Miadlikowska 2014

Accepted Manuscript
A multigene phylogenetic synthesis for the class Lecanoromycetes (Ascomy-

cota): 1307 fungi representing 1139 infrageneric taxa, 317 genera and 66
families
Jolanta Miadlikowska, Frank Kauff, Filip Högnabba, Jeffrey C. Oliver, Katalin

Molnár, Emily Fraker, Ester Gaya, Josef Hafellner, Valérie Hofstetter, Cécile
Gueidan, Martin Kukwa, Robert Lücking, Curtis Björk, Harrie J.M. Sipman,
Ana Rosa Burgaz, Arne Thell, Alfredo Passo, Leena Myllys, Trevor Goward,
Samantha Fernández-Brime, Geir Hestmark, James Lendemer, H. Thorsten
Lumbsch, Michaela Schmull, Conrad Schoch, Emmanuël Sérusiaux, David R.
Maddison, A. Elizabeth Arnold, François Lutzoni, Soili Stenroos
PII: S1055-7903(14)00129-8
DOI: http://dx.doi.org/10.1016/j.ympev.2014.04.003
Reference: YMPEV 4866
To appear in: Molecular Phylogenetics and Evolution
Received Date: 31 May 2013

Revised Date: 2 March 2014
Accepted Date: 2 April 2014
Please cite this article as: Miadlikowska, J., Kauff, F., Högnabba, F., Oliver, J.C., Molnár, K., Fraker, E., Gaya, E.,
Hafellner, J., Hofstetter, V., Gueidan, C., Kukwa, M., Lücking, R., Björk, C., Sipman, H.J.M., Burgaz, A.R., Thell,
A., Passo, A., Myllys, L., Goward, T., Fernández-Brime, S., Hestmark, G., Lendemer, J., Thorsten Lumbsch, H.,
Schmull, M., Schoch, C., Sérusiaux, E., Maddison, D.R., Elizabeth Arnold, A., Lutzoni, F., Stenroos, S., A multigene
phylogenetic synthesis for the class Lecanoromycetes (Ascomycota): 1307 fungi representing 1139 infrageneric
taxa, 317 genera and 66 families, Molecular Phylogenetics and Evolution (2014), doi: http://dx.doi.org/10.1016/
j.ympev.2014.04.003
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1 A multigene phylogenetic synthesis for the class Lecanoromycetes (Ascomycota): 1307
2 fungi representing 1139 infrageneric taxa, 317 genera and 66 families
3
4 Jolanta Miadlikowska1, Frank Kauff2, Filip Högnabba3, Jeffrey C. Oliver4, Katalin Molnár1,5,
5 Emily Fraker1,6, Ester Gaya1, Josef Hafellner7, Valérie Hofstetter1,8, Cécile Gueidan1,9, Martin
6 Kukwa10, Robert Lücking11, Curtis Björk12, Harrie J.M. Sipman13, Ana Rosa Burgaz14, Arne
7 Thell15, Alfredo Passo16, Leena Myllys3, Trevor Goward17, Samantha Fernández-Brime18, Geir
8 Hestmark19, James Lendemer20, H. Thorsten Lumbsch11, Michaela Schmull21, Conrad Schoch22,
9 Emmanuël Sérusiaux23, David R. Maddison24, A. Elizabeth Arnold25, François Lutzoni1* and
10 Soili Stenroos3*
11
1
12 Department of Biology, Duke University, Durham, NC 27708-0338, USA
2
13 FB Biologie, Molecular Phylogenetics, 13/276, TU Kaiserslautern, Postfach 3049, 67653
14 Kaiserslautern, Germany
3
15 Botanical Museum, Finnish Museum of Natural History, FI-00014 University of Helsinki,
16 Finland
4
17 Department of Ecology and Evolutionary Biology, Yale University, 358 ESC 21 Sachem Street,
18 New Haven, CT 06511, USA
7
19 Institut für Botanik, Karl-Franzens-Universität, Holteigasse 6, A-8010, Graz, Austria
10
20 Department of Plant Taxonomy and Nature Conservation, University of Gdańsk, ul. Wita
21 Stwosza 59, 80-308 Gdańsk, Poland
11
22 Science & Education, The Field Museum, 1400 S. Lake Shore Drive, Chicago, IL 60605, USA
12
23 Stillinger Herbarium, University of Idaho, Moscow, ID 83843, USA
13
24 Botanischer Garten und Botanisches Museum Berlin-Dahlem, Königin-Luise-Strasse 6-8, D-
25 14195 Berlin, Germany
14
26 Departamento de Biologıá Vegetal I, Facultad de CC. Biológicas, Universidad Complutense de
27 Madrid, E-28040-Madrid, Spain
15
28 The Biological Museums, Lund University, Box 117, SE-221 00 Lund, Sweden
16
29 BioLiq Laboratorio de Bioindicadores y Liquenología, Centro Regional Universitario
30 Bariloche, INIBIOMA, Universidad Nacional del Comahue, Bariloche, 8400RN, Argentina
17
31 Enlichened Consulting Ltd, Edgewood Blue, Box 131, Clearwater, BC V0E 1N0, Canada
1
18
1 Department of Plant Biology (Botany Unit), Facultat de Biologia, Universitat de Barcelona,
2 Av. Diagonal 643, 08028 Barcelona, Spain
19
3 Department of Biosciences, University of Oslo, PB 1066 Blindern, 0315-Oslo, Norway
20
4 Institute of Systematic Botany, The New York Botanical Garden, Bronx, NY 10458-5126,
5 USA
21
6 Harvard University Herbaria, Organismic and Evolutionary Biology, Harvard University, 22
7 Divinity Avenue, Cambridge, MA 02138, USA
22
8 National Center for Biotechnology Information, National Library of Medicine, National
9 Institutes of Health, 45 Center Drive, MSC 6510, Bethesda, MD 20892-6510, USA
23
10 Plant Taxonomy and Conservation Biology Unit, University of Liège, Sart Tilman B22, B-
11 4000 Liège, Belgium
24
12 Center for Genome Research and Biocomputing, 3021 Agriculture and Life Sciences Building,
13 Oregon State University, Corvallis, OR 97331-7303, USA
25
14 Department of Plant Sciences, The University of Arizona, 1140 E. South Campus Drive,
15 Forbes 303, Tucson, AZ 85721, USA
16
5
17 Current address: Institute of Ecology and Botany, Hungarian Academy of Sciences, 2163
18 Vácrátót, Alkotmány u. 2-4, Hungary
6
19 Current address: 1528 Bluegrass Dr., Fairbanks, AK 99709, USA
8
20 Current address: Research Station Agroscope Changins-Wädenswil ACW, 1260 Nyon,
21 Switzerland
9
22 Current address: Department of Life Sciences, Natural History Museum, Cromwell Road, SW7
23 5BD, London, England
24
25 *The order of the two senior authors is reversible and their names are listed arbitrarily in
26 alphabetical order.
27
28 Corresponding author: Jolanta Miadlikowska, Duke University, Department of Biology, Box
29 90338, 130 Science Drive, 137 Bio Sciences, Durham, NC 27708-0338, USA; E-mail:
30 jolantam@duke.edu; Phone: 919 491 7128 or 919 660 7287.
31
2
1 Abstract
2 The Lecanoromycetes is the largest class of lichenized Fungi, and one of the most
3 species-rich classes in the kingdom. Here we provide a multigene phylogenetic synthesis (using
4 three ribosomal RNA-coding and two protein-coding genes) of the Lecanoromycetes based on
5 635 newly generated and 3307 publicly available sequences representing 1139 taxa, 317 genera,
6 66 families, 17 orders and five subclasses (four currently recognized: Acarosporomycetidae,
7 Lecanoromycetidae, Ostropomycetidae, Umbilicariomycetidae; and one provisionarily
8 recognized, ‘Candelariomycetidae’). Maximum likelihood phylogenetic analyses on four
9 multigene datasets assembled using a cumulative supermatrix approach with a progressively
10 higher number of species and missing data (5-gene, 5+4-gene, 5+4+3-gene and 5+4+3+2-gene
11 datasets) show that the current classification includes non-monophyletic taxa at various ranks,
12 which need to be recircumscribed and require revisionary treatments based on denser taxon
13 sampling and more loci. Two newly circumscribed orders (Arctomiales and Hymeneliales in the
14 Ostropomycetidae) and three families (Ramboldiaceae and Psilolechiaceae in the Lecanorales,
15 and Strangosporaceae in the Lecanoromycetes inc. sed.) are introduced. The potential
16 resurrection of the families Eigleraceae and Lopadiaceae is considered here to alleviate
17 phylogenetic and classification disparities. An overview of the photobionts associated with the
18 main fungal lineages in the Lecanoromycetes based on available published records is provided.
19 A revised schematic classification at the family level in the phylogenetic context of widely
20 accepted and newly revealed relationships across Lecanoromycetes is included. The cumulative
21 addition of taxa with an increasing amount of missing data (i.e., a cumulative supermatrix
22 approach, starting with taxa for which sequences were available for all five targeted genes and
23 ending with the addition of taxa for which only two genes have been sequenced) revealed
24 relatively stable relationships for many families and orders. However, the increasing number of
25 taxa without the addition of more loci also resulted in an expected substantial loss of
26 phylogenetic resolving power and support (especially for deep phylogenetic relationships),
27 potentially including the misplacements of several taxa. Future phylogenetic analyses should
28 include additional single copy protein-coding markers in order to improve the tree of the
29 Lecanoromycetes. As part of this study, a new module (“Hypha”) of the freely available
30 Mesquite software was developed to compare and display the internodal support values derived
31 from this cumulative supermatrix approach.
3
1
2 Keywords: Classification; Cumulative supermatrix, Lecanoromycetes; Lichenized fungi;
3 Maximum likelihood; Multi-gene phylogeny; Systematics
4
5 1. Introduction
6 The Lecanoromycetes (formally introduced by Eriksson and Winka, 1997) is the third
7 largest known class of fungi (after Agaricomycetes and Dothideomycetes), with more than
8 14,000 recognized species, of which the majority (over 95%) are lichenized (Kirk et al., 2008).
9 Besides lichenicolous species, only a few truly non-lichenized lineages evolved in the
10 Lecanoromycetes, namely Odontotremataceae and Stictidaceae in the Ostropomycetidae (Baloch
11 et al., 2010, 2012; Lutzoni et al., 2001, 2004; Schoch et al., 2009; Wedin et al., 2006). Members
12 of the Lecanoromycetes typically form bi-membered symbiotic associations with coccoid and
13 filamentous green algae from the Trabouxiophyceae and Ulvophyceae, respectively (e.g.,
14 Asterochloris, Coccomyxa, Dictyochloropsis s.l., Phycopeltis, Trebouxia, and Trentepohlia) or
15 cyanobacteria from the orders Chroococcales, Nostocales, and Stigonematales (e.g., Nostoc,
16 Rhizonema, Scytonema, and Stigonema). A small fraction of species form tri-membered
17 symbioses involving two photobionts, usually a green alga and cyanobacterium, in which the
18 cyanobacterium (secondary photobiont) is located in internal or external compartments of the
19 thallus called cephalodia (overview and relevant literature is included in Gueidan et al., 2014 and
20 Voytsekhovich et al., 2011b).
21 The Lecanoromycetes are predominantly characterized by ascohymenial ascoma
22 ontogeny resulting in apothecial fruiting bodies of various structures and shapes, amyloid asci
23 with an apical thickening and two-layered wall, and a hamathecium formed by unbranched to
24 branched and anastomosing interascal hyphae (Gueidan et al., 2014). Most members of the class
25 produce a wide variety of secondary substances such as depsidones, terpenoids and xanthones
26 (e.g., Culberson and Culberson, 1994; Elix and Stocker-Wörgötter, 2008; Huneck and
27 Yoshimura, 1996), which are of great biological and systematic significance. Members of the
28 Lecanoromycetes are distributed worldwide, primarily in terrestrial habitats (rarely aquatic or
29 semiaquatic), where they grow on various substrates (tree bark, wood, leaves, rocks, soil,
30 mosses, and other lichens). An overview on the biology of the Lecanoromycetes is provided in
31 Gueidan et al. (2014) and the literature therein.
4
1 Molecular phylogenetic studies have substantially challenged the traditional classification
2 of Ascomycota that were based on, e.g., ascoma morphology and development, ascus type and
3 dehiscence, and ascospores, by revealing the non-monophyly of taxa at various taxonomic levels
4 and providing an alternative framework for taxonomic revisions and recircumscriptions (Gueidan
5 et al., 2014). Currently five subclasses and 17 orders are recognized in the Lecanoromycetes.
6 Acarosporomycetidae, Lecanoromycetidae, Ostropomycetidae, and Umbilicariomycetidae have
7 been formally described (Bendiksby and Timdal, 2013; Reeb et al. 2004; Hibbett et al. 2007),
8 whereas ‘Candelariomycetidae’ is currently a provisionary name awaiting confirmation from a
9 large-scale study with exhaustive taxon and locus sampling (Bendiksby and Timdal, 2013; Gaya
10 et al., 2012; Gueidan et al., 2014; Hodkinson and Lendemer, 2011; Lumbsch and Huhndorf,
11 2010; Miadlikowska et al., 2006; Printzen, 2010; Schmull et al., 2011).
12 Until now the majority of phylogenetic studies on the Lecanoromycetes relied on various
13 combinations of two or three ribosomal genes, i.e., nucLSU, nucSSU and mitSSU. Recently,
14 protein-coding markers (usually RPB1 and RPB2, but also beta-tubulin, GAPDH, and MCM7)
15 have been more frequently used for phylogenetic inference within Lecanoromycetes (e.g.,
16 Baloch et al., 2010; Crespo et al., 2010; Hofstetter et al., 2007; Lumbsch et al., 2007b;
17 Miadlikowska et al., 2006; Myllys et al., 2005; Otálora and Wedin, 2013; Otálora et al. 2013a;
18 Parnmen et al., 2012; Reeb et al., 2004; Rivas Plata et al., 2012a, 2012b; Schmitt et al., 2010).
19 Only five studies (Hofstetter et al., 2007; Lumbsch et al., 2004; Miadlikowska et al., 2006; Reeb
20 et al., 2004; Wedin et al., 2005) were designed specifically to reconstruct phylogenetic
21 relationships within the Lecanoromycetes at the family and higher ranks as a framework for the
22 evaluation of existing classifications. Two additional studies utilized a broad taxon sampling
23 across the Lecanoromycetes to address the phylogenetic affiliation of the genera, Lecidea s.l.
24 (Schmull et al., 2011) and Hypocenomyce s.l. (Bendiksby and Timdal, 2013). Lumbsch et al.
25 (2004) presented a Bayesian phylogeny based on a combined nucLSU and mitSSU dataset for 86
26 individuals representing 86 species and 59 genera from the Lecanoromycetes, and provided an
27 overview of coexisting classifications of the Lecanoromycetes at the order level. Reeb et al.
28 (2004) conducted a phylogenetic study addressing the circumscription and placement of the
29 Acarosporaceae based in part on Bayesian and ML analyses of combined nucSSU, nucLSU and
30 RPB2 sequences obtained from 82 species. With the study by Lutzoni et al. (2004), this was the
31 first use of RPB2 sequences to resolve relationships within the Lecanoromycetes. The study by
5
1 Wedin et al. (2005) as stated by the authors, focused on putatively basal and erroneously
2 classified groups based on morphological and preliminary phylogenetic analyses, using a similar
3 number of taxa as the previous studies (83 individuals representing 80 species and 61 genera in
4 the Lecanoromycetes).
5 The first phylogenetic study on lichen-forming fungi based on four loci (nucLSU,
6 nucSSU, mitSSU, and RPB2) was by Lutzoni et al. (2004). With the addition of RPB1 to these
7 four loci, Miadlikowska et al. (2006) were the first to use a five-locus supermatrix to resolve
8 phylogenetic relationships within Lecanoromycetes, while James et al. (2006) used a six-gene
9 phylogeny to unveil the relationships of lichen-forming fungi across the entire kingdom.
10 Hofstetter et al. (2007) provided a foundation for future multilocus phylogenetic studies for
11 Fungi by evaluating the resolving power and statistical support delivered by protein-coding
12 genes (RPB1 and RPB2) compared to the three commonly used ribosomal RNA-coding genes
13 (nucLSU, nucSSU and mitSSU). Maximum likelihood analyses performed by Hofstetter et al.
14 (2007) on each locus separately and in various combinations indicated that among available
15 markers, the optimal loci (single or combined) to use for molecular systematics of the
16 Lecanoromycetes were the protein-coding genes (RPB1 and RPB2). A gene-by-gene assessment
17 of phylogenetic informativeness (sensu Townsend 2007) within a broader, Ascomycota-wide,
18 study by Schoch et al (2009) yielded higher levels of informativeness for these two protein-
19 coding genes (and TEF1) as compared with the ribosomal genes.
20 The first implementation of a cumulative supermatrix approach was by Miadlikowska et
21 al. (2006). Three five-locus datasets for the Lecanoromycetes were assembled with a
22 progressively higher number of taxa (from 111 to 274 representing 10 orders, and 43 of the 64
23 families recognized by Eriksson at the time [2006]) and an increasing amount of missing data
24 (with the inclusion of taxa with only four and three of the five targeted genes). That study
25 showed that several non-monophyletic taxa at different ranks needed to be recircumscribed, and
26 confirmed that ascus morphology cannot be applied consistently to shape the classification
27 across the Lecanoromycetes. The authors concluded that the cumulative addition of taxa with
28 increasing amount of missing data resulted in the expected decay of phylogenetic support values,
29 but also improved statistical support for many internodes (see also Crespo et al., 2010 and Gaya
30 et al., 2012). However, the addition of 43 taxa by Schmull et al. (2011) with mainly two non-
31 protein-coding loci to the Miadlikowska et al. (2006) supermatrix, caused a rearrangement of
6
1 some phylogenetic relationships and a substantial decrease in their stability even if 5.8S
2 sequences were added to the five-locus supermatrix. A more detailed overview on the progress in
3 reconstructing phylogenetic relationships within the Lecanoromycetes and their implications for
4 classification can be found in Gueidan et al. (2014), Lumbsch et al. (2004), Lutzoni et al. (2004),
5 Miadlikowska et al. (2006), Printzen (2010), Reeb et al. (2004), and Wedin et al. (2005).
6 Despite a high number of phylogenetic studies focusing on various groups in the
7 Lecanoromycetes, a comprehensive large-scale phylogeny with a dense taxon sampling,
8 especially at the family and generic levels, coupled with the use of a larger number of protein-
9 coding genes is still needed. Numerous important relationships spanning all ranks, including the
10 class Lecanoromycetes itself, are unsettled. A robust phylogeny is essential for a major and
11 stable improvement of the classification of this class.
12 The main objectives of this study were to: 1) generate the most comprehensive phylogeny
13 for the Lecanoromycetes to date by utilizing all sequence data available in GenBank (taxa
14 represented by at least two of the five targeted genes: nucLSU, nucSSU, mitSSU, RRPB1 and
15 RPB2) and by complementing the data with new sequences to increase taxon sampling and to
16 reduce the amount of missing data; 2) evaluate the existing classification (Lumbsch and
17 Huhndorf, 2010) and recently proposed changes at the family and higher ranks; 3) resolve
18 phylogenetic placement of taxa with uncertain or unknown affinities and identify taxa that
19 require further systematic revision; and 4) revisit the distribution of photobionts across
20 Lecanoromycetes and their utility in lichen systematics and evolutionary studies.
21 Using a cumulative supermatrix approach (Miadlikowska et al., 2006; Gaya et al., 2012)
22 we assembled four multigene datasets with a progressively higher number of taxa and missing
23 data (5-gene, 5+4-gene, 5+4+3-gene and 5+4+3+2-gene datasets). Internodal support estimated
24 with maximum likelihood bootstrap analyses for each dataset was displayed graphically on the
25 optimal and most comprehensive tree using a newly developed module called “Hypha” (Oliver et
26 al., 2013), implemented in Mesquite (Maddison and Maddison, 2011). Phylogenetic confidence
27 revealed by each taxon subset of the complete supermatrix was compared and discussed in the
28 context of missing data and stability of phylogenetic relationships. Phylogenetic relationships
29 from the present, and recently published, studies and their potential implications for
30 classification of the Lecanoromycetes were examined and depicted schematically at the family
7
1 and higher ranks. Available records on photobiont-mycobiont associations across the
2 Lecanoromycetes were summarized.
3
4 2. Materials and Methods
5
6 2.1 Gene and taxon sampling
7 Sequence data were generated and gathered for five genes (three ribosomal RNA-coding
8 and two protein-coding): ca. 0.6 kb of the mitochondrial small subunit (mitSSU); ca. 1.6 kb of
9 the large subunit (nucSSU); ca. 1.4 kb of the large subunit (nucLSU); ca. 1.0-1.2 kb of the largest
10 subunit of the RNA polymerase II gene (RPB1) region A-F and ca. 1.5 kb of RPB1 region F-G
11 (two amplicons, considered as a single gene in all phylogenetic analyses); ca. 0.8-1.0 kb of the
12 second largest subunit of RNA polymerase II gene (RPB2) region 5-7 and ca. 1.0 kb of RPB2
13 region 7-11 (two amplicons, considered as a single gene in all phylogenetic analyses). Using a
14 predefined list of 649 genera currently included in the class Lecanoromycetes according to
15 Myconet (Lumbsch and Huhndorf, 2010), the WASABI database (http://
16 ocid.nacse.org/research/aftol; Kauff et al., 2007) of the Assembling the Fungal Tree of Life
17 project (AFTOL; www.aftol.org) and GenBank were searched for available sequence data,
18 including unpublished sequences generated at Duke University (Lutzoni lab) as part of the
19 AFTOL project. These sequences were complemented with sequences newly generated in the
20 Finnish Museum of Natural History, University of Helsinki (S. Stenroos' team), for the purpose
21 of this study. All database searches, sequence collection, filtering, and assembly were done using
22 automated scripts written in Biopython (Cock et al., 2009).
23 From these sequences we assembled Operational Taxonomic Units (OTUs) by combining
24 the available sequence data for each locus, giving preference to longer sequences, as well as
25 sequences for different loci obtained from the same voucher specimen, and retaining in most
26 cases a maximum of two OTUs per species with at least two of the five targeted genes. Manual
27 corrections were applied in order to remove low-quality sequence data or mislabelled sequences.
28 A total of 1307 OTUs representing 1139 infrageneric taxa (including 47 unidentified
29 specimens or doubtful identifications) and 317 genera from 66 of the 75 currently recognized
30 families, as well as all seventeen currently recognized orders and all five subclasses
31 (Acarosporomycetidae, ‘Candelariomycetidae’, Lecanoromycetidae, Ostropomycetidae and
8
1 Umbilicariomycetidae) were compiled. Members of nine families were not included in this study
2 due to a lack of DNA sequences (Biatorellaceae, Calycidaceae, and Pachyascaceae from the
3 Lecanorales) or because they were represented by only one of the five targeted genes
4 (Anamylopsoraceae, Miltideaceae, Myeloconidaceae, Phaneromycetaceae, Protothelenellaceae,
5 and Thelenellaceae from the Ostropomycetidae). In the resulting supermatrix (which includes all
6 specimens with at least 2 genes sequenced), the Acarosporomycetidae are represented by seven
7 of eight genera and the ‘Candelariomycetidae’ by four of five genera currently recognized in
8 these two subclasses. We sampled 211 genera out of approximately 393 classified within the
9 Lecanoromycetidae as follow: Caliciales (19), Lecanorales (123; including two of six genera
10 incertae sedis), Lecideales (6), Leprocaulales (1), Peltigerales (42), Rhizocarpales (2), and
11 Teloschistales (16), as well as two families of uncertain placement in the Lecanoromycetidae,
12 Sporastatiaceae (one of two genera) and the monogeneric Ropalosporaceae. From the
13 Ostropomycetidae, we included 83 of approximately 180 genera from the Baeomycetales (3),
14 Ostropales (44), Pertusariales (16), Sarrameanales (1), and Trapeliales (10), as well as four
15 families of uncertain placement, Arctomiaceae (all three genera), Hymeneliaceae (four of five
16 genera) and the monogeneric Arthrorhaphidaceae and Schaereriaceae. The
17 Umbilicariomycetidae are represented by nine of 14 genera classified in this subclass. In
18 addition, three of the 28 genera of uncertain placement within Lecanoromycetes (Lumbsch and
19 Huhndorf, 2010) were included. The outgroup consisted of ten taxa representing nine genera and
20 three classes (Lichinomycetes, Geoglossomycetes and Leotiomycetes) outside Lecanoromycetes
21 (Miadlikowska et al., 2006) for a grand total of 1317 OTUs. Our taxon/gene supermatrix for this
22 project included 6565 cells, of which 3307 included sequences retrieved from GenBank, 635
23 (16% of all sequences used for this study) included new sequences generated as part of this
24 study, and 2623 were empty, i.e., missing data. Information about sequence data used in this
25 study including sequences newly deposited in GenBank is provided in Supplemental Tables S1
26 and S2.
27
28 2.2 Molecular data acquisition
29 Herbarium material preferably not older than five years was selected for the molecular
30 study. For DNA extractions, small fragments of the specimens were removed and placed in 1.5
31 ml Eppendorf tubes. For the extractions DNeasy Blood and Tissue Kit (Qiagen) were used. The
9
1 manufacturer’s protocol was followed, except for the grinding of the thallus fragments, which
2 was done in a small amount of lysis buffer instead of liquid nitrogen. Amplification of the target
3 loci used the illustra PuReTaq Ready-To-Go™ PCR Beads (GE Healthcare) protocol. Twenty-
4 five µ l PCR samples were prepared by adding 19 µl of sterile water, 1 µl of each primer at 10
5 µM concentration, and 4 µl template DNA to the tubes containing the beads. The primers used
6 and the PCR settings are summarized in Supplemental Table S3. The reactions were run on PTC-
7 100 and PTC-200 Thermocyclers (MJ Research). PCR products were purified using the GFX
8 PCR DNA and Gel Band Purification Kit (GE Healthcare) or Montage SEQ96 Sequencing
9 Reaction Cleanup Kit (Millipore) and sequenced on a MegaBACE 1000 sequencer (Amersham).
10 Sequencing used BigDye® Terminator v1.1 Cycle Sequencing Kit and the same primers as for
11 amplification with additionnal internal sequencing primers when needed (Supplemental Table
12 S3). Alternatively, the sequencing service provided by Macrogen Inc.
13 (http://www.macrogen.com) was used. Sources for laboratory protocols and primers used for
14 generating the new sequences as part of the AFToL1 project can be found in Lutzoni et al.
15 (2004) and Hofstetter et al. (2007).
16
17 2.3 Sequence alignments and topological incongruence assessment
18 All newly generated sequences were subjected to BLAST searches for a preliminary
19 verification of their identity. They were then assembled and edited using the software package
20 Sequencher™ 4.1 (Gene Codes Corporation, Ann Arbor, MI, USA). The single gene alignments
21 were initially generated using the WASABI aligning tool (Kauff et al., 2007) or MUSCLE 3.8.31
22 (Edgar, 2004) as implemented in Seaview 4 (Galtier et al., 1996; Gouy et al., 2010). Alignments
23 including up to 1500 OTUs were manually optimized with MacClade 4.08 (Maddison and
24 Maddison, 2003), whereas alignments with higher number of OTUs were optimized with
25 Mesquite 2.75 (Maddison and Maddison, 2011). The “Nucleotide with AA color” option was
26 used for guiding the RPB1 and RPB2 alignments. The nuclear ribosomal loci were aligned
27 according to secondary structure as described in Miadlikowska et al. (2006). Ambiguously
28 aligned regions (sensu Lutzoni et al., 2000) and introns were delimited manually and excluded
29 from subsequent analyses.
30 The resulting single-locus datasets (mitSSU, 1494 sequences; nucLSU, 1347 sequences;
31 nucSSU, 689 sequences; RPB1 A-F, 551 sequences; RPB1 F-G, 106 sequences; RPB2 5-7, 392
10
1 sequences; RPB2 7-11, 358 sequences) were examined for topological incongruence among loci
2 using the program compat3 (available at www.lutzonilab.net/downloads). For each individual
3 locus, 500 bootstrap replicates (Felsenstein, 1985) were generated with RAxML 7.2.8
4 (Stamatakis, 2006; Stamatakis et al., 2008) and all pairwise comparisons between loci were
5 calculated with compat3. A conflict between two loci was assumed when a clade was supported
6 as monophyletic with a bootstrap frequency ≥75% in one tree, but supported as non-
7 monophyletic in another (Mason-Gamer and Kellogg, 1996). After removing conflicting
8 sequences, the assessment of congruence was repeated, and a total of three rounds of conflict
9 evaluation were carried out to achieve an acceptable level of topological incongruence (i.e.,
10 below 75% of bootstrap support) among loci.
11 Some of the sequences that were removed after evaluation in congruence tests, belonged
12 to misidentified taxa (for which we did not have access to specimens) or were obvious
13 contaminants. A total of 52 GenBank sequences (Supplemental Table S4) and 42 unpublished
14 sequences were excluded from the alignments, even if only some of them were revealed by
15 BLAST searches as obvious contaminants (i.e., classified outside of Lecanoromycetes). Despite
16 careful examinations and congruence analyses, our final datasets contained the following errors,
17 due to the large number of infrageneric taxa (1139) and sequences included (3942), which were
18 introduced throughout various steps during the data preparation: duplicated sequences (six
19 cases), duplicated OTUs (four cases), and erroneous sequences (very likely representing lichen-
20 associated fungi [endolichenic fungi; Arnold et al., 2009]) from GenBank (seven sequences) and
21 unpublished (ten sequences) (Supplemental Table S1; Fig. 1). Erronous sequences were flagged
22 in GenBank as such and the newly generated unpublished sequences were submitted to GenBank
23 as lichen-associated, unidentified fungi.
24
25 2.4 Datasets and phylogenetic analyses
26 The conflict-free, single-locus alignments were concatenated for the subsequent
27 phylogenetic analyses using a cumulative supermatrix approach as introduced in Miadlikowska
28 et al. (2006) to allow broad and inclusive taxon sampling. A dataset of 183 taxa with all five
29 genes was initially assembled; this 5-gene dataset was expanded with taxa for which at least four
30 of the five-targeted genes were available, resulting in a 5+4-gene dataset of 388 taxa.
31 Subsequently, taxa with at least three genes were added to form a 5+4+3-gene dataset of 764
11
1 taxa and, finally, taxa with at least two genes were added to form a 5+4+3+2-gene dataset of
2 1317 OTUs, for a total of four different datasets (with an increasing number of missing
3 sequences) that were analyzed separately (Table 1). The entire supermatrix (5+4+3+2-gene
4 dataset) was deposited in TreeBASE (accession number XXXX).
5 Maximum likelihood analyses were carried out on each of the four datasets, with nine
6 partitions assigned (nucSSU, nucLSU, mitSSU, RPB1 1st/2nd/3rd and RPB2 1st/2nd/3rd) using
7 RAxML 7.2.8. The fast bootstrap combined with the tree search algorithm (option -f a of
8 RAxML) was used, implementing a GTRGAMMAI model as provided by the program, and
9 calculating 1000 bootstrap replicates.
10 Internodal support resulting from bootstrap analyses of the four datasets were depicted on
11 the 5+4+3+2-gene phylogeny using the box scheme (Fig. 1) as implemented in the newly
12 developed “Hypha” module (Oliver et al., 2013) part of Mesquite (Maddison and Maddison,
13 2011).
14
15 3. Results and Discussion
16
17 3.1 Datasets, phylogenetic inference and confidence
18 The number of taxa in each multigene dataset was much higher than in our previous study
19 (Miadlikowska et al., 2006), ranging from 72 newly added OTUs for the 5-gene dataset, to 490
20 newly added OTUs for the 5+4+3-gene dataset (Table 1). Extra 553 OTUs were added by
21 including OTUs for which sequences were available for only two of the five genes. In our
22 previous phylogenetic study of the Lecanoromycetes (Miadlikowska et al., 2006) we did not
23 include taxa with fewer than three of the five targeted genes. This strategy elevated our number
24 of OTUs from 274 in 2006 to 1317 in this study. Of the 3942 sequences incorporated in the most
25 inclusive (5+4+3+2-gene) dataset, 635 were newly generated. This allowed us to reduce the
26 proportion of missing sequences, in each dataset that included OTUs with fewer than five genes,
27 by more than 10%, compared to Miadlikowska et al. (2006) (Table 1). The highest number of
28 new sequences was obtained for the mitochondrial ribosomal SSU (176) and nuclear ribosomal
29 LSU and SSU with163 and 137, respectively. RPB2 and RPB1 genes had the lowest sequencing
30 success (61 and 98 new complete sequences, respectively) and, consequently, these genes had
31 the highest number of missing sequences (more than half of the OTUs; Table 1). Contrary to
12
1 Miadlikowska et al. (2006), the ambiguously aligned regions were delimited only in the most
2 OTU inclusive (5+4+3+2–gene) dataset, and were not re-adjusted in the less OTU inclusive
3 datasets due to the large size of the datasets, which were difficult to handle manually. Similar to
4 other studies (Hofstetter et al., 2007; Miadlikowska et al., 2006), RPB1 and RPB2 provided the
5 greatest proportion of unambiguously aligned sites (> 75% of the sites), whereas only a small
6 fraction of the RNA coding sites (9-12%) could be aligned unequivocally due to the presence of
7 a large number of indels including spliceosomal and group I introns. A total of 7433 sites were
8 analyzed phylogenetically for the 5+4+3+2-gene dataset (Table 1).
9 Phylogenies derived from the four multigene datasets were generally concordant among each
10 other, including the 1317 OTU data with 54% of missing sequences. The cladogram of the ML
11 optimal tree resulting from the 5+4+3+2-gene analysis is presented in Fig. 1 (the phylogram
12 version of this tree is shown in the Supplemental Fig. S1) and its schematic version in Fig. 2. A
13 few cases of conflict were detected at the level of 70% and higher bootstrap support (red boxes
14 in Fig. 1) and involved: 1) terminal relationships among species (in the genera Sphaerophorus,
15 Peltigera and Trapeliopsis), 2) the sister relationship between Degelia and Parmeliella in
16 Pannariaceae (contrary to the sister relationship between Degelia and Erioderma based on the 5-
17 gene dataset), and 3) the placement of Caloplaca arnoldii in the Xanthorioideae
18 (Teloschistaceae).
19 An increasing proportion of missing data, from 0% (in the 5-gene dataset) to 54% (in the
20 5+4+3+2-gene dataset), affected bootstrap support, which frequently decreased below the
21 significance cutoff (white boxes in Figs. 1 and 2). This trend was more pronounced than in our
22 previous study (Miadlikowska et al., 2006) where we did not include taxa with fewer than three
23 genes, and where the maximum proportion of missing sequences was 37%. However, most
24 relationships remained unchanged, and many of them were highly supported by at least one of
25 the four bootstrap analyses (black boxes in Figs. 1 and 2). Certain portions of the phylogeny
26 were less stable (e.g., Parmeliaceae and Lecanoraceae in the Lecanoromycetidae, and Ostropales
27 in general) than others (e.g., Teloschistales and Peltigerales) due to a higher concentration of
28 taxa with only two (RNA-coding) genes (Figs. 3 and 4; Supplemental Table S5). Instances of
29 highly supported relationships contradicting published multigene phylogenies were very rare and
30 usually involved different combination of molecular markers used in previous studies, e.g.,
31 discrepancies in delimitation of new genera in the Collema/Leptogium complex based on MCM7
13
1 and beta-tubulin in addition to nucLSU and mitSSU loci (Otálora et al., 2013a, 2013b versus Fig.
2 1E-F). Nevertheless, adding taxa with only two or three genes sometime improved phylogenetic
3 confidence above the 70% bootstrap threshold (e.g., monophyly of Cladoniaceae, Parmeliaceae,
4 Sphaerophoraceae, and Lecanorales; Fig. 1). However, other relationships at various taxonomic
5 levels were not well supported, even if they involved taxa represented by the complete (5 loci) or
6 almost complete set of genes.
7
8 3.2 Lecanoromycetes: delimitation and relationships among subclasses (Figs. 1 and 2)
9 About half (34/65) of the backbone relationships (up to the family level exclusively) received
10 bootstrap support only below the 70% bootstrap level (i.e., no black boxes in the four-cell grid in
11 Fig. 2). However, ten of these 34 internodes were recovered by at least one of the four bootstrap
12 analyses (i.e., at least one white box per grid) and an additional 15 internodes were recovered by
13 at least two of the four bootstrap analyses (i.e., at least two white boxes per grid; Fig. 2). Only
14 nine of the 65 backbone internodes were never recoved by at least one bootstrap analysis (i.e., no
15 white or black boxes in each given grid; Fig. 2). Compared to the previous large-scale phylogeny
16 of the Lecanoromycetes reconstructed for 274 taxa (5+4+3-gene dataset; Fig. 1 in Miadlikowska
17 et al., 2006), we lost support ≥ 70% for the monophyly of the Lecanoromycetidae (with or
18 without Rhizocarpales included), Ostropomycetidae including Sarrameanales (previously
19 referred to as “Loxosporales?”), and Umbilicariomycetidae excluding the Ropalosporaceae (this
20 family was not included in the previous study). However, we did recover highly supported
21 monophyletic Ostropomycetidae-Umbilicariomycetidae-Lecanoromycetidae, Rhizocarpales,
22 Acarosporomycetidae, and Lecanorales. The subclass Lecanoromycetidae, including
23 Rhizocarpales, although not supported with bootstrap values above 70%, was recovered by three
24 of the four bootstrap analyses in this study, and was recovered with significant posterior
25 probability and bootstrap support (ML) by Bendiksby and Timdal (2013). In our phylogeny, the
26 Ostropomycetidae (including Sarrameanales), Lecanoromycetidae, and Umbilicariomycetidae
27 form a strongly supported monophyletic group (here refered to as the OSLEUM clade; Fig. 2).
28 The sister relationship between the Lecanoromycetidae and Umbilicariomycetidae, which is in
29 agreement with Miadlikowska et al. (2006) and Lutzoni et al. (2004; PP = 1.00 based on four
30 genes), was recovered by three of the four bootstrap analyses (Fig. 2), but never with bootstrap
31 support above 70%. Previous large-scale phylogenies for the Lecanoromycetes show support for
14
1 two contradicting relationships among these three subclasses: a sister relationship between the
2 Ostropomycetidae and Umbilicariomycetideae (PP = 0.98 in Lutzoni et al., 2004, for the
3 phylogeny based on three genes), and between the Lecanoromycetidae and Ostropomycetidae
4 (PP = 1.00 and ML-BP = 80% in Bendiksby and Timdal, 2013; and PP = 0.99 in Prieto et al.,
5 2012). The recognition of the Umbilicariomycetidae as a separate subclass enables the tracking
6 of this phylogenetic uncertainty.
7 The phylogenetic position of the Acarosporomycetidae and ‘Candelariomycetidae’ within the
8 Lecanoromycetes is mostly inconclusive in this study. However, we recovered the
9 Acarosporomycetidae and ‘Candelariomycetidae’ as derived from the first and second split,
10 respectively, within the Lecanoromycetes. This resolution was also shown in Miadlikowska et al.
11 (2006) with PP > 0.95 and bootstrap support above 70%, and by Reeb et al. (2004) with PP
12 >0.95. Hofstetter et al. (2007), Lumbsch et al. (2007b) and Bendiksby and Timdal (2013)
13 recovered a phylogeny with strong bootstrap support for the ‘Candelariomycetidae’ diverging
14 first, followed by the Acarosporomycetidae. In Wedin et al. (2005) the Acarosporomycetidae
15 and paraphyletic ‘Candelariomycetidae’ (sensu Bendiksby and Timdal, 2013) form a
16 monophyletic group closely related to the outgoup taxa (Eurotiomycetes; however, without
17 support). Most recently, Prieto et al. (2012) presented a Bayesian tree based on four ribosomal
18 and two protein-coding genes (MCM7 and RPB1) where Candelariales is separated from the
19 Lecanoromycetes and placed as an independent lineage among early diverging Leotiomyceta.
20 However, this placement, as well as many other deep phylogenetic relationships associated with
21 the Leotiomyceta radiation, did not receive strong support (Lutzoni et al, 2004).
22 Using Leotiomycetes, Geoglossomycetes and Lichinomycetes as outgroup taxa, as we did
23 previously (Miadlikowska et al., 2006), the delimitation of the Lecanoromycetes has revealed
24 itself to be somewhat more problematic with the addition of 1043 OTUs. The Dactylosporaceae,
25 a family classified in the Lecanorales based on morphological and anatomical characters
26 (Lumbsch and Huhndorf, 2010) was resolved within the Eurotiomycetes, close to
27 Chaetothyriales and Pyrenulales, by Schoch et al. (2009) and Rossman et al. (2010). Similar
28 results were obtained by Diederich et al. (2013) where Dactylospora was found within the
29 Eurotiomycetes, with the lichenicolous fungus Sclerococcum sphaeriale and an unidentified
30 endolichenic fungus ALr-1. Our phylogenetic analyses support the placement of Dactylospora
31 within the Lecanoromycetes, a result that is more inline with phenotypic traits (e.g., amyloid
15
1 ascus). Interestingly, Dactylospora was resolved here as sister to Strangospora (without
2 support), a genus involved in a similar discordance with respect to crossing the Lecanoromycetes
3 boundary (Figs. 1 and 2). However, this sister relationship was recovered only by the
4 phylogenetic analysis of the 5+4+3+2-gene dataset. Strangospora pinicola, tentatively classified
5 within Lecanorales (Lumbsch and Huhndorf, 2010), was placed with high support outside all
6 families sampled from the order Lecanorales in Miadlikowska et al. (2006), as the result of the
7 second split within the Lecanoromycetes (with high PP) according to Reeb et al. (2004), and
8 outside all currently recognized subclasses in the phylogeny presented here.
9 Sarea resinae represents another case of a genus currently classified within the
10 Lecanoromycetes (Trapeliaceae; Lumbsch and Huhndorf, 2010), which has been resolved
11 phylogenetically outside this class, possibly within the Leotiomycetes according to Reeb et al.
12 (2004). Our study supports the placement of this taxon outside Lecanoromycetes (Figs. 1A and
13 2), but this result is based on only two sequences for the entire genus, which have never been
14 corroborated with additional sequences from other specimens or by more genes.
15 The phylogenetic placement of Pycnoraceae by Bendiksby and Timdal (2013) (a new family
16 introduced to accommodate Pycnora) in the ‘Candelariomycetidae’ and Lecanoromycetes is
17 questionable based on our phylogenetic analyses. In this study, this family is resolved with high
18 confidence outside Lecanoromycetes, before the split of the Lecanoromycetes from the
19 Geoglossomycetes+Lichinomycetes clade (Figs. 1A and 2). In Bendiksby and Timdal (2013), the
20 ‘Candelariomycetidae’, including the Pycnoraceae, received a low bootstrap value (59%). The
21 inclusion of only one taxon for the outgroup (Geoglossum nigritum), and the use of ribosomal
22 genes only, mtSSU, nucLSU and ITS to infer relationships across the Lecanoromycetes, where
23 positional homology of many sites might be questionable but nevertheless included in the
24 phylogenetic analyses, was probably the cause of this artifactual result.
25 Biatoridium, represented by the type species (B. monasteriense) and considered currently as a
26 member of the Lecanoromycetes of uncertain affiliation, is placed with high confidence outside
27 of the class, sister to the Lichinomycetes (Fig. 1A). This confirms the results obtained by Reeb
28 et al. (2004), where Biatoridium together with Sarcosagium campestre or Thelocarpon laureri
29 were found to be sister to Lichinomycetes. Biatoridium shares morphological similarities in the
30 apothecium and ascus structure with Sarea and Strangospora (Hafellner, 1995), two other genera
31 of doubtful position within the Lecanoromycetes (Figs. 1A and 2).
16
1 The phylogenetic placement of Candelariales, Dactylosporaceae, Strangosporaceae,
2 Pycnoraceae, Biatoridium, and Sarea, as well as a robust delimitation of the Lecanoromycetes
3 must be assessed with more taxa and more than six loci (see Prieto et al., 2012) within the broad
4 phylogenetic context of the Leotiomyceta.
5 Taxonomic conclusions:
6 A. Strangospora should be removed from Lecanorales and placed in its own family,
7 Strangosporaceae, which should be classified as Lecanoromycetes incertae sedis.
8 Family: Strangosporaceae S. Stenroos, Miadl. & Lutzoni fam. nov.
9 MycoBank no.: MB XXXXX.
10 Type genus: Strangospora Körb., Parerga lichenol.: 173 (1860)
11 Diagnosis – A monogeneric family of uncertain placement in the Lecanoromycetes comprising
12 epiphytic, epixylic or bryophylous lichens with crustose and often poorly developed thalli
13 associated with chlorococcoid photobiont. Apothecia biatorine, but exciple poorly developed,
14 asci clavate, with gelatinous outer layer, a strongly thickened I+blue wall and apical dome,
15 multispored; ascospores aseptate.
16 Type species: S. pinicola (A. Massal.) Körb. 1860
17 B. Dactylosporaceae should be removed from Lecanorales and classified as Leotiomyceta
18 incertae sedis.
19
20 3.3 Acarosporomycetidae /Acarosporales/Acarosporaceae (Fig. 1A)
21 The subclass Acarosporomycetidae consists of a single order and family of nine genera
22 that are mostly saxicolous (Harris and Knudsen, 2005; Hibbett et al., 2007; Lumbsch and
23 Huhndorf, 2010; Reeb et al., 2004). We sampled seven of eight recognized genera (Lithoglypha
24 was not included) and more than doubled the number of taxa compared to Miadlikowska et al.
25 (2006; 36 versus 15 taxa). The Acarosporomycetidae is monophyletic and well supported (Fig.
26 1A). A newly sequenced Eiglera flavida (currently classified in Hymeneliaceae;
27 Ostropomycetidae inc. sed.; Lumbsch and Huhndorf, 2010) is placed with high confidence in
28 Acarosporales. Eiglera flavida represents a monotypic genus, previously classified in the family
29 Eigleraceae, which was erected by Hafellner (1984) to accommodate a species formerly placed
30 in Aspicilia or Lecanora. The genus Eiglera is distinguished from the rest of Hymeneliaceae by
31 its ascus type (I+ uniformly blue tholus versus I+ faint blue or I- tholus typical for
17
1 Hymeneliaceae), but shares with members of this family morphological, anatomical and
2 isozymic characteristics (Lutzoni and Brodo, 1995), as well as ascoma ontogenic features
3 (weakly hemiangiocarpous, relatively simple, and having paraphysoids; Lumbsch, 1997). If
4 future study confirms the phylogenetic placement of Eiglera (based on multiple individuals) as
5 derived from the first split within Acarosporomycetidae, it would justify the reinstitution of the
6 family Eigleraceae.
7 We confirm here the polyphyletic delimitation of the genera Acarospora, Sarcogyne, and
8 Pleopsidium (Reeb et al., 2004; Crewe et al., 2006; Miadlikowska et al., 2006). Delimitation of
9 these genera should be redefined in a phylogenetic context. It is very likely that Sarcogyne
10 plicata represents a member of the genus Polysporina (sister relationship with P. simplex in
11 Fig.1) as suggested also by shared morphology and substrate (Magnusson, 1935; Knudsen and
12 Kocurkova, 2011) between the two taxa. Our phylogeny supports the distinctiveness of S. plicata
13 from S. privigna (the latter being nested in the ‘Acarospora’ + Sarcogyne clade; Fig. 1A) as
14 indicated in Knudsen and Kocurkowa (2011). For future taxonomical changes within the genus
15 Acarospora, the name Myriospora that was recently recircumscribed (Arcadia and Knudsen,
16 2012) is available to accomodate the A. smaragdula group.
18 A. The genera Acarospora, Pleopsidium and Sarcogyne need taxonomic revision within the
19 phylogenetic framework of the Acarosporaceae.
20 B. If confirmed, Sarcogyne plicata should be transferred to Polysporina.
21 C. Eiglera (Hymeneliaceae, Ostropomycetidae) needs to be sampled for multi-locus sequencing
22 to confirm phylogenetic placement within Acarosporales. If this genus is derived from the first
23 divergence within this order, the family Eigleraceae should be added to the Acarosporales.
24
25 3.4 ’Candelariomycetidae’/Candelariales/Candelariaceae (Fig. 1A)
26 The subclass ‘Candelariomycetidae’ (Miadlikowska et al., 2006) and order Candelariales
27 (Hibbett et al., 2007) includes two families according to Bendiksby and Timdal (2013):
28 Candelariaceae, with four genera (Candelaria, Candelariella, Candelina and Placomaronea;
29 Lumbsch and Huhndorf, 2010; Westberg et al., 2009), and Pycnoraceae, with a single genus,
30 Pycnora (Bendiksby and Timdal, 2013; formerly the Hypocenomyce xanthococca group;
31 Hafellner and Türk, 2001). Our phylogeny supports the Candelariales as accepted in Lumbsch
18
1 and Huhndorf (2010), with a single family, Candelariaceae. The newly established family
2 Pycnoraceae (represented in our tree by two species with two loci each) was resolved here as
3 monophyletic and highly supported but not as a sister group to Candelariaceae. Instead, this new
4 family was placed, with high phylogenetic confidence (ML bootstrap support), outside of the
5 Lecanoromycetes (Fig. 1A). The alternative sister relationship of Pycnoraceae and
6 Candelariaceae received sometimes high posterior probabilities based on ribosomal loci (0.98 in
7 Wedin et al., 2005, but see Bendiksby and Timdal, 2013). It was already demonstrated, based on
8 morphological and molecular data, that the current delimitation of genera within Candelariaceae
9 does not reflect monophyletic grouping (Westberg et al., 2007, 2009). Our phylogeny confirms
10 the polyphyly of Candelariella and the necessity to revise generic circumscriptions to reflect the
11 inferred phylogenetic structure for the family Candelariaceae. As the number of spores per ascus
12 and thallus habit varies within and between closely related species, and the chemistry is uniform
13 throughout the family, a new set of characters is needed to circumscribe monophyletic genera in
14 Candelariaceae.
16 A. Candelariella is polyphyletic and needs systematic revision within the phylogenetic
17 framework of the Candelariaceae.
18 B. Inclusion of Pycnoraceae within Candelariales remains uncertain, however, its taxonomic
19 placement outside Lecanoromycetes is not supported by morpho-anatomical characters and
20 should be confirmed within a more comprehensive phylogenetic framework, particularly with
21 respect to the Leotiomycetes.
22
23 3.5 Ostropomycetidae: relationships within the subclass (Fig. 1A-D)
24 Phylogenetic relationships within Ostropomycetidae are highly unsettled, mostly because
25 fewer genes have been sequenced for many families within this subclass compared to the rest of
26 the Lecanoromycetes (Figs. 3 and 4; Supplemental Table S5). Adding more taxa without
27 sequencing more loci would not improve this situation (Figs. 3 and 4 and Supplemental Fig. S2;
28 e.g., Parnmen et al., 2012; Rivas Plata and Lumbsch, 2011; Rivas Plata et al., 2012b),
29 Consequently, delimitations of families and orders are unstable in this subclass (Baloch et al.,
30 2010; Bendiksby and Timdal, 2013; Hofstetter et al., 2007; Lumbsch et al., 2007b;
31 Miadlikowska et al., 2006; Schmull et al., 2011). Currently, six families are not assigned to any
19
1 order (incertae sedis) in Ostropomycetidae (Lendemer and Hodkinson, 2011; but see Gueidan et
2 al., 2014). The general delimitation of the Ostropomycetidae (Figs. 1A-D and 2) is in agreement
3 with the recent phylogenies; however, the addition of the Sarrameanales (Loxospora) and
4 Schaereriaceae resulted in a decrease of phylogenetic support for the monophyly of the subclass,
5 including this study (e.g., Bendiksby and Timdal, 2013; Lumbsch et al., 2007b; Schmull et al.,
6 2011, but see Miadlikowska et al., 2006).
7 The results of our phylogenetic analyses support recognition at the ordinal level of two
8 currently acepted families, the Hymeneliaceae and Arctomiaceae, in order to retain existing
9 orders in this subclass. The phylogenetic placement of the Ostropales within the
10 Lecanoromycetes has long been unclear due to the unstable backbone of the Lecanoromycetes
11 phylogeny (Lumbsch et al., 2007b). The Ostropales has been found sister to many different
12 lineages in Lecanoromycetes (Grube et al., 2004; Lücking et al., 2004; Lumbsch et al., 2004):
13 sister to the Trapeliales and Hymeneliaceae (Kauff and Lutzoni, 2002; Miadlikowska and
14 Lutzoni, 2004) or to a lineage including the Trapeliales and Baeomycetales (Miadlikowska et al.,
15 2006); sister to the Fuscideaceae, an incertae sedis family within Lecanoromycetes (Reeb et al.,
16 2004); and sister to a lineage including Anzina and Arthrorhaphis (Wedin et al., 2005). None of
17 these relationships were strongly supported. Here the order Ostropales (Thelenellaceae,
18 Protothelenellaceae, Myeloconidaceae, and Phaneromycetaceae not sampled), with
19 Arctomiaceae and Trapeliales, form a highly supported monophyletic group (Figs. 1B-D and 2).
20 However, the relationships among these three lineages are not supported. Our study confirms
21 that Hymeneliaceae and Baeomycetales, together with this trio, form a well-supported
22 monophyletic assemblage (Miadlikowska et al., 2006; Schmull et al., 2011). However,
23 Arthrorhaphidaceae is not part of this major group (which was recovered in the
24 Lecanoromycetideae, but without bootstrap support). The orders Sarrameanales (including
25 Schaereriaceae) and Pertusariales are derived from the first and second divergences within
26 Ostropomycetidae, as previously reported (Miadlikowska et al., 2006; Schmull et al., 2011).
27
28 3.5.1 Arctomiales/Arctomiaceae (Fig. 1C)
29 The order Arctomiales is proposed here to accommodate a single family, the
30 Arctomiaceae (Fries, 1860), which occupies an independent lineage outside the recognized
31 orders in the Ostropomycetidae (Figs.1C and 2). This small family consists of three genera, all of
20
1 which are associated with the cyanobacterium Nostoc as the main photobiont: Arctomia (six
