The circumscription and phylogenetic position of Bryonora (Lecanoraceae, Ascomycota), with two additions to the genus

ABSTRACT

Lecanoraceae is one of the largest families of the Lecanoromycetes, with about 30 accepted genera, many of which, however, have uncertain status and/or circumscriptions. We assess the phylogenetic position of the genus Bryonora and its segregate Bryodina for the first time, using a six-locus phylogeny comprising the Lecanoraceae as well as closely related families. We find strong support for the placement of Bryonora in the Lecanoraceae, whereas there is no support for treating Bryodina as a genus separate from Bryonora. Hence, we reduce Bryodina to synonymy with Bryonora. Further, we describe Bryonora microlepis as new to science and transfer Lecanora castaneoides to Bryonora and L. vicaria to Miriquidica. A world key to Bryonora is included.

KEYWORDS:

• Ascomycota
• Bryodina
• key
• Lecanoraceae
• new species
• 3 new taxa
• 3 new typifications

INTRODUCTION

Lecanoroid lichens form a vast, heterogeneous assemblage including crustose lichens with apothecial margins containing algal cells and, in most species, unicellular ascospores. For a long time, the type genus Lecanora Ach. represented the large majority of the described species of this group, especially after the introduction of Zahlbruckner’s highly influential but largely artificial taxonomic system (Zahlbruckner 1928). Subsequent work, from 1960 and onward, has sought to delimit more natural groups of lecanoroid lichens. Several segregates have been found to belong to other families than the Lecanoraceae, for example, Aspicilia A. Massal. (Megasporaceae), Squamarina Poelt (Squamarinaceae), and Trapelia M. Choisy (Trapeliaceae) (Wijayawardene et al. 2018). Others have been split from the genus Lecanora but were retained in the Lecanoraceae, for example, Myriolecis Clem., Palicella Rodr. Flakus & Printzen, and Protoparmeliopsis M. Choisy (Rodriguez Flakus and Printzen 2014; Wijayawardene et al. 2018; Zhao et al. 2016). Another such segregate is the genus Bryonora Poelt, which comprises 13 crustose, lichenized species mainly overgrowing dead bryophytes in arctic or alpine environments.

Bryonora was segregated from Lecanora by Poelt (1983), mainly on the basis of the well-delimited apothecial cortex composed of strongly conglutinated hyphae. The species included in Bryonora also seemed to share ecological preferences, occurring mainly above the tree limit and usually growing on soil, plant debris, or bryophytes on acidic ground. Morphologically, they are also rather similar, i.e., in their brownish color, rather large and more or less stipitate apothecia, and usually poorly developed thallus. In addition, ascospores of Bryonora generally become septate, in contrast to the species remaining in Lecanora. Poelt included six species in Bryonora, three of which were newly described by him: B. castanea (Hepp) Poelt, B. corallina Poelt, B. curvescens (Mudd) Poelt, B. rhypariza (Nyl.) Poelt, B. stipitata Poelt, and B. yeti Poelt. Poelt also suggested that the diversification center of the genus was located in the high mountains of southern Asia.

Poelt and Obermayer (1991) divided Bryonora into three infrageneric groups: section Bryonora, which included the types species of the genus (B. castanea) as well as four other species (B. curvescens, B. pruinosa, B. septentrionalis, and B. reducta) with poorly developed thallus and a cortex that does not refract polarized light (POL−); section Stipitantes, including three species (B. pulvinar, B. stipitata, and B. yeti) with subfruticose to fruticose thalli and a cortex that refracts polarized light(POL+); and section Rhyparizae, including the two species B. rhypariza and B. selenospora. This latter section was later segregated from Bryonora and placed in the new genus Bryodina Hafellner (Hafellner and Türk 2001). The thallus in B. rhypariza, the type species of Bryodina, is comparatively well developed, crust-forming, or squamulose. A further difference from species left in Bryonora is a paler apothecial margin, consisting of hyphae that are separated at the ends, not conglutinated all the way as in Bryonora. Finally, the ascospore wall is slightly thinner and the ecology seemingly differs, as B. rhypariza sometimes grows directly on rock (Hafellner and Türk 2001; Poelt and Obermayer 1991).

Since the first treatment of Bryonora, the number of species in the genus has more than doubled. In a paper addressing cyanotrophy in lichens, Poelt and Mayrhofer (1988) described two new species of Bryonora and one variety: B. reducta Poelt & Mayrh., B. selenospora Poelt. & Mayrh., and B. rhypariza var. cyanotropha Poelt & Mayrh., all from Nepal. Holtan-Hartwig (1991) showed that Lecanora castanea f. pruinosa (Th. Fr.) Th. Fr. is a distinct species and transferred it to Bryonora as B. pruinosa (Th. Fr.) Holt.-Hartw. In the same publication, he also described B. septentrionalis Holt.-Hartw from Fennoscandia and Russia. Poelt and Obermayer (1991) provided an overview of the genus and described one new species from Nepal, B. pulvinar Poelt & Oberm., with two varieties. All known species of Bryonora up to that point were from the Northern Hemisphere, but Øvstedal (Øvstedal and Lewis Smith 2001) described B. peltata Øvstedal, from the Antarctic Peninsula and Fryday and Øvstedal (2012) added a second species from the Southern Hemisphere, B. granulata Fryday, from the Falkland Islands.

In his original treatment of Bryonora, Poelt (1983) also discussed some species that he did not include in the new genus, although they showed affinities to Bryonora. One of these was Lecanora castaneoides H. Magn., which he excluded from Bryonora on account of the thin apothecium cortex, a zeorine apothecial margin, and unicellular ascospores. It was noted that a zeorine margin was unusual within Bryonora and present only in B. rhypariza. Lecanora castaneoides also seemed to have a different ecology, occurring on weathered rocks rather than on soil or plant debris on the ground. Further, Poelt (1983) excluded L. vicaria (Th. Fr.) Vain. (originally described as L. rhypariza ssp. vicaria Th. Fr.), as he found this species to deviate from Bryonora in the poorly delimited apothecium cortex and the zeorine apothecial margin.

Both Bryonora and Bryodina are generally viewed as a members of the Lecanoraceae (e.g., Wijayawardene et al. 2018; Zhao et al. 2016), but it should be noted that Poelt and Obermayer (1991) suggested, on anatomical grounds, that Bryonora (including the two species later transferred to Bryodina) was close to Protoparmelia M. Choisy, a genus that is now included in the Parmeliaceae (Wijayawardene et al. 2018). The genus Bryonora has so far received scant attention in molecular studies. An internal transcribed spacer (ITS) sequence from B. castanea was published by Grube et al. (2004) and appeared in an unresolved position in an unrooted maximum likelihood distance tree including taxa from several families. The same sequence was utilized by Zhao et al. (2016), and the branch containing B. castanea appeared as a polytomy in an unsupported clade with the genera Adelolecia Hertel & Hafellner, Frutidella Kalb, and Pyrrhospora Körb. and the Lecanora subfusca and L. subcarnea groups. Thus, the delimitation of Bryonora and Bryodina, as well as their phylogenetic positions, remains untested using comprehensive taxon sampling and multiple loci.

Our aims were to assess the phylogenetic position of Bryonora and to evaluate the delimitation of the genus, both in relation to its segregate Bryodina and with regard to species previously excluded from the genus by Poelt (1983), i.e., Lecanora castaneoides and L. vicaria. In addition, we investigated material of a previously unknown species of the genus discovered in Norway.

