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Submitted 20 January 2020
Accepted 25 June 2020
Published 3 August 2020
Corresponding author
Katarzyna Szczepańska,
katarzyna.szczepanska@upwr.edu.pl
Academic editor
Mark Young
Additional Information and
Declarations can be found on
page 21
DOI 10.7717/peerj.9555
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2020 Szczepańska et al.
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OPEN ACCESS
Taxonomic recognition of some
species-level lineages circumscribed in
nominal Rhizoplaca subdiscrepans s. lat.
(Lecanoraceae, Ascomycota)
Katarzyna Szczepańska1, Jacek Urbaniak1and Lucyna Śliwa2
1Department of Botany and Plant Ecology, Wroclaw University of Environmental and Life Sciences, Wrocław,
Poland
2Department of Lichenology, W. Szafer Institute of Botany, Polish Academy of Sciences, Kraków, Poland
ABSTRACT
Background.Rhizoplaca subdiscrepans (Nyl.) R. Sant., a saxicolous, placodioid lichen,
is considered to have a worldwide distribution in warm-temperate to boreal-arctic
areas in Asia, Europe and North America. However, recent studies have revealed that
this species includes five unrecognized species-level lineages—‘subd A, B, C, D and E’.
During research focused on the diversity of saxicolous lichens in mountainous areas of
southern Poland, some interesting representatives of the genus Rhizoplaca were found.
The main aim of our study was to determine the taxonomic status of the collected
specimens by means of molecular tools and a comparative analysis of similar herbarium
materials.
Methods. Detailed morphological, anatomical and chemical examinations of reference
material from Asia, Europe and North and South America focused primarily on
a selected group of lecanoroid taxa with a placodioid thallus. In addition, 21 new
generated sequences representing Lecanora pseudomellea, Protoparmeliopsis muralis,
Rhizoplaca opiniconensis, R. subdiscrepans s. lat.and R. phaedrophthalma were selected
for molecular study using the internal transcribed spacer region (ITS rDNA), together
with 95 available GenBank sequences mainly from the genus Rhizoplaca.
Results. Polish specimens that clustered with members of a potential species-level
lineage ‘subd E’ of Rhizoplaca subdiscrepans complex were recovered. Comprehensive
analyses of the lichen group led us to the conclusion that lineage ‘subd E’ represents
R. subdiscrepans s. str. and that the taxon appears to have a limited geographical
distribution and specific habitat preferences. Furthermore, some of the recently defined
species candidates within R. subdiscrepans s. lat.—‘subd D’ and ‘subd A’—should
be assigned to two previously known species of Rhizoplaca, namely R. opiniconensis
(Brodo) Leavitt, Zhao Xin & Lumbsch and R. phaedrophthalma (Poelt) Leavitt, Zhao
Xin & Lumbsch, respectively. These two species are characterized by phenotypic
features observed as well in analyzed specimens representing lineages ’subd D’ and
’subd A’. Moreover, the representatives of these lineages demonstrate some differences
in occupied habitat and geographical range that also correspond with the indicated
species. Additionally, it was found that Lecanora pseudomellea B.D. Ryan is a strongly
supported monophyletic lineage within Rhizoplaca, and therefore an appropriate new
combination for the species is proposed.
How to cite this article Szczepańska K, Urbaniak J, Śliwa L. 2020. Taxonomic recognition of some species-level lineages circumscribed in
nominal Rhizoplaca subdiscrepans s. lat. (Lecanoraceae, Ascomycota). PeerJ 8:e9555 http://doi.org/10.7717/peerj.9555
Subjects Biodiversity, Biogeography, Ecology, Mycology, Taxonomy
Keywords Lichenized fungi, Rhizoplaca, Cryptic species, Phylogeny, Taxonomy, Geographical
distribution, Biodiversity
INTRODUCTION
The genus Rhizoplaca Zopf belongs to a large family of lichenized fungi, Lecanoraceae. It
was first segregated out of the genus Squamaria DC. by Zopf (1905), and recognized later
by Choisy (1929), who classified it under a new name Omphalodina M. Choisy. At the same
time, Omphalodina chrysoleuca [Rhizoplaca chrysoleuca (Sm.) Zopf] was selected as the
type species of the new genus. Later Omphalodina was included in the genus Lecanora as
a section within the subgenus Placodium (Poelt, 1958); however, Leuckert, Poelt & Hahnel
(1977) proposed this taxon at a generic level once more under its older name Rhizoplaca, on
the basis of the following distinguishing features: umbilicate thalli, well-developed upper
cortex, thick lower cortex, loose medulla and cupulate hypothecium.
In subsequent molecular studies, Rhizoplaca was found to be a highly polyphyletic
genus diffuse within different clades among placodioid Lecanora spp. (Arup & Grube, 2000;
Cansaran et al., 2006). Moreover, a cryptic species-level diversity was identified within
seemingly morphologically homogeneous groups, such as Rhizoplaca chrysoleuca s. lat.
(Zhou et al., 2006;Leavitt et al., 2016), R. melanophthalma s. lat. (Leavitt et al., 2011;Leavitt
et al., 2013a) and R. macleanii s. lat. (Pérez-Ortega et al., 2012) as well as R. subdiscrepans
s. lat. (Leavitt et al., 2016). It turned out that some of the indicated lineages were, in
fact, good species candidates; these were described by Leavitt et al. (2013a) as Rhizoplaca
occulta,R. parilis,R. polymorpha,R. porterii and R. shushanii. Some additional species were
transferred into the genus Rhizoplaca by Zhao et al. (2016), e.g., Lecanora nigromarginata,
L. novomexicana,L. opiniconensis, L. phaedrophthalma and L.weberi, while others were
excluded from the genus, e.g., Rhizoplaca spidophora and R. peltata.
Currently, the genus Rhizoplaca comprises c. 24 species of lichenized fungi representing
umbilicate to placodioid growth forms (Zhao et al., 2016). The thalli predominantly
produce usnic acid in addition to various other substances. Rhizoplaca species occupy
siliceous or weakly to moderately calcareous rock or, rarely, soil, and grow mainly in open,
windy, cool and dry areas (Ryan & Nash, 1997). Species occur in the northern hemisphere
(with their centre of diversity in Central Asia and western North America), as well as in
South America and Antarctica (Leavitt et al., 2016;Zhao et al., 2016). Some of the known
species show broad ecological preferences and geographical ranges, e.g., R. chrysoleuca,
R. melanophthalma and R. subdiscrepans, whereas others have more restricted habitat
requirements and limited distributions, e.g., R. macleanii, R. maheui and R. marginalis
(Leavitt et al., 2016).
The subject of our study is Rhizoplaca subdiscrepans s. lat., a saxicolous lichen considered
to be distributed worldwide, occurring in warm-temperate to boreal-arctic areas in Asia,
Europe and North America (Ryan, 2001). In Europe it has been reported from Austria
(Hafellner & Türk, 2001), the Czech Republic (Malíček, Palice & Vondrák, 2015), Italy
(Nimis, 2016) and France (Roux, 2012), as well as Norway and Sweden (Santesson et al.,
Szczepańska et al. (2020), PeerJ, DOI 10.7717/peerj.9555 2/27
2004). It occurs on various siliceous rock types (basalt, rhyolite, granite and sandstone)
and prefers exposed, warm and dry habitats (Ryan, 2001). The species was characterized
by its yellowish-green, bullate-squamulose and polyphyllous thallus, with a pale brown
lower surface and sessile apothecia possessing orange, pruinose discs. The thallus of
R. subdiscrepans contains mainly usnic, placodiolic and pseudoplacodiolic acids (Arup &
Grube, 2000) and rarely psoromic, lecanoric and norstictic acids (Ryan, 2001) as secondary
metabolites.
Although the species was considered to be well-circumscribed, it recently proved to
be polyphyletic and several unrecognized species-level lineages have been recovered in
nominal R. subdiscrepans s. lat. by Leavitt et al. (2016). As a result of multigene analyses,
these authors proposed five candidate species defined as R. subdiscrepans ‘subd A, B, C, D,
E’, considered to be cryptic-species.
Our objective was to explain the taxonomic status of a putative representative of
R. subdiscrepans (Nyl.) R. Sant. found during extensive field studies in the foothills foreland
of the Sudety Mountains (Poland). Reference herbarium material from Asia, Europe and
North and South America was examined, and sequences of gathered specimens were placed
in a phylogenetic framework, including available GenBank sequences of placodioid lichens
of selected species of the genera Lecanora,Protoparmeliopsis and Rhizoplaca. Based on
morphological, chemical and molecular evidence, it was found that some of the recently
defined species candidates within R. subdiscrepans s. lat. should be assigned to previously
known species of the genus Rhizoplaca.
MATERIALS & METHODS
Taxon sampling
Material used in this study originated from the following herbaria: ASU, CANL (CMN),
KRAM, M, MIN, PRA, WRSL and the private herbarium of K. Szczepańska (hb.
Szczepańska). Our sampling focused on genera of lecanoroid lichens with placodioid
thalli as follows: Lecanora (L. pseudomellea), Protoparmeliopsis (P. garovaglii, P. muralis)
and Rhizoplaca (R. chrysoleuca, R. opiniconensis,R. phaedrophthalma, R. subdiscrepans);
holotypes of Lecanora pseudomellea,Rhizoplaca opiniconensis and R. phaedrophthalma
were also analysed. Unfortunately, we were unable to study the original collection of
R. subdiscrepans housed at the PC herbarium since type material at PC is unavailable for
loan and we had no opportunity to visit the herbarium. All of the specimens listed in the
text were included in the morphological, anatomical and chemical studies, but due to
the age and poor condition of some samples, it was not possible to obtain sequences and
include these in our phylogenetic analyses. New sequences generated for this study have
been deposited in GenBank.
Morphological and chemical study
All specimens were examined using light microscopy for the assessment of lobes,
areoles, squamules and apothecia, especially the morphological characters mentioned
in Leavitt et al. (2011) such as point of attachment (umbilicate/squamulose), thallus
form (polyphyllous/monophyllous), lobe morphology (distinct/indistinct), appearance
Szczepańska et al. (2020), PeerJ, DOI 10.7717/peerj.9555 3/27
of upper surface (dull/glossy), upper surface colour (yellow-green/olive), apothecia
morphology (sessile/constricted), apothecia pruinosity (pruinose/epruinose), ascospore
shape (ellipsoid/subglobose) and ascospore size. Apothecial margin (persistent/excluded),
height of hymenium, and size and shape of conidia (straight/arched) were also analysed.