2 recognized species and four newly added from the former Collema fasciculare group; Otálora
3 and Wedin, 2013), the monospecific Gregorella, and Wawea (Magain and Sérusiaux, 2012).
4 This family was always strongly supported as monophyletic in previous work (e.g., Lumbsch et
5 al., 2005; Magain and Sérusiaux, 2012), although its placement in the Ostropomycetidae was
6 never recovered with high confidence (incertae sedis in Lumbsch and Huhndorf, 2010). Our
7 phylogeny confirms the monophyletic circumscription of the family, but with bootstrap support
8 below the significance threshold because of the placement of Arctomia interfixa outside the
9 Arctomia clade derived from the first divergence within Arctomiaceae (Fig. 1C). A similar
10 position of this taxon (and the non-monophyletic delimitation of the genus Arctomia) was shown
11 recently in Magain and Sérusiaux (2012) and in Otálora and Wedin (2013), but was not revealed
12 earlier in Lumbsch et al. (2005), where the RPB1 locus was not included. Although no single
13 phylogeny incorporates all six Arctomia species, it is possible that A. interfixa (with two
14 ribosomal RNA-coding genes only, in this and previous studies) represents a different,
15 undescribed genus in the family Arctomiaceae. Recent papers (Magain and Sérusiaux 2012;
16 Otálara and Wedin 2013) have demonstrated that diversity in the Arctomiaceae is much greater
17 than previously expected as several species with collematoid thalli actually belong to that family.
18 Because of the isolated position of the Arctomiaceae between two recognized and well
19 delimited orders, the Ostropales and the Trapeliales in the Ostropomycetidae (see also Lumbsch
20 et al., 2005) and because of its unique characteristics among Ostropomycetidae (e.g., Nostoc as
21 the primary photobiont and gymnocarpous ascomata development; Henssen, 1969, Lumbsch et
22 al., 2005) we propose to recognize Arctomiaceae at the order level.
24 Order: Arctomiales S. Stenroos, Miadl. & Lutzoni ord. nov.
26 Typus: Arctomiaceae Th. Fr (1860) based on Arctomia Th. Fr. (1860)
27 Diagnosis. – Closely related to Ostropales and Trapeliales in the Ostropomycetidae, this order
28 contains a single family Arctomiaceae comprizing bryophilous crustose or fruticose, gelatinized
29 lichens associated with the cyanobacterium Nostoc. Ascomata apothecia with gymnocarpous
30 development, often with poorly developed outer wall, cylindrical asci with a well-developed
21
1 apical cap (but no ocular chamber) and a I+ mucous outer layer; 8 hyaline ascospores, elongated
2 and transversely septated, often with attenuated apices.
3 Examplar genera: Arctomia Th. Fr. 1860, Wawea Henssen & Kantvilas 1985.
4
5 3.5.2 Baeomycetales/Baeomycetaceae (Fig. 1B)
6 The order Baeomycetales (Kauff and Lutzoni, 2002; Reeb et al., 2004; Hibbett et al.,
7 2007) currently includes two families, Baeomycetaceae and the monotypic Anamylopsoraceae
8 (Hodkinson and Lendemer, 2011; Lumbsch et al., 1995). The family Baeomycetaceae, which
9 comprises three genera, Baeomyces, Phyllobaeis and the recently added Ainoa (Lumbsch et al.,
10 2007b, c; Lumbsch and Huhndorf, 2010), is well supported as a monophyletic group resulting
11 from a cladogenic event distinct from the great majority of divergences that took place during the
12 evolution of the Ostropomycetidae (Figs. 1B and 2). The classification of Anamylopsoraceae in
13 Baeomycetales (Hodkinson and Lendemer, 2011; Lumbsch et al., 2007b; Lumbsch and
14 Huhndorf, 2010) should be confirmed because Anamylopsora (not included in this study) was
15 never shown with high confidence to be closely affiliated with members of the family
16 Baeomycetaceae (Lumbsch et al., 2001).
17
18 3.5.3 Hymeneliales/Hymeneliaceae (Fig. 1B)
19 The family Hymeneliaceae (Körber 1855), including Ionaspis, Hymenelia and
20 Tremolecia (Melanolecia was not sampled; Lumbsch and Huhndorf, 2010), but without Eiglera
21 (E. flavida is sister to the Acarosporaceae), was reconstructed with strong support as a
22 monophyletic and distinct lineage in the subclass Ostropomycetidae (Figs. 1B and 2). This is the
23 only group outside the order Ostropales in which some species form symbiotic associations with
24 Trentepohlia (Lutzoni and Brodo, 1995). The family is derived from the divergence following
25 the evolutionary split of the Baeomycetales and preceding the split of Trapeliales; therefore, it is
26 flanked by two well-supported internodes. In agreement with the currently accepted orders in the
27 subclass Ostropomycetidae, the Hymeneliaceae should be recognized at the order level. The
28 phylogenetic placement of Eiglera outside of the Hymeneliaceae, sister to Acarosporaceae,
29 needs to be confirmed.
31 Order: Hymeneliales S. Stenroos, Miadl. & Lutzoni ord. nov.
22
2 Typus: Hymeneliaceae Körb. (1855) based on Hymenelia Kremp. (1852)
3 Diagnosis. – This order of uncertain placement in the Ostropomycetidae, consisting of a single
4 family (Hymeneliaceae), contains saxicolous lichens with mostly crustose, sometimes
5 evanescent thalli associated with chlorococcoid algae or Trentepohlia. Apothecia deeply
6 immersed, asci with a well-developed I+ blue or I- apical cap, usually without an ocular
7 chamber, with an outer I+ blue gelatinous layer; ascospores large, aseptate, hyaline.
8 Exemplar genera: Hymenelia Kremp. 1852, Ionaspis Th. Fr. 1871.
9
10 3.5.4 Ostropales (Fig. 1C-D)
11 Ostropales is the largest order within the class Ostropomycetidae (131 genera according
12 to Kirk et al., 2008). It includes mostly crustose lichenized and nonlichenized taxa, with high
13 species diversity in the tropics. It hosts one of the major losses of lichenization detected by
14 Lutzoni et al. (2001; Stictidaceae). Currently, ten families are recognized within this order:
15 Coenogoniaceae, Graphidaceae (including Gomphillaceae and Thelotremataceae), Gyalectaceae,
16 Myeloconidaceae, Odontotremataceae, Phaneromycetaceae, Phlyctidaceae, Porinaceae,
17 Sagiolechiaceae, and Stictidaceae (Baloch et al., 2010; Lumbsch and Huhndorf, 2010; Rivas
18 Plata et al., 2012a). Relationships among these families remain highly unsettled (e.g., see Baloch
19 et al., 2010) with the exception of the sister relationship between Gyalectaceae and
20 Sagiolechiaceae as well as the monophyly of all families excluding Porinaceae and Stictidaceae,
21 which are branching outside this major clade (Figs. 1C-D and 2).
22 The latest classification of the Graphidaceae (Rivas Plata et al., 2012a) concluded that the
23 family comprises three major clades which are delimited as subfamilies: Fissurinoideae,
24 Gomphilloideae, and Graphidoideae, the latter being composed of three major clades which are
25 recognized as tribes Graphideae, Ocellularieae, and Thelotremateae. All three recently
26 circumscribed subfamilies of mostly undetermined placement in Rivas Plata et al. (2012a),
27 received strong support in our study, except for the monophyly of the Thelotremateae and the
28 Gomphilloideae (Fig. 1C-D). The subfamilies Fissurinoideae and Gomphilloideae together form
29 a monophyletic group (but without significant support) derived from the first divergence within
30 the Graphidaceae (in agreement with Rivas Plata et al., 2012a). Considering morphological and
23
1 phylogenetic distinctness of the recently proposed taxa (subfamilies and tribes), five families
2 could be recognized instead of a broadly delimited single family Graphidaceae.
3 As reported by Rivas Plata et al. (2013), phylogenetic relationships among genera within
4 the recognized subfamilies are mostly unsupported and unsettled. The genus Thelotrema is
5 highly polyphyletic with its members distributed across the Graphidaceae, as shown also by
6 Rivas Plata et al. (2013), and is currently undergoing a taxonomical treatment by the same
7 authors. Diploschistes ocellatus, which has lecanoroid ascomata and norstictic acid as the main
8 secondary compound in its thallus, is separated from the other members of the genus (Rivas Plata
9 et al., 2013), but its phylogenetic placement did not receive significant support in our study (Fig.
10 1D) as a result of the first divergence within a clade composed of Diploschistes s. str.,
11 Wirthiotrema, Topeliopsis and two ‘Thelotrema’ species (see also Fernández-Brime et al., 2013).
12 The genera Ocellularia and Myriotrema (Ocellularieae) form a monophyletic group, which was
13 strongly supported by one of the four bootstrap analyses. The inclusion of Myriotrema within
14 Ocellularia s. l. or the expansion of Myriotrema to include Ocelluaria species outside
15 Ocellularia s. str. (Fig. 1C) would prevent the proliferation of new generic names (see Rivas
16 Plata et al., 2012b). However, the sequencing of more loci is urgently needed for a meaningful
17 and stable taxonomic revision of the Graphidaceae (see Rivas Plata et al., 2013). Members of
18 the former genus Chapsa s. l. (e.g., Astrochapsa and Pseudochapsa; Parnmen et al., 2012)
19 belong to the Thelotremateae (Rivas Plata et al., 2013). Contrary to the recent study by Rivas
20 Plata et al. (2013), Chroodiscus coccineus is resolved here outside the core of the genus (which
21 is monophyletic and well supported in our 5+4+3+2-locus phylogeny; Fig. 1D) perhaps due to an
22 erroneous nucLSU sequence obtained from GenBank (Supplemental Table S1). Dyplolabia
23 afzelii is here nested within a well-supported Fissurina clade in agreement with Rivas Plata et al.
24 (2013). Based on an extensive taxon (mostly single-locus) sampling, Fissurina s. l. encompasses
25 several morphologically unrecognized genera (Rivas Plata et al., 2013), which would favor a
26 broad circumscription of this genus.
27 The families Coenogoniaceae, Gyalectaceae, Porinaceae, and Sagiolechiaceae (here
28 represented by their respective type genus) are all monophyletic with high confidence (Fig. 1C).
29 If future studies confirm the sister relationship of Gyalectaceae and monogeneric
30 Sagiolechiaceae, recovered with high support using our 5+4+3-locus dataset (unsettled in Baloch
31 et al., 2010), both families could be merged, considering the phenotypical similarities between
24
1 Gyalecta and Sagiolechia. Petractis clausa, classified currently in Stictidaceae (Lumbsch and
2 Huhndorf, 2010), is affiliated with Phlyctis, however, its inclusion in Phlyctidaceae is not well
3 supported. In agreement with a previous study (Baloch et al., 2010), the Odontotremataceae are
4 divided into two unrelated well-supported clades: the Odontotrema s. str. clade (with the type
5 species included), and a second clade currently recognized as the genus Sphaeropezia (Baloch et
6 al., 2012), which is nested within Stictidaceae. As anticipated in Baloch et al. (2012),
7 Coccomycetella richardsonii is nested within Odontotrema (O. phacidiellum and O.
8 phacidioides) with high support (Fig. 1C). Because this phylogenetic affiliation is corroborated
9 by similarities in habit (except by its sigmoid spores) and ecology among these taxa,
10 Coccomycetella richardsonii should be considered as a member of Odontotrema. The genus
11 Stictis is confirmed here to be highly polyphyletic. A new well-supported assembly of potentially
12 unrelated taxa classified in various families (Graphidaceae, Gyalectaceae and Stictidaceae) was
13 discovered in the Ostropales (Group 1; Fig. 1C). This clade of uncertain placement in the
14 Ostropales was partially represented in Baloch at al. (2010; Gyalidea praetermissa and Petractis
15 luetkemuelleri). If justified phenotypically, this new lineage may be circumscribed as a new
16 family in the Ostropales or merged to an existing family once its affiliation is resolve with high
17 phylogenetic confidence.
19 A. Coccomycetella richardsonii should be accepted as Odontotrema species (the name
20 Odontotrema richardsonii Leight. is available).
21 B. The distinction of Sagiolechiaceae from the Gyalectaceae needs re-evaluation.
22 C. Absconditella, Petractis, Gyalidea, Stictis, and Thelotrema need systematic revisions within
23 the phylogenetic framework of the Ostropales.
24 D. Petractis nodispora and P. luetkemuelleri (Stictidaceae), Gyalidea praetermissa
25 (Graphidaceae) and Ramonia sp. (Gyalectaceae) should be accommodated in different genera
26 outside of their respective families.
27 E. The generic placements of Hemithecium implicatum, Absconditella lignicola, and
28 Cryptodiscus rhopaloides need re-evaluation with the potential of introducing new genera.
29
30 3.5.5 Pertusariales (Fig. 1A-B)
25
1 The order Pertusariales, recently retained over Agyriales (Hodkinson and Lendemer,
2 2011), includes currently seven families: Agyriaceae (monotypic family), Coccotremataceae,
3 Icmadophilaceae, Megasporaceae, Miltideaceae (not sampled in this study), Ochrolechiaceae,
4 and Pertusariaceae (Hodkinson and Lendemer, 2011; Lumbsch and Huhndorf, 2010; Schmitt et
5 al., 2010; Widhelm and Lumbsch, 2011). The families Megasporaceae, Coccotremataceae, and
6 Icmadophilaceae, as currently defined, are recovered here as monophyletic and highly supported
7 (Figs. 1A-B and 2). Relationships among families within the Pertusariales clade remain unstable,
8 with the exception of the sister relationship of Megasporaceae with Ochrolechiaceae, and the
9 early divergence of the family Icmadophilaceae from the rest of the Pertusariales (not supported
10 here, but see Miadlikowska et al., 2006).
11 The family Ochrolechiaceae (including Pertusaria dactylina, a specimen which may
12 represent a sterile isidiate Ochrolechia; see Schmitt and Lumbsch, 2004) is delimited as
13 monophyletic and well supported, without the genera Variolaria, which was formally reinstated
14 to accommodate Loxospora pustulata and two Pertusaria species not sampled in this study
15 (Lendemer et al., 2013), and Varicellaria (Pertusariaceae; Lumbsch and Huhndorf, 2010,
16 Schmitt et al., 2006, 2012), which together form a monophyletic group sister to the rest of
17 Pertusariaceae (relationships that were recovered in almost all phylogenetic analyses conducted
18 here; Fig. 1A). Varicellaria and Variolaria were segregated from Pertusaria s. l. based on
19 chemical and morphological characters shared between the two genera (e.g., the presence of
20 lecanoric acid as a major secondary compound, disciform apothecia, strongly amyloid asci, non-
21 amyloid hymenia, 1-2 spored asci, and 1- or 2- celled ascospores with thick, 1-layered walls).
22 However, their close affiliation and suggested alternative placement outside of Pertusariaceae (in
23 Ochrolechiaceae, sister to Ochrolechia) was poorly supported most of the time or not
24 reconstructed, regardless of the markers used (Nordin et al., 2010; Schmitt et al., 2006; Schmitt
25 et al., 2010; Schmitt et al. 2012). The only exception where these three genera form a highly
26 supported monophyletic group was in the study by Schmitt and Lumbsch (2004), where the
27 ingroup was restricted to Pertusariaceae and Ochrolechiaceae only; i.e., in the absence of
28 representative species from the families Megasporaceae, Icmadophilaceae, Coccotremataceae,
29 and Agyriaceae.
30 Here the family Agyriaceae is well supported as sister to the monophyletic genus
31 Pertusaria s.str. + Loxosporopsis (Schmitt and Lumbsch, 2004) as part of a monophyletic
26
1 Pertusariaceae (but without bootstrap values ≥ 70%; Fig. 1A-B). To retain the family Agyriaceae
2 (and perhaps the Miltideaceae; sister to Agyriaceae, e.g. Lendemer and Hodkinson, 2012;
3 Schmitt et al., 2012; Widhelm and Lumbsch, 2011), a new family should be created to
4 accommodate the monophyletic assembly of Varicellaria and Variolaria (Schmitt et al., 2010) or
5 otherwise the Agyriaceae should be simply subsumed within Pertusariaceae, as suggested in
6 Lendemer and Hodkinson (2012). To account for future alternative relationships among
7 members of Pertusaria s. l. and the remaining families in Pertusariales, two distinct families
8 Variolaceae and Varicellariacea could be recognized as proposed in Lendemer and Hodkinson
9 (2012). However, this would result in more monogeneric families, which is not an informative
10 taxonomic practice.
11 In the Megasporaceae clade (Fig. 1B), Sagedia is nested within Aspicilia with significant
12 support, questioning the validity of this genus. The genus Sagedia was previously reintroduced
13 by Nordin et al. (2010), although the monophyly of Aspicilia without Sagedia was not
14 significantly supported in that study (significant posterior probability only, but see Alfaro et al.,
15 2003). Based on the same two loci (nucLSU and mitSSU), Sohrabi et al. (2013) confirmed a
16 distinct relationship between Circinaria and Aspicilia s.str. (with strong support), but the
17 segregation of Sagedia remained uncertain because the monophyly of Aspicilia was not
18 supported and both genera shared a most recent common ancestor. The family affiliation of the
19 genus Aspilidea in the Pertusariales (Nordin et al., 2010) is not resolved, although its close
20 relationship with the Megasporaceae + Ochrolechiaceae clade was significantly supported (Fig.
21 1B). Interestingly, both Ochrolechia and Aspilidea host lichenicolous fungi from the genus
22 Sagediopsis (Verrucariales). In the Icmadophilaceae, the genus Siphula is not monophyletic.
24 A. Sagedia should be included in Aspicilia.
25 B. Agyrium should be transferred to the Pertusariaceae and the Agyriaceae should be
26 synonymized with the Pertusariaceae.
27 C. Siphula needs a systematic revision within the framework of the Icmadophilaceae.
28
29 3.5.6 Sarrameanales/Sarrameanaceae (Fig. 1A)
30 The recently introduced order Sarrameanales, resulting from the first divergence within
31 the Ostropomycetidae clade (Lumbsch et al., 2007b; Miadlikowska et al., 2006; Schoch et al.,
27
1 2009), which includes a single family – Sarrameanaceae (= Loxosporaceae; Kantvilas, 2004;
2 Lumbsch et al., 2007a), is monophyletic and well supported (Fig. 1A). In our phylogeny, the
3 Sarrameanaceae, exemplified by the genus Loxospora, because no sequence data are available
4 for Sarrameana, forms a clade with the non-monophyletic genus Schaereria as demonstrated
5 earlier and discussed in Schmull et al. (2011). It is very likely that S. corticola, which seems to
6 be more closely related to Loxospora than to the remaining members of its own genus, represents
7 a distinct genus outside of the Schaereriaceae (highly supported in Schmull et al., 2011).
8 However, its close morphological affinity to S. parasemella (currently Hafellnera; Houmeau and
9 Roux, 1984) may indicate that S. corticola shares a more recent common ancestor with members
10 of the genus Hafellnera. The subclass Ostropomycetidae is not well supported when the
11 Sarrameanaceae and Schaereriaceae are included (Figs. 1B and 2, but see Miadlikowska et al.,
12 2006). Although it is difficult to explain the sister relationship of Schaereria and Loxospora due
13 to substantial anatomical and chemical differences between these two genera (Hafellner, 1984;
14 Wirth, 1995; see discussion in Schmull et al., 2011), delimitation of the order Sarrameanales may
15 be extended to include Schaereria within its own family or as part of the Sarrameanaceae,
16 depending on the results of future phylogenetic studies.
18 A. Schaereria corticola should be transferred to a separate genus (perhaps closely related to
19 Hafellnera).
20 B. The Schaereriaceae and Sarrameanaceae need to be subjected to a systematic revision within
21 the phylogenetic framework of the Lecanoromycetes.
22
23 3.5.7 Trapeliales/Trapeliaceae (Fig. B-C)
24 As currently delimited, the order Trapeliales includes a single family, Trapeliaceae
25 (Hodkinson and Lendemer, 2011), formerly placed in the Agyriales (Lumbsch et al., 2001) and
26 the Baeomycetales (Lumbsch and Huhndorf, 2010). The monophyly of the Trapeliales,
27 previously demonstrated in several studies (e.g., as Agyriaceae in Miadlikowska et al., 2006),
28 received significant support in the 5+4-gene dataset analysis. The order forms a highly supported
29 monophyletic group together with the Arctomiales and Ostropales (Figs. 1B-D and 2). In
30 addition to the four genera traditionally classified in the Trapeliaceae (Orceolina, Placopsis,
31 Trapelia and Trapeliopsis; Hertel, 1970), our phylogeny confirms that Aspiciliopsis,
28
1 Placynthiella, Ptychographa, Rimularia and Xylographa belong to this family (Lumbsch et al.,
2 2001; Schmitt et al., 2003). All genera with multiple representative species, except Placopsis
3 (the only tri-membered genus within this family), are strongly supported as monophyletic, at
4 least based on two of the four datasets analyzed in this study. Based on our phylogeny, the genus
5 Sarea (S. resinae), which was provisionally placed in the Trapeliaceae (Hodkinson and
6 Lendemer, 2011), does not belong to this family and the Lecanoromycetes in general (Figs. 1A
7 and 2), as demonstrated earlier by Reeb et al. (2004). However, this result needs to be confirmed
8 with the sequencing of at least one additional specimen.
10 A. More specimens of Sarea need to be sequenced to confirm their current phylogenetic
11 placement outside Lecanoromycetes.
12
13 3.6 Umbilicariomycetidae/Umbilicariales (Fig. 1D)
14 As suggested by Miadlikowska et al. (2006), a new order, Umbilicariales (Hibbett et al.,
15 2007; Zhou and Wei, 2007), and recently a subclass, Umbilicariomycetidae (Bendiksby and
16 Timdal, 2013) were established. The current classification for Umbilicariales includes the family
17 Umbilicariaceae (Lumbsch and Huhndorf, 2010) or the families Umbilicariaceae, Elixiaceae,
18 Fuscideaceae, and Ophioparmaceae (Kirk et al., 2008). Depending on the taxon sampling, the
19 following families are included in the Umbilicariomycetidae clade: Umbilicariaceae, Elixiaceae,
20 and Fuscideaceae in Tehler and Wedin (2008); Umbilicariaceae, Ophioparmaceae, and
21 Fuscideaceae in Miadlikowska et al. (2006); and Umbilicariaceae, Elixiaceae, and
22 Ophioparmaceae in Bendiksby and Timdal (2013), a study where the subclass was formally
23 described. An additional family, the Ropalosporaceae (part of Fuscideaceae s.l.), although
24 without support, was also shown to be part of this order (Bylin et al., 2007).
25 Here Umbilicariomycetidae is monophyletic with all five families included; however, the
26 first well-supported clade (corresponding to Umbilicariales sensu Bendiksby and Timdal [2013])
27 excludes Fuscideaceae and Ropalosporaceae (Fig. 1D). Strongly supported as monophyletic, the
28 Umbilicariaceae contains two umbilicate and intermixed, genera – Umbilicaria and Lasallia. The
29 independent generic status of Lasallia separated from Umbilicaria has often been questioned; for
30 example Frey (1933) considered Lasallia as a subgenus. A recent molecular study (Hestmark et
31 al., 2011) suggested that selected species of Lasallia represent a well defined group recognizable
29
1 molecularly and morphologically (based on the average thallus size and color rather than features
2 associated with the anatomy or reproductive structures) at the same level as several other distinct
3 groups within Umbilicaria, e.g. the vellea group, the cylindrica group or the hyperborea group.
4 Overall, interspecific relationships among Umbilicaria species are not well established based on
5 existing phylogenies; however, no discrepancy was detected for significantly supported clades
6 across phylogenetic studies (Fig.1D; Hestmark et al., 2011; McCune and Curtis, 2012). Our
7 analyses revealed two well-supported clades representing the main division within this family –
8 Umbilicaria s. str. and a clade including a monophyletic Lasallia and remaining Umbilicaria
9 species (i.e., Lasallia + ‘Umbilicaria’ pro parte) (Fig. 1D). Because our phylogeny includes
10 approximately one-third of all recognized Umbilicaria species, and half of Lasallia species only,
11 a broader taxon sampling is necessary before taxonomic conclusions can be made for these two
12 genera. Based on our most comprehensive supermatrix (5+4+3+2-gene) and resulting bootstrap
13 support ≥ 70%, Umbilicariaceae includes also the epiphytic genus Xylopsora (Hypocenomyce
14 friesii group; Bendiksby and Timdal, 2013). The placement of Elixia flexella (Elixiaceae) is well
15 supported within the Umbilicariaceae + Ophioparmaceae clade and was shown to be sister to
16 Umbilicariaceae based on an extensive taxon sampling in Bendiksby and Timdal (2013). The
17 genus Fuscidea (Fuscideaceae) may contain multiple genera (not monophyletic in this
18 phylogeny; Fig. 1D) if Maronea (Fuscideaceae, see also Reeb et al., 2004 and Miadlikowska et
19 al., 2006) is recognized as a separate genus.
21 A. In addition to the Umbilicariaceae, Ophioparmaceae, and Elixiaceae, the families
22 Fuscideaceae and Ropalosporaceae should be considered as members of the Umbilicariales or as
23 separate orders in the Umbilicariomycetidae.
24 B. Lasallia should be synonymized with Umbilicaria or additional genera should be recognized
25 in the Umbilicaria/Lasallia complex based on a more extensive taxon sampling.
26 C. Maronea was confirmed as belonging to the family Fuscideaceae.
27 D. Fuscideaceae and the genus Fuscidea require a phylogenetic revision within the phylogenetic
28 framework of Umbilicariomycetidae. Fuscidea mollis may represent a separate genus in the
29 family Fuscideaceae.
30
31 3.7 Lecanoromycetidae: relationships among orders (Fig. 1D-K)
30
1 None of the phylogenetic relationships among orders of this subclass was supported by
2 bootstrap support ≥ 70%. This is due, in large part, to the addition of 1043 OTUs to the
3 supermatrix used by Miadlikowska et al. (2006), most of which are members of the
4 Lecanoromycetidae clade and are represented by only two of the five genes. However, most of
5 these deep internodes were consistently reconstructed based on different subsets of our
6 supermatrix (Figs. 1D-K and 2), and some of them were highly supported in previous studies.
7 For example, the sister group relationship of the orders Teloschistales and Caliciales (as
8 circumscribed in Gaya et al., 2012) and the close affiliation with the Lecanorales received
9 significant support in Miadlikowska et al. (2006), but was not supported in this study. Different
10 relationships among these orders were reconstructed (with significant support) by Gaya et al.
11 (2012). In that study, which was based on the same five markers but used drastically different
12 taxon sampling (focussing on Teloschistales), Teloschistales was sister to Lecanorales and both
13 formed a monophyletic clade with the Caliciales. We lost support for the sister relationships
14 between the orders Lecideales and Peltigerales (recovered from one of the four datasets) which
15 was highly supported in Miadlikowska et al. (2006; but see Gaya et al., 2012). The recently
16 introduced order Leprocaulales (Fig. 1G), with a single family Leprocaulaceae, seems to be more
17 closely related to the Teloschistales (sister to Teloschistales in all analyses, but without
18 significant support) than to the Caliciales as suggested by Lendemer and Hodkinson (2013).
19 In agreement with Miadlikowska et al. (2006), the Rhizocarpales (including Sporastatiaceae;
20 Fig 1D-E) is monophyletic and is derived from the first split in the Lecanoromycetideae clade.
21 Within Lecanoromycetidae, a few lineages (represented mostly by a single species) were found
22 to be distantly related to all recognized families: Haematomma accolens, Psorula rufonigra, and
23 Helocarpon crassipes together with Lecidea diapensiae.
24 The genus Scoliciosporum (Scoliciosporaceae) was found to be polyphyletic (Fig. 1H), with
25 S. schadeanum nested in the Ramalinaceae clade, S. intrusum in the Pilocarpaceae clade, and the
26 type species, S. umbrinum, resolved as a distinct lineage in the Lecanorales. Phenotypical traits
27 including, in part, the presence of lobaric acid, suggest that Scoliciosporum schadeanum forms a
28 distinct morphological group in the Ramalinaceae, together with other taxa not sampled in this
29 study: S. pruinosum, S. pennsylvanica, Bacidia lobarica, members of the ‘Bacidia lutescens
30 group’ sensu Ekman (1996), the Australian genus Jarmania, and Myrionora (Harris, 2009;
31 Palice et al., 2013). Although poorly supported, Scoliciosporum intrusum is shown to be closely
31
1 affiliated with members of Micarea (in the Pilocarpaceae), a relationship confirming its former
2 classification in this genus as Micarea intrusa (Th.Fr.) Coppins & H. Kilias based on phenotypic
3 similarities (Coppins, 1983).
4 The genus Haematomma (monogeneric family Haematommaceae) was revealed here as a
5 heterogenous assembly of unrelated taxa based on available sequences (Fig. 1H-I). The type
6 species, H. ochroleucum, is nested in Lecanora (Lecanoraceae, see also Ekman et al., 2008).
7 Haematomma persoonii falls within the Lecanoraceae clade but without support for its exact
8 phylogenetic position. The resolution of H. accolens, depicted as the result from the first
9 divergence within the Lecanorales clade, is possibly due to an erroneous nucSSU sequence (see
10 Supplemental Table S1). Contrary to the results presented here, the genus was shown previously
11 to be monophyletic based on nucLSU and mitSSU data (Lumbsch et al., 2008) and seems to be
12 well defined morphologically and chemically. The individual we used to represent Haematomma
13 ochroleucum (collection from Norway; Ekman 3184) might have been confused with the
14 overlooked species Lecanora thysanophora R. C. Harris. Harris et al. (2000) report that both
15 species look similar when sterile (thick, sorediate, yellow thallus occasionally sourrounded by a
16 distinct prothallus) and often share a similar chemistry (presence of atranorin, usnic acid, zeorin
17 and porphyrylic acid).
18 The monotypic Psorula rufonigra (a lichenized fungus forming a unique lichenicolous
19 association with a filamentous cyanolichen Spilonema revertens; Timdal, 2002) is separated
20 from the remaining members of Psoraceae (8 individuals representing 7 species) where it is
21 currently classified (Lumbsch and Huhndorf, 2010) and may represent an undescribed family in
22 the Lecanoromycetidae (Figs. 1F-H, 2; but see Ekman et al., 2008).
23 The type species of the genus Helocarpon (H. crassipes, Lecanoromycetes inc. sed.
24 according to Lumbsch and Huhndorf, 2010) forms, together with Lecidea diapensiae (Group 3 in
25 Schmull et al., 2011), a distinct lineage within Lecanoromycetidae (Figs. 1F, 2), to which the
26 family name Helocarpaceae (Hafellner, 1984) can be applied (see also Andersen and Ekman,
27 2005). However, it was indicated (by an anonymous reviewer) that the specimen representing H.
28 crassipes, the only collection for which sequence data is available in GenBank, was
29 misidentified. To fully circumscribe the family Helocarpaceae, reliable collections of
30 Helocarpon, Micareopsis (Lendemer et al., 2013) and other potentially affiliated taxa (Ekman et
32
1 al., 2008; Schmull et al., 2011) should be included in a comprehensive multilocus phylogenetic
2 analysis.
4 A. Scoliciosporum (Scoliciosporaceae) and Psorula (Psoraceae) need taxonomic revisions within
5 the phylogenetic framework of the Lecanoromycetidae. Scoliciosporum intrusum should be
6 reverted to Micarea intrusa.
7 B. Haematomma (Haematommaceae) phylogenetic relationships should be revisited within the
8 phylogenetic framework of the Lecanoromycetidae using more genes.
9 C. The resurrection of Helocarpaceae (Lecanoromycetidae inc. sed.) should be revisited based on
10 more complete taxon and locus sampling.
11
12 3.7.1 Caliciales and Teloschistales (Fig. 1F-H)
13 The recently resurrected order Caliciales (Gaya et al., 2012; i.e., Physciinae sensu
14 Miadlikowska et al., 2006) includes two families: Caliciaceae (with two subfamilies,
15 Calicioideae and Buellioideae) and Physciaceae. The monophyly of the families and subfamilies
16 as delimited by Gaya et al. (2012) is highly supported here (Figs. 1F-H, 2). Lopadium disciforme
17 was resolved in a sister position to the Caliciales by all phylogenetic analyses that included this
18 species, but never with bootstrap support values ≥ 70%. This phylogenetic uncertainty and
19 uniqueness is reflected by its tentative classification within the Ectolechiaceae (Kirk et al., 2008)
20 and Pilocarpaceae (Lumbsch and Huhndorf, 2010). See section 3.7.2.5 for the proposed
21 classification of Lopadium disciforme within the context of its current classification in the family
22 Pilocarpaceae.
23 In the family Caliciaceae, the genera Buellia and Cyphelium are not monophyletic.
24 Buellia dispersa and C. inquinans appear as lineages distinct from their respective genera
25 (anchored by their type species). Dimelaena radiata is closely related to Buellia tesserata (Gaya
26 et al., 2012) as corroborated by their shared chemical (3-chlorodivaricatic acid), morphological
27 (except crustose versus placodioid thallus growth), ecological and biogeographical
28 characteristics (Rico et al., 2003). Both taxa should be recognized under the same generic name
29 (Buellia or Dimelaena) depending on the future taxonomic treatments (Rico et al., 2003) and the
30 phylogenetic position of the type species D. oreina.
33
1 In the Physciaceae, the genus Physconia is reconstructed as paraphyletic with high
2 support (P. enteroxantha is placed outside of the Anaptychia+Physconia clade). Gaya et al.
3 (2012) reported that Physconia enteroxantha (and its close relative P. venusta) occupies an
4 uncertain position within this clade. The monophyly of Physconia will be retained if Anaptychia
5 is included (Helms et al., 2003), or if a separate genus is introduced to accommodate P.
6 enteroxantha and its relatives. A more comprehensive phylogeny of the Physciaceae is needed
7 before a decision can be made.
8 The recently redelimited order Teloschistales (Physciinae excluded) is restricted to four
9 families (Brigantiaeaceae, Letrouitiaceae, Megalosporaceae, and Teloschistaceae) classified in
10 two suborders, Teloschistineae and Letrouitineae (Gaya et al., 2012). The Teloschistaceae
11 includes three subfamilies: Caloplacoideae, Teloschistoideae, and Xanthorioideae (Gaya et al.,
12 2012; Arup et al., 2013). Monophyly of these taxa and their phylogenetic relationships received
13 high bootstrap support from nearly all analyses (Fig. 1G-H) and are in agreement with Gaya et
14 al. (2012). We confirm that a comprehensive taxonomic treatment is urgently needed for the
15 genera Xanthoria and Caloplaca, reconstructed as highly polyphyletic (divided into two and five
16 distinct clades, respectively) across the Teloschistaceae (Gaya et al., 2012). The recently
17 proposed 31 new and resurrected genera as a result of a phylogenetic study centered on the
18 family Teloschistaceae using ribosomal RNA-coding genes (Arup et al., 2013) requires a
19 complete reassessment using more genes (protein-coding), as some of these genera, and several
20 relationships among genera, were not strongly supported as monophyletic.
21 Several species groups previously included within Xanthoria have been recently
22 transferred to new genera (e.g., Fedorenko et al. 2009, 2012). Delimitation of the South African
23 genus Xanthodactylon is problematic in our tree because X. turbinatum is affiliated with the
24 Rusavskia clade (former Xanthoria elegans group; Kondratyuk and Kärnefelt 2003; Fig. 1G).
25 Contrary to Gaya et al. (2012), the genus Fulgensia is not monophyletic here (C.
26 xanthostigmoidea was not included in Gaya et al., 2012) and together with Seirophora, these two
27 genera are nested within the Caloplaca s. str. clade. If C. xanthostigmoidea represents C.
28 epiphyta, which shows phenotypic similarities with Fulgensia its phylogenetic placement in the
29 Fulgensia clade would support its transfer to that genus. The Caloplacoideae clade is in need of a
30 thorough revision and with the inclusion of more taxa, in combination with multiple loci, it is
31 very likely that several well-delimited groups will be transferred to new genera (e.g., C.
34
1 carphinea group; see Arup et al., 2103, for their proposal). If this is the case, the taxonomic
2 delimitation of Fulgensia and Seirophora will have to be reassessed.
3 Similarly, the clades labeled ‘Caloplaca’ II (sister to Teloschistes in the subfamily
4 Teloschistoideae and comprising some taxa from the Southern Hemisphere), ‘Caloplaca’ III
5 (including mostly species with reduced thalli), ‘Caloplaca’ IV (delimiting mainly the C. saxicola
6 group), and ‘Caloplaca’ V (including mostly maritime species or such with parasitic
7 preferences), might become subject to new generic delimitations in upcoming studies.
8 Conversely, the genera Teloschistes and Xanthomendoza are recovered as monophyletic in
9 accordance to previous phylogenies (Gaya et al. 2012, Arup et al. 2013). We accept a broad
10 delimitation of Xanthomendoza including the recently introduced generic names within this
11 genus (Fedorenko et al. 2012) because the morphological traits used to circumscribe these new
12 entities do not reflect evolutionary relationships within the Xanthomendoza clade. Our phylogeny
13 strongly supports the expansion of the family Megalosporaceae to include the genus Sipmaniella
14 (formerly Lecanoraceae) as demonstrated earlier (Gaya et. al., 2012).
16 A. The genera Buellia s.l., Dimelaena, and Physconia need taxonomic revisions within the
17 phylogenetic framework of the Caliciales (sensu Gaya et al., 2012).
18 B. The genera Caloplaca, Fulgensia, and Seirophora need taxonomic revision within the
19 phylogenetic framework of the Teloschistaceae (sensu Gaya et al., 2012).
20 C. Caloplaca verruculifera should be transferred to the genus Massjukiella.
21 D. Fulgensia and Seirophora should be synonymized with Caloplaca or smaller generic units
22 should be described to accommodate these taxa.
23 E. ‘Caloplaca’ II, III, IV, and V clades should be transferred to new genera if a narrower concept
24 for Caloplaca is applied, or to new subgenera.
25 F. Sipmaniella should be transferred from the Lecanoraceae to the Megalosporaceae.
26 G. Brigantiaeaceae should be classified within the Teloschistales.
27
28 3.7.2 Lecanorales (Fig. 1H-K)
29 The Lecanorales is the largest and most diverse order of the class Lecanoromycetes
30 (Kirk et al., 2008; Lumbsch and Huhndorf, 2010). From a total of 20 families currently
31 recognized in this order, members of three families (Pachyascaceae, Calycidiaceae and the
35
1 recently introduced Carbonicolaceae [Bendiksby and Timdal, 2013]) were not sampled in our
2 study because DNA sequences for these taxa were not available when we assembled our
3 supermatrix. Here we report that the order Lecanorales encompasses all families classified in this
4 order, with high bootstrap support, except Dactylosporaceae and Strangosporaceae, which are
5 resolved outside of the OSLEUM clade (Figs. 1A and 2).
6 Although most families in the Lecanorales were confirmed with high confidence as
7 monophyletic by our phylogenetic analyses, several families should be re-circumscribed to
8 accommodate taxa that were erroneously classified based on false interpretation of phenotypic
9 trait similarity/dissimilarity. Our results confirm the polyphyletic nature of the families
10 Lecideaceae, Pilocarpaceae, Psoraceae, and Ramalinaceae as presently delimited. A few
11 independent lineages emerged that deserve to be recognized at the family level, e.g.,
12 Ramboldiaceae and Psilolechiaceae (both introduced here).
13 All recognized families in the Lecanorales are grouped within two main sister clades: a
14 well-supported clade corresponding to the suborder Lecanorineae and a poorly supported clade
15 encompassing families classified in the suborders Psorineae + Sphaerophorineae (Tehler and
16 Wedin, 2008; Fig. 2). Scoliciosporum umbrinum (Scoliciosporaceae) and Haematomma accolens
17 both appear outside the two major clades. Both genera and their respective monogeneric families
18 are polyphyletic, with the remaining members placed in various families (highly supported in
19 some analyses) in the Lecanorales: S. intrusum in the Pilocarpaceae, Sphaerophorineae (possible
20 artifact; see Ekman et al., 2008); S. schadeanum in the Ramalinaceae, Sphaerophorineae; and
21 Haematomma ochroleucum (perhaps misidentified with Lecanora thysanophora) and H.
22 persoonii in the Lecanoraceae, Lecanorineae. This phylogenetic split of the Lecanorales into two
23 major groups was previously recovered in Ekman et al. (2008), but the Lecanorineae was poorly
24 supported in that study. The suborder Lecanorineae consists of the monophyletic Cladoniineae
25 sensu Poelt (1974), with Stereocaulaceae and Squamarinaceae sister to each other (but see
26 Ekman et al., 2008) and their close relative, Cladoniaceae (Miadlikowska et al., 2006; Myllys et
27 al., 2005; Wedin et al., 2000).
28 The family Squamarinaceae was introduced by Hafellner (1984) to accommodate a
29 single genus, Squamarina, which was later transferred to Stereocaulaceae (Lumbsch and
30 Huhndorf, 2010). A re-circumscription of the Squamarinaceae, to include the genus Herteliana
36
1 (currently in the Ramalinaceae), should be considered, based on previous results (H. taylorii
2 sister to S. lentigera in Ekman et al., 2008), and on our study confirming this result (Fig. 1H).
3 The Cladoniineae, together with Lecanoraceae, Gypsoplacaceae, Malmideaceae (and
4 other potentially affiliated taxa), and Ramboldia (Kantvilas and Elix, 1994; Ramboldiaceae
5 introduced below), form a well-supported monophyletic clade, sister to the Parmeliaceae (based
6 on the 5-gene dataset; Figs. 1H-K and 2). This large monophyletic entity is sister to a lineage
7 comprising two monogeneric families, Mycoblastaceae and Tephromelataceae, currently in
8 synonymy (Lumbsch and Huhndorf, 2010). Although the Lecanorineae was also recovered in
9 Ekman et al. (2008), a few internal relationships differ from our phylogeny, e.g., a sister
10 relationship of Parmeliaceae with Mycoblastaceae, as well as the Cladoniaceae and
11 Stereocaulaceae forming a monophyletic group.
12 The Sphaerophorineae clade includes the family Sphaerophoraceae (Wedin and Döring,
13 1999) and the non-monophyletic Psorineae (Tehler and Wedin, 2008), where Ramalinaceae and
14 Psoraceae form a well-supported clade and Pilocarpaceae are sister to Sphaerophoraceae (but
15 without bootstrap values ≥ 70%). Similar clades were reconstructed with high PP by Ekman et
16 al. (2008) and Miadlikowska et al. (2006), although the Pilocarpaceae were not included in the
17 latter study. The genus Psilolechia (including the type species) is part of a lineage distinct from
18 the Pilocarpaceae, where it was tentatively classified (Lumbsch and Huhndorf, 2010).
19 Psilolechia seems to be derived from the first split within Sphaerophorineae (based on our taxon
20 sampling) and is here recognized at the family level (Psilolechiaceae; Fig 1H). A few highly
21 supported relationships within the order Lecanorales obtained here contradict results from former
22 studies, such as the reported sister relationship between Parmeliaceae and Gypsoplacaceae by
23 Arup et al. (2007) that received 76% ML bootstrap support based on combined nucLSU and
24 nucSSU analyses.
26 Family: Ramboldiaceae S. Stenroos, Miadl. & Lutzoni fam. nov.
28 Type genus: Ramboldia Kantvilas & Elix
29 Diagnosis – A monogeneric family in the suborder Lecanorineae (Lecanorales,
30 Lecanoromycetidae) comprising species with lecideoid apothecia with an unpigmented (in some
31 species pigmented apothecia occur due to the presence of russulone and related anthraquinones)
37
1 prosoplectenchymatous exciple, a Lecanora-type ascus, sparsely branched and anastomosing
2 paraphyses, and simple, hyaline, non-halonate ascospores.
3 Ramboldia Kantvilas & Elix, Bryologist 97: 296 (1994)
4 Type species: R. stuartii (Hampe) Kantvilas & Elix 1994
5
6 Family: Psilolechiaceae S. Stenroos, Miadl. & Lutzoni fam. nov.
7 Mycobank no.: MB XXXXX.
8 Type genus: Psilolechia A. Massal.
9 Diagnosis – A monogeneric family classified in the order Lecanorales (Lecanoromycetidae,
10 Lecanoromycetes) comprising crustose lichens with effuse, ecorticate, leprose thalli formed by
11 goniocysts containing Trebouxia or stichococcoid algae. Apothecia immarginate, asci 8-spored,
12 cylindrical-clavate, with a central, elongate K/I+ dark blue tube-like structure
13 ('Röhrenstrukturen'; see Hafellner, 1984), non-amyloid ascus wall surrounded by a thin,
14 K/I+dark blue outer layer. Ascospores are oblong-ovoid to dacryoid, simple (rarely 1-septate in
15 P. leprosa), hyaline. All species of Psilolechia grow in shaded, humid places.
16 Psilolechia A. Massal., Atti Reale Ist. Veneto Sci. Lett. Arti, Sér. 3 5: 264 (1860)
17 Type species: P. lucida (Ach.) M. Choisy 1949
18
19 A. The suborder Lecanorineae includes: Cladoniaceae, Gypsoplacaceae, Lecanoraceae,
20 Mycoblastaceae, Parmeliaceae, Ramboldiaceae, Squamarinaceae, Stereocaulaceae, and
21 Tephromelataceae.
22 B. The suborder Sphaerophorineae is re-delimited to include the former Psorineae
23 (Pilocarpaceae, Psoraceae and Ramalinaceae) and Psilolechiaceae.
24 C. Scoliciosporum and Haematomma are in need of a taxonomic revision within the broad
25 phylogenetic context of the Lecanorales.
26
27 3.7.2.1 Lecanoraceae (Fig. 1I)
28 In addition to the genera currently classified in the Lecanoraceae (Lecanora, Lecidella,
29 Protoparmeliopsis, and Rhizoplaca; Lumbsch and Huhndorf, 2010), the family contains other
30 taxa that were anticipated to belong to different families based on morphological and anatomical
31 characters, e.g., Japewia tornoënsis and Frutidella currently classified in the Ramalinaceae,
38
1 Haematomma persoonii in the Haematommaceae, Glyphopeltis ligustica in the Psoraceae, and
2 Lecidea turgidula in the Lecideaceae. The placement of Japewia, Frutidella and Lecidea
3 turgidula has been shown to be problematic by Schmull et al. (2011), who also proposed that
4 Japewia and Frutidella should be recognized as members of Lecanoraceae. This result is
5 confirmed here, with high bootstrap support (Fig. 1I). The genera Lecidella and
6 Protoparmeliopsis (which are in needs of a conserved name status and representative type; see
7 Knudsen and Lendemer [2009]) are monophyletic. However, only the latter is highly supported.
8 The genus Lecanora is highly polyphyletic with its members spread across the family.
9 Here the most inclusive monophyletic entity for this genus includes the L. subfusca group,
10 considered as Lecanora s.str. (L. allophana included; Brodo and Vitikainen, 1984), as well as the
11 L. symmicta and L. rupicola groups, intermixed with other species of Lecanora and the following
12 unrelated taxa: Haematomma ochroleucum (possibly a misidentified sterile Lecanora
13 thysanophora), Pyrrhospora quernea (Lumbsch et al., 2004) and Ramboldia insidiosa (Kalb et
14 al., 2008; Fig. 1I). Lecanora glabrata, a potential member of the L. subfusca group (Arup et al.,
15 2007), came out distantly related to taxa from that group. Two other groups of Lecanora, the L.
16 dispersa and L. polytropa groups, are placed outside the monophyletic genus Lecanora as
17 circumscribed here (Fig. 1I). The well-supported monophyly of Protoparmeliopsis and its
18 phylogenetic placement within Lecanoraceae supports its re-instoration as a distinct genus from
19 Lecanora (see Lumbsch and Huhndorf, 2010). This genus, with additional Lecanora species s. l.,
20 Rhizoplaca and two members of Micarea (M. incrassata and M. assimilata) together form a
21 well-supported clade. Delimitation of genera and phylogenetic relationships within Lecanoraceae
22 are currently very unstable and a substantial improvement in resolution and support must be
23 achieved by sequencing a minimum of four to five loci before formal changes can be made.
25 A. The Lecanoraceae and the genus Lecanora need systematic revision within the phylogenetic
26 context of the Lecanorales.
27 B. Frutidella and Japewia need to be transferred from Ramalinaceae to Lecanoraceae as
28 recommended by Schmull et al. (2011).
29 C. Glyphopeltis should be transferred from Psoraceae to Lecanoraceae if its phylogenetic
30 placement in Lecanoraceae is confirmed.
39
1 D. Pyrrhospora quernea and Ramboldia insidiosa should be transferred to Lecanora within the
2 broad circumscription of that genus proposed here, once their phylogenetic placement is
3 confirmed with additional specimens.
4
5 3.7.2.2 Parmeliaceae (Fig. 1I-K)
6 The overall delimitation of genera and major clades in the speciose family Parmeliaceae
7 presented here is in agreement with the large scale and collaborative study by Crespo et al.
8 (2010). This congruence and stability might reflect the benefit of adopting a strategy combining
9 a comprehensive taxon sampling with sequencing a high number of the same genes used
10 throughout the Lecanoromycetes (Figs. 3 and 4). In some cases, our Lecanoromycetes-wide
11 study provided more resolution and support for the Parmeliaceae (Fig. 1I-K). In the parmelioid
12 group (Fig. 1J-K), the Parmotrema clade is sister to the Xanthoparmelia clade in both studies.
13 However, the genus Canoparmelia does not represent the first split in the Parmotrema clade as
14 shown in Crespo et al. (2010), but appears to be rather sister to Xanthoparmelia (with high
15 support from the 5+4+3-gene dataset). Punctelia seems to be more closely related to
16 Flavopunctelia (a former subgenus of Punctelia containing usnic acid instead of atranorin in the
17 cortex) than to lichenicolous Nesolechia, contrary to the alternative placement in Crespo et al.
18 (2010). In our trees, Punctelia subrudecta, which corresponds to P. subrudecta 2 in Crespo et al.
19 (2010), is nested (for an unknown reason) in Parmotrema with high support, rather than with
20 members of its own genus as previously reported.
21 The genus Cetrelia (Cetrelia clade in Crespo et al., 2010) is nested in a highly supported
22 Parmelina clade (uncertain placement in Crespo et al., 2010). The Parmelia clade, excluding
23 Relicina, is monophyletic and highly supported. It was resolved as sister to Parmeliopsis (highly
24 supported by our 5+4-gene analysis), a genus recognized as a separate clade in Crespo et al.
25 (2010). Relicina was nested in the Parmelia clade in Crespo et al. (2010), but occupies a separate
26 uncertain position in our phylogeny.
27 Everniastrum lipidiferum does not cluster with the remaining specimens from this genus
28 (see also Divakar et al., 2006) and is possibly affiliated with Cetrariastrum as shown in Crespo
29 et al. (2010). The newly introduced genus Montanelia, to accommodate the former Melanelia
30 disjuncta group (Divakar et al., 2012), is sister to Pleurosticta and part of the extended, highly
40
1 supported Melanohalea clade (Crespo et al., 2010), which holds an uncertain placement outside
2 the delimited parmelioid clades sensu Divakar et al. (2006).
3 Similar to previously published phylogenies (Crespo et al., 2007, 2010; Thell et al., 2009,
4 2012), the backbone structure among the “other non-parmelioid group” of Parmeliaceae
5 generally did not receive bootstrap support above 70%. However, many genera represented by at
6 least two species are shown to be monophyletic with high confidence (e.g., Alectoria,
7 Arctoparmelia, Asahinea, Brodoa, Cetraria, Evernia, Menegazzia, Platismatia, Pseudevernia,
8 Vulpicida, and Usnea) and their sister relationships are well established. For example, the
9 alectorioid group, as defined in Crespo et al. (2007; Alectoria, Pseudephebe and Sulcaria),
10 should be expanded to include also the closely related monophyletic genus Bryoria (together
11 with Bryocaulon divergens), which occupied an uncertain position in the Parmeliaceae in past
12 studies. The genus Gowardia forms a sister group relationship to Alectoria (well supported) and
13 can be kept as a separate genus following the Halonen et al. (2009) circumscription, or can be
14 treated as a member of Alectoria following Lumbsch and Huhndorf (2010). The distant
15 relationship between Usnea and Protousnea, where the latter is sister to Pannoparmelia
16 (although not highly supported), was also recovered in Crespo et al. (2010). The paraphyletic
17 relationship (without support in previous studies) between Platismatia and Imshaugia was
18 replaced here by a well-supported monophyletic relationship (Fig. 1I-J).
19 The cetrarioid core group was reconstructed as monophyletic (but not strongly supported,
20 if Melanelia is included) and in general agreement with Thell et al. (2009) and Nelsen et al.
21 (2011). Our study confirms the monophyly of the hypogymnioid group as previously
22 circumscribed (Crespo et al., 2007), possibly with the genus Evernia resulting from the first
23 phylogenetic split in this clade. Contrary to Crespo et al. (2007), we did not recover the
24 letharioid clade. Here, Letharia is resolved sister to Asahina (not supported) within a very
25 unstable part of the Parmeliaceae tree (Fig. 1J).
27 A. Everniastrum lipidiferum does not seem to belong to this genus if phylogenetic analyses of
28 sequences from other individuals of this species confirm our and previous results.
29 B. Bryocaulon divergens needs to be transferred to the genus Bryoria if phylogenetic analyses of
30 sequences from other individuals of this species confirm our results.
31
41
1 3.7.2.3 Remaining families in the Lecanorineae (Figs. 1I and 2)