MATERIALS AND METHODS

Morphology and anatomy.—

Measurements were made under a light microscope of material mounted in water, using an oil-immersion lens with a precision of 0.5 µm for measurements of finer anatomical structures (e.g., ascospores and paraphyses). Only well-developed ascospores lying outside the asci were measured. To examine color reactions of pigments and solubility of crystals, we used a 10% solution of KOH (abbreviated K), and a 10% solution of HNO₃ (abbreviated N). Apical structures of asci were examined in an iodine solution (0.3–0.4% I, abbreviated I) after pretreatment with K (abbreviated KI). Algae in the upper cortex of Bryonora microlepis were observed in chlorine-zinc-iodine (ClZnI). We checked thallus reactions with K and C, and, in addition, with an ethanolic solution of para-phenylenediamine (abbreviated Pd). We performed high-performance thin-layer chromatography (HPTLC) (following the method described by Arup et al. 1993) and TLC (Orange et al. 2010), in both cases using solvent systems B and C. Material from the following herbaria was included in the study: LD, O, S, TRH, and UPS.

DNA extraction, amplification, and sequencing.—

We extracted total DNA using a Qiagen DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) or a E.Z.N.A. Plant DNA Kit (Omega Bio-tek, Norcross, Georgia) following the manufacturers’ instructions or, alternatively, with Chelex 100 (Bio-Rad, Hercules, California) following the procedure described by Ferencova et al. (2017). For amplification and sequencing, we used the primers mrSSU1, mrSSU2, and mrSSU3R for the mitochondrial ribosomal small subunit 12S, hereafter mrSSU (Zoller et al. 1999); LRlecF and LrlecR for nuclear ribosomal large subunit 28S, hereafter LSU (Schneider et al. 2015); ITS1F, ITS5, and ITS4 for nuc rDNA ITS1-5.8-ITS2 (Gardes and Bruns 1993; White et al. 1990); MCM7-709for and MCM7-1348rev for the mini-chromosome maintenance complex 7, hereafter MCM7 (Schmitt et al. 2009); and fRPB2-5F and fRPB2-7cR with the nested primers RPB2-980F and RPB2-1554R for the RNA polymerase II complex subunit, hereafter RPB2 (Liu et al. 1999; Reeb et al. 2004). For the RNA polymerase II largest subunit, hereafter RPB1, we used the newly designed primers RPB1-Lec540for (5′-GAR ACN ATG GAY GAG CA-3´) and RPB1-Lec1950rev (5′-ACV CGA TGK CCC ATC AT-3′) with the nested primers RPB1-Lec800for (5′-ACV GTH TGY CAC AAC TG-3′) and RPB1-Lec1600rev (5′-RTC MAC WCK YTT MCC CAT-3´) or RPB1-Lec1400rev (5′-ACA TTS CCG YTY GCA CG-3′). For polymerase chain reactions (PCRs) for mrSSU, ITS, and RPB2, we followed the protocols of Svensson and Westberg (2021). For LSU, MCM7, and RPB1, we used VWR Red Taq Polymerase Master Mix (VWR International, Belgium) following the manufacturer’s protocols. For LSU, we used the following PCR thermal profile: an initial hold at 94 C for 3 min; followed by 35 cycles of denaturization at 94 C for 30 s, annealing at 57 C for 45 s, and polymerization at 72 C for 1 min 45 s; and finally a hold at 72 C for 10 min. For MCM7, we used the following PCR thermal profile: an initial hold at 94 C for 3 min; followed by 3 cycles of denaturization at 94 C for 45 s, annealing at 57 C for 45 s, and polymerization at 72 C for 1 min 45 s; then 4 cycles of 94 C for 40 s, 54 C for 40 s, and 72 C for 45 s; 4 cycles of 94 C for 40 s, 51 C for 40 s, and 72 C for 45 s; 30 cycles of 94 C for 30 s, 48 C for 30 s, and 72 C for 45 s; and finally a hold at 72 C for 10 min. For RPB1, we used the following PCR thermal profile for the first PCR: an initial hold at 94 C for 3 min; followed by 15 cycles of denaturization at 94 C for 30 s, annealing at 65 C for 45 s (decreasing 1 C each cycle), and polymerization at 72 C for 1 min 20 s; then 28 cycles of 94 C for 30 s, 50 C for 45 s, and 72 C for 1 min 20 s; and finally a hold at 72 C for 10 min. For the second PCR, using nested primers, we used the same PCR cycling thermal profile as for the first PCR. PCR products were subsequently purified with ExoCleanUp FAST (VWR International, Belgium). In addition, three ITS sequences (MW927468–MW927470) were provided by the Canadian Center for DNA Barcoding (CCDB; http://www.ccdb.ca), using the primer pairs ITS1F/ITS4 or ITS5/ITS4.

Taxon sampling.—

To determine the phylogenetic position of Bryonora, we assembled a sequence data set with a selection of species and genera from the Lecanoraceae, a couple of closely related families, and a more distant outgroup. We included sequences from five currently accepted species of Bryonora, as well as two species (Lecanora castaneoides and L. vicaria) previously considered but never included in the genus. Sequences from a potentially undescribed species of Bryonora were also included. The included specimens of B. castanea and B. rhypariza belonged to these species in the strict sense; that is, not to any of the varieties of these species that have been described from the Himalayas (Poelt 1983; Poelt and Obermayer 1991). We included various species of the genus Lecanora s.l., including species from the Lecanora subfusca group, L. polytropa group, L. subcarnea group, and L. symmicta group and L. formosa. We also included species of the genera Miriquidica Hertel & Rambold, Myriolecis, Rhizoplaca Zopf, Palicella, Protoparmeliopsis, and Lecidella Körb., which belong to the Lecanoraceae as well (Wijayawardene et al. 2018; Zhao et al. 2016). To test whether Bryonora has affinities to Protoparmelia, we included two species of this genus in addition to two other genera in the Parmeliaceae (Bryoria Brodo & D. Hawksw. and Evernia Ach.). We further included representatives from the Cladoniaceae (the genus Cladonia P. Browne) and the Stereocaulaceae (the genera Hertelidea Printzen & Kantvilas and Stereocaulon (Schreb.) Schrad.), as these families are sisters to the Lecanoraceae (Miadlikowska et al. 2014; Schmull et al. 2011). As an outgroup, we selected the Rhizocarpaceae (the genus Rhizocarpon Ramond ex DC.), as this family appears as basal to the Lecanoromycetidae (Miadlikowska et al. 2014), which includes all other families included in the analysis. For the selected genera and species, we downloaded sequences of mrSSU, LSU, ITS, MCM7, RPB1, and RPB2 from GenBank (TABLE 1).