For light microscopy, vertical, free-hand sections of apothecia were cut by a razor blade
and mounted in water. Hymenium measurements were made in water and ascospore
measurements in 10% KOH (K); the structure and coherency of paraphyses and the
solubility of granules in the epihymenium were also tested with K. At least 10 measurements
of morphological variables and measurements of 20 spores were made for each sample and
their minimum and maximum values calculated.
Chemical examinations included colour reactions and thin-layer chromatography
(TLC). Spot test reactions of thalli, apothecial margins and discs were made with
KOH, sodium hypochlorite [commercial laundry bleach] (C) and paraphenylenediamine
[solution in 95% ethyl alcohol] (PD). TLC analyses were performed in solvent systems A,
B and C using the standardized method of Culberson (1972) and following Orange, James
& White (2001).
Descriptions of the species are based on our own observations, measurements and TLC
analyses of specimens cited in this paper. The terminology used in the descriptions of the
species follows Ryan et al. (2004).
Molecular methods: DNA extraction, PCR amplification and DNA
sequencing
To infer relationships between species of lichenized fungi studied, the ITS rDNA region,
that contains ITS1, 5.8S, ITS2 sequences, was used. Total DNA was extracted from
specimens using a CTAB method (Doyle & Doyle, 1987). Dried samples were mechanically
disintegrated using Mixer Mill MM400 (Retsch; Haan, Germany). The quality of the
DNA was checked by electrophoresis on agarose gel (1%) with Simply Safe staining
chemical (Eurx, Gdańsk, Poland). The complete ITS rDNA region was amplified using
primers ITS1F (Gardes & Bruns, 1993) and ITS4 (White et al., 1990). The PCR reaction mix
included 1U Taq recombinant polymerase (Thermo-Fisher Scientific, Waltham, USA), 10x
Taq Buffer, 1 mM MgCl2, 0.5 M of each primer, 0.4 mM dNTP and 1 µl DNA template.
Amplification cycles were performed with a Veriti Thermal Cycler (Life Technologies,
Carlsbad, USA) and involved 8 min at 95 ◦C, followed by 32 cycles for 45 s at 95 ◦C
and 45 s at 52 ◦C (annealing), and 1 min at 72 ◦C, with the a final extension step of 10
min at 72 ◦C. Amplified PCR products were purified using GeneJet PCR Purification Kit
(Thermo-Fisher Scientific, Waltham, USA). This was accomplished at the Laboratory of
Molecular Biology (Environmental and Life Sciences) at Wroclaw University. Sequencing
of PCR products, post-reaction purification, forward and reverse directions reading were
done by sequencing service Genomed (Genomed, Warsaw, Poland), using an ABI 377XL
Automated DNA Sequencer (Applied Biosystems, Carlsbad, USA).
Alignment assembly and molecular phylogenetic analyses
The obtained ITS rDNA sequences were assembled and manually edited using Geneious
Pro, version 8.0. (Biomatters Ltd). Sequences of Rhizoplaca subdiscrepans (putative
Szczepańska et al. (2020), PeerJ, DOI 10.7717/peerj.9555 4/27
name for our collection) together with related species, namely Lecanora pseudomellea,
Protoparmeliopsis garovaglii, P. muralis, Rhizoplaca chrysoleuca, R. melanophthalma, R.
novomexicana, R. occulta, R. opiniconensis (as Lecanora in GeneBank), R. parilis and
R. phaedrophthalma, were included in the analysis. Our final ITS data-set included 21
sequences newly generated for this study and 95 sequences downloaded from GenBank
(Table 1). The final alignment was performed on Geneious Pro using the MAFFT algorithm
(Katoh et al., 2005), then re-checked and improved. Ambiguously aligned regions were
removed in GBlocks (Castresana, 2000). The nucleotide substitution models were separately
searched for in each partition of the ITS region (ITS1, 5.8S, ITS2) to find the best-fitting
model using the corrected Akaike information criterion (AICc) as an optimality model
criterion in a greedy algorithm search as implemented in PartitionFinder version 1.0.1
(Lanfear et al., 2012). The GTR+G model for ITS1 and ITS2 and K80 for the 5.8S partitions
were selected.
The phylogenetic reconstruction was generated using the CIPRES Scientific Gateway
(http://www.phylo.org/portal2/) (Miller, Pfeiffer & Schwartz, 2010). Maximum likelihood
(ML) bootstrap tree with simultaneous heuristic search was undertaken as implemented in
RAxML–HPC2 on XSEDE (Stamatakis, 2006) under the GTRGAMMA substitution model
and 1,000 bootstrap resamples. Bayesian inference was carried out using Markov Chain
Monte Carlo (MCMC) implemented in MrBayes 3.2.6 on XSEDE (Ronquist et al., 2012).
MrBayes was set to two independent parallel runs each initiated with four incrementally
heated chains; the run length was settled to 20M generations, and to infer convergence
the average standard deviation of split frequencies was printed every 1000th generation
discarding the first 50% of the trees sampled as a burn-in fraction. The analyses were
stopped after 1M generations when the standard deviation had dropped below 0.01. The
resulting phylogenetic trees were visualized in Figtree software (Rambaut, 2014). The
alignment of ITS sequences described here is deposited in TreeBASE with number TB2:
S26365.
RESULTS
Results of the morphological and chemical studies are presented in the taxonomic section
under particular species descriptions. All taxa representing R. subdiscrepans s. lat. appear
to be semi-cryptic, with slight morphological variety and some overlapping features.
Nevertheless, some noticeable differences are visible in their chemistry, appearance
of apothecia and marginal lobes, as well as in the shape and size of ascospores and
conidia. The major distinguishing characters of Rhizoplaca subdiscrepans, R. opiniconensis,
R. phaedrophthalma, and R. pseudomellea are summarized in Table 2.
The final alignment matrix contained 538 bp and Protoparmeliopsis was selected as
the outgroup (Leavitt et al., 2016;Zhao et al., 2016). The topology of the tree was similar
to that presented by Leavitt et al. (2016). Our newly generated sequences from Poland,
Canada and the USA were resolved within R. subdiscrepans s. lat. as defined by Leavitt
et al. (2016) (Fig. 1). The first group of samples (Rhizoplaca subdiscrepans s. str. 21 and
22) were recovered within the clade ‘subd E’ with high bootstrap support (BS) =96
Szczepańska et al. (2020), PeerJ, DOI 10.7717/peerj.9555 5/27
Table 1 The species and specimens treated in current study with locality and GenBank accession numbers. Newly generated sequences are in
boldface.
Species Locality GenBank (ITS)
Accesion number
Protoparmeliopsis garovaglii 1 Austria AF189718
Protoparmeliopsis garovaglii 2 USA, Utah, Leavitt 199 (BRY-C) KU934537
Protoparmeliopsis garovaglii 3 USA, Idaho, Leavitt 078 (BRY-C) KU934540
Protoparmeliopsis garovaglii 4 Poland, Szczepańska 1240 (KRAM), L21 MK084624
Protoparmeliopsis garovaglii 5 Bolivia, Flakus 17529 (KRAM), L88 MK084625
Protoparmeliopsis garovaglii 6 Bolivia, Flakus 21175 (KRAM), L89 MK084626
Protoparmeliopsis garovaglii 7 Bolivia, Flakus 21118 (KRAM), L90 MK084627
Protoparmeliopsis garovaglii 8 Peru, Flakus 9603 (KRAM), L92 MK084628
Protoparmeliopsis garovaglii 9 Peru, Flakus 9540 (KRAM), L91 MK084629
Protoparmeliopsis garovaglii 10 USA, Idaho, Leavitt 109 (BRY-C) KU934549
Protoparmeliopsis muralis 1 Romania, J.-S. Hur (KOLRI), SK 765 KP059048
Protoparmeliopsis muralis 2Czech Republic, Lipno nad Vltavou, Szczepańska 1263, isolate
L97
MN931719
Protoparmeliopsis muralis 3 USA, Utah, Leavitt 143 (BRY-C) KT453726
Protoparmeliopsis_muralis 4 Germany, Saxony, Scholz barcode M0275697 (M), DNA 9890 KT818623
Protoparmeliopsis muralis 5 USA, Utah, Leavitt 077 (BRY-C) KU934552
Protoparmeliopsis muralis 6 Russia, Chelyabinsk, Vondrák 9405 (PRA) KU934556
Protoparmeliopsis muralis 7 Russia, Kizilskoe, Vondrák 9417 (PRA) KU934557
Protoparmeliopsis muralis 8 Russia, Chelyabinsk, Vondrák 9414 (PRA) KU934558
Protoparmeliopsis muralis 9 Russia, Orenburg, Vondrák 106b (PRA) KU934560
Rhizoplaca melanophthalma 1 USA, Nevada, LLS (EA 15-123A), BRY C55051 HM577272