2 Initially the family Gypsoplacaceae was established for a single species –Gypsoplaca
3 macrophylla (Timdal, 1990). Here, this genus is nested within a monophyletic group (highly
4 supported by the 5+4-gene analysis) encompassing several mostly corticolous members of
5 ‘Lecidea’ and the saxicolous Miriquidica garovaglii (Lecanoraceae). In Schmull et al. (2011),
6 these ‘Lecidea’ species were reconstructed with a very low confidence as part of Group 1 in the
7 Lecanoraceae and Group 2 in the Pilocarpaceae. Recently the family Malmideaceae has been
8 recognized to accommodate mostly corticolous species from the tropics previously classified in
9 the Lecidea piperis- and Lecanora granifera-groups (Kalb et al., 2011) and a very distinctive
10 new genus, Savoromala from Madagascar (Ertz et al., 2013). In our phylogeny, Lecidea
11 floridensis, a member of the new genus Malmidea together with L. cyrtidia (see Ertz et al. 2013),
12 and another unidentified species of the genus Lecidea represent a highly supported
13 Malmideaceae (Fig. 1I). A sister relationship between Malmideaceae and Gypsoplacaceae,
14 revealed in this study (uncertain placement in the Lecanorales in Ertz et al. [2013]), did not
15 receive strong support. Incomplete sampling within the Malmideacea and lack of obvious
16 morphological similarities with Gypsoplaca and other members of Lecideaceae s.l. (e.g., Lecidea
17 nylanderi) do not support a formal expansion of the family Gypsoplacaceae.
18 Within the Cladoniaceae, Metus and Pycnothelia form a sister group closely related to
19 Cladonia, whereas the remaining genera Cladia (including Ramalinora glaucolivida) and
20 Pilophorus show unsettled placements in the family. The phylogenetic placement of the genus
21 Hertelidea in the Stereocaulaceae (Lumbsch and Huhndorf, 2010) is confirmed for the first time
22 based on molecular data.
24 A. The taxonomy of ,members of the families Malmideaceae and Gypsoplacaceae, and their
25 close relatives, needs to be reassessed within a more comprehensive phylogenetic context.
26
27 3.7.2.4 Ramalinaceae (Fig. 1H)
28 In its current circumscription, the family Ramalinaceae is not monophyletic. The following
29 genera should be excluded from the family and placed elsewhere in the Lecanorales: Herteliana
30 (type species placed in the Squamarinaceae), Schadonia (one representative placed in the
31 Pilocarpaceae), Sipmaniella (one representative placed in the Megalosporaceae), Frutidella, and
42
1 Japewia (type species placed in the Lecanoraceae; Schmull et al., 2011). The remaining genera
2 classified in the Ramalinaceae form a well-supported monophyletic group, including the
3 monogeneric Crocyniaceae and the family Megalariaceae (type species sampled) as well as
4 selected members of the Catillariaceae, Lecideaceae, Pilocarpaceae and Scoliciosporaceae. A
5 similar phylogenetic delimitation of the family, but with different taxon samplings was shown in
6 previous studies (Andersen and Ekman, 2005; Ekman et al., 2008; Reese Næsborg et al., 2007;
7 Miadlikowska et al., 2006; Schmull et al., 2011), with the exception of Byssolecania variabilis,
8 which was resolved within Pilocarpaceae (where it is currently classified) based on mitSSU and
9 nucLSU sequences (Nelsen et al., 2008; Andersen and Ekman, 2005), and Scoliciosporum
10 schadeanum (Scoliciosporaceae), which was never sampled before. In light of this and
11 previously published phylogenies, the family Crocyniaceae should be synonymized with
12 Ramalinaceae. It is uncertain if the entire family Megalariaceae is nested within Ramalinaceae
13 because the genus Tasmidella was not sampled (Catillochroma was recently synonymized with
14 Megalaria by Fryday and Lendemer, 2010; but see Lücking et al., 2011).
15 Our study revealed a non-monophyletic delimitation of the genera Bacidina, Lecania,
16 Mycobilimbia (Reese Naesborg et al., 2007), and Toninia, although high support was received
17 only for the placement of L. chloritiza outside the genus Lecania s.str., previously reported in
18 Sérusiaux et al. (2012). There are many validly published genera within Ramalinaceae that were
19 never sequenced that could potentially accommodate L. chlorotiza, L. naegelii and L. falcata
20 (Sérusiaux et al., 2012) outside of the genus Lecania s.str. (Fig.1H). The genus Bilimbia
21 (Lecanoromycetes inc. sed.) belongs to the Ramalinaceae and should retain its distinct status
22 from Mycobilimbia. Our data revealed two main lineages within the Ramalinaceae s.l.,
23 corresponding to the Ramalinaceae s.str. (poorly supported) and the Bacidiaceae (highly
24 supported), as circumscribed in many earlier papers (the most recent being Sérusiaux et al.
25 2012).
27 A. Herteliana should be transferred from the Ramalinaceae to the resurrected Squamarinaceae.
28 B. Japewia and Frutidella should be removed from the Ramalinaceae and classified within
29 Lecanoraceae.
30 C. Mycobilimbia needs a systematic revision within the phylogenetic context of the
31 Lecanoromycetidae, and should be transferred from the Lecideaceae to the Ramalinaceae.
43
1 D. Bilimbia can now be classified within the the Ramalinaceae.
2 E. Bacidina, Toninia and Lecania need systematic revisions within the context of the family
3 Ramalinaceae.
4 F. Crocynia should be transferred to the Ramalinaceae and the family Crocyniaceae
5 synonymized with Ramalinaceae.
6 G. The genus Megalaria should be transferred from the Megalariaceae to the Ramalinaceae and
7 the Megalariaceae should be synonymized with the latter family.
8 H. The phylogenetic placement of Byssolecania should be revisited within the context of the
9 Lecanorales and based on a more extensive sampling of this genus.
10
11 3.7.2.5 Remaining families in the Sphaerophorineae + Psorineae clade (Figs. 1H and 2)
12 The well-delimited Psoraceae contains two monophyletic genera, Psora and
13 Protoblastenia (poorly supported), as well as Micarea sylvicola (Ekman et al., 2008), a current
14 member of the Pilocarpaceae. The genus Micarea should be subjected to a phylogenetically
15 based taxonomic revision, as it consists of unrelated taxa placed in the Lecanoraceae (Fig. 1I)
16 and Psoraceae (Fig. 1H), whereas the core of this genus (the former Micareaceae) belongs to the
17 Pilocarpaceae (Ekman et al., 2008). Protomicarea limosa (Psoraceae) belongs to
18 Sphaerophoraceae as already demonstrated by Ekman et al. (2008).
19 Here we report that the family Pilocarpaceae (highly supported based on two of four
20 datasets; Fig. 1H) also includes members of the Ramalinaceae (Schadonia fecunda; see also
21 Ekman et al., 2008) and Scoliciosporaceae (Scoliciosporum intrusum; lacking morphological
22 justification for this phylogenetic placement). The Pilocarpaceae is revealed as highly
23 heterogeneous, with many members resolved outside this family, closely affiliated with families
24 in the Lecanorales (Psoraceae, Ramalinaceae, and Lecanoraceae) and with Lecideaceae, or as
25 separate lineages. For example, the genus Psilolechia, classified tentatively in the Pilocarpaceae
26 (Lumbsch and Huhndorf, 2010) is placed outside of recognized families in Sphaerophorineae
27 (with high support in some analyses) as the first evolutionary split within this clade (but not
28 supported). It is recognized here in its own family (Psilolechiaceae; Fig. 1H). The inclusion of
29 Catillaria erysiboides (Catillariaceae) in Psilolechiaceae did not receive significant support, and
30 therefore should be revisited in future studies. Another putative member of the Pilocarpaceae
31 (Lumbsch and Huhndorf, 2010), the genus Lopadium (represented here by L. disciforme),
44
1 occupies an uncertain position in the Lecanoromycetidae outside the Pilocarpaceae and perhaps
2 the Lecanorales in general (appears closely related to the Caliciales but not supported; Figs. 1F
3 and 2). Its isolated position within the Lecanoromycetidae may justify resurrecting the family
4 Lopadiaceae (Hafellner, 1984).
6 A. The family Lopadiaceae (Lecanoromycetidae inc. sed.) should be resurrected to accommodate
7 Lopadium s.str. if our results are confirmed with additional sequences from additional species.
8 B. Micarea is not monophyletic and needs a phylogenetically-based revision within the
9 Lecanorales framework.
10 C. The Catillariaceae and Catillaria are not monophyletic and need a systematic revision within
11 the Lecanoromycetidae framework. New genera should be created to accommodate C.
12 scotinoides in the Ramalinaceae, C. erysiboides in the Psilolechiaceae, and C. modesta in the
13 Lecideaceae if these results are confirmed with additional specimens from these species.
14 D. A new genus should be created to accommodate Micarea sylvicola in the Psoraceae if further
15 studies confirm this result.
16 E. Psilolechia should be removed from the Pilocarpaceae and classified together with Catillaria
17 erysiboides (under a new genus name, if confirmed with additional data) in Psilolechiaceae
18 (Lecanorales).
19 F. Protomicarea should be transferred from the Psoraceae to the Sphaerophoraceae.
20 G. More individuals of Miriquidica should be included in phylogenetic analyses before
21 transferring the genus from the Lecanoraceae to the Gypsoplacaceae.
22 H. Glyphopeltis may belong to the Lecanoraceae, not the Psoraceae, but additional data are
23 needed, including from the type species.
24
25
26 3.7.3 Lecideales (Fig. 1E)
27 The order Lecideales was recently resurrected to accommodate a single family,
28 Lecideaceae, now restricted to saxicolous species from the genera Lecidea s.str. (sensu Hertel)
29 including the type species (L. fuscoatra), and Porpidia (Schmull et al., 2011). Our phylogeny
30 confirms with high support the monophyletic delimitation of Lecideaceae sensu Schmull et al.,
31 (2011). Several newly added saxicolous species of Lecidea (e.g., L. plana) and members of
45
1 Porpidia (e.g., P. carlottiana, P. macrocarpa, P. melinodes, P. contraponenda, P. crustulata), as
2 well as the genus Bellemerea (including B. alpina, the type species) and Lecidoma demissum
3 (part of unsupported Group 3 in Schmull et al., 2011) cluster without support within the
4 Lecideaceae (Lumbsch and Huhndorf, 2010). Moreover, the genus Arthrorhaphis
5 (Arthrorhaphidaceae) considered a member of uncertain placement in the Ostropomycetidae
6 (Lumbsch and Huhndorf, 2010) and shown as such in previous phylogenies (e.g., Miadlikowska
7 et al., 2006; Lumbsch et al., 2007b), is unexpectedly found here to be affiliated with the
8 Lecideaceae; however, this is not supported. Surprisingly, two other morphologically distinct
9 taxa, Leimonis erratica (classified and placed in the Pilocarpaceae, but not supported in Schmull
10 et al., 2011) and Catillaria modesta (Catillariaceae) are also part of the extended Lecideaceae,
11 but without support. This and previous studies have already demonstrated that the genera
12 Porpidia and Lecidea s.str. are not monophyletic and therefore must be redefined based on a
13 more comprehensive and stable phylogeny for this group. Contrary to the phylogenetic studies
14 focusing on the former family Porpidiaceae (Buschbom and Mueller, 2004; Buschbom and
15 Barker, 2006), an individual of Porpidia speirea (different specimen and markers used) is nested
16 in the Lecidea s.str. clade (Miadlikowska et al., 2006; Schmull et al., 2011), indicating a
17 misidentification or heterogeneity for this species. Two potential members of the Lecideaceae,
18 the monotypic genus Romjularia and its closest relative Mycobilimbia berengeriana (possibly
19 Romjularia) are separated from the remaining members of Lecideaceae, but without high
20 confidence.
22 A. Lecidea, Mycobilimbia, and Porpidia are not monophyletic and need systematic revisions
23 within the phylogenetic framework of the Lecanoromycetidae.
24 B. The order Lecideales should be accepted to accommodate the family Lecideaceae s.str.,
25 although its final delimitation must be assessed with more loci to improve phylogenetic
26 confidence.
27
28 3.7.4 Leprocaulales (Fig. 1G)
29 Leprocaulaceae was originally created for two genera, Halecania (not represented in this
30 study) and Leprocaulon (Lendemer and Hodkinson, 2013). It comprised morphologically
31 diverse, primarily sterile, asexually reproducing lichens that produce pannarin, argopsin, and
46
1 usnic acid. Fertile taxa have Halecania-type asci, lecanorine apothecia, and hyaline ascospores
2 that resemble those of Lecania. Based on our results, the circumscription of the Leprocaulaceae
3 could be extended to include the monotypic genus Speerschneidera (S. euploca; Lendemer and
4 Hodkinson, 2013; Nelsen et al., 2008) and Solenopsora candicans (Catillariaceae), two taxa
5 forming a robust monophyletic group with Leprocaulon quisquiliare (Fig. 1G), as well as
6 Catinaria atropurpurea (not included in our study, but see Ekman et al., 2008).
7 Although included in the study by Lendemer and Hodkinson (2013), S. euploca was
8 excluded from their taxonomic conclusions because of its nucLSU sequence (AY300862) which
9 blasts with 100% identity to a sequence of Catinaria atropurpurea from the Ramalinaceae
10 (AY756347), the family in which S. euploca has traditionally been classified (but currently
11 classified in the Lecanorales inc. sed.; Lumbsch and Huhndorf, 2010). The high similarity of the
12 nucLSU sequences between these two genera can be explained because C. atropurpurea does not
13 cluster with the Ramalinaceae but instead forms a sister relationship with Halecania alpivaga,
14 currently a member of the Leprocaulaceae (Ekman et al., 2008).
15 The genus Solenopsora comprises approximately 19 published species (Kirk et al., 2008)
16 of diverse morphology ranging from crustose with immersed to emergent apothecia, as for
17 example in S. candicans, to squamulose or small foliose with substipitate apothecia, as seen in S.
18 holophaea (the type species of the genus). Despite the broad range of gross morphology, all
19 species of Solenopsora are united in having eight-spored, Catillaria type asci, simple paraphyses
20 with internally brown pigmented, clavate apices, and hyaline, one-septate, non-halonate
21 ascospores. On the basis of these anatomical characters, the genus is classified in the family
22 Catillariaceae (Lumbsch and Huhndorf, 2010; Verdon and Rambold, 1998).
24 A. Speerschneidera should be transferred from the Lecanorales inc. sed. to the Leprocaulaceae
25 (Leprocaulales).
26 B. Proper delimitations of the genera Solenopsora, Halecania, and Catinaria require further
27 taxonomic revisionary studies.
28
29 3.7.5 Peltigerales (Fig. 1E-F)
30 Currently, Peltigerales includes ten families (Spribille and Muggia, 2012) grouped in two
31 suborders, Collematineae and Peltigerineae (Miadlikowska and Lutzoni, 2004). The
47
1 Collematineae consists of four families (Coccocarpiaceae, Collemataceae, Pannariaceae and
2 Placynthiaceae), whereas the Peltigerineae includes six families (Lobariaceae, Massalongiaceae,
3 Nephromataceae, Peltigeraceae, and the most recently added Koerberiaceae and Vahliellaceae;
4 Miadlikowska and Lutzoni, 2004; Muggia et al., 2011; Spribille and Muggia, 2012; Wedin et al.,
5 2007, 2011). The first four families listed above for Peltigerineae form a well-supported clade
6 (Fig. 1F), to which Vahliellaceae is sister (but without support values ≥ 70%), whereas
7 Koerberiaceae is resolved here as sister to Collematineae (a relationship also not well supported).
8 The monophyly of Peltigerineae with Vahliellaceae and Koerberiaceae was supported only by
9 posterior probabilities in Spribille and Muggia (2012; see Alfaro et al. 2003). Therefore, these
10 two families hold an uncertain phylogenetic placement in the order Peltigerales. However, the
11 family Koerberiaceae seems to be more closely related to the suborder Collematineae than
12 Peltigerineae (Fig. 1E; see also Muggia et al., 2011).
13 Within Peltigerineae, the sister relationship between Peltigeraceae and Lobariaceae, as
14 well as their close affiliation with the Nephromataceae and Massalongiaceae are confirmed
15 (Miadlikowska et al., 2006) with strong bootstrap support for 11 of 12 internodal bootstrap
16 values reported here (Fig. 1F). These phylogenetic relationships contradict (sometimes with
17 conflicting high support values) the results from other studies on the Peltigerales based on a
18 different taxon sampling (Muggia et al., 2011; Wedin et al., 2009; Wedin et al., 2007; Wiklund
19 and Wedin, 2003; Spribille and Muggia, 2012) and on a different selection of loci (exclusively
20 ribosomal except RPB1 in Wedin et al., 2009). Here for the first time, the monophyly of the
21 Collematineae, including all families traditionally included in this suborder, received high
22 bootstrap support. We recovered a stable sister relationship between the Collemataceae and the
23 Placynthiaceae. This clade is closely related to the Coccocarpiaceae (significantly supported
24 based on two of four datasets), a family of unresolved (e.g., Muggia et al., 2011; Spribille and
25 Muggia, 2012) or unsupported (Wedin et al., 2009) phylogenetic placement in past studies.
26 Except for Lobaria and Pseudocyphellaria, all genera in the Lobariaceae, including
27 newly described genera (Moncada et al., 2013) are monophyletic and for the most part received
28 at least one bootstrap value ≥ 70%. Our results do not support the recognition of Anomalobaria
29 as a separate genus (nested in Lobaria; Fig. 1F). In the study by Moncada et al. (2013) based on
30 combined ribosomal loci, the monophyly of Lobaria without Anomalobaria (former
31 Pseudocyphellaria anomala group) received only 47% of ML bootstrap support, suggesting that
48
1 the split between these two genera may be artificial. Three representatives of the former Lobaria
2 s.l. (L. hallii, L. meridionalis, L. pseudoglaberrima) and Pseudocyphellaria rainierensis – none
3 of which was included in Moncada et al. (2013) – cannot be assigned to newly circumscribed
4 genera with certainty based on this phylogeny. A close relationship of P. rainierensis with
5 members of Lobarina was shown in Miadlikowska and Lutzoni (2004), but without support.
6 Reading errors within the conserved parts of the nucLSU sequence of P. rainieriensis
7 (AF401963) identified in Moncada et al. (2013), did not effect the phylogenetic placement of this
8 taxon because they were part of ambiguous regions excluded from all analyses. To avoid the
9 recognition of additional genera (for example to accommodate L. meridionalis) and to allow the
10 classification of existing species of Lobaria, a broad delimitation of this genus (Lobaria s. l. in
11 Fig. 1F) to reinclude newly proposed Ricasolia, Lobariella, Lobarina and perhaps
12 Pseudocyphellaria rainierensis should be reconsidered.
13 An unexpectedly high level of support was obtained for intrageneric relationships within
14 the genus Peltigera (Peltigeraceae). The latest global phylogeny for Peltigera based on
15 concatenated nucLSU sequences and phenotypic characters (54 individuals from 40 putative
16 species) allowed the circumscription, with high confidence, of eight sections within the genus;
17 however, their relationships were largely unsettled (Miadlikowska and Lutzoni, 2000). Our
18 5+4+3+2-gene dataset contains fewer individuals and fewer taxa than sampled in 2000 (33
19 individuals from 30 putative species), but half of them are represented by more than three genes
20 including part of RPB1 or RPB2. All sections, except section Peltigera (the P. canina group), are
21 monophyletic, although one (section Polydactylon) is not well supported and three others
22 (sections Chloropeltigera, Phlebia and Retifoveatae) are represented by only one species.
23 Contrary to Miadlikowska and Lutzoni (2000), all sorediate members from the P. canina group
24 (section Peltigera I in Fig. 1F) are separated from the remaining species of that section (section
25 Peltigera II in Fig. 1F) by sections Retifoveatae and Horizontales (sister to Peltigera II clade in
26 Fig. 1F). This phylogenetic relationship was reconstructed and supported based only on the
27 5+4+3-gene dataset. All tri-membered Peltigera species (part of three sections: Chloropeltigera,
28 Phlebia and Peltidea) share a most recent common ancestor (high support in the 5+4-gene
29 dataset), suggesting a single, or two at the most (depending on the placement of the bi-membered
30 species P. malacea and P. frippi in the section Peltidea) independent acquisitions of the green
31 photobiont Coccomyxa during the evolutionary history of the genus (Miadlikowska and Lutzoni,
49
1 2004). Section Polydactylon forms a well supported monophyletic clade with this tri-membered
2 sections. The aquatic species P. hydrothyria is part of this clade, but its phylogenetic placement
3 remains uncertain. Another aquatic Peltigera species (Peltigera sp. nov. from Chile), which will
4 be formally described as part of a revisionary work on the section Peltigera in a separate
5 publication, is nested in the P. canina section (Peltigera II clade; Fig. 1F).
6 Our results highly support the recent recircumscription of the genera Collema and
7 Leptogium (Otálora et al., 2013a, 2013b) and their sister relationship in the absence of
8 Paracollema (not sampled in this study; Fig. 1E-F). Strongly supported monophyletic
9 circumscription of Scytinium, Lathagrium, and Enchylium (based on the combined nucLSU,
10 mitSSU, MCM7, and beta-tubulin in Otálora et al., 2013a, 2013b) was not revealed in this study.
11 Moreover, members of these three newly delimited genera of the former Collema/Leptogium
12 complex are intermixed with high bootstrap support (mostly from the 5+4+3+2-gene dataset).
13 Placement of Pseudoleptogium as the first split in Collemataceae is in agreement with Otálora et
14 al., 2013a, b.
15 Our results support the exclusion of Physma and Staurolemma from Collemataceae
16 (despite their homiomerous thallus and lack of detectable secondary metabolites shared with
17 members of Collema and Leptogium), as well as their transfer to the Pannariaceae (Otálora et al.,
18 2010a; Spribille and Muggia, 2012; Wedin et al., 2009). A single member of the genus
19 Ramalodium included in our analyses is nested within the genus Staurolemma (but see Wedin et
20 al., 2009). Both genera share asci without apical amyloid structure, but the phylogenetic identity
21 of Ramalodium needs to be confirmed with a broader sampling. Degelia durietzii and D. gayana
22 (type species) are more closely related to Leioderma and Erioderma than to D. plumbea, which
23 is placed sister to Parmeliella (Muggia et al., 2011).
25 A. Anomalobaria, Lobariella, Lobarina and Ricasolia could be re-included in the genus Lobaria
26 to prevent the erection of more new genus names to accommodate the newly proposed
27 classification within Lobaria s.l.
28 B. The identity and phylogenetic placement of the specimen representing Pseudocyphellaria
29 rainierensis should be reassessed based on additional samples.
30 C. Degelia needs to be subjected to a systematic revision within the phylogenetic framework of
31 Pannariaceae.
50
1 D. Leptochidium should be transferred from Placynthiaceae to Massalongiaceae (see also
2 Muggia et al., 2011).
3 E. The identity and phylogenetic placement of Ramalodium succulentum within Staurolemma
4 should be reassessed based on additional samples.
5 F. Steinera should be removed from Coccocarpiaceae and Koerberia from Placynthiaceae to be
6 classified, together with Vestergrenopsis, in the family Koerberiaceae (Spribille and Muggia,
7 2012), a family that should be recognized as an incertae sedis within Peltigerales.
8 G. The newly established genera Scytinium, Lathagrium, and Enchylium need to be reassessed
9 because they are likely non-monophyletic.
10
11 3.7.6 Rhizocarpales (Fig. 1D-E)
12 The order Rhizocarpales, introduced by Miadlikowska et al. (2006), includes a single family
13 (Rhizocarpaceae) and the genus Sporastatia (formerly in the Catillariaceae) recently classified in
14 a separate family, Sporastatiaceae, outside of the Rhizocarpales by Bendiksby and Timdal
15 (2013). In the past, the sister relationship between the genera Sporastatia and Rhizocarpon
16 (Rhizocarpaceae) was demonstrated in several phylogenies (Buschbom and Mueller, 2004;
17 Lutzoni et al., 2004; Miadlikowska et al., 2006; Reeb et al., 2004); however, none of them
18 included the genus Toensbergia (formerly Hypocenomyce or Pycnora leucococca). Here we
19 confirm, with strong support from all four analyses, the close relationship between Rhizocarpon
20 and Sporastatia (Fig. 1D). Overall we also recovered the same phylogenetic placement of this
21 order within Lecanoromycetes, but without support values ≥ 70% (Fig. 2). The phylogenetic
22 position of the Rhizocarpales as the first split within the Lecanoromycetidae (as defined here),
23 although often reconstructed, has rarely been supported (Miadlikowska et al., 2006). This is the
24 second time that we show that Catolechia wahlenbergii (the type species of the genus) is nested
25 within Rhizocarpon (see Miadlikowska et al., 2006).
27 A. The monotypic genus Catolechia should be subsumed within Rhizocarpon.
28 B. The genus Sporastatia should be transferred from the Catillariaceae to the Rhizocarpaceae or,
29 alternatively, be accommodated in Sporastatiaceae (together with Toensbergia; Bendiksby and
30 Timdal, 2013) as the second family in the Rhizocarpales.
31
51
1 3.8 Photobiont distribution across Lecanoromycetes
2 A comprehensive overview on the photobionts associated with members of Lecanoromycetes
3 (Fig. 2), based on available data, is included in Gueidan et al. (2014), Miadlikowska et al.
4 (2006), Voytsekhovich et al. (2011b) and literature cited therein. The most frequently reported
5 photobionts across the Lecanoromycetes are coccoid green algae (Chlorophyta) representing the
6 following genera: Trebouxia, Asterochloris, Dictyochloropsis s.l., Coccomyxa, Myrmecia,
7 Pseudococcomyxa, Stichococcus and Chlorella. For many lichens, the photobiont is reported as
8 an unidentified unicellular alga (e.g., in the Pilocarpaceae, Gypsoplacaceae, Rhizocarpaceae, and
9 Acarosporaceae), reflecting the difficulty to identify these algae and generally the state of the
10 systematic classification of lichen photobionts (Fig. 2). In the case of Trebouxia, which is one of
11 the most common chlorobionts reported for the Lecanoromycetidae, Umbilicariomycetidae,
12 ‘Candelariomycetidae’ and selected lineages in Ostropomycetidae (e.g., Graphidaceae,
13 Pertusariales), its identity needs to be verified in many groups as it may represent the closely
14 related genus Asterochloris or other unidentified chlorococcoid genera. Filamentous green algae
15 such as Trentepohlia and Phycopeltis (Trentepohliaceae) are restricted to most lineages in the
16 Ostropales and family Hymeneliaceae, all in the subclass Ostropomycetidae. These lichens are
17 usually common in humid and shaded habitats, on trees predominantly in tropics (members of
18 the Ostropales) or on rocks (Hymeneliaceae).
19 Many lineages across the Lecanoromycetes are associated with one photobiont type, a
20 trend especially visible in trentepohlioid lichens in the Ostropales, where only a few have
21 switched to the coccoid type, (e.g., Phlyctidaceae or Graphidaceae), or have lost their photobiont
22 completely (Stictidaceae and Odontotremataceae s.str.; Lutzoni et al., 2001; Wedin et al., 2005,
23 2006; Baloch et al., 2012). The same applies to the large lineage comprising the Parmeliaceae
24 and affiliated families in the Lecanorineae or in the Umbilicariales, which are strictly associated
25 with Trebouxia/Asterochloris as the main photobiont according to published records. The
26 multilocus evolutionary study by Dal Grande et al. (2014) demonstrated that the algal genus
27 Dictyochloropsis associated with macrolichens from the family Lobariaceae (Peltigerales) is
28 polyphyletic and that members of a single clade (a putative new genus) are involved in symbiotic
29 associations with mycobionts from this family and other lichen genera in Lecanoromycetes
30 (Brigantaea and Megalospora [Teloschistales], Phlyctis [Ostropales], and Biatora
31 [Ramalinaceae, Lecanorales]. Usually a single photobiont has been reported to be associated
52
1 with a mycobiont in bi-membered symbiosis (e.g., Dal Grande et al, 2014); however, recently
2 multiple green algae in addition to their main photobiont were isolated from the thalli of selected
3 species of Micarea and Placynthiella in Ukraine (del Hoyo et al., 2011, Voytsekhovich et al.,
4 2011c).
5 Cyanobacteria as primary photobionts are restricted to the order Peltigerales in the
6 Lecanoromycetes, and two unrelated lineages in the Ostropomycetidae: Arctomiaceae (most of
7 which grow on bryophytes in Arctic and subarctic areas) and in Petractis clausa, the only
8 cyanobacteria-associating member of this genus as currently circumscribed (a genus which is
9 primarily associated with Trentepohlia, as are most chlorolichens in the order Ostropales).
10 Nostoc is present in all families of the order Peltigerales, but only in a few of them as the
11 exclusive primary cyanobiont (for example in the Collemataceae, Massalongiaceae,
12 Nephromataceae, Peltigeraceae, and Vahliellaceae). In addition to Nostoc, other filamentous
13 cyanobacteria are associated with mycobionts in all lineages of Collematineae (except
14 Collemataceae) and a single family (Lobariaceae) in the suborder Peltigerineae. The only other
15 lichen lineage with cyanobacteria as the primary photobiont (the class Lichinomycetes) is
16 associated with various filamentous and coccoid cyanobacteria (e.g., Gleocapsa and Anacystis)
17 other than Nostoc, except for Lemphollema, which is associated with the latter.
18 Tri-membered associations with cyanobacteria as secondary photobionts (within
19 cephalodia) in addition to green algae, are very rare outside of the Peltigerales suborder
20 Peltigerineae (Lobariaceae, Peltigeraceae, and Nephromataceae) and the Pannariaceae
21 (Collematineae). They can be found in other genera within the Lecanoromycetidae such as in the
22 genera Pilophorus (Cladoniaceae), Stereocaulon (Stereocaulaceae), Amygdalaria, and
23 Rhizocarpon (R. hensseniae; not sampled in this study), as well as in several genera in the
24 Pilocarpaceae. Cephalodia can also be found in the subclass Ostropomycetidae, for example in
25 the genera Coccotrema (Coccotremataceae, Pertusariales) and Placopsis (Trapeliaceae,
26 Trapeliales). Micarea assimilata and M. incrassata, two of the cephalodiate members of the
27 Pilocarpaceae, are shown with high confidence to belong to the Lecanoraceae based on two out
28 of the four datasets analyzed (Fig. 1I). All tri-membered lichens in the Peltigerales contain
29 exclusively Nostoc as the secondary photobiont despite the presence of other types of
30 cyanobacteria as the primary photobionts in members of many families in this order. With a few
31 exceptions, for example Placopsis from Trapeliaceae (Raggio et al., 2012) and Stereocaulon
53
1 from Sterocaulaceae (Voytsekhovich et al, 2011b), Nostoc was rarely reported to be an accessory
2 photobiont outside of the Peltigerales (instead, filamentous cyanobacteria are present).
3 According to the mycobiont phylogeny (Fig. 2), cyanobacteria were acquired independently
4 several times as the secondary photobiont in the predominantly chlorococcoid lichen lineages
5 outside of the Peltigerales, twice in the Lecanorales (Lecanoromycetidae), and twice in the
6 Ostropomycetidae (Trapeliaceae and Coccotremataceae). With the exception of tripartite
7 Pannariaceae having Myrmecia as the primary photobiont (Oksner, 1974; Spribille and Muggia,
8 2012), none of the other members of the Collematineae form symbiotic associations with green
9 algae, whereas in the Peltigerineae, Coccomyxa/Pseudococcomyxa are the primary photobiont in
10 several lineages within a clade comprising the Nephromataceae, Peltigeraceae and Lobariaceae.
11 In the latter family, Dictyochloropsis and other green algae are also involved as primary
12 photobionts (Fig. 2). In the genus Peltigera, the acquisition of Coccomyxa/Pseudococcomyxa
13 took place only once, leading to the diversification of tri-membered species within a single well
14 supported clade (sections Chloropeltigera, Peltidea, and Phlebia) together with two bi-
15 membered taxa, P. malacea and P. frippii; often expressing a high level of specificity toward
16 Nostoc (O’Brien et al., 2013). If the recent discovery that some bi-membered cyanobacterial
17 lichens (Pseudocyphellaria, Sticta and Peltigera) are cryptic tri-membered symbioses in which
18 the mycobiont has two ‘co-primary photobionts’, a cyanobacterium (dominant) and a green alga
19 (Henskens et al, 2013), is confirmed, and the identity of the cryptic algae revealed, this might
20 change profoundly our understanding of the evolution of symbiotic associations in Peltigerales
21 and other groups of lichens.
22 It is very likely that Scytonema, reported from many lichens as a primary cyanobiont
23 based on visual observations, represents another genus including a novel symbiotic lineage,
24 Rhizonema, which was identified in Stereocaulon, Coccocarpia and basidiolichens (Dictyonema
25 and Acantholichen) collected in the tropics. Rhizonema was predicted to be also associated with
26 members of the Pannariaceae, Bacidina spp., and selected genera in the Pilocarpaceae as an
27 accessory photobiont (Lücking et al., 2009).
28 In corroboration with phenotypic synapomorphies, photobionts can be helpful in the
29 delimitation of taxa at the family and higher ranks. For example the Teloschistales, Letrouitineae
30 and Megalosporaceae are associated with Dictyochloropsis (Gaya pers. comm.), contrary to the
31 members of the Teloschistaceae, which are associated with Trebouxia. In the order Lecanorales,
54
1 members of the family Psilolechiaceae have a strong preference for stichococcoid algae, a group
2 of photobionts most probably corresponding to the green algal genus Diplosphaera (see Thüs et
3 al., 2011). Photobiont-mycobiont associations can also explain in part relationships and
4 evolutionary history of Lecanoromycetes as suggested earlier by Rambold et al. (1998).
5 However, since 2006 no significant progress has been made, with the exception of Lücking et al.
6 (2009), Otalora et al. (2010), O’brien et al. (2013) and Dal Grande et al. (2014) toward an
7 inventory of chloro- and cyanobionts in the Lecanoromycetes using molecular markers. A good
8 example to follow is the survey of photobionts across Verrucariales (Eurotiomycetes) completed
9 recently by Thüs et al. (2011).
10
11 4. Conclusions
12 Figure 5 represents a revised classification based on published phylogenies and our
13 results. Our broad scale phylogeny (Fig. 1) demonstrated that monophyly of taxa (genera,
14 families, and orders) in the Lecanoromycetes should be assessed phylogenetically within a
15 broader taxonomic framework than is assumed based on their current classifications. Examples
16 include remnants of the Zahlbruckner’s families waiting for comprehensive taxonomical
17 treatments such as Catillariaceae and Lecideaceae, which are spread across the
18 Lecanoromycetidae; members of Ramalinaceae and Pilocarpaceae placed in different families in
19 the Lecanorales; and genera with members scattered in the Lecanoromycetidae. To reach this
20 goal, a drastic improvement in the robustness of future phylogenies on the Lecanoromycetes is
21 needed.
22 We predict, that expanding our datasets by adding taxa with a single gene will reduce the
23 stability of phylogenetic relationships (Fig. 4) due to the lack of characters for these taxa (see
24 Wiens and Morrill, 2011) paired with an increasing number of very short internodes
25 (Supplemental Fig. S1). This was already demonstrated in this study by adding taxa with two
26 genes to the 5+4+3-gene dataset used in Miadlikowska et al. (2006), as well as in the study by
27 Schmull et al. (2011). Although previous studies reported a poor relationship between intermodal
28 support and the amount of missing data when adding taxa under certain conditions (if the
29 remaining characters are invariant or saturated and/or when obvious rate heterogeneity is
30 ignored, e.g., Pyron and Wiens, 2011; Wiens and Morrill, 2011), we noticed that in the case of
31 Lecanoromycetes, bootstrap support for the clades containing mainly taxa with two loci only was
55
1 generally lower compare to clades with more genes. This difference can be explained in part by
2 the fact that our datasets contained a maximum of five loci (versus 12 in Pyron and Wiens, 2011)
3 and only two of them were single copy protein coding genes (versus 10 in Pyron and Wiens,
4 2011). Most individuals in our dataset (except the complete 5-gene) were represented exclusively
5 by ribosomal loci, mainly nucLSU and mitSSU (RPB1 and RPB2 were absent in 59% and 67%,
6 respectively, of all OTUs included in this study), which provided only 20% of the complete set
7 of the targeted 7,000 bp (5-gene dataset; Table 1) versus 12,712 bp in Pyron and Wiens (2011).
8 Ribosomal loci, although commonly used for reconstrucing phylogenies of various groups in
9 Lecanoromycetes and fungi in general, have relatively low phylogenetic informativeness (sensu
10 Townsend 2007), and therefore provide less phylogenetic resolving power for resolving
11 relationships across the Lecanoromycetes and Ascomycota compared to protein-coding genes
12 (Hofstetter et al., 2007; Schoch et al., 2009; Miadlikowska et al., in prep). By using a cumulative
13 stepmatrix approach (Miadlikowska et al., 2006; Gaya et al., 2012) we were able to monitor the
14 joint impact of the increasing number of taxa and missing sequences on the stability of the nodes
15 (Fig. 1).
16 Many of the clades that were weakly supported in this study (including the most complete
17 dataset) were also poorly supported in other studies focusing on specific groups witin the
18 Lecanoromycetes, and thus potentially with less missing data. The same is true for strongly
19 supported clades. Sequencing efforts for phylogenetic studies of Lecanoromycetes and fungi in
20 general cannot be limited anymore to the easily amplified ribosomal RNA-coding genes, but
21 should be extended to include protein-coding genes such as MCM7, RPB1 and RPB2 for at least
22 one representative from each genus. In systematic biology, new molecular markers, such as
23 MCM7 (e.g., for the Ostropomycetidae in Schmitt et al., 2010; Collemataceae in Otálora et al.,
24 2013a, 2013b) and markers derived from the AFTOL 2 project (Miadlikowska et al., in prep.),
25 are rarely developed and sequenced as part of studies on Lecanoromycetes. Yet, protein-coding
26 genes have been shown to have a major positive impact on phylogenetic resolution and
27 confidence for Lecanoromycetes (Reeb et al., 2004; Hofstetter et al., 2007), but can also
28 contribute to highly supported conflicting relationships (see Otálora et al, 2013a, 2013b versus
29 this study).
30 Currently, the need for more genes (characters) is higher than for more taxa for large-
31 scale phylogenetic studies within Lecanoromycetes (Fig. 4 and Supplemental Fig. S2). This
56
1 issue will be addressed, increasingly, with genomic sequencing directly from (environmental)
2 genomic DNA obtained from lichen thalli (the feasibility of which has been demonstrated by
3 McDonald et al., 2013). Nevertheless, in parallel to sequencing more genes, taxon sampling
4 should be increased to include species (with type species preferably) from genera and families,
5 which were never subjected to phylogenetic analyses (e.g., Biatorellaceae) or are
6 underrepresented in existing phylogenies (e.g., Catillariaceae). Phylogenetic studies of lichens
7 within the broader context of Leotiomyceta, including newly described classes (e.g.,
8 Xylonomycetes in Gazis et al., 2012 and Coniocybomycetes in Prieto et al., 2012), is also needed
9 for a stable and meaningful delimitation of the Lecanoromycetes and the accurate phylogenetic
10 placement of several lichenized lineages that do not belong to this class (Figs. 1 and 2). It is also
11 within the broader context of the Leotiomyceta that a better understanding of the origin of lichen
12 symbiosis can be achieved.
13 In large-scale phylogenetic studies (i.e., high number of taxa and genes) the only viable
14 support values currently available to estimate phylogenetic confidence seems to come from non-
15 parametric bootstrap datasets, especially when analyzed with maximum likelihood as the
16 optimization criterion. Conflicts that receive high bootstrap values with ML are rarely observed
17 across phylogenetic studies of the Lecanoromycetes. However, this is not the case for internodes
18 with high posterior probabilities, where most well supported conflicts are observed. Phylogenetic
19 trees including large numbers of taxa have high numbers of very short internodes. Simulation
20 studies have shown that current Bayesian methods can give high posterior probabilities for
21 wrong relationships (Alfaro et al. 2003, Lewis et al. 2005), especially for short internodes.
22 Moreover, current implementations of Bayesian phylogenetic methods cannot be completed on
23 datasets with large number of taxa, due to a lack of convergence by Monte Carlo Markov chains.
24 As shown here with our cumulative supermatrix approach (Miadlikowska et al. 2006, Gaya et al.
25 2012), different combinations of taxa and genes can yield different levels of supports (Figs. 1
26 and 2). Therefore, phylogenetic studies can be more inclusive, in terms of taxa with fewer genes,
27 and more conclusive, if multiple data sets (such as the four data sets used here; i.e., 5-gene, 5+4-
28 gene, 5+4+3-gene and 5+4+3+2-gene datasets) are used for bootstrap analyses instead of a single
29 ML bootstrap analysis.
30
31
57
1 Acknowledgements
2 We thank T. Milledge and J. Pormann for their assistance with the Duke DSCR computer cluster,
3 L. Bukovnik (Duke IGSP) for assistance with DNA sequencing, members of the Lutzoni lab for
4 helpful comments and suggestions, M. McMullen (Duke University Herbarium) for curating
5 lichen specimens and proofreading the manuscript, W. R. Buck and collaborators in the
6 Assembling the Fungal Tree of Life (AFTOL) project for providing lichen specimens, R. Harris
7 and T. Ahti for verifying identifications of selected specimens, and reviewers for helpful
8 comments on the manuscript. This publication was supported by NSF awards DEB-0228668 and
9 DEB-0919455 to FL, DEB-0732984 to FL and JM, DEB-0640996 to AEA and the Academy of
10 Finland (grant 211171) to SS. Additional financial support comes from NSF award DEB-
11 1025930 to JM and FL.
12
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9 Lichen. 38, 419-427.
10 Timdal, E., 2002. Psorula. In: Nash III, T.H., Ryan, B.D., Diederich, P., Gries, C., Bungartz, F.,
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16 Verdon, D., Rambold, G., 1998. A new species in the genus Solenopsora (Catillariaceae,
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19 vzaimootnosheniya s mikobiontom. LAP LAMBERT Academic Publishing, Saarbrucken,
20 Germany.
21 Voytsekhovich, A., Mikhailyuk, T.I., Darienko, T.M., 2011a. Lichen photobionts. 1:
22 Biodiversity, ecophysiology and co-evolution with the mycobiont. Algologia 21, 3-26.
23 Voytsekhovich, A., Mikhailyuk, T.I., Darienko, T.M., 2011b. Lichen photobionts. 2: origin and
24 correlation with mycobiont. Algologia 21, 151-177.
25 Voytsekhovich, A., Dymytrova, L., Nadyeina, O. 2011c. Photobiont composition of some taxa of
26 the genera Micarea and Placynthiella (Lecanoromycetes, lichenized Ascomycota) from
27 Ukraine. Folia Cryptog. Estonica 48, 135-148.
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4 Wedin, M., Döring, H., Gilenstam, G., 2006. Stictis s. lat. (Ostropales, Ascomycota) in northern
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9 Ascomycota). Lichenologist 39, 61-67.
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3
4 Figure captions
5 Figure 1. Phylogenetic relationships among 1307 putative members of the Lecanoromycetes
6 based on maximum likelihood analyses of the combined mitSSU, nucLSU, nucSSU, RPB1 and
7 RPB2 sequences (5+4+3+2-locus dataset) and 10 species used as outgroup (Geoglossomycetes,
8 Lichinomycetes and Leotiomycetes). Numbers in parentheses after taxon names indicate the
9 numbers of genes in our supermatrix for each OTU. Numbers (0, 1, 2) after taxon names indicate
10 presence of multiple specimens from the same taxon. Stars indicate type species. The four-box
11 grids associated with each internode indicate maximum likelihood bootstrap support based on
12 different datasets. Black boxes indicate bootstrap support ≥70%; white boxes indicate bootstrap
13 support <70%, blue boxes indicate cases where internodal support is not applicable due to at
14 least one of the (usually two) immediately downstream branches being absent (due to missing
15 taxa) or the node was not recovered by the bootstrap analysis (not present in the majority-rule
16 consensus tree with all compatible groupings included; 1% treshold); and red boxes indicate
17 conflicting relationships at bootstrap support ≥70%. If in disagreement, the current classification
18 is provided in square brackets. Taxa in red represent obvious phylogenetic misplacements
19 resulting, most likely, from erroneous sequences (see also Supplemental Table S1), misidentified
20 specimens, or stochastic error. Porina guentheri (2) and Ostropa barbara 0 (4) are represented
21 by erronous sequences; Placynthiella oligotropha 0 (3) is represented by an erroneous RPB1
22 sequence; Chroodiscus coccineus is represented by an erronous nucLSU sequence;
23 Haematomma accolens 0 (3) is represented by an erroneous nucSSU sequence; and H.
24 ochroleucum 0 (2) might represent sterile Lecanora thysanophora. Punctelia subrudecta 0 (2),
25 Hypotrachyna aff. 1 (2), and Lecanora conizaeoides 0 (3), are represented by correct sequences,
26 according to the BLAST searches, and therefore the reason for their unexpected placements is
27 unknown.
28
29 Figure 2. Schematic cladogram derived from the 5+4+3+2-gene phylogeny (Fig. 1) depicting
30 relationships among major phylogenetic lineages (families and above) reconstructed within the
31 Lecanoromycetes. If in disagreement, the current classification is provided in square brackets.
76
1 Bootstrap support is shown according to the legend and explanation provided for Fig. 1. Colored
2 branches indicate dominant type of primary photobiont and colored horizontal ovals indicate all
3 photobiont groups reported in association with mycobionts within order/family/phylogenetic
4 lineage based on available records for selected members classified in these taxa, even if not
5 present in the tree. Some records of Trebouxia may represent Asterochloris. Presence of
6 secondary photobionts (different genera or families of cyanobacteria) is indicated by vertical
7 ovals. Selected literature used for the photobiont assignment: Backor et al., 2010; Baloch et al.,
8 2010; Beck, 2002; Brodo, 1990; Brodo et al. 2001; Coppins and Purvis, 1987; Dal Grande et al.,
9 2014; Engelen et al., 2010; Fernández-Mendoza et al., 2011; del Hoyo et al, 2011; Friedl and
10 Büdel, 2008; Gueidan et al., 2014; Höggnaba et al., 2009; Kantvilas, 2004; Lücking et al., 2009;
11 Miadlikowska et al., 2006; Muggia et al., 2011; Nash et al. 2002, 2004; Otálora et al., 2010b,
12 2013c; Peksa and Skaloud, 2011; Purvis et al., 1992; Raggio et al., 2012; Skaloud and Peksa,
13 2010; Spribille and Muggia, 2012; Tschermak-Woess, 1988; Voytsekhovich, 2013;
14 Voytsekhovich et al., 2011a, b.
15
16 Figure 3. Comparison between average number of genes (right Y axis and red line) across
17 lineages in the Lecanoromycetes (Fig. 2) and stability of reconstructed phylogenies
18 approximated by the percentage of internodes with BS-ML ≥ 70% (blue shade; corresponding to
19 black boxes in Fig. 1), percentage of internodes with BS-ML < 70% (orange shade;
20 corresponding to white boxes in Fig. 1), and percentage of missing internodes in bootstrap
21 analyses (green shade; corresponding to blue boxes in Fig. 1) (Supplemental Table S2).
22 Numbers in parentheses after taxon names refer to the number of internodes (grids) included in
23 each clade. Average number of sequenced genes are also provided for larger taxonomic entities
24 (orders and subclasses) associated with horizontal brackets.
25
26 Figure 4. Strong positive correlation (p < 2.2e-16) between average number of genes included in
27 our supermatrix and the percentage of internodes with BS-ML ≥ 70%, based on results from
28 bootstrap analyses on 5-gene, 5+4-gene, 5+4+3-gene, and 5+4+3+2-gene datasets (i.e.
29 correlation between blue shade and red line in Fig. 3).
30
77
1 Figure 5. Schematic representation of phylogeny and classification of the class Lecanoromycetes
2 based on Gueidan et al. (2014) and modified according to relationships resulting from this study
3 and other most recent studies (see References). Phylogenetic relationships among families,
4 orders and subclasses were compiled based on published phylogenies and are shown as resolved
5 if reported with posterior probability ≥ 95% and/or maximum likelihood bootstrap ≥70% in
6 multiple studies (in most cases). The number of recognized families in each order is provided in
7 parentheses after the order name. Bars across branches indicate families with unknown
8 placement (not sampled, not monophyletic, not supported) in the Lecanoromycetes,
9 Lecanoromycetidae and Ostropomycetidae. Stars after family names indicate that they are not
10 represented in our datasets due to lack of DNA sequences (Pachyascaceae and Calycidiaceae) or
11 are represented by a single locus in GenBank. Question marks indicate that Dactylosporaceae
12 and Strangosporaceae may be placed outside of the Lecanoromycetes (as an independent lineage
13 in the Eurotiomycetes; according to Diederich et al., 2013; Schoch et al., 2009; Rossman et al.,
14 2010), as well as Pycnoraceae (according to this study). Sarea resinae (Trapeliales,
15 Lecanoromycetidae) and Biatoridium (Lecanoromycetes inc. sed.) are not depicted here.
16
17 Supplemental Figure S1. Phylogram representing phylogenetic relationships among 1307
18 putative members of the Lecanoromycetes based on maximum likelihood analyses of the
19 combined mitSSU, nucLSU, nucSSU, RPB1 and RPB2 sequences (5+4+3+2-locus dataset) and
20 10 species used as outgroup (Geoglossomycetes, Lichinomycetes and Leotiomycetes). Numbers
21 (0, 1, 2) after taxon names indicate presence of multiple specimens from the same taxon. The
22 four-box grids associated with each internode indicate maximum likelihood bootstrap support
23 based on different datasets. Black boxes indicate bootstrap support ≥70%; white boxes indicate
24 bootstrap support <70%, blue boxes indicate cases where internodal support is not applicable due
25 to at least one of the (usually two) immediately downstream branches being absent (due to
26 missing taxa) or the node was not recovered by the bootstrap analysis (not present in the
27 majority-rule consensus tree with all compatible groupings included; 1% treshold); and red boxes
28 indicate conflicting relationships at bootstrap support ≥70%.
29
30 Supplemental Figure S2. Non-significant correlation (p = 0.055) between the number of
31 internodes within each recognized lineage (Fig. 2) and the percentage of internodes with BS-ML
78
1 ≥ 70%, based on results from bootstrap analyses on 5-gene, 5+4-gene, 5+4+3-gene, and
2 5+4+3+2-gene datasets.
3
4
79
Xanthoparmelia sublaevis (2)
Xanthoparmelia conspersa 0 (5)*
Xanthoparmelia angustiphylla (2)
Above threshold Threshold values Xanthoparmelia lineola (2)
Below threshold Xanthoparmelia conspersa 1 (4)*
5+4+3+2-gene 70 70 5+4+3-gene Xanthoparmelia atticoides (2)
Missing or inapplicable Xanthoparmelia isidiovagans (2)
Conflict at ML-BP ≥70% 5+4-gene 70 70 5-gene Xanthoparmelia semiviridis 1 (4)
Xanthoparmelia semiviridis 0 (3)
Xanthoparmelia tinctina (2)
Xanthoparmelia exornata (3)
Xanthoparmelia subverrucigera (2)
Xanthoparmelia verrucigera (2)
Xanthoparmelia notata (2)
Xanthoparmelia subincerta (2)
Xanthoparmelia transvaalensis (2)
K Xanthoparmelia delisei (2)
Xanthoparmelia pokornyi (2)
Xanthoparmelia clade
Xanthoparmelia pulloides (2)
Xanthoparmelia pulla (2)
Xanthoparmelia aff. glabrans (2)
Xanthoparmelia loxodes 0 (2)
Xanthoparmelia ovealmbornii (2)
Xanthoparmelia azaniensis (3) Xanthoparmelia
Xanthoparmelia patagonica (2)
Xanthoparmelia subprolixa (2)
Xanthoparmelia glabrans (2)
Xanthoparmelia loxodes 1 (2)
Xanthoparmelia stenophylla (2)
Xanthoparmelia subdiffluens (2)
Xanthoparmelia protomatrae (2)
Xanthoparmelia mougeotii (3)
Xanthoparmelia tegeta (2)
Xanthoparmelia brachinaensis (2)
Xanthoparmelia digitiformis (2)
Xanthoparmelia aff. delisei (2)
Xanthoparmelia crespoae (2)
Xanthoparmelia lithophiloides (2)
Xanthoparmelia subspodochroa (2)
Xanthoparmelia murina (2)
Xanthoparmelia norcapnodes (2)
Xanthoparmelia lithophila (2)
Xanthoparmelia hottentotta (3)
Xanthoparmelia hueana (2)
Xanthoparmelia saxeti (3)
Xanthoparmelia scotophylla (2)
Canoparmelia caroliniana (5) Canoparmelia [Parmotrema clade]
Canoparmelia texana (3)*
Parmotrema pilosum (2)
Parmotrema fistulatum (2)
Punctelia subrudecta (2)
Parmotrema subcaperatum (2)
Parmotrema haitiense (3)
Parmotrema subtinctorium (2)
LECANOROMYCETIDAE
Parmotrema tinctorum 1 (4)

