Fungal Diversity DOI 10.1007/s13225-015-0354-5
Towards a revised generic classification of lecanoroid lichens (Lecanoraceae, Ascomycota) based on molecular, morphological and chemical evidence Xin Zhao 1 & Steven D. Leavitt 2 & Zun Tian Zhao 1 & Lu Lu Zhang 1 & Ulf Arup 3 & Martin Grube 4 & Sergio Pérez-Ortega 5,6 & Christian Printzen 7 & Lucyna Śliwa 8 & Ekaphan Kraichak 9 & Pradeep K. Divakar 10 & Ana Crespo 10 & H. Thorsten Lumbsch 2
Received: 3 September 2015 / Accepted: 16 November 2015 # School of Science 2015
Abstract The phylogenetic relationship of lecanoroid lichens is studied using two data sets: 1) a 2-locus data set including 251 OTUs representing 150 species, and 2) a 6-locus data set with 82 OTUs representing 53 species. The genus Lecanora as currently circumscribed is shown to be highly polyphyletic and several genera, including Adelolecia, Arctopeltis, Bryonora, Carbonea, Frutidella, Lecidella, Miriquidica, Palicella, Protoparmeliopsis, Pyrrhospora, and Rhizoplaca are nested within Lecanora sensu lato. A core group of Lecanora is supported as monophyletic and includes species of the L. carpinea, L. rupicola, and L. subcarnea groups, and a core group of the L. subfusca group. Three monophyletic clades that are well supported in our analyses and well characterized by phenotypical characters are accepted here: 1)
Myriolecis to accommodate the Lecanora dispersa group and Arctopeltis; 2) Protoparmeliopsis for the L. muralis group; and 3) Rhizoplaca is emended to include three placodioid taxa previously classified in Lecanora (L. novomexicana. L. opiniconensis, L. phaedrophthalma), whereas R. aspidophora and R. peltata are excluded from Rhizoplaca. The latter is transferred into Protoparmeliopsis. Lecidella is strongly supported as a monophyletic group. Our studies indicate the presence of additional clades of species currently placed in Lecanora sensu lato that warrant taxonomic recognition but additional data will be necessary before the circumscription of these entities is fully understood. 37 new combinations are proposed into the genera Myriolecis (30), Protoparmeliopsis (2), and Rhizoplaca (5).
Electronic supplementary material The online version of this article (doi:10.1007/s13225-015-0354-5) contains supplementary material, which is available to authorized users. * H. Thorsten Lumbsch
[email protected]
5
Departamento Biogeoquímica y Ecología Microbiana, Museo Nacional de Ciencias Naturales, Madrid, Spain
Xin Zhao
[email protected]
6
Present address: Real Jardín Botánico, Plaza de Murillo 2, 28014 Madrid, Spain
Ekaphan Kraichak
[email protected]
7
Department of Botany and Molecular Evolution, Senckenberg Research Institute and Natural History Museum Frankfurt, Senckenberganlage 25, D-60325 Frankfurt am Main, Germany
College of Life Sciences, Shandong Normal University, Jinan 250014, People’s Republic of China
8
Laboratory of Lichenology, W. Szafer Institute of Botany, Polish Academy of Sciences, Lubicz 46, 31–512 Kraków, Poland
9
Department of Botany, Faculty of Science, Kasetsart University, 10900 Bangkok, Thailand
10
Departamento de Biología Vegetal II, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain
1
2
Science & Education, The Field Museum, Chicago, IL, USA
3
Biological Museum, Lund University, Box 117, SE-22100 Lund, Sweden
4
Institute of Plant Sciences, Karl-Franzens-University Graz, Holteigasse 6, 8010 Graz, Austria
Fungal Diversity
Keywords Arctopeltis . Classification . Generic concept . Lecanora . Lichenized fungi . Myriolecis . Rhizoplaca . Protoparmeliopsis . Rhizoplaca . Taxonomy
Introduction The classification of lichenized fungi has changed dramatically over the last decades and the family Lecanoraceae is a prime example of these changes. Traditionally, this family has included crustose lichens with apothecial margins containing algal cells and asci containing hyaline, non-septate ascospores (Zahlbruckner 1907). The core genus Lecanora represented the large majority of species diversity in the family. However, already a first seminal paper by Eigler (1969) distinguished several groups by using microscopic characters of ascomata within the large genus Lecanora and demonstrated the heterogeneity of the genus. Subsequent studies on ascus-types resulted in exclusion of aberrant taxa before molecular sequence data became readily available (Hafellner 1984). In its widest sense, the core genus of the family was a very heterogeneous assemblage including species now classified in different families and even subclasses, such as Trapelia Choisy (Hertel 1969, 1970; Lumbsch et al. 2001a; Lumbsch et al. 2001b) or Aspicilia Massal. (Clauzade and Roux 1984; Nordin et al. 2010) which are currently placed in different orders within Ostropomycetidae (Lumbsch and Huhndorf 2010; Miadlikowska et al. 2014; Resl et al. 2015). Even after the exclusion of these aberrant taxa, the circumscription of the family and its core genus Lecanora remained unclear. A number of molecular studies have included taxa from Lecanoraceae and discussed their phylogenetic placement in Lecanorales, with Lecanoraceae being sister to a clade including Cladoniaceae and Stereocaulaceae (Ekman and Wedin 2000; Lendemer and Hodkinson 2013; Miadlikowska et al. 2006; Schmull et al. 2011). Cladoniaceae (Lumbsch et al. 2004). or a clade consisting of Gypsoplacaceae and Malmideaceae, which formed a sister group to a clade including Cladoniaceae and Stereocaulaceae (Miadlikowska et al. 2014). The family currently includes over 25 genera (Lumbsch and Huhndorf 2010) and about 770 described species (Jaklitsch et al. 2015). It includes lichenized and lichenicolous species with a crustose thallus, apothecial ascomata with or without a thalline margin; an often poorly developed true excipulum except in biatorine and lecideine taxa, 8-spored to rarely multispored asci of the Lecanora-type, and simple or rarely 1–5-septate, hyaline, cylindrical, oblong-ellipsoidal, ellipsoidal or globose ascospores (Lumbsch 2004). While our knowledge of the phylogenetic placement of the family and its circumscription is largely reflected in the current classification, the current circumscription of the core genus of the family does not reflect our knowledge of the phylogeny in this group. Several studies have