Rhizoplaca melanophthalma 2 USA, Utah, LLS (EA 18-143), BRY C55052 HM577273
Rhizoplaca melanophthalma 3 USA, Colorado, LLS (EA 18-145), BRY C550 HM577274
Rhizoplaca melanophthalma 4 USA, Utah, LLS (EA 22-177), BRY C55054 HM577275
Rhizoplaca melanophthalma 5 USA, Idaho, LLS (EA 41-403), BRY C55055 HM577276
Rhizoplaca melanophthalma 6 USA, Wyoming, SDL, BRY C55056 HM577277
Rhizoplaca melanophthalma 7 USA, Wyoming, SDL, BRY C55057 HM577278
Rhizoplaca melanophthalma 8 USA, Utah, SDL, LLS, GS, BRY C55058 HM577279
Rhizoplaca novomexicana 1 USA, Utah, SDL, LLS, GS, BRY C55024 HM577255
Rhizoplaca novomexicana 2 USA, Utah, SDL, LLS, GS, BRY C55025 HM577256
Rhizoplaca novomexicana 3 USA, Utah, SDL, LLS, GS, BRY C55026 HM577257
Rhizoplaca occulta 1 USA, Utah, Juab Co.: West of Goshen, LLS (EA 18-140), BRY
C55074
HM577305
Rhizoplaca occulta 2 USA, Idaho, Butte Co.: placeSalmon Challis National Forest,
LLS (EA 37-356), BRY C55075
HM577306
Rhizoplaca occulta 3 USA, Nevada, White Pine Co.: Humboldt-Toiyabe N.F., SDL,
LLS, BRY C55076
HM577307
Rhizoplaca occulta 4 USA, Idah, NA AF159942
Rhizoplaca occulta 5 USA, Idaho, NA AF159944
Rhizoplaca chrysoleuca A1 Russia, Vondrák 9979 (PRA) KU934563
Rhizoplaca chrysoleuca A2 Russia, Vondrák 10021 (PRA) KU934564
(continued on next page)
Szczepańska et al. (2020), PeerJ, DOI 10.7717/peerj.9555 6/27
Table 1 (continued)
Species Locality GenBank (ITS)
Accesion number
Rhizoplaca chrysoleuca A3 Russia, Vondrák 10126 (PRA) KU934566
Rhizoplaca chrysoleuca A4 Russia, Vondrák 10040 (PRA) KU934567
Rhizoplaca chrysoleuca B1 Russia, Altay, Vondrák 9981 (PRA) KU934568
Rhizoplaca chrysoleuca B2 Russia, Altay, Vondrák 9993 (PRA) KU934569
Rhizoplaca chrysoleuca B3 Russia, Altay, Vondrák 10023 (PRA) KU934570
Rhizoplaca chrysoleuca B4 Russia, Altay, Vondrák 10059 (PRA) KU934572
Rhizoplaca chrysoleuca C Russia, Altay, Vondrák 10017 (PRA) KU934573
Rhizoplaca opiniconensis 1 USA, Arizona, Apache Co., Nash III 27196 & Ryan (ASU) AF159928
Rhizoplaca opiniconensis 2 USA, Wisconsin, Leavitt 12-005 (F) KU934881
Rhizoplaca opiniconensis 3 USA, Wisconsin, Leavitt 12-007 (F) KU934882
Rhizoplaca opiniconensis 4 Russia, Altay, Vondrák 10013 (PRA) KU934883
Rhizoplaca opiniconensis 5 Russia, Altay, Vondrák 10029 (PRA) KU934884
Rhizoplaca opiniconensis 6 Russia, Altay, Vondrák 10034 (PRA) KU934885
Rhizoplaca opiniconensis 7 Russia, Altay, Vondrák 10128 (PRA) KU934886
Rhizoplaca opiniconensis 8Canada, Ontario, Leeds Co., Brodo 25117 (CANL, holotype) MN931720
Rhizoplaca opiniconensis 9Canada, Ontario, Wetmore 88134 (MIN), isolate L30 MN931721
Rhizoplaca opiniconensis 10 USA, Minnesota, Otter Tail Co., Wetmore 74475 (MIN), isolate
L35
MN931722
Rhizoplaca parilis 1 USA, Utah, LDP, BRY C55077 HM577308
Rhizoplaca parilis 2 USA, Utah, LDP, BRY C55078 HM577309
Rhizoplaca parilis 3 USA, Utah, LDP, BRY C55079 HM577310
Rhizoplaca parilis 4 USA, Utah, LDP, BRY C55080 HM577311
Rhizoplaca parilis 5 USA, Utah, LDP, BRY C55081 HM577312
Rhizoplaca phaedrophthalma 1 China,Tibet, s. coll, s. herb. AF159938
Rhizoplaca phaedrophthalma 2 USA, Utah, Leavitt 734 (BRY-C) HM577230
Rhizoplaca phaedrophthalma 3 USA, Utah, Leavitt 735 (BRY-C) HM577231
Rhizoplaca phaedrophthalma 4 USA, Utah, Leavitt 1023 (BRY-C) HM577232
Rhizoplaca phaedrophthalma 5 Russia, state unknown, Vondrák 9406 & Frolov (PRA)aKU934870
Rhizoplaca phaedrophthalma 6 Russia, state unknown, Vondrák 9406 (PRA) KU934871
Rhizoplaca phaedrophthalma 7 USA, Nevada, Leavitt 9052 (F) KU934872
Rhizoplaca phaedrophthalma 8 Russia, Orenburg, Vondrák 9384 (PRA) KU934873
Rhizoplaca phaedrophthalma 9 Russia, Orenburg, Vondrák 9406 (PRA) KU934875
Rhizoplaca phaedrophthalma 10 Russia, Chelyabinsk, Vondrák 9405 (PRA) KU934876
Rhizoplaca phaedrophthalma 11 Canada, British Columbia, Ryan 31877 (ASU), isolate L48 MN931728
Rhizoplaca phaedrophthalma 12 Canada, British Columbia, Ryan 31889 (ASU), isolate L49 MN931729
Rhizoplaca phaedrophthalma 13 USA, Idaho, Twin Falls Co., Ryan 32870 (ASU), isolate L50 MN931730
Rhizoplaca phaedrophthalma 14 USA, Montana, Gallatin Co., Wetmore 80571 (MIN), isolate
Lec10
MN931732
Rhizoplaca phaedrophthalma 15 USA, Montana, Park Co., Wetmore 80979 (MIN), isolate L16 MN931726
Rhizoplaca phaedrophthalma 16 USA, Oregon, Deschutes Co., Wetmore 95059 (MIN), isolate
L12
MN931723
Rhizoplaca phaedrophthalma 17 USA, Oregon, Malheur Co., Wetmore 5101 (MIN), isolate L14 MN931724
Rhizoplaca phaedrophthalma 18 USA, Wyoming, Park Co., Wetmore 80866 (MIN), isolate Lec9 MN931731
Rhizoplaca phaedrophthalma 19 USA, Wyoming, Park Co., Wetmore 81235 (MIN), isolate L15 MN931725
(continued on next page)
Szczepańska et al. (2020), PeerJ, DOI 10.7717/peerj.9555 7/27
Table 1 (continued)
Species Locality GenBank (ITS)
Accesion number
Rhizoplaca phaedrophthalma 20 USA, Wyoming, Park Co., Wetmore 81446 (MIN), isolate L17 MN931727
Rhizoplaca polymorpha 1 USA, Utah, LDP, BRY C55091 HM577322
Rhizoplaca polymorpha 2 USA, Utah, LDP, BRY C55092 HM577323
Rhizoplaca polymorpha 3 USA, Idaho, SDL, HCL, JHL, BRY C55094 HM577325
Rhizoplaca polymorpha 4 USA, Idaho, SDL, HCL, JHL, BRY C55095 HM577326
Rhizoplaca polymorpha 5 USA, Utah, SDL et al., F 11-026 JX948194
Rhizoplaca pseudomellea 1USA, California,Tulare Co., Wetmore 51151 (MIN), isolate L93 MN931734
Rhizoplaca pseudomellea 2USA, California, Tuolumne Co., Ryan 24402 (MIN), isolate L94 MN931735
Rhizoplaca pseudomellea 3USA, Oregon, Harney Co., Wetmore 95079 (MIN), isolate Lec7 MN931736
Rhizoplaca pseudomellea 4USA, Oregon, Harney Co., Wetmore 95084 (MIN), isolate Lec8 MN931737
Rhizoplaca pseudomellea 5USA, Oregon, Lake Co., Ryan 28456 (ASU), isolate L40 MN931733
Rhizoplaca subdiscrepas B1 China, Yanbian Korean Autonomous Prefecture, s. coll, s. herb. KP226212
Rhizoplaca subdiscrepans B2 Kazahztan, Karkaralinsk, Seaward s.n. (BRY-C) KU934877
Rhizoplaca subdiscrepans B3 Russia, Altay, Vondrák 10001 (PRA) KU934878
Rhizoplaca subdiscrepans B4 Russia, Chelyabinsk, Vondrák 10049 (PRA) KU934879
Rhizoplaca subdiscrepnas C Russia, Altay, Vondrák 9975 (PRA) KU934880
Rhizoplaca subdiscrepans s.s.1 Russia, state unknown, Vondrák jv5 (PRA) KU934887
Rhizoplaca subdiscrepans s.s.2 Russia, Orenburg, Vondrák 440_Irikla4 (PRA) KU934888
Rhizoplaca subdiscrepans s.s.3 Russia, Orenburg, Vondrák 442_Irikla4 (PRA) KU934889
Rhizoplaca subdiscrepans s.s.4 Russia, Orenburg, Vondrák 9384 & Frolov (PRA)aKU934890
Rhizoplaca subdiscrepans s.s.5 Russia, Orenburg, Vondrák 9385 & Frolov (PRA)aKU934891
Rhizoplaca subdiscrepans s.s.6 Russia, Bashkortostan, Vondrák 9411 & Frolov (PRA)aKU934892
Rhizoplaca subdiscrepans s.s.7 Russia, Orenburg, Vondrák 9415 & Frolov (PRA)aKU934893
Rhizoplaca subdiscrepans s.s.8 Russia, Orenburg, Vondrák 9416 & Frolov (PRA)aKU934894
Rhizoplaca subdiscrepans s.s.9 Russia, Chelyabinsk, Vondrák 9418 & Vondráková (PRA)aKU934895
Rhizoplaca subdiscrepans s.s.10 Russia, Orenburg, Vondrák 9422a & Frolov (PRA)aKU934896
Rhizoplaca subdiscrepans s.s.11 Russia, Orenburg, Vondrák 9422b & Frolov (PRA)aKU934897
Rhizoplaca subdiscrepans s.s.12 Russia, Chelyabinsk, Vondrák 9408 & Vondráková (PRA)aKU934898
Rhizoplaca subdiscrepans s.s.13 Russia, Orenburg, Vondrák 9412 & Vondráková (PRA)aKU934899
Rhizoplaca subdiscrepans s.s.14 Russia, Orenburg, Vondrák 9420 & Frolov (PRA)aKU934900
Rhizoplaca subdiscrepans s.s.15 Russia, Orenburg, Vondrák 9420b & Frolov (PRA)aKU934901
Rhizoplaca subdiscrepans s.s.16 Russia, Orenburg, Vondrák 9420c & Frolov (PRA)aKU934902
Rhizoplaca subdiscrepans s.s.17 Russia, Orenburg, Vondrák 9420d & Frolov (PRA)aKU934903
Rhizoplaca subdiscrepans s.s.18 Ukraine, Prague, Vondrák 9843 (PRA) KU934904
Rhizoplaca subdiscrepans s.s.19 Russia, state unknown, Vondrák jv4 (PRA) KU934905
Rhizoplaca subdiscrepans s.s.20 Russia, Orengurg, Vondrák 9384a (PRA) KU934906
Rhizoplaca subdiscrepans s.s.21 Poland, Sudety Mts foreland, Szczepańska 923 (KRAM), isolate
11pLecanora_new
MN931738
Rhizoplaca subdiscrepans s.s.22 Poland, Sudety Mts foreland, Szczepańska 967 (KRAM), isolate
13pLecanora_new
MN931739
Notes.
aSpecimens examined (available for study in PRA).