Parmotrema martinicanum (3)
Parmotrema robustum (2)
Parmotrema perlatum (3)
Parmotrema austrosinense (5) Parmotrema
Parmotrema clade
Parmotrema tinctorum 0 (5)
Parmotrema perforatum (3)*
Parmotrema reticulatum 0 (2)
Parmotrema hypoleucinum (2)
Parmotrema crozalsianum (3)
Parmotrema carneopruinatum (2)
Parmotrema crinitum (3)
Parmotrema reticulatum 1 (4)
Parmotrema clavuliferum (2)
Parmotrema cetratum (3)
Parmotrema pseudoreticulatum (2)
Flavoparmelia baltimorensis 0 (2)
Flavoparmelia baltimorensis 1 (5)
Flavoparmelia caperata (4)* Flavoparmelia
Flavoparmelia springtonensis (3)
Flavoparmelia soredians (3)
Austroparmelina pruinata (2)
Punctelia rudecta (5)
Punctelia hypoleucites (5)
Punctelia borreri (3)* Punctelia
Punctelia pseudocoralloidea (3)
Punctelia subflava (2)
Flavopunctelia flaventior 1 (5)* Flavopunctelia
PARMELIACEAE PART 3 Flavopunctelia flaventior 0 (4)*
Nesolechia oxyspora (2)*
Remotrachyna ciliata (3)
Remotrachyna scytophylla (2)
Remotrachyna adducta (2)
Remotrachyna koyaensis (2)
Remotrachyna crenata (2) Remotrachyna
Remotrachyna infirma (2)
Remotrachyna incognita (2)
Remotrachyna flexilis (2)
Remotrachyna costaricensis (2)
Bulbothrix aff. hypocraea (2)
Bulbothrix isidiza (2)
Bulbothrix decurtata (2)
Bulbothrix tabacina (2)
Bulbothrix setschwanensis (2) Bulbothrix II
Parmelina clade
Bulbothrix meizospora (3)