focused on phylogenetic relationships within Lecanora (Arup and Grube 1998, 1999, 2000; Grube et al. 2004; Papong et al. 2013; Pérez-Ortega et al. 2010; Śliwa et al. 2012) or provided evidence for distinct clades in the family that were described as new genera in or outside of Lecanoraceae (Kondratyuk et al. 2014; Rodriguez Flakus and Printzen 2014). Generally, the molecular data did not support groups as monophyletic that were based on growth forms, such as the umbilicate genera Arctopeltis Poelt and Rhizoplaca Zopf, which were found to be nested within the L. dispersa group (Arup and Grube 1998) and different clades among placodioid Lecanora spp. (Arup and Grube 2000). respectively. However, other groups, some of them already circumscribed by Eigler (1969) based on a combination of anatomical and chemical characters, were supported by various authors and among others by molecular evidence. This includes Lecanora sensu stricto (Lumbsch and Elix 2004; Lumbsch et al. 1996; Papong et al. 2013). the L. dispersa group (Arup and Grube 1998; Pérez-Ortega et al. 2010; Śliwa 2007; Śliwa et al. 2012), the L. polytropa-, L. symmicta-groups, and the L. varia group in a restricted sense (Pérez-Ortega et al. 2010; Printzen and May 2002), the L. rupicola group within Lecanora sensu stricto (Grube et al. 2004; Leuckert and Poelt 1989). the Placodium group in a restricted sense (Arup and Grube 1998; Pérez-Ortega et al. 2010; Ryan and Nash 1993a, 1993b, 1997b), and the genera Lecanoropsis Choisy (=L. saligna group) (Choisy 1949; Pérez-Ortega et al. 2010; Printzen 2001; van den Boom and Brand 2008), and Protoparmeliopsis Choisy (=L. muralis group) (Choisy 1929; Kondratyuk et al. 2014; Pérez-Ortega et al. 2010). Recently, molecular studies have also suggested that morphologically well distinguished and established genera, such as Adelolecia Hertel & Hafellner, Frutidella Kalb, Japewia Tønsberg, Lecidella Körber, Palicella Rodr. F l a k u s & P r i n t z e n , P y r r h o s p o r a K ö r b e r, a n d Scoliciosporum Massal. are nested within Lecanora sensu lato (Kondratyuk et al. 2014; Miadlikowska et al. 2014; Rodriguez Flakus and Printzen 2014; Schmull et al. 2011). This manuscript focuses on the generic delimitation of species currently placed in Lecanora sensu stricto and clades that are potentially nested within Lecanora sensu lato. We assembled two data sets to address this issue: 1) a concatenated data set of ITS and mtSSU rDNA sequences to circumscribe groups of Lecanora sensu lato and related genera, and 2) a concatenated data set of six loci (two nuclear ribosomal, one mitochondrial ribosomal, and three nuclear protein-coding genes) for selected taxa from the groups identified in analyses of the two-marker dataset. Using the results of those two analyses we propose changes towards a new generic classification of lecanoroid lichens that will better reflect our current understanding of the evolution of this group of lichenized fungi.
Fungal Diversity
Materials and methods Taxon sampling For ‘analysis 1’, species belonging to nine groups of Lecanora sensu lato including Lecanora dispersa-, L. intumescens-, L. polytropa-, L. rupicola-, L. saligna-, L. subcarnea-, L. subfusca-, L. symmicta-, L. varia groups and 13 related genera (Adelolecia, Bryonora, Carbonea, Frutidella, Japewia, Lecidella, Miriquidica, Palicella, Protoparmeliopsis, Pyrrhospora, Ramboldia, Rhizoplaca, Scoliciosporum) were included. Ramboldia was selected as outgroup based on previous results (Miadlikowska et al. 2014). The taxa we selected from our samples and GenBank to represent these groups were listed in Online Resource 1. A total of 251 OTUs (Operational Taxonomic Units) representing 150 species were compiled for the two-locus analysis. For ‘analysis 2’, we selected representative taxa from some groups identified in analysis 1 for which fresh material was available and sequencing for at least three of the six selected loci was successful. This includes species of the L. dispersa group; L. formosa, L. muralis group (=Protoparmeliopsis); L. polytropa group; L. subcarnea group, L. subfusca group, L. symmicta group; and ten Lecidella species. Three Miriquidica species from GenBank were also included. According to Arup and Grube (2000). Rhizoplaca is not monophyletic and formed three clades. We included Lecanora novomexicana, R. chrysoleuca, R. haydenii, R. marginalis , R. melanophthalma, R. parilis, R. peltata, R. porterii, R. shushanii and R. subdiscrepans which represent those clades. Nine Ramboldia species were also included. Three Parmeliaceae were included as outgroup. A total of 82 OTUs representing 53 taxa were compiled for the six-locus analysis (Online Resource 2).