Szczepańska et al. (2020), PeerJ, DOI 10.7717/peerj.9555 8/27
and posterior probability (PP) =1 together with specimens from Ukraine and Russia
(Rhizoplaca subdiscrepans s. str. 18 and 19 respectively). Sequences of herbarium specimens
marked as R. phaedrophthalma were nested within the clade ‘subd A’ (BS =80 and PP =
1), together with the sequence of R. phaedrophthalma published by Arup & Grube (2000)
and Zhao et al. (2016), while R. opiniconensis (BS =100 and PP =1) nested within the
clade ‘subd D’ of Leavitt et al. (2016). Moreover, the sequence of the type collection of
R. opiniconensis (Rhizoplaca opiniconensis 10; see Fig. 2A), another sample identified by
Irwin Brodo, who described R. opiniconensis as a new taxon (Brodo, 1986), as well as the
sequence of R. opiniconensis analysed by Arup & Grube (2000) and Zhao et al. (2016),were
placed within the lineage of candidate species ‘subd D’. Lecanora pseudomellea, included
for the first time in a phylogenetic analysis of Rhizoplaca, formed a strongly supported
monophyletic lineage (BS =100 and PP =1) within the latter genus.
Taxonomy
Rhizoplaca opiniconensis (Brodo) Leavitt, Zhao Xin & Lumbsch, in Zhao et al., Fungal
Diversity 78: 302. 2016. ≡Lecanora opiniconensis Brodo, Mycotaxon 26: 309. 1986.
Figs. 2A,2B.
Type: [CANADA], ONTARIO: Leeds Co., Snake Island, in Lake Opinicon, Queen’s
University Biological Station, Chaffeys Locks, on S-facing rockface, 1.0–1.5 m above water,
2 Feb. 1985, I.M. Brodo 25117 (holotype, CANL!).
Thallus lichenized, placodioid, polyphyllous, usually distinctly rounded, irregular when
older, thick, loosely attached to the substrate, 2–5 cm diam. or more. Marginal lobes
distinctly developed, short, 0.5–1 mm long and 0.2–0.6 mm wide, plane to slightly
convex, broadened and crenate-incised at the tips. Thallus centre squamulose-areolate to
squamulose-bullate, irregularly cracked, thick, loosely attached to the substrate. Squamules
crowded, flat to strongly convex, irregular, 0.2–1.5 mm diam. Upper surface smooth, dull,
yellowish-green to yellowish-orange (more evident in herbarium material), marginal lobes
usually darker than the thallus centre. Apothecia numerous, sessile to constricted, 0.2–2
mm diam. circular, older apothecia irregular and distorted. Margin thin, concolourous
with thallus or paler, flexuose, crenate, usually persistent, sometimes excluded, disc pale
yellowish-brown to yellowish-orange, epruinose, plane, rarely convex, dull. Hymenium
colourless, 60–70 µm high, hypothecium colourless. Epihymenium orange-brown to
olive-brown, with small granules dissolving in K. Ascospores 8 per ascus, hyaline, simple,
ellipsoid, 10–12 ×6–7 µm. Pycnidia immersed, simple with bluish-black ostioles, conidia
hyaline, filiform, mostly arched 15–25 ×0.5 µm.
Chemistry.Thallus K–, C–, KC+ yellow, PD–, medulla K+ yellow, C–, KC+ yellow, PD–.
Secondary metabolites detected by TLC: isousnic (+/−), usnic (+) and placodiolic (+)
acids, as well as some unidentified terpenoids (+/−).
Ecology and distribution.On quartzite and volcanic rocks, at higher elevations (c. 1,200–
3,000 m), in shaded places near streams, in North America and Eastern Asia.
Comments. Characteristic features of R. opiniconensis are the mostly arched conidia and
thallus colour, which changes to a more orange colour in herbarium specimens. Some
specimens are morphologically similar to R. subdiscrepans s. str., in which case proper
Szczepańska et al. (2020), PeerJ, DOI 10.7717/peerj.9555 9/27
Table 2 Overview of the distinguishing features of the Rhizoplaca subdiscrepans s. str., R. opiniconensis, R. phaedrophthalma and R. pseudomellea.
Character Species
R. subdiscrepans s.s. (R. subdiscrepans E’)aR. opiniconensis (R. subdiscrepans D’)aR. phaedrophthalma (R. subdiscrepans
A’)aR. pseudomellea
Thallus
morphology polyphyllous placodioid, yellow-green, glossy
marginal lobes distinctly developed, discrete
or contiguous to slightly overlapping, convex,
broadened, flabellate and crenate-incised at tips
thallus centre squamulose-bullate, composed of
convex subunits squamules convex, sinusoid and
plicate
polyphyllous, placodioid, yellow-green,
with orange tint in older collections, dull polyphyllous placodioid, yellow-green,
dull placodioid, orange-brown to reddish or
rusty brown, glossy
marginal lobes distinctly developed,
slightly convex, broadened, flabellate and
crenate-incised at tips
marginal lobes weakly developed, slightly
convex, broadened and flabellate at tips marginal lobes distinctly developed, long,
slightly convex, minutely broadened and
greenish-black to black at tips
thallus centre squamulose-areolate to
squamulose-bullate thallus centre squamulose-areolate to
squamulose-bullate thallus center areolate
squamules flat to strongly convex, irregu-
lar squamules flat to slightly convex, irregular areoles convex, rounded to irregular
Apothecia sessile, disc pale yellowish-brown, plane, epru-
inose, margin persistent sessile to constricted, disc pale yellowish-
brown, plane, epruinose, margin persis-
tent
sessile, disc reddish-brown, strongly con-
vex, epruinose, margin excluded in older
apothecia
sessile, disc orange-brown to reddish-
brown, plane, epruinose margin excluded
in older apothecia
Ascospores ellipsoid 10–12 ×6–7 µm ellipsoid 10–12 ×6–7 µm subglobose 8–10 ×5–7 µm ellipsoid 10–12 ×6–7 µm
Conidia filiform, mostly straight 10–25 ×0.5 µm filiform, mostly arched 15–25 ×0.5 µm filiform, mostly straight 15–30 ×0.5 µm filiform, straight 10–25 ×0.5 µm
Chemistry isousnic (+/−), usnic (+), placodiolic (+), fatty
acids (+/−), terpenoids (+)isousnic (+/−), usnic (+), placodiolic
(+), terpenoids (+/−)usnic (+), placodiolic (+), terpenoids
(+/−)isousnic (+/−), usnic (+/−), psoromic
(+/−), fatty acids (+/−)
Ecology siliceous rock at lower elevations, in dry and
sunny sites siliceous rock at higher elevations, in
shaded sites near streams siliceous rock at higher elevations, in semi-
arid areas siliceous rock at higher elevations, in dry
and sunny sites
Geography Europe and Western Asia North America and East-Central Asia North America and Westen and Central
Asia North America
Locus classicus Europe (Switzerland) North America (Canada) Asia (Nepal) North America (USA)
Notes.
aCandidate species by Leavitt et al. (2016).