Bulbothrix hypocraea (2)
Parmelinella wallichiana (2)*
Bulbothrix goebelii (2)
Bulbothrix suffixa (2)
Bulbothrix coronata (2)
Bulbothrix apophysata (2) Bulbothrix I
Bulbothrix aff. goebelii 0 (2)
Bulbothrix aff. goebelii 1 (2)
Cetrelia olivetorum (2) Cetrelia [Cetrelia clade]
Cetrelia chicitae (2)
Anzia colpodes 0 (2)* Anzia
Anzia colpodes 1 (2)*
Myelochroa aurulenta 1 (3)*
Myelochroa aurulenta 0 (5)*
Myelochroa irrugans (3) Myelochroa
Myelochroa metarevoluta (3)
Parmelina pastillifera (2)
Parmelina coleae (2)
Parmelina tiliacea 1 (3)*
Parmelina tiliacea 0 (5)* Parmelina
Parmelina quercina (3)
Parmelina sp. 1 (2)
LECANORALES Parmelina sp. 0 (2)
Hypotrachyna revoluta (3)
Hypotrachyna britannica (2)
Hypotrachyna neodissecta (2)
Parmelinopsis horrescens (3)*
Parmelinopsis afrorevoluta (2)
Hypotrachyna aff. taylorensis (2)
Parmelinopsis neodamaziana (2)
Parmelinopsis subfatiscens (3) Parmelinopsis +
Hypotrachyna clade
Parmelinopsis cryptochlora (2) Hypotrachyna II
Hypotrachyna exsecta (2)
Parmelinopsis minarum 0 (3)
Hypotrachyna booralensis (2)
Hypotrachyna polydactyla (2)
Hypotrachyna osseoalba (2)
Hypotrachyna immaculata (2)
Parmelioid group Hypotrachyna pseudosinuosa (2)
Hypotrachyna caraccensis (4)
Hypotrachyna imbricatula (2)
Hypotrachyna sinuosa (3)
Hypotrachyna laevigata (2)
Hypotrachyna degelii (5) Hypotrachyna I
Hypotrachyna rockii (2)
Hypotrachyna taylorensis (2)
Hypotrachyna endochlora (2)
Everniastrum sorocheilum (2)
Everniastrum nepalense (3) Everniastrum
Everniastrum vexans (2)
Everniastrum cirrhatum (2)*
Everniastrum lipidiferum (2) = not Everniastrum
Cetrariastrum dubitans (2) Cetrariastrum
Cetrariastrum andense (2)
Relicina subnigra (3)
Relicina sydneyensis (2) Relicina/Parmelia clade
Melanohalea clade
Melanelixia fuliginosa 1 (4)
J Melanelixia fuliginosa 0 (3)
Melanelixia subaurifera (3) Melanelixia
Melanelixia subargentifera (3)
Melanelixia glabra (3)*
Melanohalea olivacea (3)
Melanohalea subelegantula (2) Melanohalea
Pleurosticta acetabulum 0 (5)* Pleurosticta
Pleurosticta acetabulum 1 (3)*
Montanelia disjuncta (3)
Parmelia saxatilis 0 (4)*
Parmelia saxatilis 1 (3)*
Parmelia discordans (3)
Parmelia ernstiae (2) Parmelia
Parmelia serrana (3)
Parmelia squarrosa (3)
Parmelia sulcata (3)
Parmeliopsis hyperopta (3)
Parmeliopsis ambigua 0 (5)* Parmeliopsis
Parmeliopsis ambigua 1 (3)*
LECANOROMYCETIDAE
Flavocetraria nivalis 1 (3) Flavocetraria