Table 1. Sequence data used for the phylogenetic analysis, with GenBank accession numbers and voucher information

Taxon	Voucher/source	mrSSU	ITS	LSU	MCM7	RPB1	RPB2
Bryonora castanea 1	Sweden, Westberg PAD321 (UPS)	OM417201	OM423658	OM423613		OM417054	OM417053
Bryonora castanea 2	Austria, Hafellner 33126 (GZU)		AY541238
Bryonora castaneoides 1	Sweden, Svensson 3156 (UPS)	OM417202	OM423659	OM423614	OM417052	OM417058
Bryonora castaneoides 2	Sweden, Westberg KAR156 (UPS)		OM423660	OM423615
Bryonora castaneoides 3	Sweden, Arup L13343 (LD)		OM423661
Bryonora curvescens 1	Sweden, Westberg UCDLR150 (UPS)	OM417203		OM423616
Bryonora curvescens 2	Sweden, Svensson 3490 (UPS)	OM417204	OM423662		OM417051	OM417055
Bryonora curvescens 3	Norway, Holien 14945 (TRH)		MW927468
Bryonora microlepis 1	Norway, Haugan & Timdal 9931 (O)	OM296131	OM296137
Bryonora microlepis 2	Norway, Klepsland 625 (holotype, O)	OM296130	OM296136
Bryonora microlepis 3	Norway, Timdal 13869 (O)		MW927469
Bryonora microlepis 4	Norway, Haugan 8628 (O)		OM296135
Bryonora pruinosa 1	Sweden, Westberg et al. PL101 (UPS)	OM417205	OM423663			OM417057
Bryonora pruinosa 2	Norway, Westberg s.n. (UPS)	OM417206	OM423664
Bryonora rhypariza 1	Sweden, Arup L14139 (LD)	OM417207	OM423665
Bryonora rhypariza 2	Sweden, Svensson 3397 (UPS)	OM417208	OM423666		OM417050	OM417056
Bryonora rhypariza 3	Norway, Bendiksby et al. 12441 (O)	OM296134
Bryonora septentrionalis 1	Sweden, Westberg et al. PL272 (UPS)	OM417209	OM423667	OM423617
Bryonora septentrionalis 2	Norway, Haugan 8351 (O)	OM296132	OM296138
Bryonora septentrionalis 3	Canada, Timdal & Rui 18013 (O)		MW927470
Bryoria nadvornikiana	Sweden, Hermansson 14179 (UPS), Myllys et al. 2011	HQ402664	HQ402719	KR995384	KU668465	KU668551
Cladonia coniocraea	Spain, MACB 93729, Pino-Bodas et al. 2013		KC526130				KC526069
Cladonia mitis	Finland, Myllys 240513-1 (H), Myllys et al. 2014		KJ947957		KJ948081
Cladonia stygia	Finland, Stenroos 5200 (TUR), Miadlikowska et al. 2014	KJ766374				KJ766847
Cladonia sulcata	Tasmania, Lumbsch 19975i (F), Parnmen et al. 2010	GQ500949	GQ500913	GQ500959	HM441295
Evernia prunastri	Belgium, Ertz 7596 (BR, DUKE), Miadlikowska et al. 2014	KJ766389	HQ650611	KJ766557			KJ766953
Hertelidea botryosa	U.S.A., McCune 28534 (H), Miadlikowska et al. 2014	KJ766403		KJ766568		KJ766856
Lecanora allophana	Sweden, Ekman 3434 (BG), Andersen and Ekman 2005	AY567710		AY853376
Lecanora caesiorubella	Australia, Lumbsch 19974k (F), Schoch et al. 2012		JN943728	JN939508	JN992711	JN987920
Lecanora flavopalllida	Australia, Lumbsch 19972d (F), Kirika et al. 2012	JQ782673	JN943723	JN939516	JN992722	JN987925	KT453938
Lecanora formosa	China, ZX YN0188, Zhao et al. 2016	KT453820	KT453769	KT453774	KT453877		KT453979
Lecanora horiza	Spain, Zhao et al. 2016	KT453821	KT453772		KT453876	KT453903
Lecanora imshaugii	U.S.A., Lumbsch 19273b (F), Kirika et al. 2012	JQ782681	JQ782717		KT453886	KT453904
Lecanora intricata	Austria, Arup L97031 (LD)		AF070022	DQ787345
Lecanora intumescens	Norway, Ekman 3162 (BG), Andersen and Ekman 2005	AY567715		AY300841		AY756386
Lecanora polytropa	U.S.A., Lutzoni et al. 06.04.05-11 (DUKE), Miadlikowska et al. 2006	DQ986807	HQ650643	DQ986792			DQ992418
Lecanora saligna	U.S.A., Leavitt 5702 (BRY), Leavitt et al. 2016		KU934539		KU934910	KU935293	KU935036
Lecanora symmicta	Germany, Printzen 9999a (FR), Rodriguez Flakus and Printzen 2014	KJ152466		KJ152453		KJ152452	KJ152487
Lecidella effugiens	China, Zhao 20141148-2, Zhao et al. 2016	KT453833	KT453747	KT453786	KT453883		KT453941
Lecidella patavina	China, Zhao 20140501-2, Zhao et al. 2016	KT453845	KT453767	KT453799	KT453879	KT453910	KT453967
Miriquidica complanata	Poland, Szczepanska 935 (herb. Szczepanska), Singh et al. 2015	KP822512	KF562187	KF562179	KF562169	KF601233
Miriquidica garovaglii	Poland, Kossowska 221 (herb. Kossowska), Singh et al. 2015	KP822514		KP796395	KP822387
Miriquidica gyrizans	U.S.A., Fryday 10175 (MSC), Spribille et al. 2020	MN508282	MN483126	MN460217
Miriquidica instrata	U.S.A., Spribille s.n. 2010 (GZU), Spribille et al. 2020	MN508311	JN009720	MN460241	JN009746
Miriquidica leucophaea	Poland, Kossowska 1339 (herb. Kossowska), Singh et al. 2015	KP822515	KP822310		KP822388	KP822195
Miriquidica nigroleprosa 1	Poland, Kossowska 158 (herb. Kossowska), Singh et al. 2015	KP822520		KP796401	KP822393
Miriquidica nigroleprosa 2	Poland, Kossowska 128 (herb. Kossowska), Singh et al. 2015	KP822518	KP822312	KP796399	KP822391	KP822198
Miriquidica vicaria 1	Sweden, Nordin 7784 (UPS)	OM417210	OM423668
Miriquidica vicaria 2	Norway, Haugan 10185 (O)	OM296133	OM296139
Miriquidica vicaria 3	Sweden, Nordin 7884 (UPS)		OM423669
Miriquidica vicaria 4	Sweden, Nordin 7794 (UPS)		OM423670
Myriolecis dispersa	U.S.A., Leavitt 12-002, Zhao et al. 2016		KT453733		KT453865	KU935287	KU935029
Myriolecis schofieldii	U.S.A., Spribille 39188 (MSC), Spribille et al. 2020	MN508274	MN483119	MN460213
Palicella glaucopa	Chile, Rodriguez Flakus 2101 (FR), Rodriguez Flakus and Printzen 2014	KJ152473	KJ152484	KJ152456		KJ152443	KJ152490
Palicella schizocromatica	Canada, Spribille 16850A (herb. Spribille), Rodriguez Flakus and Printzen 2014	KJ152465		KJ152457			KJ152489
Protoparmelia badia	Norway, Haugan 8617 (O), Singh et al. 2017	KY012808		KY066281		KY012842
Protoparmelia oelagina	Norway, Tønsberg 41328 (BG), Singh et al. 2017	KY012836	KY066272	KY066317	KY012805
Protoparmeliopsis muralis	U.S.A., Leavitt 143 (BRY-C), Zhao et al. 2016	KT453822	KT453726	KT453776	KT453870	KU935306	KT453928
Rhizocarpon hochstetteri	Finland, Pykälä 22715 (H?), Miadlikowska et al. 2014	KJ766485		KJ766652
Rhizocarpon oederi	U.S.A. Spribille 36629 (MSC), Spribille et al. 2020	MN508296	MN483144	MN460228	MN437622
Rhizocarpon sphaerosporum	Spain, Lumbsch s.n. (F), Wedin et al. 2005	AY853340		AY853390		DQ870991
Rhizocarpon suomiense	Norway, Holtan-Hartwig & Timdal 4917 (O), Ihlen and Ekman 2002	AF483181	AF483613
Rhizocarpon superficiale	U.S.A., Lutzoni et al. 06.08.05-17 (DUKE), Miadlikowska et al. 2006	DQ972988				DQ973060	DQ973074
Rhizoplaca chrysoleuca	U.S.A., BRY 55000, Zhao et al. 2016	KT453856	HM577233	KT453812	HM577385	KT453899	KT453935
Rhizoplaca parilis	Kyrgyzstan, Lommi s.n. (H), Leavitt et al. 2013		JX948193		KU934984	KU935392	KU935138
Stereocaulon paschale	U.S.A.(?), Reeb STEPAS (DUKE), Bhattacharya et al. 2000			AF279413			AY641078
Stereocaulon pileatum	Norway, Tønsberg 27339 (BG), Andersen and Ekman 2005	AY567718	AF517927	AY756335		AY756426
Stereocaulon sp.	Iceland, Resl s.n. 2014 (GZU), Spribille et al. 2020	MN508303	MN483154	MN460234	MN437627

Note. Newly obtained sequences are in boldface.