Szczepańska et al. (2020), PeerJ, DOI 10.7717/peerj.9555 10/27
Rhizoplaca subdiscrepans s.s.4
Rhizoplaca melanophthalma 8
Rhizoplaca pseudomellea 1
Protoparmeliopsis muralis 4
Rhizoplaca chrysoleuca A4
Rhizoplaca opiniconensis 6
Rhizoplaca phaedrophthalma 8
Protoparmeliopsis garovaglii 8
Rhizoplaca phaedrophthalma 4
Rhizoplaca subdiscrepas B1
Rhizoplaca melanophthalma 3
Rhizoplaca subdiscrepans s.s.6
Rhizoplaca melanophthalma 5
Rhizoplaca subdiscrepans s.s.10
Rhizoplaca subdiscrepans s.s.5
Rhizoplaca subdiscrepans s.s.9
Rhizoplaca occulta 5
Rhizoplaca melanophthalma 4
Rhizoplaca subdiscrepans s.s.3
Rhizoplaca opiniconensis 2
Protoparmeliopsis garovaglii 6
Rhizoplaca polymorpha 1
Rhizoplaca chrysoleuca A2
Rhizoplaca pseudomellea 4
Rhizoplaca phaedrophthalma 3
Rhizoplaca phaedrophthalma 12
Rhizoplaca phaedrophthalma 20
Rhizoplaca subdiscrepans s.s.15
Protoparmeliopsis garovaglii 4
Rhizoplaca novomexicana 2
Rhizoplaca occulta 2
Rhizoplaca phaedrophthalma 1
Rhizoplaca occulta 4
Rhizoplaca phaedrophthalma 9
Rhizoplaca chrysoleuca A3
Rhizoplaca melanophthalma 1
Rhizoplaca melanophthalma 2
Rhizoplaca phaedrophthalma 16
Protoparmeliopsis muralis 3
Rhizoplaca subdiscrepans s.s.13
Protoparmeliopsis garovaglii 7
Rhizoplaca occulta 3
Rhizoplaca phaedrophthalma 14
Rhizoplaca phaedrophthalma 7
Rhizoplaca melanophthalma 7
Rhizoplaca opiniconensis 9
Protoparmeliopsis garovaglii 10
Rhizoplaca subdiscrepans s.s.21
Rhizoplaca subdiscrepans s.s.11
Protoparmeliopsis garovaglii 3
Rhizoplaca opiniconensis 10
Rhizoplaca subdiscrepans s.s.7
Rhizoplaca subdiscrepans s.s.18
Rhizoplaca phaedrophthalma 6
Rhizoplaca phaedrophthalma 5
Rhizoplaca chrysoleuca B4
Rhizoplaca parili 4
Rhizoplaca opiniconensis 4
Rhizoplaca subdiscrepans s.s.16
Rhizoplaca phaedrophthalma 13
Protoparmeliopsis muralis 5
Rhizoplaca subdiscrepas B3
Rhizoplaca parili 3
Rhizoplaca phaedrophthalma 10
Rhizoplaca phaedrophthalma 11
Rhizoplaca subdiscrepans s.s.2
Protoparmeliopsis muralis 1
Protoparmeliopsis muralis 2
Rhizoplaca subdiscrepans s.s.17
Rhizoplaca subdiscrepans s.s.14
Rhizoplaca subdiscrepas B4
Rhizoplaca opiniconensis 3
Rhizoplaca opiniconensis 8
Rhizoplaca polymorpha 5
Rhizoplaca subdiscrepans s.s.12
Protoparmeliopsis muralis 8
Rhizoplaca opiniconensis 5
Protoparmeliopsis garovaglii 2
Protoparmeliopsis muralis 6
Rhizoplaca chrysoleuca A1
Rhizoplaca phaedrophthalma 19
Rhizoplaca subdiscrepans s.s.19
Protoparmeliopsis garovaglii 1
Protoparmeliopsis garovaglii 9
Rhizoplaca polymorpha 3
Rhizoplaca chrysoleuca B1
Protoparmeliopsis muralis 7
Rhizoplaca chrysoleuca B2
Rhizoplaca opiniconensis 7
Protoparmeliopsis muralis 9
Rhizoplaca parili 1
Rhizoplaca subdiscrepans C
Rhizoplaca novomexicana 1
Rhizoplaca polymorpha 4
Rhizoplaca parili 2
Rhizoplaca phaedrophthalma 2
Rhizoplaca subdiscrepans s.s.8
Rhizoplaca parili 5
Rhizoplaca pseudomellea 2
Rhizoplaca occulta 1
Rhizoplaca chrysoleuca C
Rhizoplaca chrysoleuca B3
Protoparmeliopsis garovaglii 5
Rhizoplaca polymorpha 2
Rhizoplaca opiniconensis 1
Rhizoplaca subdiscrepans s.s.1
Rhizoplaca subdiscrepans s.s.20
Rhizoplaca phaedrophthalma 17
Rhizoplaca phaedrophthalma 15
Rhizoplaca novomexicana 3
Rhizoplaca pseudomellea 3
Rhizoplaca melanophthalma 6
Rhizoplaca pseudomellea 5
Rhizoplaca phaedrophthalma 18
Rhizoplaca subdiscrepas B2
Rhizoplaca subdiscrepans s.s.22
/0.97
/1
96/1
/0.99
/1
80/0.99
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97/1
94/1
70/1
78/0.99
82/0.97
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70/0.97
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Figure 1 Bayesian inference of phylogenetic relationship within Rhizoplaca subdiscrepans s. lat. based
on ITS rDNA sequences. High bootstrap support values are shown above thickened branches and bold
numbers representing clades (ML –BP ≥70%, Bayesian analysis –PP ≥0.9).
Full-size DOI: 10.7717/peerj.9555/fig-1
Szczepańska et al. (2020), PeerJ, DOI 10.7717/peerj.9555 11/27
Figure 2 Habitus of Rhizoplaca species treated. Habitus of Rhizoplaca species treated. (A) R. opinico-
nensis, I.M. Brodo 25117, CANL (holotype). (B) R. opiniconensis,J. Schuster 3098, ASU. (C) R. phaedroph-
thalma, F. Lobbichler, M 122-86/40 (holotype). (D) R. phaedrophthalma,C.M. Wetmore 81235, MIN. (E)
R. pseudomellea, B.D. Ryan 13609, ASU (holotype). (F) R. pseudomellea,C.M. Wetmore 95084, MIN. (G)
R. subdiscrepans, K. Szczepańska 967, WRSL. (H) Thallus of R. subdiscrepans in natural habitat. Bars =one
mm (C, G), two mm (A–B, D–F, H). (Photos: K. Szczepańska, K. Wilk).
Full-size DOI: 10.7717/peerj.9555/fig-2
Szczepańska et al. (2020), PeerJ, DOI 10.7717/peerj.9555 12/27
identification of both taxa may be difficult. However, in contrast to R. subdiscrepans s.
str., R. opiniconensis does not produce fatty acids and was genetically confirmed in North
American and Asian but not from European specimens. Additionally, the species has also
specific habitat preferences.
Specimens examined. CANADA. ONTARIO: West edge of Marathon, shore of Lake
Superior and back on rock ridges with black spruce and scattered quaking aspen, alt. 629
ft, 5 Aug. 2002, C.M. Wetmore 88134 (MIN). MEXICO. MEXICO: Baja California Sur,
below top of Sierra Agua Verde (part of the Sierra San Francisco), on rhyolite, N-facing
cliff, alt. c. 1300 m, 1 Jan. 1998, T.H. Nash III 40100 (ASU); Chihuahua, along secondary
dirt road to Casa Grandes from Bavispe, Sonora, oak woodland area with large outcrops
of rhyolite, on rhyolite, alt. c. 2050 m, 18 July 1994, T.H Nash III 36458 (ASU). USA.
ARIZONA: Apache Co., Apache National Forest, Mt Baldy Wilderness area, trail from
Phelp’s cabin along East Fork of Little Colorado River, on rock, alt. 3001 m, 1 July 1990,
T.H Nash III 27051 (ASU); Apache Co., Petrified Forest National Park, south of I-40, east
of Petrified Forest Rd, near Crystal Forest Trail, on sandstone, alt. 1669 m, 14 May 1990,
W.C. Davis 735 (ASU); Cochise Co., Chiricahua Mountains, Colorado National Forest, cliff
area above Mormon Springs in Mormon Canyon, oak woodland, on exposed cliff-face, alt.
c. 2070 m, 24 Nov. 1995, T.H. Nash III 37156 (ASU); Cochise Co., Chiricahua Mountains,
Chiricahua National Monument, along the Loop Trail above Rhyolite Canyon and below
‘‘Heart of the Rocks’’, oak-pine forest, on rhyolite, alt. c. 2000 m, 5 June 1998, T.H. Nash
III 41767 (ASU); Cochise Co., Chiricahua Mountains, Chiricahua National Monument,
just before junction of trail to Heart of Rocks and trail to Massai Point, steep NE-facing
part of rhyolite boulders under tall rock formation, alt. c. 2020 m, 5 June 1993, B.D. Ryan
30861 (ASU); Graham Co., E side of the Pinaleno Mountains, along trail above Shannon
Campground, mixed conifer-oak forest, on acidic rock, alt. c. 2000 m, 15 July 1994, T.H
Nash III 36056 (ASU); Pima Co., east side of Baboquivari Peak, on exposed granite, alt. c.
1920 m, 13 Oct. 1990, T.H. Nash III 27444 (ASU); Pima Co., Santa Catalina Mountains,
Coronado National Forest, upper part of Sabino Canyon, near Marshall Gulch, granite
boulders in mixed conifer forest, on granite, alt. c. 2310 m, 3 June 1998, T.H. Nash III
41709 (ASU). MINNESOTA: Cook Co., Grand Portage State Forest, Lake Superior shore
NE of Horseshoe Bay (16 miles SW of Grand Portage), shrubby wet cobble shore, alt. 603
ft, 28 Sept. 2001, C. Reschke 1567 (MIN); Cook Co., Grand Portage National Monument,
north side of Mt Rose, rock cliffs and large rock blocks below with some white birch and
white spruce, alt. 867 ft, 13 July 2012, C.M. Wetmore 100545 (MIN); Pope Co., Glacial
Lakes State Park, 5 miles S of Starbuck, Prairie area with border woods on esker 0.1 mile
NE of Mountain Lake, 7 Aug. 1993, J.P. Schuster 3098 (ASU); Lake of the Woods Co.,
Clementson (8 miles east of Baudette), at mouth of Rapid River to Rainy River, on hillside
with bur oak, ash, white birch and balsam poplar, also on rocks above rapids, 18 Aug.
1994, C.M. Wetmore 74873 (MIN); Otter Tail Co., Inspiration Peak State Scenic Wayside
Park, 12 miles west of Parkers Prairie, on hill with grass and rock on top and on hillside
with red oak and bur oak, 14 Aug. 1994, C.M. Wetmore 74475 (MIN); Washington Co.,
Lost Valley Prairie Scientific and Natural Area, prairie area with limestone outcroppings
and cedar trees, surrounded by aspen and mixed hardwoods, 3 miles NNW of junction of
Szczepańska et al. (2020), PeerJ, DOI 10.7717/peerj.9555 13/27
St Croix and Mississippi Rivers, 3 miles NNE of Hastings, 5 Aug. 1990, J.P. Schuster 2460
(KRAM). NEW MEXICO: Cibola Co, Cibola National Forest, Zuni Canyon 0.2 miles SW
of Log Chute (8.5 miles SW of Grants), small N-S canyon with rocks, douglas fir, oak and
few ponderosa pine, alt. 7434 ft, 17 June 2010, C.M. Wetmore 99445 (MIN); Cibola Co., El
Malpais National Conservation Area above Ranger Station (12.8 miles S of Grants), ridge
S of ranger station with juniper, sage and rocks, alt. 6837 ft, 15 June 2010, C.M. Wetmore
99205 (KRAM); Cibola Co, El Malpais National Conservation Area, 9 miles S of Grants,
lava beds with scattered juniper, alt. 6553 ft, 15 June 2010, C.M. Wetmore 99234 (MIN);
McKinley Co., Cibola National Forest, 0.5 mile E of Quaking Aspen campground on USFS
162 (15 miles SE of Gallup), valley with rock ledges, ponderosa pine, juniper and oak,
alt. 7735 ft, 18 June 2010, C.M. Wetmore 99527 (KRAM); San Miguel Co., near town of
Villanueva (42 miles SW of Santa Fe), hillside and ridge near dam with juniper and pinyon
pine, alt. 5900 ft, 11 June 2010, C.M. Wetmore 98925 (KRAM).
Rhizoplaca phaedrophthalma (Poelt) Leavitt, Zhao Xin & Lumbsch, in Zhao et al., Fungal
Diversity 78: 302. 2016 ≡Lecanora phaedrophthalma Poelt, Mitt. Bot. Staatss. München 2:
483. 1958.