To obtain fungal sequences apothecia were used for extracting the total DNA with the Prepease DNA Isolation Kit (USB, Cleveland, OH, USA) and following the leaf extraction protocol. Amplification of the targeted loci used the Ready-To-Go PCR Beads (GE Healthcare) protocol. 25 μl PCR samples were prepared by adding 23 μl of sterile water, 1 μl of each primer at 10 μM concentration, and 0.5 μl template DNA to the tubes containing the beads. A specific forward primer for amplifying RPB2 gene of Lecidella was designed for this study. The primers used and the PCR settings are summarized in Table 1. PCR products were purified using Exo SAP-IT (USB, Cleveland, OH, USA), following the manufacturers’ instructions. Sequencing was performed using BigDye Terminator v3.1 (Applied Biosystems, Foster City, CA, USA) and the same primers as for amplification. Products were then run on an ABI 3730 automated sequencer according to established protocols (Applied Biosystems) at the Pritzker Laboratory for Molecular Systematics at the Field Museum, Chicago, IL, USA. Sequence alignments Contigs were assembled and edited using the program Geneious v6.1.2 (Biomatters Ltd., Auckland, NZ). Sequences of each locus were aligned using the program MAFFT v7 (Katoh et al. 2009; Katoh and Toh 2008). For ITS sequences, we used the L-ING-i alignment algorithm with the remaining parameters set to default values. For nucLSU, the G-ING-i algorithm and the Bleave gappy regions^ were selected. We used the E-ING-i algorithm for mtSSU and RPB1, and the G-ING-i algorithm for MCM7 and RPB2, with the remaining parameters set to default values. Ambiguous positions were removed using Gblocks (Castresana 2000) with using the less stringent selection option. Summaries of alignment information for the two datasets are provided (Tables 2 & 3).
Molecular methods Phylogenetic analysis Molecular data were generated and gathered for six different loci: the internal transcribed spacer (ITS), nuclear large subunit (nucLSU), mitochondrial small subunit (mtSSU), the minichromosome maintenance complex component 7 (MCM7), the largest subunit of the RNA polymerase II gene (RPB1) and the second largest subunit of RNA polymerase II gene (RPB2). Sequences of eight Lecanora, nine Lecidella, one Letharia, one Ramboldia and nine Rhizoplaca species, for a total of 53 specimens were newly generated for this study. These sequences were complemented with sequences available from GenBank (Online Resource 1 & 2). We assembled OTUs by combining the available sequence data for each locus, as well as sequences for different loci obtained from the same voucher specimen, and retaining at least three of six targeted loci.
The single-locus alignments were concatenated using Geneious for subsequent phylogenetic analyses. Maximum likelihood (ML) analyses were carried out on both datasets using the locus-specific model partitions (ITS and mtSSU for analysis 1; ITS, nucLSU, mtSSU, MCM7, RPB1 and RPB2 for analysis 2) in RAxML v8.1.11 (Stamatakis 2006). A search combining 200 separate ML searches was conducted, implementing a GTRGAMMA model, and 1000 pseudoreplicates to evaluate bootstrap support for each node. In addition to ML bootstrap support (BS) the six locus concatenated alignment was subjected to a Bayesian analysis (B/MCMC) using MrBayes v3.2.3 (Huelsenbeck and Ronquist 2001). Nucleotide substitution models for the six loci for the Bayesian analysis were selected by jModelTest
Fungal Diversity Table 1
Primer information and PCR settings used for this paper
Loci/PCR info
ITS
nucLSU
mtSSU
MCM7
PCR primers
ITS1f1 ITS4a2
AL1R3 AL2R4 LR55 LR65
mrSSU16 mrSSU2r6 mrSSU3r6 MSU17 MSU77
LecMCM7f8 gRPB1a10 LecMCM7r8 fRPB1c11 MCM7-709f9 MCM71348r9
Lecidella_RPB2f (5′-DGATGTGCGAG AYCGAGART3′) RPB2-6f12 RPB2-7cr12
94 °C 10 min 34 cycles 95 °C 45 s 50 °C 45 s 72 °C 1 min30sec none
94 °C 10 min 34 cycles 94 °C 45 s 50 °C 50s 72 °C 1 min none
94 °C 10 min 34 cycles 94 °C 45 s 50 °C 50s 72 °C 1 min None
94 °C 10 min 34 cycles 94 °C 45 s 50 °C 50s 72 °C 1 min none
72 °C 10 min
72 °C 5 min
72 °C 5 min
72 °C 5 min
Initial denaturation 95 °C 5 min Phase 1
Phase 2
Final extension
10 95 66 72 34 95 56 72 72
95 10 cycles 95 °C 30s 66 °C 30s °C 1 min30s 72 34 cycles 95 °C 30s 56 °C 30s °C 1 min30s 72 °C 10 min 72
°C 5 min cycles °C 30s °C 30 sec °C 1 min30sec cycles °C 30s °C 30 sec °C 1 min30sec °C 10 min
RPB1
RPB2
1