Sequence alignment and partitioning scheme.—

For the non-protein-coding markers (mrSSU, ITS, and LSU), we estimated separate alignments using PASTA (Mirarab et al. 2015), with the mask option activated, MAFFT (algorithm L-INS-i) as the aligner, OPAL for the pairwise merging, and FastTree as the tree estimator, with GTR+Γ as the model for molecular evolution. PASTA is an iterative method that optimizes the alignment using maximum likelihood; hence, we did not conduct any subsequent filtering of ambiguous regions of the resulting alignments. However, inspection of the alignment of LSU revealed problems due to the presence of long introns in several species. We therefore estimated the LSU data set using MAFFT with the algorithm E-INS-i, which is designed to handle sequence data sets with multiple conserved domains divided by long gaps (Katoh et al. 2019). From the MAFFT alignment of LSU, we removed one intron from Evernia prunastri (226 bp), three from Lecanora polytropa (50, 203, and 236 bp), one from Miriquidica complanata (60 bp), and one from M. gyrizans (79 bp). We then estimated the LSU alignment using PASTA, as above. To avoid potential problems with missing data, the ends of all the PASTA alignments were trimmed.

For the protein-coding genes (MCM7, RPB1, and RPB2), we estimated alignments with MAFFT using the algorithm E-INS-i (Katoh et al. 2019). After aligning the sequences, we identified several noncoding introns in the RPB1 and RPB2 alignments, which we removed before any further analysis was performed. No such introns were detected in the alignment of MCM7. The first 65 bp of our newly generated RPB1 sequences for Bryonora were, although apparently protein-coding, not easily alignable with the rest of the RPB1 alignment and ended with a stop codon. We therefore removed the first 65 bp of the RPB1 for Bryonora before analysis.

We checked for incongruence among markers by performing a separate maximum likelihood analysis of each alignment using IQ-TREE with 500 nonparametric bootstrap replicates (Nguyen et al. 2015). The six single-marker trees from these analyses were then compared to identify any conflicting results, defined as conflicting clades with >75% bootstrap support. We found no such conflicts and therefore decided to concatenate the six alignments into one. The final alignment is included in SUPPLEMENTARY MATERIAL 1.

We assessed the division of the concatenated alignment into partitions using PartitionFinder 2.1.1. (Lanfear et al. 2017), which also allows for simultaneous estimation of models of molecular evolution for the partitions. We restricted the estimation to models implemented in MrBayes 3.2.6., used AICc (Akaike Information Criterion with small sample correction) for model selection, assumed linked branched lengths (= edge-proportional model), and used the “greedy” algorithm (Lanfear et al. 2012). We assessed the division of the concatenated alignment into 14 partitions: mrSSU, LSU, ITS1, 5.8S, ITS2, and independent 1st, 2nd, and 3rd codon positions for each of the three protein-coding genes MCM7, RPB1, and RPB2.

The best model fit was achieved when the 1st codon positions of MCM7 and RPB1 were merged into one partition and when the 2nd and 3rd codon positions of RPB1 and RPB2 were likewise merged into a single partition each. For the resulting 11 partitions, GTR+Γ was selected as the best model for ITS1 and RPB2 1st; GTR+Γ+I for mrSSU, LSU, RPB1 1st + MCM7 1st, RPB1 2nd + RPB2 2nd, and MCM7 3rd; SYM+Γ for ITS2; SYM+Γ+I for RPB1 3rd + RPB2 3rd; HKY+Γ+I for MCM7 2nd; and K80+Γ+I for 5.8S.

Phylogenetic analysis.—

We performed phylogenetic analyses on the concatenated, partitioned alignments, implementing the models of molecular evolution from PartitionFinder using MrBayes 3.2.6. (Ronquist et al. 2012). We used flat Dirichlet priors for the substitution rates and state frequencies, an exponential (1) distribution for the gamma shape parameter, a compound Dirichlet prior (α = 1, β = 0.1) for branch lengths, and uniform distributions for invariant sites and topology. We performed two runs of four Markov chain Monte Carlo (MCMC) chains each, three heated and one cold. For initial test runs, we set the temperature of the heated chains to 0.20 but reduced this to 0.10 to improve swapping rates. The sample frequency was set to every 100th generation. The analysis was halted when convergence was reached, which was defined as an average standard deviation of split frequencies below 0.01. The fraction of trees discarded as burn-in was set to 25%. We considered a posterior probability of 0.95 or higher as high support.

In addition to the Bayesian analysis, we also performed maximum likelihood analyses of the concatenated alignment with IQ-TREE, using the same partitioning scheme and models of molecular evolution as for the Bayesian analysis. As in the Bayesian analysis, we used edge-proportional partition models. We assessed branch support by running 500 nonparametric bootstrap replicates. We considered a bootstrap value above 85% as high support.

RESULTS

We generated 50 new sequences (TABLE 1). The final alignment included 68 terminals, 215 sequences, and had 4784 characters, 1763 of which were parsimony-informative. The analysis halted after 11.4 million generations, resulting in a posterior of 85 681 samples. The majority-rule consensus tree is shown in FIG. 1, with bootstrap values from the maximum likelihood analysis above 70% added.

Figure 1. Majority-rule consensus tree based on a Bayesian MCMC analysis of mrSSU, LSU, ITS, MCM7, RPB1, and RPB2, showing the phylogenetic position of Bryonora in the Lecanoraceae. Branch support is given as posterior probability (PP)/bootstrap support (BS). Bootstrap support values are from a corresponding maximum likelihood analysis. Only BS values >70% are shown. GenBank accession numbers and voucher information are given in TABLE 1.

We found high support (posterior probability [PP] = 1, bootstrap [BS] = 100) for a monophyletic Bryonora, including two main clades: one clade (PP = 1, BS = 100) with five of the seven species, including the type species of both Bryonora (B. castanea) and Bryodina (B. rhypariza), and a second clade (PP = .85, BS = 86) with Bryonora pruinosa and B. septentrionalis (FIG. 1). The phylogenetic position of Bryonora is firmly within the Lecanoraceae, in a highly supported (PP = 1, BS = 88) clade together with, for example, the Lecanora subfusca group and the genus Palicella.

The two candidates for inclusion in Bryonora, Lecanora castaneoides and L. vicaria, were recovered in different parts of the tree. L. castaneoides was found nested within Bryonora, whereas L. vicaria was sister to Miriquidica leucophaea (PP = 1, BS = 83) in a monophyletic Miriquidica (PP = 1, BS = 100). The two species are revised below. Finally, the suspected undescribed species was recovered in Bryonora as well and is newly described as Bryonora microlepis below.