Figs. 2C,2D.
Type: [NEPAL], NÖRDL. ZENTRALNEPAL: Manangbhot (oberes Marsyandi –Tal) Felsen
nördlich Banphag, alt. 4100 m, 13 June 1955, Fr. Lobbichler (holotype, M 122–86/40!).
Thallus lichenized, placodioid, polyphyllous, rounded to irregular when older, 2–5 cm diam.
Marginal lobes usually weakly developed, 0.5–1.2 mm long and 0.2–0.7 mm wide, short,
plane to slightly convex, broadened at the tips and flabellate. Thallus centre squamulose-
areolate to squamulose-bullate, irregularly cracked, thick, loosely attached to the substrate.
Squamules crowded, flat to slightly convex, irregular, 0.5–2 mm diam., usually with bluish-
black tinges on the margins and underside towards edges. Upper surface smooth, dull,
pale and light yellow, pruinose in parts, marginal lobes concolourous with thallus centre.
Apothecia numerous, sessile, dispersed to grouped mainly in the centre, 0.5–1.2 mm diam.,
circular, older irregular. Margin thin, smooth, concolourous with thallus, slightly raised
when young, then excluded, disc orange-brown to reddish-brown, usually contrasting
with the thallus, epruinose, strongly convex, dull. Hymenium colourless 50–60 µm high,
hypothecium colourless. Epihymenium orange-brown, with small granules dissolving in K.
Ascospores 8 per ascus, hyaline, simple, subglobose, 8–10 ×5–7 µm. Pycnidia immersed,
simple with bluish-black ostioles, conidia hyaline, filiform, mostly straight, 15–30 ×0.5
µm.
Comments. The main characteristic feature of R. phaedrophthalma is the morphology of
apothecia, which are strongly convex, with reddish-brown, epruinose discs and excluded
margin. Additionally, mature specimens of the species, in contrast to R. opiniconensis and
R. subdiscrepans s. str., are usually poorly placodioid, with weakly define marginal lobes. R.
phaedrophthalma seems to be also characterized by the size of its conidia, which are slightly
larger than in other species treated, as well as by subglobose rather than ellipsoid spores.
Concerning secondary metabolites produced by the species, neither isousnic acid nor fatty
acids were seen in TLC, unlike from the thalli of other taxa.
Szczepańska et al. (2020), PeerJ, DOI 10.7717/peerj.9555 14/27
Specimens examined. CANADA. BRITISH COLUMBIA: Buse Hill, on exposed rock near
top of hill, alt. c. 950 m, 27 Aug. 1994, B.D. Ryan 31877 (ASU); near Quilchena Hotel,
alt. c. 625 m, 28 Aug. 1994, B.D. Ryan 31889 (ASU). RUSSIA. ORENBURG REGION:
Surroundings of water reservoir ‘‘Iriklinskoe vodokhranilishche’’, Iriklinskiy, vill. Vishevoe,
rocks 2 km E of village, in valley of stream, alt. 250–270 m, on acidic schist, 10 June 2011,
J. Vondrák 9406 & Frolov (PRA, two samples). USA. COLORADO: Park Co., Pennsylvania
Mt, Pike National Forest, on rock, c. 3688 m, 1 June 1990, M.A. Thomas 32870 (ASU).
IDAHO: Twin Falls Co., Hagerman Fossil Beds National Monument, 0.5–1 km E of S end
of 500 East Rd, on basalt, alt. c. 900 m, 10 Sept 1998, B.D. Ryan 32870 (ASU). MONTANA:
Gallatin Co., Yellowstone National Park, below Black Butte along highway 191 near NW
corner of park, on talus slope and along highway, alt. 6700 ft, 15 July 1998, C.M. Wetmore
80571 (MIN); Park Co., Yellowstone National Park, Grazing enclosure 1 mile W of Garsiner
at northern edge of park. Open grassland on knoll with sagebrush and rock outcrop, alt.
5300 ft, 21 July 1998, C.M. Wetmore 80979 (MIN). OREGON: Deschutes Co., 6 miles W
of Redmond, Juniper and sage area with lots of loose soil, alt. 3020 ft, 18 July 2006, C.M.
Wetmore 95059 (MIN); Malheur Co., along US20 above Malheur River 13 miles SW of
Harper, Sage brush prairie on north slope, alt. 2790 ft, 19 July 2006, C.M. Wetmore 5101
(MIN). WYOMING: Park Co., West and of Lamar Canyon, 6 miles E of Tower Junction,
on south facing hillside with large granitic glacial erratics and scattered douglas fir, alt. 6500
ft, 19 July 2006, C.M. Wetmore 81235 (MIN); Park Co., Yellowstone National Park. Pebble
Creek Trail at rocks above cliffs, below switchback, 0.25 miles up from camp-ground, alt.
6900 ft, 27 July 1998, C.M.Wetmore 81446 (MIN); Park Co., Yellowstone National Park,
Sheepeater Cliffs, 5.5 miles S of Mammoth, on south-facing columnar cliffs and talus above
stream, alt. 7200 ft, 20 July 1998, C.M. Wetmore 80866 (MIN).
Rhizoplaca subdiscrepans (Nyl.) R. Sant., The Lichens of Sweden and Norway: 278. 1984. ≡
Squamaria chrysoleuca var. subdiscrepans Nyl., Flora 44: 718. 1861. ≡Lecanora subdiscrepans
(Nyl.) Stizenb., Berichtüber die Tätigkeit der St. Gallischen Naturwissenschaftlichen
Gesellschaft 1880-1881: 341. 1882.
Figs. 2G,2H.
Type: [EUROPE], Helvetiae et Tyroliae (PC).
Thallus lichenized, placodioid, polyphyllous, usually rounded, thick, convex, loosely
attached to the substrate, 1–5 cm or more in diam. Marginal lobes distinctly developed,
short, 1–2.5 mm long and 0.5–1.5 mm wide, discrete or contiguous to slightly overlapping,
convex, broadened and crenate-incised at the tips. Thallus centre squamulose-bullate,
composed of crowded strongly convex subunits. Squamules rounded, sinusoid and plicate,
0.25–1 mm diam. Upper surface smooth, glossy, greenish-yellow, in shade becoming
greyish-green, marginal lobes concolourous with thallus centre, upper cortex interspersed
with brownish granules soluble in K. Apothecia usually numerous and grouped, 0.5–2.5
mm diam., rounded, sessile, adnate. Margin prominent, rather thick, concolourous with
thallus, shiny, entire to crenate when older, disc plane, bright orange, yellowish-orange to
brownish-orange, matte, epruinose. Hymenium colourless, 50–80 µm high, hypothecium
colourless, paraphyses simple or weakly branched with swollen apices, subhymenium well-
differentiated, pale grey. Epihymenium orange-brown, interspersed with small granules
Szczepańska et al. (2020), PeerJ, DOI 10.7717/peerj.9555 15/27
dissolving in K. Ascospores 8 per ascus, hyaline, simple, ellipsoid, 11–12 ×6–7 µm.
Pycnidia immersed, simple with bluish-black ostioles, conidia hyaline, filiform, mostly
straight 10–2 ×0.5 µm.
Chemistry. Thallus K+ yellow, C+ pale yellow, KC+ yellow, PD–, medulla K+ yellow, C+
yellow, KC+ yellow, PD–. Secondary metabolites detected by TLC: isousnic (+/−), usnic
(+) and placodiolic (+) acids, as well as unidentified fatty acid (+/−) and unidentified
terpenoids (+).
Ecology and distribution. On volcanic and granite rocks in warm and sunny places, at
lower elevations (200–400 m), and very rare at higher elevations (c. 800 m), in Europe
(Poland and Ukraine) and Western Asia.
Comments. Thallus morphology of specimens representing R. subdiscrepans s. str. is rather
homogeneous and similar to R. opiniconensis. Both species have a placodioid thallus,
squamulose-bullate in the centre, however R. opiniconensis usually has an orange tint to the
thallus, especially visible in older collections. Nevertheless, R. subdiscrepans s. str. produces
fatty acids that have not been found in other taxa of R. subdiscrepans s. lat.
Specimens examined. HUNGARY: Montes Matra, region montis Kékes, in rupibus
Saskö dictis, on andesitic rock, alt. 880 m, 6 June 1974, G. Kiszely & A. Vezda (KRAM,
two specimens). POLAND. SUDETY MTS: Przedgórze Sudeckie foreland, Wzgórza
Strzegomskie Hills, Góra Krzyzowa Mt, alt. 354 m, on basalt rocks in open place, 4
Oct. 2013, K. Szczepanska 923,967 (KRAM, hb. Szczepanska). RUSSIA. CHELYABINSK
REGION: Kizilskoe, Obruchevka (c. 20 km E of Kizilskoe), Mt Razbornaya, c. 8 km W of
village, alt. c. 450 m., on granite outcrops and stones, 25 June 2011, J. Vondrák 9408 &
Vondráková (PRA); Kizilskoe, Obruchevka (c. 20 km E of Kizilskoe), Mt Razbornaya, c. 8
km W of village, alt. c. 450 m., on sun-exposed granite outcrops and stones, 25 June 2011,
J. Vondrák 9418 & Vondráková (PRA). Orenburg region: surroundings of water reservoir
‘‘Iriklinskoe vodokhranilishche’’, Iriklinskiy, vill. Chapaevka, volcanic and limestone rocks
on opposite slope of lake W of village, alt. 270–290 m, on sunny volcanic rock, 11 June 2011,
J. Vondrák 9384, 9385, 9415 & Frolov (PRA), 9422 & Frolov (PRA, two samples); Kuvandik,
vill. Maloe Churaevo (c. 25 km N of Kuvandic) camp c. 2 km W of illage, steppes and
Quercus robur-Tilia cordata-Ulmus laevis woodland areas around camp, alt. 250–500 m,
on sun-exposed siliceous outcrops, 27 June 2011, J. Vondrák 9412 & Vondráková (PRA);
surroundings of water reservoir ‘‘Iriklinskoe vodokhranilishche’’, vill. Chapaevka, on
opposite slope of lake, volcanic rock in valley of stream ‘‘Verkhnaya Orlovka’’, alt. 270–300
m, on sun-exposed siliceous outcrops, 11 June 2011, J. Vondrák 9416 & Frolov (PRA), 9420
& Frolov (PRA, four samples). REPUBLIC OF BASHKORTOSTAN: surroundings of water
reservoir ‘‘Iriklinskoe vodokhranilishche’’, vill. Tashtugay, rocks in valley of river Tanalik,
c. 4 km S of village, alt. 260–300 m, on acid schist, 10 June 2011, J. Vondrák 9411 & Frolov
(PRA). SWEDEN. Ngermanland: Vibyggeråpar., Värna, Valaberget (Mt c. 32 km SSE of
Örnskoldsvik), steep southern slope facing the sea, on steep rocks to S, alt. 10 m, 11 Aug.