(Gardes and Bruns 1993) 2 (Larena et al. 1999) 3 (Döring et al. 2000) 4 (Mangold et al. 2008) 5 (Vilgalys and Hester 1990) 6 (Zoller et al. 1999) 7 (Zhou and Stanosz 2001) 8 (Leavitt et al. 2011) 9 (Schmitt et al. 2009) 10 (Stiller and Hall 1997) 11 (Matheny et al. 2002) 12 (Liu et al. 1999)
v2.1.7 (Darriba et al. 2012; Guindon and Gascuel 2003) (Table 3). A Bayesian analysis was run for 10,000,000 generations with four independent chains and sampling every 1000th tree. All model parameters were unlinked. Two independent Bayesian runs were conducted to ensure that stationarity was reached and the runs converged at the same log-likelihood level (Nylander et al. 2008). After discarding the burn-in, the remaining 7500 trees of each run were pooled to calculate a 50 % majority rule consensus tree. The clades that received bootstrap support ≥70 % under ML and posterior probabilities (pp) ≥0.95 were considered well supported (Hillis and Bull 1993). Phylogenetic trees were visualized using FigTree v1.4.2 (Rambaut 2009). Since the species of Lecanora as currently circumscribed did not form a monophyletic lineage, we used alternative hypothesis testing to evaluate whether our data set rejects monophyly of the genus. For this we performed the Shimodaira– Hasegawa (SH) test (Shimodaira and Hasegawa 1999) and the expected likelihood weight (ELW) test following Strimmer & Rambaut (2002). using the program Tree-PUZZLE v5.2 (Schmidt et al. 2002). The program used the concatenated dataset to compare the best tree from the alternative hypotheses with the unconstrained ML tree. These trees were inferred Table 2
Characteristics of loci used in the 2-locus data set
Alignments
ITS
mtSSU
Total
Number of sequences Newly generated sequences Number of sites (including gaps) Missing sequences/the percentages
243 40 737 8/3 %
110 35 555 141/56 %
353 75 1292 149/30 %
in Tree-PUZZLE, using the same nucleotide substitution model as described above.
Results A total of 203 DNA sequences were newly generated for this study, including 41 ITS, 30 nucLSU, 36 mtSSU, 25 MCM7, 31 RPB1 and 40 RPB2 sequences which were listed in bold in Online Resource 1 & 2. The two data sets used for this study were deposited in TreeBase (ID# 18,171). For ‘analyses 1’, 353 sequences were analyzed (Online Resource 1). The concatenated matrix consisted of 1292 aligned nucleotide position characters (Table 2). Since phylogenies derived from the ML and B/MCMC analyses were generally concordant with each other, the ML phylogram of the concatenated analysis is shown in the Online Resource 3. A cartoon tree, summarizing the tree is shown in Fig. 1. The genus Lecanora as currently circumscribed in highly polyphyletic with species of the genera Adelolecia, Bryonora, Carbonea, Frutidella, Lecidella, Miriquidica, Protoparmeliopsis, Pyrrhospora, and Rhizoplaca nested within. Well supported monophyletic groups include the L. dispersa group (incl. Arctopeltis), Protoparmeliopsis (=L. muralis group), the L. varia group, Carbonea, the L. symmicta group, Palicella (=L. filamentosa group), the L. rupicola group, and the L. intumescens group. The genus Miriquidica was monophyletic but not well supported. Surprisingly, L. straminea was nested among the members of the L. dispersa group as was L. flotoviana. The identity of the latter taxon is, however, ambiguous since the sequence
Fungal Diversity Table 3
Characteristics of loci used in the 6-locus data set
Alignments
ITS
nucLSU
mtSSU
MCM7
RPB1
RPB2
Total
Number of sequences
76
65
61
48
62
52
364
Newly generated sequences
35
30
33
25
31
39
193
488 6/7 %
536 17/21 %
548 21/26 %
539 34/41 %
697 20/24 %
704 30/37 %
3512 128/26 %
GTR + I + G
SYM + G
GTR + I + G
HKY + I + G
GTR + I + G
SYM + I + G
Number of sites (including gaps) Missing sequences/the percentages Nucleotide substitution models
from GenBank most likely represent L. flotoviana auct. (=L. semipallida) not L. flotoviana Spreng. A few species did not cluster within one of the groups and their phylogenetic relationships remain unclear. This includes the placodioid species L. dispersoareolata, L. nashii, L. bipruinosa, L. formosa, and L. pringlei, and the crustose L. subintricata, L. saxigena, L. cinereofusca, and L. physciella. The latter has a wellsupported relationship to Miriquidica. A sequence of Sedelnikovaea baicalensis clusters with Lecanora achariana in the L. muralis group (Online Resource 3). The taxa and sequences used in ‘analysis 2’ are listed in Online Resource 2. The concatenated matrix consisted of 364 