TAXONOMY

Bryonora castaneoides (H. Magn.) Arup, M. Svensson & M. Westb., comb. nov. FIG. 2A–B

Figure 2. A–B. Bryonora castaneoides. A. Habit of form strongly associated with cyanobacteria and a thallus of Vestergrenopsis showing scattered thallus areoles “floating” on a carpet of Stigonema. Note apothecia in upper left hand corner that grow directly on the thallus of Vestergrenopsis (Du Rietz 889c, UPS L-115750). B. Habit of cracked areolate thallus and apothecia not strongly associated with cyanobacteria and cyanolichens (Westberg ULR223, UPS L-880215). Bars = 1 mm.

MycoBank MB842776

≡ Lecanora castaneoides H. Magn., Ark Bot 33A:107. 1946.

Typification: SWEDEN. LYCKSELE LAPPMARK: Tärna par., Björkfors, by a brook at 600 m, 12 Jul 1924, A.H. Magnusson 8143 (UPS L-189715!, lectotype, designated here, MBT10005453; S L2044!, isolectotype).

= ?Lecanora erythrophaea H. Magn., Ark Bot 33A:110. 1946.

Typification: [SWEDEN. LYCKSELE LAPPMARK:] “Tärna: Umfors, rocks on shore of Överuman, mixed with Ionaspis odora and Pyrenopsis sp., [no date] hb. Stm [= Stenholm]” (cited from protolog, material not located).

Thallus variable, from more or less (±) invisible or discernible only around the apothecia to thin, ± confluent to cracked areolate, without a well-defined cortex, K−, C−, Pd−. When associated with cyanophilous lichens or free-living Stigonema, the thallus may be better developed, consisting of scattered areoles, sometimes appearing on a carpet of cyanobacteria and then often with well-defined cortex, 12–20 µm thick, of ± isodiametric cells. Areoles 0.1–0.9 mm diam, usually 0.05–0.2(–0.4) mm thick, even to finely scurfy or minutely granular, beige, brownish to grayish beige, dark gray to brown; medulla with crystals not refracting polarized light, not dissolving in K; photobiont trebouxioid with cells 7–14 µm diam, but sometimes colonies of Nostoc or Stigonema occur within or are clearly associated with the thallus.

Apothecia common, zeorine, scattered or often aggregated, round to angular from compression or irregular, slightly immersed but more commonly adnate to sessile, 0.3–0.6(–1.0) mm diam; disc ± flat to convex, sometimes ± flexuous, dark reddish brown to medium brown, slightly glossy, rarely thinly white pruinose; thalline margin usually deeply suppressed below the proper margin and not possible to see from above, but occasionally conspicuous, even, to 50 µm thick, at base of apothecium, cortex 12–20 µm, well defined, of ± isodiametric cells; proper margin up to 75 µm thick but suppressed and invisible in convex apothecia, concolorous with disc or slightly darker, somewhat glossy, level with disc or somewhat prominent; hymenium (55–)60–70 µm thick, hyaline with only upper part brown; epihymenium brown, without any crystals; paraphyses simple to slightly branched, 2.0–2.5 µm wide with upper cell slightly wider, 3.0–4.0(–4.5) µm; hypothecium hyaline, 70–150 µm thick, consisting of short and irregular to almost isodiametric, thick-walled hyphae; excipulum pale with brown outer part, consisting of radiating, branching hyphae with narrow cell lumina, often with crystals refracting polarized light (POL+), dissolving in K but not in N; amphithecium with crystals not refracting polarized light (POL−), not or partly dissolving in K, but not in N; asci 8-spored, 46–58 × 13–16 µm, of the Lecanora type; ascospores 0–1(–3)-septate, hyaline, narrowly ellipsoid to ± cylindrical, (11–)13–15.4–18(–21) × 4–4.8–5.5(–6) µm, length/width ratio 2.2–3.2–5.0 (n = 91, 9 specimens). Pycnidia not seen.

Chemistry: No lichen substances detected by HPTLC.

Ecology and distribution: Bryonora castaneoides grows directly on siliceous to calciferous rocks, often on various types of slate. It usually grows intermixed with other crustose lichens, often with species of Placynthium and Vestergrenopsis, or seemingly associated with free-living cyanobacteria, mainly of the genera Nostoc and Stigonema. Most of the known localities are near fresh water of creeks and lakes that become inundated or sprayed from time to time. The association with cyanobacteria or lichens containing cyanobacteria seems to stimulate thallus growth and development as well as apothecial growth: a well-defined thallus cortex seems to develop only when abundant cyanobacteria are associated with the lichen thallus. Cells of Nostoc are sometimes even found inside the thallus of B. castaneoides together with the normal trebouxioid algae.

So far, B. castaneoides is known only from Norway (including Svalbard) and Sweden, but it is likely that the species has been overlooked and that the distribution is wider. All localities are within the Scandinavian mountain range or in arctic areas.

Notes: We located two syntypes of Lecanora castaneoides in UPS and S, of which we designate the largest collection (UPS L-189175) as the lectotype. Poelt (1983) cited an “isotype” in M without giving further collection data, but no material matching the protologue can presently be found there (A. Beck, pers. comm., 2021).

Magnusson described Lecanora erythrophaea in the same work as L. castaneoides (Magnusson 1946), but he did not compare the two species. According to Magnusson (1946), the holotype of L. erythrophaea was 1.2 × 0.6 cm and grew intermixed with other lichens in a collection made by Carl Stenholm and kept in his personal herbarium. However, the holotype of L. erythrophaea could not be located in GB, where Stenholm’s herbarium is now lodged (E. Larsson, pers. comm., 2020). Lecanora erythrophaea has only been reported once after its description (Magnusson 1952). The specimen that this report was based on (UPS L-789780!) is Bryonora castaneoides, which is probably why Santesson included L. erythrophaea in the synonymy of L. castaneoides in the Fennoscandian checklist (Santesson 1993; Santesson et al. 2004). As the description of L. erythrophaea is similar to that of L. castaneoides, we also tentatively include L. erythrophaea in the synonymy of L. castaneoides.

Lecidea hilarescens Nyl., described based on material from Greenland (Nylander 1862), was suggested to be related to B. castaneoides by Printzen (1995). The holotype of L. hilarescens (H-Nyl 21207!) has discrete, ± white thallus areoles, rather light, flesh-colored apothecia, ascospores 9 × 3–4 µm, and an epihymenium with brown crystals that dissolve in K. In our opinion, L. hilarescens is not likely to belong in Bryonora and may be a member of the Lecanora polytropa group.

Within the genus Bryonora, B. castaneoides is well characterized by the combination of saxicolous habit, ellipsoid, 0–1-septate ascospores, and a lack of lichen substances. The most closely related species in our phylogeny were B. rhypariza and B. microlepis, both species that, apart from several other differences in anatomy and ecology, have secondary compounds (norstictic acid in the former and stictic acid in the latter). The most similar species in the genus appears to be B. reducta, which was described from a single collection from a high-elevation (3200 m above sea level [a.s.l.]) locality in Nepal by Poelt and Mayrhofer (1988). Like B. castaneoides, it has uniformly brown apothecia, no secondary chemistry, and grows in a mat of Stigonema (Poelt and Mayrhofer 1988; Poelt and Obermayer 1991). The ascospores in B. reducta differ from those of B. castaneoides by being somewhat thicker and dumbbell-shaped (Poelt and Mayrhofer 1988; Poelt and Obermayer 1991) and are unlike any ascospores we have seen in the examined material of B. castaneoides. It is possible that B. reducta represents a later synonym for B. castaneoides, but given the difference in ascospore shape and the large distance between the locality for B. reducta and the known distribution of B. castaneoides, more data are, in our opinion, needed to allow for firm conclusions.