Flavocetraria nivalis 0 (5)
Nephromopsis ornata (3)
Ahtiana aurescens (2)
Nephromopsis komarovii (2) Nephromopsis +
Cetrarioid core group

Nephromopsis ahtii (3) Ahtiana + Arctocetraria
Arctocetraria andrejevii (3)*
Nephromopsis laureri (3)
Tuckermannopsis ciliaris (4)*
Tuckermannopsis chlorophylla (4) Tuckermannopsis
Masonhalea richardsonii (4)*
Dactylina arctica (5)*
Porina guentheri (2) = contaminant
Vulpicida pinastri 0 (5)
Vulpicida pinastri 1 (4) Vulpicida
Vulpicida pinastri 2 (2)
Vulpicida juniperinus (3)*
Cetraria islandica 0 (5)*
Cetraria islandica 1 (4)* Cetraria
Cetraria aculeata (2)
Melanelia stygia (3)*
Melanelia hepatizon (3) Melanelia
Protousnea magellanica (3)*
Pannoparmelia angustata (3)*
Asahinea chrysantha (3)* Asahinea
Asahinea scholanderi (3)
Letharia columbiana (3)
PARMELIACEAE PART 2 Hypogymnia physodes 1 (5)*
Hypogymnia physodes 0 (5)*
Hypogymnia vittata 0 (4) Hypogymnia
Hypogymnioid group
Hypogymnia vittata 1 (3)
Hypogymnia farinacea (3)
Hypogymnia hultenii (3)
Brodoa oroarctica (3)*
Brodoa atrofusca (3) Brodoa
Brodoa intestiniformis (3)
Pseudevernia furfuracea 1 (4)*
Pseudevernia furfuracea 0 (4)* Pseudevernia
Pseudevernia consocians (5)
Arctoparmelia centrifuga (4)* Arctoparmelia
Arctoparmelia separata (5)
Evernia prunastri 1 (5)*
Evernia prunastri 0 (5)*
Evernia prunastri 2 (3)* Evernia
Evernia mesomorpha (4)
Evernia esorediosa (3)
Psiloparmelia denotata (2) Psiloparmelia
Psiloparmelia sp. (2)
Everniopsis trulla (3)*
Cornicularia normoerica (4)*
Allantoparmelia alpicola (3)*
Oropogon sperlingii (2)
Usnea antarctica 1 (3)
Usnea antarctica 0 (5)
Usnea aurantiacoatra (2)
Usnea sphacelata (4)
Usnea trachycarpa (3)
Usnea longissima (2) Usnea
Usnea florida 0 (3)*
Usnea florida 1 (2)*
Usnea dasypoga (2)
Usnea strigosa (5)
Hypotrachyna aff. (2)
Platismatia glauca 1 (5)*
Platismatia glauca 2 (4)* Platismatia
Platismatia glauca 0 (5)*
Platismatia norvegica (3)
LECANORALES
Imshaugia aleurites 1 (4)*
Imshaugia aleurites 2 (3)* Imshaugia
Imshaugia aleurites 0 (4)*
PARMELIACEAE PART 1 Alectoria ochroleuca 0 (4)
Alectoria ochroleuca 2 (4) Alectoria
Alectoria ochroleuca 1 (4)
Alectorioid group
Alectoria sarmentosa (4)*
Gowardia nigricans (3)*
Pseudephebe pubescens 0 (5)* Pseudephebe
Pseudephebe pubescens 1 (4)*
Sulcaria sulcata (2)*
Sulcaria virens (2) Sulcaria
Bryoria trichodes 2 (4)*
Bryocaulon divergens (4) = Bryoria Bryoria
Bryoria capillaris (4)
Bryoria fuscescens (3)
Bryoria fremontii (3)
Menegazzia terebrata (5)*
Menegazzia myriotrema (2) Menegazzia
Menegazzia confusa (2)
Cetrariella delisei (3)*
Coelopogon epiphorellus (3)
Lethariella cashmeriana (3)
Protoparmelia badia 0 (3)*
Protoparmelia badia 1 (3)* Protoparmelia
Lecanora conizaeoides 0 (3)
Lecanora paramerae (3)
Lecanora hybocarpa 1 (3)
Lecanora hybocarpa 0 (5)
Lecanora argopholis (2)
Lecanora glabrata (2)
Lecanora filamentosa (2)
Lecanora fuscescens (2)
Ramboldia insidiosa (3) = not Ramboldia
Lecanora sp. (3)
Lecanora sulphurea 1 (3)
I Lecanora sulphurea 0 (3)
L. symmicta group
Lecanora symmicta (4)
Lecanora strobilina (5) Lecanora
Pyrrhospora quernea (3)*
Lecanora rupicola (2) L. rupicola group
Lecanora carpinea (2)
Lecanora thysanophora (3)
Haematomma ochroleucum* (2) = L. thysanophora?
Lecanora intumescens (4)
Lecanora oreinoides (2)
Lecanora caesiorubella (4) L. subfusca group
Lecanora cinereofusca (2) = Lecanora s. str.
Lecanora campestris (2)
Lecanora allophana (2)*
Lecidea turgidula 0 (3)
Lecidea turgidula 1 (3) ‘Lecidea’ [Lecideaceae]
LECANOROMYCETIDAE
Lecidella elaeochroma (4)

Lecidella meiococca (3) Lecidella
Lecidella euphorea (4)
Lecidella stigmatea (2)
Frutidella caesioatra (3)* Frutidella [Ramalinaceae]
Frutidella pullata (3)
Glyphopeltis ligustica (2) [Psoraceae]
Protoparmeliopsis achariana 0 (5)
Protoparmeliopsis muralis 1 (3)* Protoparmeliopsis
Protoparmeliopsis muralis 0 (4)*
LECANORACEAE Lecanora contractula (5) L. dispersa group
Lecanora perpruinosa (2)
Lecanora valesiaca (2)
Rhizoplaca melanophthalma (2)
Lecanora intricata (2)
Lecanora polytropa (4)
Micarea incrassata (3) [Pilocarpaceae] = not Micarea ‘Lecanora’
Lecanora conizaeoides 1 (4) + ‘Micarea’
Micarea assimilata (2) [Pilocarpaceae] = not Micarea
Lecanora concolor (3)
Japewia tornoënsis (2)* [Ramalinaceae]
Haematomma persoonii (3) [Haematommataceae]
Lecidea sp. 0 (4) = not Lecidea
MALMIDEACEAE Lecidea floridensis (2) = Malmidea
Lecidea cyrtidia (3) = not Lecidea
Gypsoplaca macrophylla 0 (3)* Gypsoplaca
GYPSOPLACACEAE Gypsoplaca macrophylla 1 (2)*
Lecidea roseotincta (2) [Lecideaceae]
Lecidea nylanderi (2) [Lecideaceae]
Miriquidica garovaglii (4) [Lecanoraceae]
Cladia muelleri 0 (3)
Cladia muelleri 1 (3)
Cladia beaugleholei (3)
Cladia fuliginosa (2)
Cladia sullivanii (2)
Cladia inflata (2)
Cladia aggregata (2)*
Cladia schizopora (2)
Cladia retipora 1 (3) Cladia
Cladia retipora 0 (3)
Cladia corallizon (2)
Cladia ferdinandii (2)
CLADONIACEAE Cladia deformis (2)
Cladia moniliformis (2)
Cladia dumicola (2)
Cladia glaucolivida (2)
LECANORINEAE Cladonia caroliniana (5)
Cladonia digitata (3)
Cladonia stygia (3)
Cladonia rangiferina (4)
Cladonia rotundata (2) Cladonia
Cladonia sulcata (2)
Cladonia stipitata (5)
Cladonia gracilis (2)
Cladonia peziziformis (3)
Metus conglomeratus 1 (3)* Metus
Metus conglomeratus 0 (4)*
Pycnothelia papillaria 1 (4)* Pycnothelia
[CLADONIINEAE] Pycnothelia papillaria 0 (5)*
Pilophorus cereolus (3)
Pilophorus strumaticus (3) Pilophorus
Stereocaulon tomentosum (4)
Stereocaulon paschale 1 (3)*
Stereocaulon intermedium (2) Stereocaulon Part 2
LECANORALES STEREOCAULACEAE PART 2 Stereocaulon vesuvianum (2)
Stereocaulon paschale 0 (5)*
Stereocaulon pileatum (3)
Stereocaulon sorediiferum (3)
Stereocaulon urceolatum (2) Stereocaulon Part 1
STEREOCAULACEAE PART 1 Stereocaulon tornense (3)
Stereocaulon rivulorum (2)
Lepraria finkii 0 (5)
Lepraria finkii 1 (3)
Lepraria finkii 2 (3) Lepraria
Lepraria bergensis (3)
Lepraria incana (3)*
Hertelidea botryosa (4)*
Squamarina lentigera (4)
SQUAMARINACEAE Squamarina cartilaginea (5) Squamarina
Squamarina gypsacea (5)
Herteliana taylorii (3)* [Ramalinaceae]
Ramboldia laeta (2)
Ramboldia arandensis (2)
RAMBOLDIACEAE Ramboldia russula (2) Ramboldia [Lecanoraceae]
Ramboldia gowardiana (4)
Ramboldia elabens (4)
Mycoblastus sanguinarius 0 (5)*
MYCOBLASTACEAE Mycoblastus sanguinarius 1 (3)* Mycoblastus
Mycoblastus affinis (4)
Tephromela atra 1 (4)*
TEPHROMELATACEAE Tephromela atra 2 (5)* Tephromela
Tephromela atra 0 (5)*
Lecania inundata (2)
Lecania turicensis (2)
Lecania atrynoides (4)
Lecania atrosanguinea (2)
Lecania aipospila (2)
Lecania rabenhorstii (2)
Lecania nylanderiana (2)
Lecania fructigena (4) Lecania s. str.
Lecania erysibe (2)
Lecania cyrtella 2 (3)
Lecania hutchinsiae (2)
Lecania cyrtellina (2)
Bilimbia sabuletorum 0 (5)*
H Bilimbia sabuletorum 1 (3)* Bilimbia
Bilimbia lobulata (2) [Lecanoromycetes i. s.]
Bilimbia microcarpa (2)
Lecania naegelii (2) = not Lecania
Mycobilimbia carneoalbida (2) [Lecideaceae]
Thamnolecania brialmontii (2)
Catillaria scotinodes (2) [Catillariaceae]
Mycobilimbia tetramera 1 (2) *
Mycobilimbia tetramera 0 (5) * Mycobilimbia [Lecideaceae]
Biatora meiocarpa (2)
Biatora subduplex (4) Biatora
RAMALINACEAE Biatora vernalis (3)*
Scoliciosporum schadeanum (2) [Scoliciosporaceae] = not Scoliciosporum
LECANOROMYCETIDAE
s.str. Crocynia pyxinoides (5) [Crocyniaceae]