sequences and 3512 aligned nucleotide position characters (Table 3). Since phylogenies derived from the ML and B/ MCMC analyses were generally concordant with each other, the ML phylogram of the concatenated analysis is shown in Fig. 2. Lecanoraceae sensu stricto is strongly supported as monophyletic (99 % BS/1.0 pp), whereas Lecanoraceae sensu lato, including the genus Miriquidica, is only moderately supported (76 % BS/0.95 pp). Lecanora, as currently circumscribed, is highly polyphyletic and its monophyly significantly rejected in both alternative hypothesis tests (all tests P < 0.001). Lecidella species form a monophyletic group with a strong support (100 % BS/1.0 pp). Nine major clades can be seen within Lecanoraceae sensu lato: Miriquidica, the Lecanora symmicta group, Rhizoplaca, Myriolecis (=the Lecanora dispersa group), Protoparmeliopsis (=Lecanora muralis group), the Lecanora subfusca group, the L. subcarnea group, Lecanora formosa (these three groups representing Lecanora s.str.), and Lecidella. Ramboldia forms a sister group to Lecanoraceae and its monophyly is strongly supported. A core group of Lecanora sensu stricto – incl. The Lecanora subfusca group, the L. subcarnea group, and Lecanora formosa – forms a well-supported monophyletic group including ten species in our analysis. When more than one sample of a species was included, they usually clustered together in a monophyletic group, with the notable exception of L. caesiorubella. The genus Lecidella forms a sister-group relationship with Lecanora sensu stricto. Within Lecidella two main clades can be seen, one including samples of Lecidella patavina and L. stigmatea. Both species are characterized by hyaline
hypothecia and lack chlorinated xanthones. The other clade in Lecidella, consisting of L. carpathica, L. effugiens, L. elaeochroma, L. aff. elaeochroma, L. elaechromoides, L. euphorea, and L. tumidula, includes species with a pigmented hypothecium and often contain chlorinated xanthones. L. enteroleucella is somewhat intermediate in having a hyaline hypothecicum but containing chlorinated xanthones. The Lecanora sensu stricto + Lecidella clade has a strongly supported sister-group relationship to a clade consisting of four groups: the Lecanora polytropa group, Rhizoplaca sensu stricto, the L. dispersa group, and Protoparmeliopsis (=L. muralis group). The L. polytropa group is only represented by two species, whereas the placement of L. sulphurea is not supported. Rhizoplaca sensu stricto forms a well-supported monophyletic group including the R. chrysoleuca- and R. melanophthalma-groups, and L. novomexicana. This clade is sister to a clade including the L. dispersa group and Protoparmeliopsis (=L. muralis group). Both groups are strongly supported as is their sister-group relationship. Rhizoplaca peltata falls into the Protoparmeliopsis clade in addition to species currently placed in Protoparmeliopsis and Lecanora garovaglii.
Discussion This study confirms previous studies and results from phylogenetic studies on distantly related groups of lichenized fungi that morphologically characters are unstable over evolutionary time and hence have only a limited value to circumscribe taxa at generic level (Arup and Grube 1998, 2000; Blanco et al. 2004; Crespo et al. 2007; Ekman 2001; Högnabba 2006; Lumbsch et al. 2014; Lumbsch et al. 2010; Nelsen et al. 2014; Parnmen et al. 2012; Stenroos and DePriest 1998; Tehler and Irestedt 2007; Wedin and Döring 1999). The genera Arctopeltis and Rhizoplaca that were primarily distinguished based on their umbilicate thallus were confirmed not to represent distinct, monophyletic lineages. Arctopeltis was found to be nested within the L. dispersa group and Rhizoplaca was found to be highly polyphyletic. The fact that umbilicate growth evolved several time independently within the family raises the question of the adaptive
Fungal Diversity
Fig. 1 Cartoon tree showing major clades of lecanoroid lichens. The cartoon tree is drawn using FigTree using ML tree of the 2-locus data set. Nodes with ML-bootstrap support 70 % or higher were indicated on the branch
value of this growth form that should be addressed in a separate study. Our study clearly demonstrates that Lecanora as currently circumscribed is highly polyphyletic. However, a core group of Lecanora sensu stricto, which corresponds to Lecanora sensu stricto as previously circumscribed (Lumbsch 1999; Lumbsch et al. 1995; Lumbsch et al.