Additional specimens examined: NORWAY. SØR-TRØNDELAG: Oppdal, Blesebekken, 1.4 km SE of Kongsvoll, on rocks at the brook, 1030 m, 5 Aug 1964, Tibell 2259 (UPS L-138332); SVALBARD: Hopen, Husdalen, 1982, Skye (UPS L-204360). SWEDEN. JÄMTLAND: Åre par., Handöl Rapids in river Handölan, W of Lake Ånnsjön, on exposed, flat rock on the shore, ca. 0.2 m above the water level, 29 Jul 1994, Owe-Larsson H94-89 (UPS L-588742); LULE LAPPMARK: Jokkmokk par., Padjelanta National Park, along the Nordkalotten trail ca. 3.6 km S of the Staloluokta cabins, at small lake in the river Viejevágge, on Placynthium on sloping rock surface of slate at the waterfall, alt. 665 m, 28 Jul 2004, Arup L04065 (LD); LYCKSELE LAPPMARK: Tärna par., Ume älv, Över-Umans sydvästligaste vik, “Elyna-ön,” alt. 530 m, svagt sluttande häll, övergeolitoral, [= Ume river, the southwestern most bay of Lake Över-Uman, the ”Elyna-island”, alt. 530 m, gently sloping rock surface on the shore] 17 Aug 1960, Du Rietz 889c (UPS L-115750); “ön 138,” SV-sidan, strandklippor av kalkfri glimmerskiffer, [= the island “138”, SW-side, mica-schistose rocks on the shore] 29 Aug 1960, Du Rietz 2169c (UPS L-119225) [HPTLC: nil]; Vilasund, shore of lake Över-Uman below Mårtensson’s boarding house, alt. 520 m, on medium-sized boulder in the middle geolitoral zone, 25 Aug 1963, Du Rietz 499d (LD, O); ibid., 28 Aug 1963, Du Rietz 588a (UPS L-128417) [HPTLC: nil]; TORNE LAPPMARK: Jukkasjärvi par., Björkliden, 675 m W of the railway station, on exposed, sloping slate rocks on E side the creek Rákkasjohka, elev. ca. 520 m, 6 Aug 2013, Arup L13343 (LD); Abisko National Park, sekt. 7, Abiskojaures östra strand, på strandblock, [= the eastern shore of Lake Abiskojaure, on boulder by the shore] 14 Aug 1943, Santesson (UPS L-123023); Vassitjåkko, on moist stone, ca. 700 m, 26 Jul 1921, Magnusson 5920 (LD, O, S); Karesuando par., S of Maunu, just E of the bridge over the stream Kiltijoki, on rock by the stream, 29 Aug 2019, Westberg & Odelvik KAR156 (UPS L-989349); Kopparåsen, regios subalpina, among boulders, 700 m, 1 Aug 1921, Magnusson 6133b (UPS L-789780); ÅSELE LAPPMARK: Vilhelmina par., 1.9 km NE of Fatmomakke Church, rapids in semiopen, old growth coniferous/deciduous forest, on schistose rock, 27 Jul 2017, Westberg ULR223 (UPS L-880215) [HPTLC: nil]; ibid., Svensson 3156 (UPS).

Bryonora microlepis Haugan & Timdal, sp. nov. FIG. 3A–E

Figure 3. A–E. Bryonora microlepis. A–C. Habit of sterile thallus. Note the pale margin of the lobes and squamules. D–E. Section through squamule, stained in lactophenol cotton blue. A, C–E. Timdal 13869, O L-201412. B. Klepsland 625, holotype, O L-175933. Bars: A–C = 1 mm; D–E = 50 µm.

MycoBank MB842775

Typification: NORWAY. OPPLAND: Vågå, Jønndalen, S slope of Mt. Raudnebb, 61.9532°N, 9.0950°E (± 7 m), 890 m alt., eroded rock outcrop and stabilized sandy soil in S-facing scree, 7 Aug 2011, Jon T. Klepsland 625 (O L-175933!, holotype [TLC: stictic acid]).

Diagnosis: Thallus composed of scattered to conglomerate, up to 0.6 mm diam, rounded, plane to convex, shiny brown, gray-edged squamules containing stictic acid.

Etymology: The name refers to the characteristic, small thallus squamules.

Thallus up to ca. 3 cm diam or irregularly spreading, squamulose, attached by rhizohyphae; hypothallus not seen; squamules small, up to 0.6 mm diam, rounded, plane to convex, first dispersed, later forming conglomerate cushions, adnate to ascending, sometimes partly imbricate, brown, usually gray-edged, shiny, usually with a network of shallow fissures in the cortex, K+ yellow, Pd+ orange; thallus anatomy of the “Kegelrinden” type (Poelt 1958), i.e., with conical cortex sections extending into the center of the squamule flanked by pockets of algal layer and medulla (FIG. 3D–E); cortex without remnants of algae (ClZnI!) or crystals (POL−), composed of strongly conglutinated, anticlinally oriented, thick-walled, up to 5 µm wide hyphae with thread-shaped lumina in inner part and shorter, angular to rounded lumina in upper part; algal layer and medulla composed of loosely interwoven hyphae, containing crystals dissolving in K, KI−; photobiont unicellular green algae, up to 10 µm diam. Apothecia and pycnidia not seen.

Chemistry: Stictic acid detected by TLC.

Ecology and distribution: Bryonora microlepis grows on calcareous mineral soil in small crevices of sloping rock faces at sun-exposed sites, on south- to west-facing slopes or boulderscrees in open north boreal forest to low alpine areas. Most of the known localities harbor a range of rare or red-listed lichens typical of xeric and calcareous habitats. The collection from Møre og Romsdal deviates by being from a lowland locality on a rocky lake shore, but also here the species grows on calciferous substrate, probably weakly influenced by trickling water. The species is known from six localities in the continental parts of South Norway; five are from the upper valleys east of the mountain chain and one from an inner fjord area of western Norway. All localities are in rain-shadow areas, and the annual precipitation is mostly below 500 mm.

Notes: The species differs from the other species of the genus in the thallus being composed of small, gray-edged squamules that are first dispersed and later form conglomerate cushions. The “Kegelrinden” thallus anatomy is unique within the genus, and so is the presence of stictic acid. It might be mistaken for a Miriquidica, since gray-edged squamules are common in that genus, as is the occurrence of stictic acid. No stictic acid–containing Miriquidica is known to grow on soil, however.

Additional specimens examined: NORWAY. HEDMARK: Folldal, Tverrgjelet, 62.1521°N, 9.7195°E (± 707 m), 1160–1200 m alt., on calcareous soil on S-facing slope, 18 Jul 1996, Haugan 4974 (O L-29315); OPPLAND: Dovre, Grimsdalen, Buåi, 62.0658°N, 9.5749°E (± 7 m), 1090 m alt., S-facing, steep slope, calcareous rock, 4 Aug 2005, Timdal 10536 (O L-148965); Nord-Fron, Kvikne, Halvhuda, 61.5741°N, 9.6074°E (± 7 m), 645 m alt., calciferous rock outcrop in grazed meadow, 28 May 2011, Haugan & Timdal 9931 (O L-173267) [TLC: stictic acid]; Vang, Smådalsfjellet, S of Sletteskaret, 61.063°N, 8.5595°E (± 10 m), 1270 m alt., rock outcrop in the alpine zone, 9 Sep 2015, Timdal 13869 (O L-201412); MØRE OG ROMSDAL: Molde, Hestneset, 62.455°N, 8.2434°E (± 7 m), 151 m alt., sun-exposed rock along lake shore, 25 Apr 2009, Haugan 8628 (O L-161432) [TLC: stictic acid].