Rolfidium coccocarpioides (3)
Phyllopsora sp. (4)
Ramalina complanata 0 (4)
Ramalina complanata 1 (4)
Ramalina farinacea (5)
Ramalina pollinaria (2) Ramalina
Ramalina fastigiata 1 (3)
Ramalina fastigiata 0 (4)
Cliostomum griffithii (3)
Niebla cephalota (4)
Lopezaria versicolor (5)*
Megalaria grossa (4)* [Megalariaceae]
RAMALINACEAE s.l. Toninia sedifolia 0 (4)
Toninia sedifolia 1 (4)
Bacidina phacodes (4)*
Bacidina arnoldiana (5) Toninia + Bacidina
Bacidina chloroticula (2)
BACIDIACEAE Toninia cinereovirens (4)*
Bacidina californica (3)
Lecania chlorotiza (2) = not Lecania
Byssolecania variabilis (3) [Pilocarpaceae]
Bacidia schweinitzii 1 (3)
Bacidia schweinitzii 0 (5)
Bacidia suffusa (3) Bacidia
Bacidia rubella (3)
Bacidia rosella (5)*
Psora saviczii (3)
Psora decipiens 1 (3)*
Psora decipiens 0 (4)* Psora
PSORACEAE Psora rubiformis (5)
Psora testacea (3)
Protoblastenia szaferi (3)
Protoblastenia rupestris (3)* Protoblastenia
Protoblastenia calva (5)
Micarea sylvicola (3) [Pilocarpaceae] = not Micarea
Fellhanera subtilis (2)
Fellhanera bouteillei (4) Fellhanera
Nelsenium usnicum (3)
Schadonia fecunda (2) [Ramalinaceae]
Calopadia foliicola (3)
LECANORALES Calopadia phyllogena (3) Calopadia
PILOCARPACEAE Byssoloma leucoblepharum (4) Byssoloma
Byssoloma subdiscordans (4)
Micarea adnata (4)
SPPHAEROPHORINEAE (INCL. PSORINEAE) Micarea micrococca (2)
Micarea denigrata (4) Micarea
Micarea alabastrites (3)
Micarea doliiformis (2)
Scoliciosporum intrusum (2) [Scoliciosporaceae] = not Scoliciosporum.
Sphaerophorus globosus 1 (5)*
Sphaerophorus globosus 0 (5)* Sphaerophorus
Sphaerophorus fragilis (4)
SPHAEROPHORACEAE Leifidium tenerum (2)*
Neophyllis melacarpa (3)*
Protomicarea limosa (3)* [?Psoraceae]
Psilolechia lucida 0 (4)*
PSILOLECHIACEAE Psilolechia lucida 1 (3)* Psilolechia [?Pilocarpaceae]
Psilolechia leprosa (3)
Catillaria erysiboides (2) [Catillariaceae]
SCOLICIOSPORACEAE Scoliciosporum umbrinum 0 (3)* Scoliciosporum
LECANORALES Scoliciosporum umbrinum 1 (3)*
Haematomma accolens (3) = [Haematommaceae]
Caloplaca scopularis 0 (4)
TELOSCHISTALES Caloplaca marina (3)
TELOSCHISTACEAE PART 2 Caloplaca scopularis 1 (3) ‘Caloplaca’ V
Caloplaca coronata (4)
Massjukiella candelaria (5)
Massjukiella polycarpa 0 (5)* Massjukiella
Massjukiella polycarpa 1 (5)*
Caloplaca verruculifera (4)
Xanthoria aureola (5)
Xanthoria parietina 1 (3)* Xanthoria s. str.
Xanthoria calcicola (4)
Xanthoria parietina 0 (5)*
Caloplaca arnoldii subsp. obliterata (5)
Caloplaca holocarpa (2) ‘Caloplaca’ IV
Caloplaca decipiens (2)
Rusavskia elegans 0 (5)*
Rusavskia sorediata (2) Rusavskia
G Xanthodactylon turbinatum (2)
Xanthodactylon flammeum 0 (2) Xanthodactylon
Xanthodactylon flammeum 1 (2)
Xanthokarrooa karrooensis (2)*
Jackelixia ligulata (2)
Caloplaca cf. squamosa (4)
Caloplaca ochracea (3) ‘Caloplaca’ III
XANTHORIOIDEAE Xanthomendoza poeltii (5)
Xanthomendoza montana (3)
Xanthomendoza fulva (2)
Xanthomendoza borealis (3) Xanthomendoza
Xanthomendoza weberi (2)
Xanthomendoza fallax 1 (4)
Xanthomendoza fallax 0 (5)
Xanthomendoza ulophyllodes (4)
TELOSCHISTOIDEAE Xanthomendoza mendozae (4)*
Teloschistes flavicans (5)*
Teloschistes exilis 0 (5)
Teloschistes exilis 1 (5)
Teloschistes chrysophthalmus 0 (5) Teloschistes
Teloschistes chrysophthalmus 1 (3)
Teloschistes hosseusianus (5)
Xanthoria andina (4)
Caloplaca chilensis (5) ‘Caloplaca’ II + ‘Xanthoria’
LECANOROMYCETIDAE
Seirophora contortuplicata (2)

Seirophora lacunosa (4)
TELOSCHISTACEAE PART 1 Caloplaca thallincola (4)
Caloplaca xantholyta (3)
Caloplaca chlorina (2)
Caloplaca isidiigera (4) C. cerina group = Caloplaca
Caloplaca thracopontica (3) Caloplaca s. str. + Seirophora
Caloplaca cerina (3)*
Caloplaca chalybaea (5) C. variabilis group
Caloplaca carphinea (4)
CALOPLACOIDEAE Caloplaca scoriophila (5) C. carphinea group
Caloplaca furax (4)
TELOSCHISTINEAE Caloplaca scotoplaca (5)
Fulgensia bracteata (3)
Caloplaca xanthostigmoidea (2)
Fulgensia fulgens (5)
Caloplaca gomerana 1 (5) ‘Caloplaca’ I + Fulgensia
Caloplaca gomerana 0 (2)
Caloplaca flavorubescens (4)
Caloplaca cinnamomea (5)
Caloplaca crenularia (4)
Ioplaca pindarensis (2)
TELOSCHISTALES Megalospora tuberculosa 0 (5)
Megalospora tuberculosa 1 (5)
MEGALOSPORACEAE Megalospora sulphurata (4)* Megalospora
Megaloblastenia marginiflexa (4)
Sipmaniella sulphureofusca (3)* [Lecanoraceae]
Brigantiaea leucoxantha 1 (2)
Brigantiaea cf. leucoxantha (4)
BRIGANTIAEACEAE Brigantiaea cf. tricolor (4)*
Brigantiaea
Brigantiaea leucoxantha 0 (2)
LETROUITINAE Brigantiaea sorediata (3)
Brigantiaea fuscolutea (5)
Letrouitia domingensis (5)*
TELOSCHISTALES LETROUITIACEAE Letrouitia parabola (5) Letrouitia
Letrouitia vulpina (5)
Leprocaulon quisquiliare (3) [Lecanoromycetes i. s.]
LEPROCAULALES LEPROCAULACEAE/LEPROCAULALES Solenopsora candicans (5) [Catillariaceae]
Speerschneidera euploca (3)* [Lecanorales i. s.]
Buellia zoharyi (2)
CALICIALES Buellia punctata 0 (4)
Buellia frigida (4)
Buellia punctata 1 (4)
Buellia stillingiana (4)
Buellia disciformis (3)* Buellia
Buellia aethalea (2)
Buellia tesserata (4)
Dimelaena radiata (4)
CALICIACEAE Buellia dialyta (4)
Tholurna dissimilis (3)*
Thelomma ocellatum (2)
LECANOROMYCETIDAE
Texosporium sancti-jacobi (2)*

Cyphelium inquinans (3) = not Cyphelium
BUELLIOIDEAE Buellia dispersa (2) = not Buellia
Pyxine sp. (3)
Pyxine subcinerea (5) Pyxine
Pyxine sorediata (4)*
Dirinaria applanata (4)
Diplotomma alboatrum (3)*
Diplotomma epipolium (3) Diplotomma
Diploicia canescens (2)*
CALICIOIDEAE Cyphelium tigillare 1 (3)* Cyphelium
Cyphelium tigillare 0 (3)*
Calicium adspersum (2) Calicium
Calicium viride (3)*
Physcia stellaris (3)
Physcia dubia (4)
CALICIALES Physcia caesia (3)
Physcia aipolia (3)
Physcia
with Lopadiaceae Physcia aff. aipolia (5)
Physcia adscendens (2)
Heterodermia vulgaris (4)
Heterodermia hypoleuca (2) Heterodermia
Tornabea scutellifera 1 (3) Tornabea
PHYSCIACEAE PART 2 Tornabea scutellifera 0 (2)
Anaptychia palmulata 0 (5)
Anaptychia palmulata 1 (3) Anaptychia
Anaptychia runcinata (2)
Physconia perisidiosa (3)
Physconia muscigena (5) Physconia
Physconia grisea (2)
Physconia enteroxantha (3) = not Physconia
PHYSCIACEAE PART 1 Rinodina tephraspis (5)
Hyperphyscia adglutinata (2)
Phaeophyscia orbicularis (5)*
CALICIALES Lopadium disciforme (4)/ LOPADIACEAE [?PILOCARPACEAE]
Psorula rufonigra (2)* [Psoraceae]
Lecidea diapensiae (2) [Lecideaceae]
Helocarpon crassipes (3)* [Lecanoromycetes i. s.]
Pseudocyphellaria sp. 1 (2)
PELTIGERALES Pseudocyphellaria sp. 0 (2)
Pseudocyphellaria intricata (2)
Pseudocyphellaria lechleri (2)
Pseudocyphellaria neglecta (4)
Pseudocyphellaria perpetua 1 (3)
Pseudocyphellaria perpetua 0 (3)
Pseudocyphellaria coriifolia (3) Pseudocyphellaria
Pseudocyphellaria crocata (2)*
Pseudocyphellaria pilosella (2)
Pseudocyphellaria cf. intricata 1 (2)
Pseudocyphellaria cf. intricata 0 (2)
Pseudocyphellaria freycinetii (3)
Pseudocyphellaria argyracea (2)
Pseudocyphellaria divulsa (3)
Sticta weigelii (2)
Sticta beauvoisii (4)
Sticta canariensis (3)
Sticta fuliginosa (4)
Sticta limbata (3) Sticta
F Sticta gaudichaudia (2)
Sticta hypochroa (3)
Sticta caulescens (2)
Sticta filix (2)
Ricasolia quercizans 1 (4)
Ricasolia quercizans 0 (4) Ricasolia
Ricasolia virens (3)
Ricasolia amplissima (4)*
Lobariella pallida 1 (5) Lobariella
Lobariella pallida 0 (5)
Lobaria pseudoglaberrima (3)
Lobaria meridionalis (2)
LECANOROMYCETIDAE
Lobarina oregana (2)

Lobarina scrobiculata (5)* Lobarina
Pseudocyphellaria rainierensis (3)
Lobaria hallii (4) Lobaria s. l.
Lobaria macaronesica (2)
Lobaria sachalinensis (2)
LOBARIACEAE Lobaria immixta (2)
Lobaria pulmonaria (5)*
Lobaria orientalis (2) Lobaria s. str.
Anomalobaria anomala 0 (5)*
Anomalobaria anomala 1 (3)*
Lobaria linita (2)
Lobaria retigera (2)
Lobaria pseudopulmonaria (2)
Parmostictina obvoluta (2)
Parmostictina mallota (2) Parmostictina
Parmostictina dubia (2)
Parmostictina hirsuta (2)*
Crocodia aurata (3)*
Crocodia clathrata (2) Crocodia
Podosticta endochrysoides (2)
Podosticta berberina (2) Podosticta
Peltigera praetaxtata (4)
Peltigera canina 0 (4)*
Peltigera canina 1 (3)*
Peltigera cinnamomea (3)
Peltigera rufescens (2)
Peltigera laciniata (2)
Peltigera degenii (5) Peltigera II
Peltigera membranacea (4)
Peltigera monticola (2)
Peltigera ponojensis (3)
Peltigera kristinssonii (3)
Peltigera sp. nov. (5)
Peltigera continentalis (2)
LECANOROMYCETIDAE
Peltigera retifoveata (2) Retifoveatae

Peltigera elisabethae (2)
Peltigera horizontalis (3) Horizontales
Peltigera polydactyloides (2)
Peltigera neckeri (3) Peltigera
Peltigera extenuata (2)
Peltigera didactyla (2) Peltigera I (sorediated)
Peltigera sorediifera (2)
Peltigera pulverulenta (2)
Peltigera hymenina (2)
Peltigera occidentalis (2)
Peltigera polydactylon (3) Polydactylon
Peltigera scabrosa (2)
Peltigera neopolydactyla (3)
Peltigera hydrothyria (2)
Peltigera aphthosa (4)
Peltigera britannica (3) Peltidea
PELTIGERACEAE Peltigera malacea (3)
Peltigera venosa (2) Phlebia
Peltigera leucophlebia (4) Chloropeltigera
Solorina saccata 2 (2)
Solorina saccata 0 (3)
PELTIGERINEAE Solorina saccata 1 (2)
Solorina bispora (4) Solorina
ae Solorina spongiosa (3)
with Vahliellace Solorina crocea 0 (3)*
Solorina crocea 1 (2)*
Nephroma parile (5)
NEPHROMATACEAE Nephroma bellum (4)
Nephroma arcticum (5)* Nephroma
Nephroma antarcticum (2)
Nephroma resupinatum (3)
Massalongia carnosa 0 (4)*
Massalongia carnosa 2 (2)* Massalongia
Massalongia carnosa 1 (3)*
MASSALONGIACEAE Polychidium muscicola 1 (4)* Polychidium
Polychidium muscicola 0 (5)*
Leptochidium albociliatum 1 (2)* Leptochidium [Placynthiaceae]
Leptochidium albociliatum 0 (4)*
Vahliella leucophaea (3)* [Pannariaceae]/VAHLIELLACEAE
Leptogium austroamericanum (2)
Leptogium phyllocarpum (2)
Leptogium corticola (2)
Leptogium cyanescens (5)
COLLEMATACEAE PART 2 Leptogium brebissonii (2) Leptogium Part 2
PELTIGERALES Leptogium burnetiae (2)
Leptogium saturninum (5)
Leptogium azureum (4)*
Leptogium furfuraceum (3) Leptogium Part 1
Leptogium pseudofurfuraceum (3)
Collema flaccidum (3)
Collema subnigrescens (2)
Collema nigrescens (2)*
Collema curtisporum (3) Collema
Collema furfuraceum 1 (2)
Collema furfuraceum 0 (5)
Collema subconveniens (4)
Callome multipartita (2)*
Scytinium magnussonii (2)
COLLEMATACEAE PART 1 Scytinium subaridum (2)
Scytinium tenuissimum (2)
Enchylium conglomeratum (2)
Scytinium fragrans (2)
Scytinium microphylloides (2)
Scytinium imbricatum (2)
Scytinium lichenoides (5)
Scytinium pulvinatum (2)
Scytinium gelatinosum (3)
Lathagrium undulatum (3) Scytinium + Lathagrium
PELTIGERALES Lathagrium auriforme (2)
Enchylium polycarpon (2)
+ Enchylium
Scytinium fragile (2)
Scytinium schraderi (2)
Scytinium turgidum (2)
LECANOROMYCETIDAE
Scytinium plicatile (3)

Scytinium parvum (2)
Lathagrium cristatum (4)
Enchylium tenax (2)*
Scytinium callopismum (2)
Pseudoleptogium diffractum (3)*
Placynthium nigrum 2 (2)*
Placynthium nigrum 1 (4)*
PLACYNTHIACEAE Placynthium nigrum 0 (5)* Placynthium
Placynthium flabellosum (3)
Coccocarpia domingensis (3)
COCCOCARPIACEAE Coccocarpia erythroxyli (5) Coccocarpia
LECANOROMYCETIDAE
Coccocarpia palmicola (4)

Fuscopannaria leucosticta (4)*
Fuscopannaria ahlneri (3)
Fuscopannaria praetermissa (3) Fuscopannaria
Fuscopannaria mediterranea 0 (4)
Fuscopannaria mediterranea 1 (3)
PANNARIACEAE Fuscopannaria ignobilis (5)
Protopannaria pezizoides 2 (2)*
Protopannaria pezizoides 1 (3)* Protopannaria
COLLEMATINEAE Protopannaria pezizoides 0 (5)*
Leciophysma finmarkicum (3)* Leciophysma
Leciophysma furfurascens (4)
Physma pseudoisidiatum (2)
Physma byrsaeum (3) Physma [Collemataceae]
Physma radians (3)
Fuscoderma applanatum (3)* Fuscoderma
wi Fuscoderma amphibolum (2)
th Psoroma hypnorum (4)*
Ko
er Staurolemma weberi (2)
be Ramalodium succulentum (3) = Staurolemma? Staurolemma
r iac Staurolemma sp. (3) [Collemataceae]
ea Staurolemma omphalarioides (2)
e Pannaria conoplea (2)
Pannaria rubiginosa (3)* Pannaria
Pannaria sp. (4)
Pannaria rubiginella (3)
Parmeliella appalachensis 0 (4)
Parmeliella appalachensis 1 (4) Parmeliella
Parmeliella triptophylla (5)*
Parmeliella nigrocincta (2)
Degelia plumbea 1 (5)
Degelia plumbea 0 (5) “Degelia”
Erioderma verruculosum (5)
Erioderma leylandii (4) Erioderma
Degelia durietzii (3) = not Degelia
Leioderma erythrocarpum (2)
Degelia gayana (2)*
PELTIGERALES KOERBERIACEAE Koerberia biformis (2) [Placynthiaceae]
Steinera glaucella (2) [Coccocarpiaceae]
Romjularia lurida (5) [Lecideaceae]
? Mycobilimbia berengeriana (3) [Lecideaceae]
Lecidea fuscoatra 2 (4)*
Lecidea fuscoatra var. grisella (4)*
Lecidea fuscoatra 1 (4)*
LECANOROMYCETIDAE
LECIDEACEAE S. STR./ Lecidea fuscoatra 0 (5)*

Porpidia speirea (5)
LECIDEALES Lecidea lapicida (4) Lecidea s. str.
Lecidea silacea 1 (4)
Lecidea silacea 0 (5)
Lecidea confluens (2)
Lecidea laboriosa (5)
Lecidea atrobrunnea (3)
Lecidea auriculata (5)
Porpidia melinodes 0 (2)
Porpidia melinodes 1 (2)
Porpidia flavicunda 0 (2)
Porpidia flavicunda 1 (2) Porpidia II
Porpidia albocaerulescens 1 (4)
Porpidia albocaerulescens 0 (5)
Porpidia carlottiana (2)
Arthrorhaphis citrinella 0 (4)* Arthrorhaphis
E Arthrorhaphis citrinella 1 (2)*
Arthrorhaphis citrinella 2 (2)* [Arthorhaphidaceae]
Placynthiella oligotropha (3) [Trapeliaceae]
Lecidoma demissum (5)*
LECIDEALES Lecidea plana (3) = not Lecidea
Lecidea tessellata (3) = not Lecidea
LECIDEALES Leimonis erratica (4)* [Pilocarpaceae]
Bellemerea alpina (2)* Bellemerea
Bellemerea diamarta (3)
Porpidia macrocarpa (2)
Lecidea sp. 1 (2) Porpidia I
Porpidia crustulata (2)
Porpidia contraponenda (2)
Catillaria modesta (2) [Catillariaceae]
Rhizocarpon geographicum s. l. (4)*
RHIZOCARPALES Rhizocarpon norvegicum (2)
Rhizocarpon macrosporum (3)
Rhizocarpon disporum (3)
Rhizocarpon Part 2
RHIZOCARPACEAE PART 2 Rhizocarpon geminatum (2)
Rhizocarpon superficiale (4)
Catolechia wahlenbergii 1 (5)* [?Rhizocarpaceae] = Rhizocarpon
Catolechia wahlenbergii 0 (5)* [?Rhizocarpaceae] = Rhizocarpon
RHIZOCARPACEAE PART 1 Rhizocarpon hochstetteri (3)
RHIZOCARPALES Rhizocarpon oederi (4) Rhizocarpon Part 2
Rhizocarpon geographicum (3)*
Sporastatia polyspora 0 (4)
Sporastatia polyspora 1 (4) Sporastatia [Catillariaceae;
RHIZOCARPALES LECANOROMYCETIDAE Sporastatia testudinea 1 (4)*
Sporastatia testudinea 0 (5)*
Sporastatiaceae]
Umbilicaria subcalvescens (2)
Umbilicaria calvescens (2)
Umbilicaria dichroa (2)
Umbilicaria haplocarpa (2)
Umbilicaria subglabra (2)
Umbilicaria hirsuta (3)
Umbilicaria grisea (2)
Umbilicaria crustulosa (2)
Umbilicaria spodochroa (5)
Umbilicaria vellea (4)
Umbilicaria cinereorufescens (2)
Umbilicaria leprosa (3) Umbilicaria s. str.
Umbilicaria aprina (5)
UMBILICARIACEAE Umbilicaria africana (2)
Umbilicaria dendrophora (2)
Umbilicaria proboscidea (2)
UMBILICARIOMYCETIDAE
Umbilicaria decussata (3)

Umbilicaria arctica (5)
Umbilicaria nylanderiana (2)
Umbilicaria polyphylla (3)
Umbilicaria hyperborea (3)*
Umbilicaria rigida (4)
Umbilicaria lyngei (3)
Lasallia hispanica (3)
Lasallia pustulata 1 (2)*
Lasallia pustulata 0 (5)*
Lasallia papulosa (5)
Lasallia pennsylvanica (4) Lasallia + ‘Umbilicaria’
Umbilicaria mammulata (5)
Umbilicaria esculenta (2)
Umbilicaria muehlenbergii 1 (5)
Umbilicaria muehlenbergii 0 (4)
ae Xylopsora friesii (2)* [Ophioparmaceae]
e ace eae Elixia flexella (2)*/ELIXIACEAE
d
i ac Ophioparma ventosa 1 (3)*
us c or Ophioparma ventosa 0 (5)* Ophioparma
u t F l os p Ophioparma lapponica (5)
tho pa OPHIOPARMACEAE Boreoplaca ultrafrigida 0 (4)* Boreoplaca
wi d Ro Boreoplaca ultrafrigida 1 (2)*
an Hypocenomyce scalaris 1 (3)
Hypocenomyce scalaris 2 (5) Hypocenomyce
Hypocenomyce scalaris 0 (5)
UMBILICARIALES Fuscidea austera (4)
Fuscidea cyathoides (4) Fuscidea
Fuscidea lygaea (3)
UMBILICARIALES Maronea chilensis (4) Maronea [?Fuscideaceae]
FUSCIDEACEAE Maronea constans (3)
UMBILICARIOMYCETIDAE Fuscidea mollis (2) = not Fuscidea
Ropalospora chlorantha (3)/ROPALOSPORACEAE
Thelotrema monospermum (2)
OSTROPALES Thelotrema saxatile (2)
Thelotrema pachysporum (2)
Thelotrema monosporum (2)
Thelotrema lepadinum 0 (4)*
Thelotrema lepadinum 1 (3)*
Thelotrema suecicum (3) Thelotrema s. str.
Thelotrema subtile (3)
Thelotrema porinoides (2)
Thelotrema nureliyum (3)
Thelotrema diplotrema (2)
Thelotrema gallowayanum (2)
Thelotrema porinaceum (2)
Leucodecton subcompunctum (3)
Chroodiscus argillaceus (2)
Chroodiscus defectus (2) Chroodiscus
Chroodiscus australiensis (2)
Pseudochapsa phlyctidioides (2)
Astrochapsa meridensis (2)
Astrochapsa pseudophlyctis (2) Astrochapsa
Diploschistes cinereocaesius 0 (4)
Diploschistes cinereocaesius 1 (5)
THELOTREMATEAE Diploschistes muscorum (3)
Diploschistes rampoddensis (3) Diploschistes s. str.
Diploschistes diacapsis (3)
Diploschistes scruposus (3)*
Diploschistes thunbergianus (3)
Diploschistes actinostomus (5)
Thelotrema bicinctulum (2) = not Thelotrema
OSTROPOMYCETIDAE
Wirthiotrema glaucopallens (2)* Wirthiotrema

Wirthiotrema trypaneoides (2)
Topeliopsis decorticans (2) Topeliopsis
Topeliopsis muscigena (2)*
D Diploschistes ocellatus 1 (2)
Diploschistes ocellatus 0 (4) Diploschistes
Diploschistes ocellatus 2 (2)
Thelotrema sp. (2) = not Thelotrema
Phaeographis sp. (2)
Sarcographa ramificans (2) Sarcographa
Sarcographa fenicis (2)
Platygramme caesiopruinosa 0 (2)*
Platygramme caesiopruinosa 1 (2)* Platygramme
Platygramme australiensis (2)
Phaeographis lobata (2)
Phaeographis lecanographa (2) Phaeographis (P. lobata clade)
Phaeographis brasiliensis (2) Phaeographis
Phaeographis intricans (2)
Phaeographis caesioradians (2) (P. intricans clade)
Allographa cinerea (2)
GRAPHIDACEAE PART 2 Allographa ruiziana (2) Allographa
Allographa pavoniana (2)
Platythecium grammitis (5)*
Chroodiscus coccineus (2)* [ Thelotremateae]
Diorygma pruinosum (2)
Diorygma circumfusum (2) Diorygma
Diorygma junghuhnii (2)
Diorygma sipmanii (2)
GRAPHIDEAE Graphis scripta 1 (3)*
Graphis scripta 0 (5)*
Graphis cleistoblephara (2)
Graphis cf. gracilescens (2) Graphis Part 2
Graphis caesiella (2)
Graphis implicata (2)
Graphis aff. caesiella (2)
Graphis sp. 1 (2)
Graphis sp. 0 (2) Graphis Part 1
Glyphis substriatula (2)
OSTROPALES Glyphis cicatricosa (2)* Glyphis
Glyphis scyphulifera (2)
Ocellularia thelotremoides (2)
Ocellularia perforata (2)
Ocellularia massalongoi (2)
GRAPHIDOIDEAE Ocellularia diacida (2)
Ocellularia papillata (2)
Ocellularia cavata (2)
Ocellularia aurantia 1 (2) Ocellularia s. str.
Ocellularia aurantia 0 (2)
Ocellularia chiriquiensis (2)
Ocellularia sp. (2)
OCELLULARIEAE Ocellularia postposita (2)
Ocellularia minutula (3)
Myriotrema halei (3) Myriotrema
GRAPHIDACEAE PART 1 Myriotrema olivaceum (3)*
Ocellularia inturgescens (2) = not Ocellularia
Ocellularia profunda 0 (2) = not Ocellularia
Ocellularia profunda 1 (2) = not Ocellularia
Acanthothecis aurantiaca (2) [Thelotremateae]
Fissurina triticea (2)
Fissurina insidiosa (5)
Fissurina marginata (2)
Fissurina sp. 1 (2) Fissurina + Dyplolabia
Fissurina sp. 2 (2)
FISSURINOIDEAE
C Fissurina sp. 0 (5)
Dyplolabia afzelii (2)
Fissurina aff. humilis (2)
Gyalideopsis vulgaris (2)
Echinoplaca strigulacea (4)
Gyalidea fritzei (2)
Gyalidea hyalinescens (5) Gyalidea
GOMPHILLOIDEAE Tricharia sp. (2)
Gyalecta herculana (3)
Gyalecta hypoleuca (4)
Gyalecta schisticola (3)
GYALECTACEAE Gyalecta jenensis (4)*
Gyalecta fagicola (2)
Gyalecta russula (3) Gyalecta
Gyalecta geoica (2)
Gyalecta truncigena (3)
Gyalecta flotowii (3)
Gyalecta friesii (3)
Gyalecta ulmi (4)
Gyalecta leucaspis (2)
Sagiolechia rhexoblephara 0 (2)*
SAGIOLECHIACEAE Sagiolechia rhexoblephara 1 (2)* Sagiolechia
Sagiolechia protuberans (3)*
Petractis nodispora (2) [Stictidaceae] = not Petractis
Petractis luetkemuelleri (3) [Stictidaceae] = not Petractis
GROUP 1 Ramonia sp. (2) [Gyalectaceae]
Gyalidea praetermissa (2) [Graphidaceae] = not Gyalidea
Coenogonium luteum (4)
Coenogonium disjunctum (2) Coenogonium
COENOGONIACEAE Coenogonium leprieurii (4)
Coenogonium pineti (3)
Phlyctis agelaea (2)* Phlyctis
PHLYCTIDACEAE Phlyctis argena (5)
Petractis clausa (2)* [Stictidaceae]
OSTROPOMYCETIDAE
Odontotrema phacidiellum (2)