1996; Lumbsch et al. 1997; Papong et al. 2013) based on morphological and chemical characters, is strongly supported as a monophyletic clade. It includes species containing atranorin, and forming lecanorine, to aspicilioid, rarely also almost biatorine or lecideoid apothecia, oxalate crystals in the amphithecial cortex (reduced in aspicilioid taxa), and filiform conidia. In
Fungal Diversity
Fig. 2 Maximum Likelihood phylogeny of Lecanoraceae inferred from the concatenated 6-locus data data set, showing 10 major groups of Lecanoraceae sensu lato. Branches in bold received maximum
likelihood bootstrap support values equal or above 70 % and posterior probabilities equal or above 0.95
its restricted sense, the genus includes a core group of the L. subfusca group [including species with hyaline and pigmented hypothecium (Papong et al. 2013)], the almost biatorine L. flavopallida (Guderley et al. 1998). species with pruinose apothecia belonging to the saxicolous L. rupicola and L. subcarnea groups (Dickhäuser et al. 1995; Leuckert and Poelt 1989) and corticolous
pruinose species (Imshaug and Brodo 1966; Lumbsch et al. 1997). as well as the lecideoid Lecanora formosa (Knoph and Leuckert 2000). Our 2-locus analyses revealed the presence of a number of clades that will require taxonomic recognition at the generic level but we do not feel confident to propose changes for a number of those clades here since we were either unable to
Fungal Diversity
include them in our 6-locus analysis or only very few samples and taxa for those clades were included. This includes the L. varia-, L. saligna-, L. polytropa-, and the L. symmicta-groups. This and previous studies, as well as phenotypical characters, (Arup and Grube 1998; Pérez-Ortega et al. 2010; Printzen 2001; Printzen and May 2002) support that these groups do not belong to Lecanora s.str. However, additional data are necessary to fully understand the circumscription of these clades before they can be accepted as separate genera. The unresolved relationships among species of those groups and among the groups themselves strongly suggest to base future taxonomic changes on multilocus genetic data and multiple individuals per species. Some species did not cluster with any of the groups, among those L. dispersoareolata¸ which previously clustered with the L. polytropa group, and L. bipruinosa, which clustered with species here placed in Rhizoplaca (Arup and Grube 1998; Pérez-Ortega et al. 2010). The phylogenetic position of Lecanora nashii was also unresolved in a previous study (Grube and Blaha 2003). whereas L. subintricata had a sister-group relationship with the L. polytropa group (Pérez-Ortega et al. 2010). The closely related L. cinereofusca and L. saxigena also did not have a strongly supported relationship in a previous analysis (Lendemer and Harris 2014). but morphologically the species fit very well into Lecanora s. str. (Brodo 1984). Based on phenotypical characters L. pringlei was already shown to be unrelated to other placodioid taxa of Lecanora sensu lato (Ryan and Nash 1997a). Our current analyses failed to identify a closely related group. In all these cases additional sequences will be necessary to identify the phylogenetic relationships of these species. The fact that a sequence of Sedelnikovaea baicalensis clusters with Lecanora achariana in the L muralis group (Online Resource 3) in our analysis underlines that the taxonomic status of that genus requires additional studies. When originally described (Kondratyuk et al. 2014) Sedelnikovaea was hypothesized to be distantly related to Lecanoraceae. Monophyly of Lecidella and its placement i n Lecanoraceae is strongly supported in our analyses in agreement with a previous study (Miadlikowska et al. 2014) and three clades currently placed in Lecanora were supported as monophyletic and well-supported at least in the 6-locus analysis. These are the L. dispersa group (incl. Arctopeltis and L. straminea), the L. muralis group (=Protoparmeliopsis), and the genus Rhizoplaca s. str. Since these groups have repeatedly been found in molecular analyses (Arup and Grube 1998, 2000; Pérez-Ortega et al. 2010; Śliwa et al. 2012) and are supported by phenotypical characters (Arup and Grube 2000; Eigler 1969; Śliwa 2007) we propose here to accept them at generic level and propose the necessary new combinations below.