Miriquidica vicaria (Th. Fr.) Arup, Haugan & Timdal, comb. nov.FIG. 4D

Figure 4. A–D. Bryonora castanea, B. pruinosa, B. rhypariza, and Miriquidica vicaria. A. Habit of B. castanea with apothecium margin more or less concolorous with disc (Kristinsson 24861, UPS L-996123). B. B. pruinosa with its apothecial margin typically darker than the disc (Hellbom & Hellbom s.n., UPS L-7552246). C. B. rhypariza differs from most species in the genus by a well-developed thallus and conspicuous thalline margin (Du Rietz s.n., UPS L-118215). D. Habit of Miriquidica vicaria, in this case rather similar to a Bryonora, but differing in a well-developed thallus where the margin of the areoles is often paler than the center and in exclusively having simple ascospores (Nordin 7784, UPS L-721906). Bars = 1 mm.

MycoBank MB842777

≡ Lecanora rhypariza ssp. vicaria Th. Fr., Lichenogr Scand 1(1):271. 1871.

≡ Lecanora vicaria (Th. Fr.) Vain., Meddeland Soc Fauna Fl Fenn 10:208. 1883.

Typification: [SWEDEN. HÄRJEDALEN: Tännäs par.,] Funäsdalen, 1867, P.J. Hellbom s.n. (UPS L-023730!, lectotype, designated here, MBT10005454; isolectotypes G G00292207 [image!], UPS L-023729!).

Thallus up to 4 cm diam, squamulose, dispersed to continuous; hypothallus not seen; squamules roundish, up to 4 mm diam and 1.5 mm thick, weakly to strongly convex, beige to greenish brown, gray- or pale-edged, soon becoming cerebriform, i.e., consisting of a conglomeration of squamule sections, each with a paler edge, esorediate, K+ red, Pd+ orange; cortex up to 200 µm thick, composed of anticlinally arranged, branching and anastomosing, thick-walled hyphae with cylindrical lumina, containing crystals dissolving in K with yellow effusion and subsequent precipitation of red, needle-shaped crystals (POL+); algae green, coccoid, up to 13 μm diam; medulla lacking crystals, K−, Pd−, KI−. Apothecia indistinctly lecanorine when young, soon becoming biatorine with a narrow or more or less excluded thalline margin, round to irregular, plane to convex, pale brown when young, later dark brown to blackish, up to 1.5 mm diam, immersed as young but later adnate; proper exciple slightly raised or level with disc, 25–70(–90) µm thick, paler to darker than disc, brown to pale in the rim, pale brown in inner part; hymenium 50–70(–75) µm thick, hyaline; epihymenium yellowish green-brown, K+ olive-green, N−; hypothecium pale, –350 µm thick; paraphyses mainly simple, sometimes anastomosing, 1.0–1.5 wide with tip to 3 µm wide; asci 8-spored, of the Lecanora type, ascospores simple, narrowly ellipsoid, (8.5–)10–11.4–13.0–(15.0) × (4.0–)5–5.1–6.0–(6.5) μm (n = 80).

Chemistry: Norstictic acid (major) and connorstictic acid (minor) detected by TLC.

Ecology and distribution: The species is terricolous, muscicolous, or saxicolous on dry siliceous to somewhat calciferous rock. It is known from open subalpine to low alpine areas. Miriquidica vicaria is so far only known from Norway and Sweden.

Notes: We located three collections of Miriquidica vicaria from which a lectotype could be designated. The collection UPS L-023730 is originally from T. Fries’ herbarium and is hence the specimen on which the description of Lecanora rhypariza var. vicaria was based. It is also the largest and best developed, and we therefore designate this specimen the lectotype. A possible later synonym to M. vicaria is Lecanora rubida Wirth, which was described from France (Nordin 2015; Wirth 1981). An evaluation of the status of L. rubida is pending further studies of more material.

In our phylogenetic reconstruction (FIG. 1), the species is resolved well inside the Miriquidica clade, as the sister of M. leucophaea (Flörke ex Rabenh.) Hertel & Rambold. Miriquidica leucophaea is somewhat similar to M. vicaria but differs in forming a strictly saxicolous, areolate, usually gray thallus on a black hypothallus, having lecideine apothecia, and in containing miriquidic acid instead of norstictic acid. The muscicolous M. squamulosa Fryday, known from the subantarctic Campbell Island, is the only other muscicolous Miriquidica species containing norstictic acid. This species is characterized by being cephalodiate, having convex squamules with lobulate margins, and having immersed, lecideine, black apothecia (Fryday 2008). Miriquidica ventosa (Vain.) Timdal, known from Finland and Russia, is also muscicolous or saxicolous and has gray-edged squamules, but the squamules are concave and sorediate and the species contains miriquidic acid (Andreev 2004; Owe-Larsson and Rambold 2001; Timdal 1993). Other Miriquidica species containing norstictic acid are strictly saxicolous, forming a typical areolate thallus (not with gray-edged squamules), and have black, lecideine apothecia. M. vicaria may also be confused with Psorinia conglomerata (Ach.) Gotth. Schneid., but that species has biatorine, black apothecia even when young, a more greenish epihymenium, and, at least partly, 1-septate ascospores. The asci of P. conglomerata have a well-developed ocular chamber, i.e., they are more similar to the Bacidia type. There are two chemotypes of P. conglomerata: (i) atranorin and stictic acid (major) and (ii) atranorin and norstictic acid (major) (Timdal 1992); atranorin does not occur in M. vicaria.

Additional specimens examined: NORWAY. SOGN OG FJORDANE: Årdal, Sletterust, along Mannsbergelvi, 61.2753°N, 8.0230°E, 1060 m alt., muscicolous on calciferous rock, 13 Jun 2011, Haugan 10185 (O L-173700) [TLC: norstictic acid, connorstictic acid]; TROMS: Målselv, Mt. Rostafjell, Rostaaksla, 69.0470°N, 19.5341°E, 700 m alt., 26 May 1984, Timdal 4134 (O L-17360) [TLC: norstictic acid]. SWEDEN. JÄMTLAND: Undersåker par., Gåsåliden, close to path from Edsåsen, on pebbles on gravelly ground, alt. 850 m, 63.27833°N, 13.12662°E, 24 Aug 2015, Nordin 7884 (UPS L-723005); Mt. Middagsvalen, SW slope, ca. 1.5 km NW of Vallbo, on soil and mosses, alt. 845 m, 63.13912°N, 13.04358°E, 21 Aug 2015, Nordin 7784 (UPS L-721906), on bryophytes, alt. 855 m, 63.13983°N, 13.04343°E, 21 Aug 2015, Nordin 7784 (UPS L-721936); LYCKSELE LAPPMARK: Tärna par., Ume älv, Över-Umans sydvästligaste vik, [= Ume river, the southwesternmost bay of Lake Över-Uman] 66.06–66.07°N, 14.32–14.33°E, alt., 530 m, 29 Aug 1960, Du Rietz 2194b (UPS: L-119263); TORNE LAPPMARK: Jukkasjärvi par., Björkliden, N side of the river Rakkasjohka, just N of the trail from Björkliden Fjällby to Njula, alt. 495 m, 68.40528°N, 18.66981°E, 6 Aug 2013, Westberg (UPS L-794945).

A WORLD KEY TO THE GENUS BRYONORA

In previous works (Poelt 1983; Poelt and Obermayer 1991), three Bryonora species (B. castanea, B. rhypariza, and B. pulvinar) were divided into varieties. We have kept these varieties in the key, as they may, in some cases, represent distinct species and at any rate deserve further study. Species that do not occur in the Nordic countries have not been studied by us, and data for the key are thus partly compiled from the literature (Fryday and Øvstedal 2012; Holtan-Hartwig 1991; Øvstedal and Lewis Smith 2001; Poelt 1983; Poelt and Mayrhofer 1988; Poelt and Obermayer 1991).

