: The PCR parameters are presented for each marker.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
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