Taxonomy Myriolecis Clements. The Genera of Fungi: 79 (1909). Type species: Myriolecis sambuci (Pers.) Clem. ≡ Lichen sambuci Pers., Ann. Bot. 1: 26 (1794) ≡ Lecanora sambuci (Pers.) Nyl., Lich. Scand.: 168 (1861). =Arctopeltis Poelt, Int. J. Mycol. Lichenol. 1: 147 (1983). Type species: Arctopeltis thuleana Poelt. The genus Myriolecis includes species most common on calciferous rocks and bark. While most species have a crustose and often inconspicuous thallus, a few taxa form placodioid to umbilicate thalli. The species either contain chlorinated xanthones, often accompanied by depsidones or lack secondary metabolites. The genus has a worldwide distribution but is most diverse in temperate to Arctic-alpine regions of the northern Hemisphere. Most species are currently placed in the L. dispersa group, which has been shown to form a clade separate from Lecanora sensu stricto and being congeneric with Arctopeltis thuleana. The oldest available generic name is Myriolecis and we here propose to resurrect the name to accommodate the species of this clade. The following new combinations are necessary: Myriolecis agardhiana (Ach.) Śliwa, Zhao Xin & Lumbsch, comb. nov. (MB 814228) Basionym: Lecanora agardhiana Ach., Syn. Lich.: 152 (1814). Myriolecis albescens (Hoffm.) Śliwa, Zhao Xin & Lumbsch, comb. nov. (MB 814262) Basionym: Psora albescens Hoffm., Deutschl. Fl. 2: 165 (1796) ≡ Lecanora albescens (Hoffm.) Flörke in Flotow, Flora 11: 633 (1828). Myriolecis andrewii (de Lesd.) Śliwa, Zhao Xin & Lumbsch, comb. nov. (MB 814263) Basionym: Lecanora andrewii de Lesd. in M’Andrew, Trans. Proc. Bot. Soc. Edinburgh 26 (2): 184 (1913). Myriolecis antiqua (J.R. Laundon) Śliwa, Zhao Xin & Lumbsch, comb. nov. (MB 814264) Basionym: Lecanora antiqua J.R. Laundon, Lichenologist 42(6): 631 (2010). Myriolecis carlottiana (Lewis & Śliwa) Śliwa, Zhao Xin & Lumbsch, comb. nov. (MB 814265) Basionym: Lecanora carlottiana Lewis & Śliwa, Bryologist 115(3): 376 (2012). Myriolecis contractula (Nyl.) Śliwa, Zhao Xin & Lumbsch, comb. nov. (MB 814266) Basionym: Lecanora contractula Nyl., Not. Sällsk. Fauna Fl. Fenn. Förh. NS 8: 126 (1866). Myriolecis crenulata (Hook.) Śliwa, Zhao Xin & Lumbsch, comb. nov. (MB 814267) Basionym: Lecanora crenulata Hook. in Smith, Engl. Fl. 5: 190 (1833). Myriolecis dispersa (Pers.) Śliwa, Zhao Xin & Lumbsch, comb. nov. (MB 814268) Basionym: Lichen dispersus Pers., Neue Ann. Bot. 1: 27 (1794) ≡ Lecanora dispersa (Pers.) Sommerf., Suppl. Fl. Lapp.: 96 (1826). Myriolecis expectans (Darb.) Śliwa, Zhao Xin & Lumbsch, comb. nov. (MB 814269) Basionym: Lecanora
Fungal Diversity
expectans Darb., Nation. Antarct. Exped. 1901–1904 5: 5 (1910). Myriolecis flowersiana (H. Magn.) Śliwa, Zhao Xin & Lumbsch, comb. nov. (MB 814270) Basionym: Lecanora flowersiana H. Magn., Acta Hort. Gotob. 19(2): 38 (1952). Myriolecis fugiens (Nyl.) Śliwa, Zhao Xin & Lumbsch, comb. nov. (MB 814271) Basionym: Lecanora fugiens Nyl., Flora 56: 289 (1873). Myriolecis hagenii (Ach.) Śliwa, Zhao Xin & Lumbsch, comb. nov. (MB 814277) Basionym: Lichen hagenii Ach., Lichenogr. Suec. Prodr.: 57 (1799) ≡ Lecanora hagenii (Ach.) Ach., Lich. Univ.: 367 (1810). Myriolecis invadens (H. Magn.) Śliwa, Zhao Xin & Lumbsch, comb. nov. (MB 814278) Basionym: Lecanora invadens H. Magn., Lich. Central Asia: 87 (1940). Myriolecis juniperina (Śliwa) Śliwa, Zhao Xin & Lumbsch, comb. nov. (MB 814279) Basionym: Lecanora juniperina Śliwa in Nash et al., Lich. Fl. Sonoran 2: 231 (2004). Myriolecis mons-nivis (Darb.) Śliwa, Zhao Xin & Lumbsch, comb. nov. (MB 814280) Basionym: Lecanora mons-nivis Darb., Wissenschaftliche Ergebnisse der Schwedischen Südpolar-Expedition 1901–1903 4(2): 9 (1912). Myriolecis percrenata (H. Magn.) Śliwa, Zhao Xin & Lumbsch, comb. nov. (MB 814281) Basionym: Lecanora percrenata H. Magn., Lich. Central Asia: 88 (1940). Myriolecis perpruinosa (Fröberg) Śliwa, Zhao Xin & Lumbsch, comb. nov. (MB 814282) Basionym: Lecanora perpruinosa Fröberg, Calcicolous Lich. Őland: 50 (1989). Myriolecis persimilis (Th. Fr.) Śliwa, Zhao Xin & Lumbsch, comb. nov. (MB 814283) Basionym: Lecanora hagenii [ssp.] persimilis Th. Fr., Lichenogr. Scand. 1: 251 (1871) ≡ Lecanora persimilis (Th. Fr.) Nyl., Flora 59: 577 (1876). Myriolecis poeltiana (Clauzade & Cl. Roux) Śliwa, Zhao Xin & Lumbsch, comb. nov. (MB 814284) Basionym: Lecanora poeltiana Clauzade & Cl. Roux, Beihefte zur Nova Hedwigia 79: 188 (1984). Myriolecis pruinosa (Chaub.) Śliwa, Zhao Xin & Lumbsch, comb. nov. (MB 814285) Basionym: Lecanora pruinosa Chaub. in Saint-Amans, Fl. agen.: 495 (1821). Myriolecis reuteri (Schaer.) Śliwa, Zhao Xin & Lumbsch, comb. nov. (MB 814286) Basionym: Lecanora reuteri Schaer., Enum. Crit. Lich. Europ.: 59 (1850) ≡ Patellaria reuteri (Schaer.) Trevis., Rev. Period. Lav. Imp. Reale Acad., Padova 1(3): 256 (1852). Myriolecis salina (H. Magn.) Śliwa, Zhao Xin & Lumbsch, comb. nov. (MB 814287) Basionym: Lecanora salina H. Magn., Bot. Not.: 229 (1926). Myriolecis schofieldii (Brodo) Śliwa, Zhao Xin & Lumbsch, comb. nov. (MB 814288) Basionym: Lecanora schofieldii Brodo, Botany 88(4): 352 (2009).