• 1. On rock or on other lichens on rock 2

• 1′. On soil, mosses, or plant debris 4

• 2. Apothecium sections K+ red (norstictic acid); ascospores subglobose to kidney-shaped or spirally twisted, 15–19 × 7–9 µm; in Himalaya B. selenospora

• 2′. Apothecium sections K− (without norstictic acid); ascospores ellipsoid or dumbbell-shaped 3

• 3. Ascospores often dumbbell-shaped, (12–)14–16(–19) × 5–7 µm; Himalaya B. reducta

• 3′. Ascospores ellipsoid, (11–)13–18(–21) × 4–5.5(–6) µm; Norway, Sweden, Svalbard B. castaneoides

• 4. Thallus composed of scattered to conglomerate, shiny brown, gray-edged squamules; containing stictic acid; apothecia unknown B. microlepis

• 4′. Thallus various, not of gray-edged squamules; not containing stictic acid; usually apotheciate 5

• 5. Apothecium sections K+ red (norstictic acid) 6

• 5′. Apothecium sections K− (without norstictic acid) 11

• 6. Thallus well-developed and apothecia with a distinct thalline margin 7

• 6′. Thallus usually poorly developed and apothecia without a distinct thalline margin, appearing biatorine 9

• 7. Thallus on cyanobacteria; apothecium margin usually suppressed B. rhypariza var. cyanotropha

• 7′. Thallus not on cyanobacteria; apothecium margin usually thick and conspicuous 8

• 8. Ascospores 20–32 µm long; often on soilB. rhypariza var. lamaina

• 8′. Ascospores (14–)16–23(–26) µm long; usually growing on Grimmia or AndreaeaB. rhypariza var. rhypariza

• 9. Ascospores less than 25 µm long B. castanea var. castanea

• 9.′ Ascospores more than 25 µm long 10

• 10. Ascospores (25–)29–39(–42) × (4-)4.5–7(−7.5) µm; widely distributed B. curvescens

• 10′. Ascospores (22–)25–33(–40) × (6–)7–10(–12) µm; Himalaya B. castanea var. euryspora

• 11. Thallus of distinctly elongated, podetium-like units 12

• 11′. Thallus crustose, squamulose, or indistinct 15

• 12. Medulla KC−; thallus units capitate; apothecia unknown B. corallina

• 12′. Medulla KC+ reddish (soon disappearing); thallus units not capitate; usually richly fertile 13

• 13.Thallus with punctiform or slightly elongate pseudocyphellae, strongly shiny, of rod-shaped units B. stipitata

• 13′.Thallus without pseudocyphellae but with deep furrows, matt to somewhat shiny, dwarf fruticose or with somewhat flattened units 14

• 14. Ascospores simple or 1(–3)-septate, (16–)20–25(–28) µm long B. pulvinar var. pulvinar

• 14′. Ascospores simple, 12(–15) µm long B. pulvinar var. microspora

• 15.Thallus effuse on soil, of flattish areoles that break down into ± sorediate granules; apothecia 0.3–0.4 mm diam; with perlatolic acid; South America B. granulata

• 15′.Thallus poorly developed to granulose, not breaking into sorediate granules; apothecia normally larger, up to 4 mm diam; without perlatolic acid 16

• 16.Ascospores 19–39 µm long B. septentrionalis

• 16′.Ascospores up to 19 µm long 17

• 17. Thallus with protocetraric acid; Southern Hemisphere B. peltata

• 17′.Thallus without protocetraric acid; Northern Hemisphere 18

• 18.Apothecium margin darker than the disc; disc often pruinose; without lobaric acid; widely distributed in the Northern Hemisphere B. pruinosa

• 18′.Apothecium margin paler than the disc; disc not pruinose; with lobaric acid; Himalaya B. yeti

DISCUSSION

This is the first study to assess the phylogenetic placement and the circumscription of Bryonora in a multilocus, phylogenetic context. Although uncertainties remain concerning the delimitation of the Lecanoraceae, as well as relationships within the family, we found strong support for the inclusion of Bryonora. Conversely, we found no indication of the previously hypothesized, close relationship between Bryonora and Protoparmelia.

Bryonora was recovered as a highly supported, monophyletic group that is sister to a clade that includes species of Lecanora in the strict sense (L. allophana, L. horiza, and L. imshaugii), thus representing the core of the family. Although relationships within the Lecanoraceae are not always supported, it is highly unlikely that future revisions of family boundaries would result in the transfer of Bryonora to another family. This is in contrast to the position of the genus Miriquidica in our analysis, which, although not fully supported, is well outside the Lecanoraceae. This genus has been viewed as a member of this family ever since its description (Hertel and Rambold 1987), but it is notable that when not “forced” into the Lecanoraceae by the choice of outgroup (as in, e.g., Zhao et al. 2016), Miriquidica has also ended up outside the family in other phylogenies (Miadlikowska et al. 2014; Spribille et al. 2020). The phylogenetic position of Miriquidica deserves further study, preferably using more markers, as its position is usually unresolved.

The presence or absence of a zeorine apothecial margin has been an important character for the delimitation of Bryonora. Although Poelt (1983) included B. rhypariza in Bryonora, he explicitly excluded two other species (Lecanora castaneoides and L. vicaria) from the genus mainly due to their having a zeorine apothecial margin. Subsequently, Poelt and Obermayer (1991) introduced an infrageneric division of Bryonora where the two species with a zeorine margin, B. rhypariza and B. selenospora (described in 1988), formed their own section (sect. Rhyparizae), which was later accommodated in the new genus Bryodina (Hafellner and Türk 2001). The argument in favor of recognizing Bryonora species with a zeorine apothecial margin as a lineage separate from the rest of the genus received some support in our analysis, as B. rhypariza is sister to a clade containing one of the previously excluded zeorine species, B. castaneoides (plus B. microlepis, with unknown apothecia). At the same time, the type species of Bryonora (B. castanea) and Bryodina (B. rhypariza) are both part of the same highly supported subclade within Bryonora. Recognizing Bryodina as genus distinct from Bryonora would thus imply describing a new, third genus for Bryonora pruinosa and B. septentrionalis, creating a generic division that would have low information content and unclear boundaries. In our opinion, Bryodina is therefore best treated as a synonym of Bryonora.

The seven Bryonora species included in our phylogeny include all species currently known from Europe, but sequence data for the additional eight species from the Himalayas and the Southern Hemisphere were not available. This includes the three species with fruticose to subfruticose thalli that were included in the section Stipitantes by Poelt and Obermayer (1991). Whether the morphological characteristics of this group indicate a separate evolutionary trajectory within or outside Bryonora remains to be evaluated. Likewise, the status of the varieties of B. castanea, B. rhypariza, and B. pulvinar described by Poelt (1983) and Poelt and Obermayer (1991) warrants further study, as several of them may represent distinct species.

ACKNOWLEDGMENTS

We wish to thank Mika Bendiksby for providing us with 11 ITS and mrSSU sequences and Stefan Ekman for comments on the manuscript. We thank three anonymous reviewers for their constructive comments.

DISCLOSURE STATEMENT

No potential conflict of interest was reported by the author(s).

Supplementary material

Supplemental data for this article can be accessed on the publisher’s Web site.

Additional information

Funding

Research by M.S. is financially supported by the Swedish Taxonomy Initiative (grant no. 2016-206 4.3), and the sequencing at Canadian Center for DNA Barcoding (CCDB) was funded by the Norwegian Barcode of Life.