ODONTOTREMATACEAE S. STR. Coccomycetella richardsonii (2) = Odontotrema Odontotrema s. str.
Odontotrema phacidioides (2)*
Porina aenea (3)
Porina byssophila (2)
PORINACEAE Porina epiphylla (3) Porina
Porina lectissima (2)
Porina internigrans (2)
Cryptodiscus foveolaris (3)
OSTROPALES Cryptodiscus tabularum (2)
Cryptodiscus gloeocapsa 1 (2) Cryptodiscus
Cryptodiscus gloeocapsa 0 (2)
Cryptodiscus pini (2)
Cryptodiscus pallidus (2)*
Xyloschistes platytropa (2)*
Absconditella lignicola (2)
Cyanodermella viridula (2)*
Absconditella sp. (3)
Absconditella sphagnorum (3)* Absconditella
STICTIDACEAE Sphaeropezia lyckselensis 0 (2)
Sphaeropezia lyckselensis 1 (3)
Sphaeropezia diffindens (2) Sphaeropezia
Sphaeropezia arctoalpina (3)
Sphaeropezia capreae 0 (3)
Sphaeropezia capreae 1 (2)
Schizoxylon albescens (2)
Ostropa barbara 1 (4)*
Stictis brunnescens (2) = not Stictis
Carestiella socia (2)*
Stictis populorum (4) = not Sictis
Stictis urceolatum 1 (2)
Stictis urceolatum 0 (3) Stictis s. str.
OSTROPALES Stictis radiata (4)*
Acarosporina microspora (5)
Cryptodiscus rhopaloides (2) = not Cryptodiscus
Gregorella humida 0 (3)* Gregorella
Gregorella humida 1 (2)*
ARCTOMIACEAE/ Wawea fruticulosa (3)*
ARCTOMIALES
ARCTOMIALES Arctomia delicatula (3)* Arctomia
Arctomia teretiuscula (2)
Arctomia interfixa (2) = not Arctomia
Placopsis cribellans (3)
TRAPELIALES Placopsis santessonii (3)
Placopsis lambii (2)
Placopsis argillacea (2)
Placopsis sp. (2) Placopsis
Placopsis perrugosa (4)
Placopsis pycnotheca (2)
Placopsis gelida (5)*
Placopsis contortuplicata (2)
Placopsis parellina (2)
Placopsis rhodophthalma (3)
TRAPELIACEAE PART 2/ Orceolina kerguelensis (5)* Orceolina
Orceolina antarctica (2)
TRAPELIALES PART 2 Aspiciliopsis macrophthalma (2)*
Placopis bicolor 0 (2)
Trapelia glebulosa 0 (4)
Trapelia chiodectonoides (3) Trapelia Part 2
Trapelia coarctata (2)*
Trapelia placodioides 0 (5)
Trapelia placodioides 1 (3) Trapelia Part 1
Trapelia glebulosa 1 (3)
Placynthiella uliginosa (4) Placynthiella
Placynthiella icmalea (4)
Trapeliopsis flexuosa 1 (3)
Trapeliopsis flexuosa 0 (5)
Trapeliopsis granulosa 1 (4)
TRAPELIACEAE PART 1/ TRAPELIALES Trapeliopsis granulosa 0 (4) Trapeliopsis
Trapeliopsis glaucopholis (3)
TRAPELIALES PART 1 Trapeliopsis percrenata (3)
Trapeliopsis pseudogranulosa (2)
Xylographa soralifera (3) Xylographa
TRAPELIALES Xylographa parallela (3)*
Rimularia psephota (3)
Ptychographa xylographoides (4)*
Ionaspis lacustris 0 (4)
Ionaspis lacustris 2 (4) Ionaspis
HYMENELIACEAE/ Ionaspis lacustris 1 (2)
HYMENELIALES Ionaspis odora (3)
HYMENELIALES Hymenelia epulotica 1 (4)
Hymenelia epulotica 0 (4) Hymenelia
Tremolecia atrata (2)
Phyllobaeis erythrella 1 (5)
Phyllobaeis erythrella 0 (3) Phyllobaeis
BAEOMYCETACEAE/ Phyllobaeis imbricata (4)*
BAEOMYCETALES BAEOMYCETALES Baeomyces placophyllus (5) Baeomyces
Baeomyces rufus (4)*
Ainoa geochroa (2)*
Aspicilia epiglypta (2)
PERTUSARIALES Aspicilia dudinensis (2)
Aspicilia indissimilis (2)
Aspicilia laevata (2)
Aspicilia caesiocinerea 0 (5)
Aspicilia cinerea 0 (5)*
Aspicilia cinerea 1 (3)*
Sagedia zonata (2)*
Sagedia simoensis (2)
Sagedia mastrucata (2)
Aspicilia sp. (4) Aspicilia + Sagedia
Aspicilia dendroplaca (2)
Aspicilia mashiginensis (2)
Aspicilia rivulicola (2)
Aspicilia nicrapensis (2)
Aspicilia supertegens (2)
Aspicilia permutata (2)
Aspicilia verruculosa (2)
Aspicilia caesiocinerea 1 (3)
Aspicilia haeyrenii (2)
MEGASPORACEAE Circinaria hispida 0 (3)
Circinaria hispida 1 (3)
Circinaria izcoana (3)
Circinaria calcarea (2)
Circinaria emiliae (2) Circinaria
Circinaria elmorei (2)
Circinaria leprosescens (3)
Circinaria gibbosa (2)
Megaspora verrucosa 1 (3)* Megaspora
Megaspora verrucosa 0 (4)*
Lobothallia radiosa 0 (5)
Lobothallia radiosa 1 (3)
B Lobothallia recedens (2)
Lobothallia alphoplaca (3)*
Lobothallia
Lobothallia melanaspis (2)
Lobothallia farinosa (2)
Aspilidea myrinii (2)*
OSTROPOMYCETIDAE
Ochrolechia pallescens (3)

Ochrolechia balcanica (3)
Ochrolechia upsaliensis (3)
Ochrolechia szatalaensis (3)
Ochrolechia parella (5)
Ochrolechia subviridis (2)
Ochrolechia frigida (3)
Ochrolechia juvenalis (4)
OCHROLECHIACEAE Ochrolechia turneri (3)
Ochrolechia oregonensis (3) Ochrolechia
Ochrolechia tartarea (2)*
Ochrolechia androgyna (3)
Pertusaria dactylina (5)
Ochrolechia trochophora (4)
Ochrolechia yasudae (5)
Ochrolechia subpallescens (3)
Ochrolechia sp. (5)
Ochrolechia africana (2)
Ochrolechia peruensis (2)
Coccotrema maritimum (4)
Coccotrema pocillarium (3)
COCCOTREMATACEAE Coccotrema cucurbitula (4) Coccotrema
Coccotrema coccophorum (3)
Pertusaria gibberosa (3)
Pertusaria leioplaca (3)
Pertusaria mesotropa (3)
Pertusaria pustulata (3)
Pertusaria subverrucosa (3)
Pertusaria coccodes (3)
Pertusaria kalelae (2)
Pertusaria werneriana (2)
Pertusaria tejocotensis (2)
PERTUSARIACEAE Pertusaria cf. quartans (2)
Pertusaria flavicans (2)
PART 2 Pertusaria rupicola (2)
Pertusaria hymenea (2)
Pertusaria carneopallida (2) Pertusaria s. str.
Pertusaria flavicunda (2)
Pertusaria graphica (2)
Pertusaria lecanina (3)
Pertusaria sp. (2)
Pertusaria pentelici (2)
Pertusaria lactescens (2)
OSTROPOMYCETIDAE Pertusaria plittiana (3)
Pertusaria subobductans (2)
Pertusaria pertusa (5)*
Pertusaria coronata (3)
Pertusaria paramerae (3)
Pertusaria rhodiensis (2)
Pertusaria laeviganda (2)
Pertusaria hermaka (2)
Pertusaria oculata (2)
PERTUSARIALES Pertusaria pupillaris (2)
Pertusaria excludens (2)
Loxosporopsis corallifera (2)*
Agyrium rufum 0 (3)* Agyrium/AGYRIACEAE
Agyrium rufum 1 (3)*
Pertusaria amara 1 (5)
PERTUSARIALES Pertusaria cf. amara (2)
Pertusaria albescens (3)
OSTROPOMYCETIDAE
Pertusaria mammosa (3)
PERTUSARIACEAE Pertusaria corallophora (3)
Pertusaria corallina (3) ‘Variolaria’
PART 1 Pertusaria scaberula (4)
Pertusaria erythrella (3)
Pertusaria subventosa (3)
Pertusaria ophthalmiza (3)
Pertusaria panyrga (3)
Varicellaria hemisphaerica (5)
Varicellaria lactea (3)
Varicellaria rhodocarpa 0 (3) Varicellaria [Ochrolechiaceae]
Varicellaria rhodocarpa 1 (2)
Varicellaria velata (3)
Icmadophila ericetorum 1 (3)*
Icmadophila ericetorum 0 (5)* Icmadophila
ICMADOPHILACEAE Icmadophila ericetorum 2 (3)*
Siphula ceratites (5)*
Dibaeis baeomyces 1 (4)* Dibaeis
Dibaeis baeomyces 0 (5)*
Siphula fastigiata (2) = not Siphula
PERTUSARIALES Thamnolia subuliformis (4) Thamnolia
Thamnolia vermicularis (5)*
Loxospora ochrophaea (4) Loxospora
SARRAMEANACEAE INCL. SARRAMEANALES Loxospora cismonica (3)
SARRAMEANALES Schaereria corticola (3)
SCHAERERIACEAE Schaereria dolodes 0 (3) Schaereria [Schaereriaceae]
OSTROPOMYCETIDAE Schaereria dolodes 1 (2)
Schaereria fuscocinerea (2)
Candelariella vitellina (3)*
Candelariella aurella (3) Candelariella
Candelariella coralliza (2)
CANDELARIACEAE/CANDELARIALES Candelariella reflexa (5)
CANDELARIALES Candelina submexicana (2)
Candelaria concolor 0 (4)* Candelaria
Candelaria concolor 1 (2)*
Candelariella terrigena 0 (4)
‘CANDELARIOMYCETIDAE’ Candelariella terrigena 1 (2)
Candelariella placodizans (3)
Dactylospora haliotrepha 1 (3)*
Dactylospora haliotrepha 0 (4)*
DACTYLOSPORACEAE Dactylospora mangrovei (5) Dactylospora [Lecanorales]
Dactylospora imperfecta (2)
Dactylospora lobariella (2)
Strangospora pinicola 2 (4)*
STRANGOSPORACEAE Strangospora pinicola 0 (3)* Strangospora [Lecanorales i. s.]
Strangospora pinicola 1 (3)*
Acarospora complanata (4)
Acarospora umbilicata (4)
Acarospora hilaris (4)
Acarospora schleicheri 0 (5)*
Acarospora schleicheri 1 (4)*
Acarospora sp. 0 (2) Acarospora s. str.
Acarospora clauzadeana (3)
Acarospora cf. dissipata (2)
ACAROSPOROMYCETIDAE
ACAROSPORACEAE Acarospora peliscypha (3)

Acarospora bullata 0 (4)
Acarospora moenium (2)
Acarospora canadensis (3)
Sarcogyne regularis 1 (3)
Sarcogyne regularis 0 (4) ‘Acarospora’ + Sarcogyne
Acarospora bullata 1 (2)
Acarospora macrospora (3)
Sarcogyne privigna (3)
Acarospora cervina (3)
Acarospora laqueata (5)
Glypholecia scabra (4)
Sarcogyne similis (3)
Pleopsidium flavum 1 (3)*
Pleopsidium flavum 0 (4)*
Pleopsidium cf. chlorophanum 0 (2)
Pleopsidium cf. chlorophanum 1 (2) Pleopsidium s. str.
Pleopsidium chlorophanum 2 (3)
Acarospora fulvoviridula (2)
Myriospora smaragdula (2)*
Myriospora scabrida (3)
A ACAROSPORALES Acarospora molybdina (2)
Pleopsidium gobiense 0 (5)
Pleopsidium gobiense 1 (3)
Sarcogyne plicata (2) = Polysporina?
Polysporina simplex (4)*
Thelocarpella gordensis (2)*
ACAROSPOROMYCETIDAE Eiglera flavida (4)*/ EIGLERACEAE [HYMENELIACEAE; Ostropomycetidae]
Peltula umbilicata (5)
LICHINOMYCETES Peltula auriculata (5)
GEOGLOSSOMYCETES Trichoglossum hirsutum (5)
Outgroup
Biatoridium monasteriense (3)* [Lecanoromycetes i. s.]
PYCNORACEAE [CANDELARIALES; ‘CANDELARIOMYCETIDAE’] Geoglossum nigritum (5)

Pycnora sorophora (2) [?Lecanoraceae]
Pycnora praestabilis (3) [Ophioparmaceae] Pycnora
Sarea resinae (2) [Trapeliaceae; Lecanoromycetes][Leotiomycetes]
Cudoniella clavus (5)
Ostropa barbara 0 (4) = contaminant
Mollisia cinerea (5)
LEOTIOMYCETES Chlorociboria aeruginosa (5) Outgroup
Botryotinia fuckeliana (5)
Dermea acerina (5)
Leotia lubrica (5)
Above threshold Threshold values

Below threshold 5+4+3+2-gene 70 70 5+4+3-gene
Missing or inapplicable
Conflict at ML-BP ≥70% 5+4-gene 70 70 5-gene
Parmeliaceae
PHOTOBIONT: Lecanoraceae
Coccoid green algae Gypsoplacaceae + Malmideaceae(+‘Lecidea’+Miriquidica)
Trebouxia Cladoniaceae
Asterochloris Stereocaulaceae
LECANORINEAE
Trentepohlia or Phycopeltis [CLADONIINEAE] Squamarinaceae (+Herteliana)
(Trentepohliaceae) Ramboldiaceae
Chlorella, Pseudochlorella Mycoblastaceae
Protococcus, Radiococcus
Tephromelataceae
Cystococcus/Leptosira like
LE
Ramalinaceae (+Bilimbia+Mycobilimbia p.p.+Crocynia
Myrmecia +Megalaria+Byssolecania)
CA
Psoraceae
Stichococcoid algae
Dictyochloropsis s.l. NO Pilocarpaceae (+Schadonia)
RA SPHAEROPHORINEAE (INCL. PSORINEAE)
Sphaerophoraceae (+Protomicarea)
Coccomyxa, LE
Pseudococcomyxa
S Psilolechiaceae
?
Cyanobacteria Scoliciosporaceae/S. umbrinum
Nostoc Haematomma accolens = not Haematomma
Scytonema, Stigonema, TELOSCHISTINEAE Teloschistaceae
Hyphomorpha, Calothrix, TELOSCHISTALES Megalosporaceae (+Sipmaniella)
Tolypothrix, Dichothrix, Brigantiaeaceae
other filamentous taxa LETROUTINAE
Letrouitiaceae
Gloeocapsa, Anacystis, LEPROCAULALES/Leprocaulaceae
other coccoid taxa ?S
E Caliciaceae
Rhizonema I C IAL
A L Physciaceae
Non-lichenized C
Lopadiaceae/Lopadium disciforme
living on a cyanolichen Psorula rufonigra
primary secondary Helocarpon crassipes? + Lecidea diapensiae
LE Lobariaceae
CA Peltigeraceae
PELTIGERINEAE
N Nephromataceae
O
RO Massalongiaceae (+ Leptochidium)
M Vahliellaceae/Vahliella leucophaea
Y
CE
T PELTIGERALES OLLEMA T IN E AE Collemataceae
ID C Placynthiaceae
A
E Coccocarpiaceae
Pannariaceae (+ Staurolemma+Physma)
? Koerberiaceae
Romjularia + Mycobilimbia p.p.
LECIDEALES/Lecideaceae s. str.
RHIZOCARPALES/Rhizocarpaceae (+ Catolechia+Sporastatiaceae)
Umbilicariaceae (+ Xylopsora + Elixiaceae)
Ophioparmaceae
UMBILICARIOMYCETIDAE/UMBILICARIALES Fuscideaceae
Ropalosporaceae/Ropalospora chlorantha
Graphidaceae
Gyalectaceae
OS
Sagiolechiaceae
LE
OS Coenogoniaceae
Above threshold
UM
T Group 1 (+‘Petractis’+Ramonia
Below threshold RO +‘Gyalidea’)
PA Phlyctidaceae (+ Petractis)
Missing or inapplicable LE
S Odontotremataceae s. str.
Threshold bootstrap values:
LE
Porinaceae
5+4+3+2-gene 70 70 5+4+3-gene Stictidaceae
CA
5+4-gene 70 70 5-gene ARCTOMIALES /Arctomiaceae

NO
TRAPELIALES/Trapeliaceae
HYMENELIALES /Hymeneliaceae
RO
BAEOMYCETALES/Baeomycetaceae
PE
MY
RT Megasporaceae
US Ochrolechiaceae
CE
AR
IA Coccotremataceae
OSTROPOMYCETIDAE LE
TE
S Pertusariaceae (+Agyriaceae)
S
Icmadophilaceae
SARRAMEANALES/Sarrameanaceae (+Schaereriaceae)
Candelariaceae
‘CANDELARIOMYCETIDAE’/CANDELARIALES Dactylosporaceae
Strangosporaceae
ACAROSPOROMYCETIDAE/ACAROSPORALES/Acarosporaceae + Eigleraceae
Geoglossomycetes
Lichinomycetes
Pycnoraceae [Candelariales; Candelariomycetidae]
Sarea resinae [Trapeliaceae, Lecanoromycetes;Leotiomycetes]
Leotiomycetes
Leotia lubrica
Percentage of internodes
0
10
20
30
40
50
60
70
80
90
100
Parmeliaceae (272)
Lecanoraceae (49)
Gypsoplacaceae (7)
Cladoniaceae (30)
Stereocaulaceae (15)
Squamarinaceae (5)
Ramboldiaceae (4)
Mycoblastaceae (2)
Tephromelataceae (2) BS-ML≥70%
Ramalinaceae (54)
Psoraceae (8)
Lecanorales [3.3]
Pilocarpaceae (13)
Sphaerophoraceae (5)
Psilolechiaceae (3)
Scoliciosporaceae (1)
Teloschistaceae (64)
Megalosporaceae (4)
Brigantiaeaceae (5)
Letrouitiaceae (2)
BS-ML<70%
Leprocaulales (2)
Caliciaceae (25)
Physciaceae (19)
Teloschistales [4.0]
Lecanoromycetidae [3.4]
Helocarpaceae (1)
Lobariaceae (53)
Peltigeraceae (39)
Nephromataceae (4)
Massalongiaceae (6)
Collemataceae (39)
Placynthiaceae (3)
Coccocarpiaceae (2)
Pannariaceae (35)
Peltigerales [3.1]
Koerberiaceae (2)
internode absent
Romjularia + Mycobilimbia p.p. (1)

Lecideales (33)
Rhizocarpales (14)
Umbilicariaceae (33)
Ophioparmaceae (7)
Fuscideaceae (5)
Graphidaceae (99)
Gyalectaceae (11)
Sagiolechiaceae (2)
Umbilicariales [3.5]/
Coenogoniaceae (3)
Umbilicariomycetidae
Group 1 (3)
Phlyctidaceae (2)
Odontotremataceae s. str. (2)
Ostropales [2.6]
Porinaceae (4)
Stictidaceae (26)
Arctomiales (5)
Trapeliales (33)
Hymeneliales (6)
Baeomycetales (5)
Megasporaceae (36)
Ochrolechiaceae (18)
Ostropomycetidae [2.9]
Coccotremataceae (3)
Pertusariaceae (51)
Icmadophilaceae (8)
Sarrameanales (8)
Pertusariales [3.2]
Candelariales (9)
Dactylosporaceae (4)
Strangosporaceae (2)
average no. of genes in the 5+4+3+2-gene dataset
Acarosporales (37)
0
1
2
3
4
5
0.5
1.5
2.5
3.5
4.5
Average no. of genes

100
Percentage of internodes with BS-‐ML≥70%
y = 26.044x -‐ 45.129
90 R² = 0.72638
80
70
60
50

40
30
20
10
0
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5
Average no. of genes
Eigleraceae ACAROSPORALES (2) ACAROSPOROMYCETIDAE
Acarosporaceae
Candelariaceae CANDELARIALES (1) ‘CANDELARIOMYCETIDAE’
Pycnoraceae?
Dactylosporaceae?
Strangosporaceae nom. nov?
Sarrameanaceae incl. Schaereriaceae SARRAMEANALES (1)
Icmadophilaceae
Miltideaceae
Pertusariaceae incl. Agyriaceae PERTUSARIALES (6)
Coccotremataceae
OSTROPOMYCETIDAE
Ochrolechiaceae
Megasporaceae
Baeomycetaceae BAEOMYCETALES (2)
Anamylopsoraceae
Hymeneliaceae HYMENELIALES NOM. NOV. (1)
Protothelenellaceae*
Trapeliaceae TRAPELIALES (1)
Arctomiaceae ARCTOMIALES NOM. NOV. (1)
Thelenellaceae*
Graphidaceae
Gyalectaceae
Sagiolechiaceae
Coenogoniaceae
Phlyctidaceae OSTROPALES (10)
Odontotremataceae
Porinaceae
Stictidaceae
Myeloconidaceae*
Phaneromycetaceae*
Ropalosporaceae
Fuscideaceae
Ophioparmaceae UMBILICARIOMYCETIDAE
Umbilicariaceae UMBILICARIALES (3)
Elixiaceae
Rhizocarpaceae incl. Sporastatiaceae RHIZOCARPALES (1)
Lecideaceae LECIDEALES (1)
Pannariaceae
Coccocarpiaceae
Placynthiaceae
Collematineae
Collemataceae
Koerberiaceae PELTIGERALES (10)
Vahliellaceae
Massalongiaceae Peltigerineae
Nephromataceae
Lobariaceae
Peltigeraceae
LECANOROMYCETIDAE
Lopadiaceae
Leprocaulaceae LEPROCAULALES (1)
Physciaceae CALICIALES (2)
Caliciaceae
Brigantiaeaceae
Letrouitiaceae Letrouitineae
Teloschistaceae TELOSCHISTALES (4)
Megalosporaceae Teloschistineae
Pachyascaceae*
Biatorellaceae
Calycidiaceae*
Catillariaceae
Scoliciosporaceae
Psilolechiaceae nom. nov.
Pilocarpaceae
Sphaerophoraceae Sphaerophorineae incl. Psorineae
Psoraceae
Ramalinaceae incl. Crocyniaceae and Megalariaceae
Tephromelaceae
Mycoblastaceae LECANORALES (21)
Parmeliaceae
Squamarinaceae
Stereocaulaceae Lecanorineae
Cladoniaceae
Carbonicolaceae
Ramboldiaceae nom. nov.
Gypsoplacaceae + Malmideaceae
Lecanoraceae incl. Haematommaceae
TABLE I. Summary of datasets including number of OTUs, number of analyzed sites (before slash), total number of sites (after slash),
and the percentage of analyzed sites (in parentheses) for each gene separately, and combined, in comparison with datasets used in
Miadlikowska et al. (2006). The 5-gene dataset includes taxa for which complete or partial data from all five genes (nucSSU, nucLSU,
mitSSU, RPB1 and RPB2) were available. The 5+4-gene, 5+4+3-gene, and 5+4+3+2-gene datasets include taxa for which at least four,
three, or two genes were included, respectively.
Newly
added 5+4+3+2-
Missing 5-gene 5-gene 5+4-gene 5+4-gene 5+4+3-gene 5+4+3-gene
OTUs or gene
Datasets sequences1 dataset/ dataset/ dataset/ dataset/ dataset/ dataset/
sequences dataset/
2006 this study 2006 this study 2006 this study
to this study
GenBank1
No. of
OTUs/percent 183 388 764 1317
148 OTUs NA 111/0% 188/26% 274/37%
of missing (+72)/0% (+200)/10% (+490)/25% (+553)/54%
data
1125/7445 1494/15913 1085/72281494/15913 1071/7215 1494/15913 1494/15913
nucSSU 137 OTUs 711
(15%) (9%) (15%) (9%) (15%) (9%) (9%)
1141/5162 1061/9363 1121/5151 1061/9363 1122/5096 1061/9363 1061/9363
nucLSU 163 OTUs 126
(22%) (11%) (22%) (11%) (22%) (11%) (11%)
471/2635 429/3642 437/2691 429/3642 445/2862 429/3642 429/3642
mitSSU 176 OTUs 130
(17%) (12%) (16%) (12%) (15%) (12%) (12%)
98 (113*) 778 2688/3159 2676/3172 2676/3243 2676/3172 2673/3229 2676/3172 2676/3172
RPB1
OTUs (2007*) (85%) (84%) (82%) (84%) (83%) (84%) (84%)
61 (81^) 878 1932/2291 1773/2307 1851/2349 1773/2307 1803/2409 1773/2307 1773/2307
RPB2
OTUs (1864^) (84%) (77%) (79%) (77%) (75%) (77%) (77%)
7357/20692 7433/34397 7170/20662
7433/34397 7114/20811 7433/34397 7433/34397
Combined 635 seq. 2623
(35%) (22%) (35%) (22%) (34%) (22%) (22%)
1
*: RPB1 (A-F) and RPB1 (F-G) separately; ^: RPB2 (5-7) and RPB2 (7-11) separately; : in the most inclusive (5+4+3+2-gene) dataset
assembled for this study
Parmeliaceae
PHOTOBIONT: Lecanoraceae
Coccoid green algae Gypsoplacaceae + Malmideaceae(+‘Lecidea’+Miriquidica)
Trebouxia Cladoniaceae
Asterochloris Stereocaulaceae
LECANORINEAE
Trentepohlia or Phycopeltis [CLADONIINEAE] Squamarinaceae (+Herteliana)
(Trentepohliaceae) Ramboldiaceae
Chlorella, Pseudochlorella Mycoblastaceae
Protococcus, Radiococcus
Tephromelataceae
Cystococcus/Leptosira like
LE
Ramalinaceae (+Bilimbia+Mycobilimbia p.p.+Crocynia
Myrmecia +Megalaria+Byssolecania)
CA
Psoraceae
Stichococcoid algae
Dictyochloropsis s.l. NO Pilocarpaceae (+Schadonia)
RA SPHAEROPHORINEAE (INCL. PSORINEAE)
Sphaerophoraceae (+Protomicarea)
Coccomyxa, LE
Pseudococcomyxa
S Psilolechiaceae
?
Cyanobacteria Scoliciosporaceae/S. umbrinum
Nostoc Haematomma accolens = not Haematomma
Scytonema, Stigonema, TELOSCHISTINEAE Teloschistaceae
Hyphomorpha, Calothrix, TELOSCHISTALES Megalosporaceae (+Sipmaniella)
Tolypothrix, Dichothrix, Brigantiaeaceae
other filamentous taxa LETROUTINAE
Letrouitiaceae
Gloeocapsa, Anacystis, LEPROCAULALES/Leprocaulaceae
other coccoid taxa ?S
E Caliciaceae
Rhizonema I C IAL
A L Physciaceae
Non-lichenized C
Lopadiaceae/Lopadium disciforme
living on a cyanolichen Psorula rufonigra
primary secondary Helocarpon crassipes? + Lecidea diapensiae
LE Lobariaceae
CA Peltigeraceae
PELTIGERINEAE
N Nephromataceae
O
RO Massalongiaceae (+ Leptochidium)
M Vahliellaceae/Vahliella leucophaea
Y
CE
T PELTIGERALES OLLEMA T IN E AE Collemataceae
ID C Placynthiaceae
A
E Coccocarpiaceae
Pannariaceae (+ Staurolemma+Physma)
? Koerberiaceae
Romjularia + Mycobilimbia p.p.
LECIDEALES/Lecideaceae s. str.
RHIZOCARPALES/Rhizocarpaceae (+ Catolechia+Sporastatiaceae)
Umbilicariaceae (+ Xylopsora + Elixiaceae)
Ophioparmaceae
UMBILICARIOMYCETIDAE/UMBILICARIALES Fuscideaceae
Ropalosporaceae/Ropalospora chlorantha
Graphidaceae
Gyalectaceae
OS
Sagiolechiaceae
LE
OS Coenogoniaceae
Above threshold
UM
T Group 1 (+‘Petractis’+Ramonia
Below threshold RO +‘Gyalidea’)
PA Phlyctidaceae (+ Petractis)
Missing or inapplicable LE
S Odontotremataceae s. str.
Threshold bootstrap values:
LE
Porinaceae
5+4+3+2-gene 70 70 5+4+3-gene Stictidaceae
CA
5+4-gene 70 70 5-gene ARCTOMIALES /Arctomiaceae

NO
TRAPELIALES/Trapeliaceae
HYMENELIALES /Hymeneliaceae
RO
BAEOMYCETALES/Baeomycetaceae
PE
MY
RT Megasporaceae
US Ochrolechiaceae
CE
AR
IA Coccotremataceae
OSTROPOMYCETIDAE LE
TE
S Pertusariaceae (+Agyriaceae)
S
Icmadophilaceae
SARRAMEANALES/Sarrameanaceae (+Schaereriaceae)
Candelariaceae
‘CANDELARIOMYCETIDAE’/CANDELARIALES Dactylosporaceae
Strangosporaceae
ACAROSPOROMYCETIDAE/ACAROSPORALES/Acarosporaceae + Eigleraceae
Geoglossomycetes
Lichinomycetes
Pycnoraceae [Candelariales; Candelariomycetidae]
Sarea resinae [Trapeliaceae, Lecanoromycetes;Leotiomycetes]
Leotiomycetes
Leotia lubrica
1 1. Large-scale molecular phylogenetic synthesis of the third largest class of
2 fungi.
3 2. Based on a cumulative supermatrix approach of > 1100 species

4 representing all orders.
5 3. Up to ca. 8 kb from four nuclear and one mitochondrial genes were

6 sequenced per species.
7 4. Comprehensive revision of classification including 74 taxonomic

8 conclusions.
9 5. A new module “Hypha” of the Mesquite software is introduced.

10
11
80

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Accepted Manuscript

A multigene phylogenetic synthesis for the class Lecanoromycetes (Ascomy-

Jolanta Miadlikowska, Frank Kauff, Filip Högnabba, Jeffrey C. Oliver, Katalin

To appear in: Molecular Phylogenetics and Evolution

Received Date: 31 May 2013

Parmotrema tinctorum 1 (4)

Bulbothrix meizospora (3)

Flavocetraria nivalis 1 (3) Flavocetraria

Cetrarioid core group

Lecidella elaeochroma (4)

s.str. Crocynia pyxinoides (5) [Crocyniaceae]

Seirophora contortuplicata (2)

Texosporium sancti-jacobi (2)*

Lobarina oregana (2)

Peltigera retifoveata (2) Retifoveatae

Scytinium plicatile (3)

Coccocarpia palmicola (4)

LECIDEACEAE S. STR./ Lecidea fuscoatra 0 (5)*

Umbilicaria decussata (3)

Wirthiotrema glaucopallens (2)* Wirthiotrema

Odontotrema phacidiellum (2)

Ochrolechia pallescens (3)

ACAROSPORACEAE Acarospora peliscypha (3)

PYCNORACEAE [CANDELARIALES; ‘CANDELARIOMYCETIDAE’] Geoglossum nigritum (5)

Above threshold Threshold values

5+4-gene 70 70 5-gene ARCTOMIALES /Arctomiaceae

Romjularia + Mycobilimbia p.p. (1)

Average no. of genes

5+4-gene 70 70 5-gene ARCTOMIALES /Arctomiaceae

3 2. Based on a cumulative supermatrix approach of > 1100 species

5 3. Up to ca. 8 kb from four nuclear and one mitochondrial genes were

7 4. Comprehensive revision of classification including 74 taxonomic

9 5. A new module “Hypha” of the Mesquite software is introduced.

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