1986, R. Moberg 6937 (KRAM).
Szczepańska et al. (2020), PeerJ, DOI 10.7717/peerj.9555 16/27
New combination
Rhizoplaca pseudomellea (B.D. Ryan) Szczepanśka, Rodriguez-Flakus & Śliwa, comb. nov.
≡Lecanora pseudomellea B.D. Ryan, Bryologist 96: 295. 1993.
MB 835038
Figs. 2E,2F.
Type: [USA]. CALIFORNIA: Alpine Co., 1 mi E of Monitor Pass, along Calif. Hwy 89, 16
mi E of Markleeville, alt. 2440 m, field of small rocks among Sagebrush, 24 June 1985, B.D.
Ryan 13609 (holotype, ASU!).
Thallus lichenized, placodioid, usually distinctly rosette, moderately attached to the
substrate, 1–4 cm diam. Marginal lobes distinctly developed, 1–3.5 mm long and 0.7–
2 mm wide, slightly convex, smooth, contiguous, sinusoid, minutely broadened and
grey, greenish-black to black at the tips. Thallus centre areolate, areoles convex, bullate,
rounded to irregular, smooth or slightly rough, 0.5–2 mm diam. Upper surface smooth,
glossy, yellowish-brown, orange-brown to reddish- or rusty brown, marginal lobes paler,
yellowish, except darker tips. Apothecia grouped in the thallus centre, 0.5–2.5 mm diam.,
rounded to irregular when older, sessile. Margin thin, concolourous with thallus, shiny,
flexuose, slightly raised when young, becoming excluded, disc concolourous with thallus or
darker, reddish-brown, glossy, plane to slightly convex, epruinose. Hymenium colourless,
60–70 µm high, with colourless hypothecium. Epihymenium orange-brown, interspersed
with small granules dissolving in K. Ascospores 8 per ascus, hyaline, simple, ellipsoid, 10–12
×6–7 µm. Pycnidia punctiform, globose, immersed, simple with bluish-black ostioles, c.
260 µm diam., conidia hyaline, filiform, 10–25 ×0.5 µm.
Chemistry.Thallus K–, C–, KC–, PD–, medulla K–, C–, KC–, PD–. Secondary metabolites
detected by TLC: isousnic ( +/−), usnic (+/−), psoromic (+/−) and fatty acids (+/−).
Ecology and distribution. On granite and volcanic rocks, in dry and sunny stations, at
higher elevations (1,500–2,500 m) in North America.
Comments.R. pseudomellea is characterized mainly by its distinct rosette thallus with long,
sinusoid, darker at the tips marginal lobes and orange-brown to reddish-brown, glossy
upper surface. Compared to other species treated here, R. pseudomellea has a darker colour,
longer and much less convex marginal lobes, as well as a distinctly areolate not squamulose
thallus centre. However, its apothecia are usually very similar in colour and morphology
to R. phaedrophthalma.R. pseudomellea has very variable secondary chemistry. The most
often secondary metabolite occurring in the thallus is isousnic acid, but it may also contains
psoromic acid.
Specimens examined. USA. CALIFORNIA: Kern Co., Eastern slope of the southern Sierra
Nevada, BLM land on the divide between Oil Canyon and Pine Tree Canyon, along 4WD
road which also serves as part of the Pacific Crest Trail, in a pinyon pine and scrub oak
woodland, on white shale-like rocks, alt. c. 6150 ft, 19 Apr. 1997, J.R. Shevock 15124 (ASU).
OREGON: Harney Co., Steen Mountain (68 ml SSE of Burns), NW slope near snow patch,
alt. 8700 ft, 18 July 2006, C.M. Wetmore 95084, 95079 (MIN); Lake Co., Fremont National
Forest, Along Dairy Creek, SE part of Gearhart Wilderness, alt. c. 1915 m, 27 Aug 1991, B.D.
Ryan 28456 (ASU); Lake Co., Fremont National Forest, Palisade Rocks, SE part of Gearhart
Wilderness, c. 1950 m, 11 Sept. 1991, B.D. Ryan 28652 (ASU); Tulare Co., Sequoia National
Szczepańska et al. (2020), PeerJ, DOI 10.7717/peerj.9555 17/27
Park, Milk Ranch Peak at border of park E of headquarters, around peak with white fir,
incense cedar, pines and rocks, alt. 6100 ft, 21 May 1984, C.M. Wetmore 51151 (MIN);
Tuolumne Co., Winter Sports Area, N of Hwy 108, 5 km SW of Fraser Flat Campground,
alt. 1700 m, on volcanic rock, pine, oak, 13 Aug. 1989, B.D. Ryan 24402 (MIN).
DISCUSSION
Rhizoplaca subdiscrepans s. lat. was found to be highly polyphyletic by Leavitt et al. (2016),
and as a result of multigene analyses, these authors delimited five cryptic species-level
lineages within the species complex, defining them as R. subdiscrepans ‘subd A, B, C, D
and E’. In parallel, another paper was published concerning the generic classification
of lecanoroid lichens and including some representatives of the genus Rhizoplaca (Zhao
et al., 2016). In the latter authors’ phylogenetic analyses, they also took into account
several placodioid species previously classified in Lecanora, e.g., L. opiniconensis and L.
phaedrophthalma. Among others, they included in their analysis single sequences of the
two latter species, published by Arup & Grube (2000) but not treated by Leavitt et al. (2016).
As a result, Zhao et al. (2016) found a core group of Rhizoplaca formed a monophyletic
group together with the mentioned species and transferred these species along with some
others to Rhizoplaca.
Both R. opiniconensis and R. phaedrophthalma were described as new taxa based on
morphological and chemical features in the second half of the 20th century. However, it
is presently known that morphological characters may not be sufficient for detecting
taxa (Bickford et al., 2007;Crespo & Pérez-Ortega, 2009). On the other hand, careful
morphological analysis of distinct phylogenetic lineages may lead to the recognition
of some previously overlooked characters (Kroken & Taylor, 2001;Del Prado et al., 2007;
McCune & Altermann, 2009;Frolov et al., 2016).
Species candidates within R. subdiscrepans s. lat. were considered to be cryptic by Leavitt
et al. (2016), but in our morphological analysis, an attempt was made to identify potential
diagnostic features for samples representing different clades. In contrast to recognized
candidate species within the R. melanophthalma complex, which were highly variable
(Leavitt et al., 2011), the morphology of R. subdiscrepans s. lat. representatives seem to be
rather similar, especially in the case of clades ‘subd E’ (conspecific with R. subdiscrepans s.
str.) and ‘subd D’ (conspecific with R. opiniconensis). The most common and characteristic
features of all examined samples were a placodioid, polyphyllous, yellow-green thallus with
squamulose-bullate centre, visible marginal lobes, and sessile apothecia with pale yellowish
to brown, epruinose discs. However, despite their apparently similar morphology, the
samples are heterogeneous and vary in the appearance of their apothecia and marginal
lobes, as well as in the shape and size of ascospores and conidia. These characters have been
shown to be diagnostic in the case of the Parmeliaceae family (Argüello et al., 2007;Divakar
et al., 2010).
In our study, R. opiniconensis seems to be characteristic in its arched conidia and thallus
colour that becomes distinctly more orange in herbarium material. Whereas the main
characteristic feature of R. phaedrophthalma (conspecific with ‘subd A’) is the morphology
Szczepańska et al. (2020), PeerJ, DOI 10.7717/peerj.9555 18/27
of the apothecia, which are strongly convex with reddish-brown discs and an excluded
thalline margin. Furthermore, some differences in the latter species are the size of the
conidia, which are slightly larger than in the other discussed taxa, and ascospores that
are distinctly smaller and subglobose rather than ellipsoid. Additionally, in contrast to
other analysed representatives of R. subdiscrepans s. lat., thalli of mature specimens of R.
phaedrophthalma are usually poorly placodioid, with weakly developed marginal lobes.
In the above morphological studies, we noted that none of the representatives of R.
subdiscrepans s. lat. had orange and pruinose apothecial discs, which are mentioned in the
literature as characteristic for R. subdiscrepans (Ryan, 2001). These characters, as well as the
greyish-green tint of the upper surface of the thallus, are more appropriately applied to R.
chrysoleuca s. lat. than to the R. subdiscrepans complex, an assumption that is consistent with
the opinion of Cansaran et al. (2006). However, according to Zheng, Sheng & An (2007),
apothecial discs and their pruinosity do not indicate proper phylogenetic relationships
among Rhizoplaca species, so this issue requires further research.
Chemical characters that are widely used for species delimitation in lichenology are not
always good taxonomic indicators, especially when they do not correspond with molecular
data (Hawksworth, 1976;Culberson, 1986;Ryan & Nash, 1997;Divakar et al., 2010;Thell
et al., 2017;Ossowska et al., 2018;Zakeri et al., 2019). This fact may apply among others
to the genus Rhizoplaca for which high chemical variability has been shown (Wei & Wei,
2005;Zhou et al., 2006;Leavitt et al., 2013a). On the other hand, in many cases chemistry
may be successfully used as a character to support the circumscription of particular lichen
taxa (Elix, Corush & Lumbsch, 2009;Leavitt, Johnson & St. Clair, 2011;Molina et al., 2011;
Spribille, Klug & Mayrhofer, 2011;Onut-Brännström, Johannesson & Tibell, 2018;Mark et
al., 2019). This was demonstrated within the R. melanophthalma species complex in the
case of R. parilis S. Leavitt., Fernandez-Mendoza, Lumbsch, Sohrabi & L. St. Clair (Leavitt
et al., 2013a).
With this in mind, we carefully analysed the content of secondary metabolites in the thalli
of R. subdiscrepans s. lat. representatives sampled herein. The survey indicated such lichen
substances as isousnic, usnic and placodiolic acids, as well as fatty acids and terpenoids,
but some differences in their presence in particular recognized species could be observed.