Myriolecis semipallida (H. Magn.) Śliwa, Zhao Xin & Lumbsch, comb. nov. (MB 814289) Basionym: Lecanora semipallida H. Magn., Lich. Central Asia: 89 (1940). Myriolecis straminea (Ach.) Śliwa, Zhao Xin & Lumbsch, comb. nov. (MB 814290) Basionym: Lecanora straminea Ach., Lich. Univ.: 430 (1810). Myriolecis sverdrupiana (Øvstedal) Śliwa, Zhao Xin & Lumbsch, comb. nov. (MB 814291) Basionym: Lecanora sverdrupiana Øvstedal, Nova Hedwigia 37: 685 (1983). Myriolecis thuleana (Poelt) Śliwa, Zhao Xin & Lumbsch, comb. nov. (MB 814292) Basionym: Arctopeltis thuleana Poelt, Int. J. Mycol. Lichenol. 1: 147 (1983) ≡ Lecanora thuleana (Poelt) Śliwa, Bryologist 112: 271 (2012). Myriolecis torrida (Vain.) Śliwa, Zhao Xin & Lumbsch, comb. nov. (MB 814293) Basionym: Lecanora torrida Vain., Ark. Bot. 8(4): 45 (1909). Myriolecis wetmorei (Śliwa) Śliwa, Zhao Xin & Lumbsch, comb. nov. (MB 814294) Basionym: Lecanora wetmorei Śliwa in Nash et al., Lich. Fl. Sonoran 2: 283 (2004). Myriolecis zosterae (Ach.) Śliwa, Zhao Xin & Lumbsch, comb. nov. (MB 814295) Basionym: Lecanora subfusca [ v a r. ] z o s t e r a e A c h . , S y n . M e t h . L i c h . : 1 5 8 (1814) ≡ Lecanora zosterae (Ach.) Nyl., Flora 59: 577 (1876). Protoparmeliopsis Choisy. Bull. Soc. bot. Fr. 76: 524 (1929). Type species: Protoparmeliopsis muralis (Rabenh.) Choisy. Species currently placed in the L. muralis group and Rhizoplaca peltata have been shown to form a clade separate from Lecanora sensu stricto. The clade includes placodioid or umbilicate species growing on siliceous rocks or on soil. The species have their center of distribution in semi-arid regions of the northern Hemisphere, especially at higher altitudes. The generic name Protoparmeliopsis is available for this clade and numerous species have previously been combined into that genus (Hafellner et al. 2005; Hafellner and Türk 2001; Kondratyuk et al. 2013; Santesson et al. 2004). The following new combinations are necessary: Protoparmeliopsis garovaglii (Körb.) Arup, Zhao Xin & Lumbsch, comb. nov. (MB 814296) Basionym: Placodium garovaglii Körb., Parerga lichenol. (Breslau) 1: 54 (1859). Note: Sometimes the epitheton is given as Bgarovaglioi^ (e. g., Index Fungorum). However, the Latinized version of the Italian name Garovaglio is Garovaglius and hence its genitive form should be Garovaglii. Protoparmeliopsis peltata (Ramond) Arup, Zhao Xin & Lumbsch, comb. nov. (MB 814297) Basionym: Lichen peltatus Ramond in Lamark & de Candolle, Fl. franç., Edn 3 (Paris) 2: 377 (1805). Rhizoplaca Zopf. Justus Liebigs Ann. Chem. 340: 291 (1905). Type species: Rhizoplaca opaca (Ach.) Zopf (≡Rhizoplaca chrysoleuca [Sm.] Zopf).
Fungal Diversity
In its current circumscription Rhizoplaca was found to be polyphyletic (Arup and Grube 2000). but a core group of species form a monophyletic group together with the placodioid Lecanora novomexicana, L. opiniconensis, and L. phaedrophthalma, which are transferred to Rhizoplaca below. The morphologically close species L. nigromarginata H. Magn. and L. weberi Ryan are also transferred to Rhizoplaca although no molecular data are currently available. The species in the genus have a placodioid to umbilicate thalli and contain usnic acid, dibenzofurans, despides, depsidones, and terpenoids. The genus occurs in the northern Hemisphere, South America, and Antarctica, but is absent from Australasia. It has its center of distribution in western North America. Rhizoplaca nigromarginata (H. Magn.) Leavitt, Zhao Xin & Lumbsch, comb. nov. (MB 814298) Basionym: Lecanora nigromarginata H. Magn., Ann. Crypt. Exot. 5: 23 (1932). Rhizoplaca novomexicana (H. Magn.) Leavitt, Zhao Xin & Lumbsch, comb. nov. (MB 814299) Basionym: Lecanora novomexicana H. Magn., Ann. Crypt. Exot. 5: 26 (1932). Rhizoplaca opiniconensis (Brodo) Leavitt, Zhao Xin & Lumbsch, comb. nov. (MB 814300) Basionym: Lecanora opiniconensis Brodo, Mycotaxon 26: 309 (1986). Rhizoplaca phaedrophthalma (Poelt) Leavitt, Zhao Xin & Lumbsch, comb. nov. (MB 814301) Basionym: Lecanora phaedrophthalma Poelt, Mitt. Bot. Staatss. München 2: 483 (1958). Rhizoplaca weberi (Ryan) Leavitt, Zhao Xin & Lumbsch, comb. nov. (MB 814302) Basionym: Lecanora weberi Ryan, Mycotaxon 36: 10 (1932).
Acknowledgments The project was financially supported by the National Natural Science Foundation of China (31170187, 31570017). We thank the China Scholarship Council for supporting the first author’s study at the Field Museum (Chicago). Jolanta Miadlikowska is thanked for kind assistance with transferring isolates (Duke University). SPO was supported by grant CTM2012-38222-C02-02 from the Spanish Ministry of Economy and Competitiveness.
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