Among the secondary metabolites detected by TLC, neither isousnic acid nor fatty acids
have been found in the thalli of R. phaedrophthalma, whereas the presence of fatty acids
was observed only in specimens of R. subdiscrepans s. str., including those samples from
Poland. None of the examined samples of R. subdiscrepans s. lat. contained psoromic,
lecanoric or norstictic acids, as mentioned in the literature (Ryan, 2001).
Rhizoplaca subdiscrepans has until recently been considered to be distributed worldwide.
However, phylogenetic analyses showed some slight differences in the geographical range of
cryptic lineages within this species complex (Leavitt et al., 2016). All of the clades identified
by Leavitt et al. (2016) included representatives on the Asian continent. Nonetheless,
individuals belonging to clades ‘subd A’ and ‘subd D’ also occurred in North America,
whereas clade ‘subd E’ had a European distribution. The results of our study correspond
with theirs.
Szczepańska et al. (2020), PeerJ, DOI 10.7717/peerj.9555 19/27
Rhizoplaca opiniconensis (= ‘subd D’) has been genetically confirmed as occurring in
North America and is not found in Europe. The species epithet, however, is reported
here for the first time from outside North America, i.e., from East-Central Asia (Altay).
It should be noted that based on detailed habitat analysis of available samples it seems
this taxon is hygrophytic, preferring mountain habitats in higher elevations, localized in
shaded and moist places close to water resources. Rhizoplaca phaedrophthalma, same as R.
opiniconensis, has not been noted from Europe, its centre of distribution being located in
North America and Western and Central Asia, with the locus classicus in Nepal, which is
in accord with the distribution pattern of clade ‘subd A’(Leavitt et al., 2016). The species
prefers different habitat conditions than R. opiniconensis, since it occurs mostly on siliceous
rock in semi-arid areas but also at higher elevations.
Finally, R. subdiscrepans s. str. (= ‘subd E’) besides Eastern Asia has been recorded
in Europe (Poland and Ukraine) and has not been confirmed in North America. This
is important in the light of the fact that R. subdiscrepans s. str.has been described from
Switzerland in Europe. The distribution pattern of the species supports our concept that
it is indeed conspecific with clade ‘subd E’ (Leavitt et al., 2016). R. subdiscrepans occupies
specific habitats, occurring namely in warm, dry and sunny places in lower elevations,
usually on volcanic rocks with a southern exposure. It is the only taxon of the discussed
group of lichens with such ecological preferences. It is worth noting here that analysed
samples of R. subdiscrepans s. str. are very similar in morphology to representatives
of R. opiniconensis. Nevertheless, the species seems to be distinguishable based on its
different geographical range and habitat preferences. These characters are often mentioned
in literature as a supporting species recognition, even when there is a lack of evident
phenotypic differences or they are insufficient to circumscribe the species (Crespo et al.,
2002;Argüello et al., 2007;Divakar et al., 2010;Onut-Brännström, Johannesson & Tibell,
2018).
The above data can also be compared to the R. melanophthalma species complex
presented in Leavitt et al. (2013b). Two of the identified phylogenetic lineages therein
had intercontinental distributions, while four were found exclusively in North America.
In this case, phylogenetic analyses, as well as geographical distribution, provided the
basis for delimitation and description of new taxa (Leavitt et al., 2013a). In addition, R.
melanophthalma s. str. was circumscribed and shown to be represented by a clade with the
widest geographical range that also includes Europe.
During our study, a few sequences were generated for specimens representing Lecanora
pseudomellea B.D. Ryan, a placodioid taxon occurring only in North America. The species
forms a strongly supported monophyletic lineage within the Rhizoplaca and was therefore
transferred to this genus. Characteristic features of this taxon are an orange-brown to
reddish-brown, glossy upper surface, as well as long, sinusoid, darker at the tips, marginal
lobes. Compared to other taxa discussed above, R. pseudomellea has a darker colour, longer
and much less convex marginal lobes, as well as a distinctly areolate, not squamulose,
thallus centre. It has a very variable secondary chemistry and the occurrence of compounds
varies widely between samples. The most common substance occurring in the thallus is
Szczepańska et al. (2020), PeerJ, DOI 10.7717/peerj.9555 20/27
isousnic acid, but the species may also contain psoromic acid, which is not present in the
other taxa of the discussed group.
CONCLUSIONS
Despite the development of modern phylogenetic methods, species delimitation within
lichenized fungi is still problematic. Implementation of integrative taxonomy and
incorporation of various data, such as genetic, morphological, chemical, geographical
and ecological data, usually deliver some resolution. An increasing number of analysed
samples can also become more informative with these tools; these often lead to the
conclusion that molecularly defined units are semi-cryptic rather than cryptic, as in the
case of our study.
In our study based on molecular and phenotypic data, as well as in reference to
previously described species, names are proposed for three lineages, the ‘subd A, D and E’
of R. subdiscrepans s. lat. delimited by Leavitt et al. (2016) and recognized respectively as
R. phaedrophthalma,R. opiniconensis (supported by the placement of the type sequence in
our phylogeny) and R. subdiscrepans s. str. Furthermore, we suggest transferring Lecanora
pseudomellea to the genus Rhizoplaca with a proposal for a new combination—Rhizoplaca
pseudomellea.
The geographical conclusion of our survey is that R. subdiscrepans s. str. appears to be
mostly a European taxon with a range extended to Western Asia, whereas R. opiniconensis
has a broader distribution than previously recorded, as it occurs not only in North America
but also in Asia.
ACKNOWLEDGEMENTS
The curators of ASU, CANL (CMN), MIN, PRA and WRSL are gratefully acknowledged
for the loan of specimens. Sincere thanks are due to Pamela Rodriguez-Flakus (Kraków)
for performing phylogenetic analyses, Ulf Arup (Lund) for providing data on voucher
specimen for GenBank sequence of R. opiniconensis, Steven D. Leavitt (Chicago) for
valuable help with voucher specimens of R. subdiscrepans, Adam Flakus and Karina Wilk
(Kraków) for support with TLC analyses and helpful assistance with photos respectively
and Prof. Mark R. D. Seaward (Bradford) for a language revision and valuable comments
on the manuscript.
ADDITIONAL INFORMATION AND DECLARATIONS
Funding
This work was supported by statutory funds of Wroclaw University of Environmental and
Life Sciences and its Leading Research Groups support project (2020–2025, Art. 387/3) as
well as by statutory funds of the W. Szafer Institute of Botany, Polish Academy of Sciences
and National Science Centre, Poland, project 2016/21/B/NZ8/02463 granted to L`
S. The
funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Szczepańska et al. (2020), PeerJ, DOI 10.7717/peerj.9555 21/27
Grant Disclosures
The following grant information was disclosed by the authors:
Wroclaw University of Environmental and Life Sciences.
Leading Research Groups support project (period 2020–2025).
National Science Centre, Poland: 2016/21/B/NZ8/02463.
W. Szafer Institute of Botany, Polish Academy of Sciences.
National Science Centre, Poland: 2016/21/B/NZ8/02463.
Competing Interests
The authors declare there are no competing interests.
Author Contributions
•Katarzyna Szczepańska conceived and designed the experiments, performed the
experiments, analyzed the data, prepared figures and/or tables, authored or reviewed
drafts of the paper, and approved the final draft.
•Jacek Urbaniak and Lucyna Śliwa performed the experiments, analyzed the data, prepared
figures and/or tables, authored or reviewed drafts of the paper, and approved the final
draft.
DNA Deposition
The following information was supplied regarding the deposition of DNA sequences:
The ITS sequences are available at GenBank: MN931719 to MN931739.
The GenBank accession number, species, and location of the specimen (herbarium
collection number) of each studied specimen are as follows:
MN931719 Protoparmeliopsis muralis, Szczepańska 1263 (hb. Szczepańska)
MN931720 Rhizoplaca opiniconensis, Brodo 25117 (CANL) Canadian Museum of
Nature
MN931721 Rhizoplaca opiniconensis, Wetmore 88134 (MIN) University of Minnesota
MN931722 Rhizoplaca opiniconensis, Wetmore 74475 (MIN)
MN931728 Rhizoplaca phaedrophthalma, Ryan 31877 (ASU) Arizona State University
MN931729 Rhizoplaca phaedrophthalma, Ryan 31889 (ASU)
MN931730 Rhizoplaca phaedrophthalma, Ryan 32870 (ASU)
MN931732 Rhizoplaca phaedrophthalma, Wetmore 80571 (MIN)
MN931726 Rhizoplaca phaedrophthalma, Wetmore 80979 (MIN)
MN931723 Rhizoplaca phaedrophthalma, Wetmore 95059 (MIN)
MN931724 Rhizoplaca phaedrophthalma, Wetmore 5101 (MIN)
MN931731 Rhizoplaca phaedrophthalma, Wetmore 80866 (MIN)
MN931725 Rhizoplaca phaedrophthalma, Wetmore 81235 (MIN)
MN931727 Rhizoplaca phaedrophthalma, Wetmore 81446 (MIN)
MN931734 Rhizoplaca pseudomellea, Wetmore 51151 (MIN)
MN931735 Rhizoplaca pseudomellea, Ryan 24402 (MIN)
MN931736 Rhizoplaca pseudomellea, Wetmore 95079 (MIN)
MN931737 Rhizoplaca pseudomellea, Wetmore 95084 (MIN)
MN931733 Rhizoplaca pseudomellea, Ryan 28456 (ASU)
Szczepańska et al. (2020), PeerJ, DOI 10.7717/peerj.9555 22/27
MN931738 Rhizoplaca subdiscrepans, Szczepańska 923 (KRAM)
MN931739 Rhizoplaca subdiscrepans, Szczepańska 967 (KRAM) W. Szafer Institute of
Botany, Polish Academy of Sciences.
Data Availability
The following information was supplied regarding data availability:
The alignment of ITS sequences are available at TreeBASE: http://purl.org/phylo/
treebase/phylows/study/TB2:S26365?x-access-code=cecc71c8fa6e3378ec581a812b7b976b&
format=html.
Supplemental Information
Supplemental information for this article can be found online at http://dx.doi.org/10.7717/
peerj.9555#supplemental-information.
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