MycoKeys 106: 327–354 (2024)
DOI: 10.3897/mycokeys.106.123484

Research Article

A phylogenetic study of Micarea melaeniza and similar-looking
species (Pilocarpaceae) unveils hidden diversity and clarifies
species boundaries and reproduction modes
Annina Kantelinen1 , Måns Svensson2 , Jiří Malíček3 , Jan Vondrák3,4 , Göran Thor5 , Zdeněk Palice3 ,Stanislav Svoboda3 , Leena Myllys1
Botany and Mycology Unit, Finnish Museum of Natural History, University of Helsinki, P.O. Box 7, FI-00014 Helsinki, Finland
Museum of Evolution, Uppsala University, Norbyvägen 16, SE-752 36 Uppsala, Sweden
3 Czech Academy of Sciences, Institute of Botany, Zámek 1 252 43, Průhonice, Czech Republic
4 Department of Botany, Faculty of Science, University of South Bohemia, CZ-37005 České Budějovice, Czech Republic
5 Department of Ecology, Swedish University of Agricultural Sciences, P.O. Box 7044, SE-750 07 Uppsala, Sweden
Corresponding author: Annina Kantelinen (annina.kantelinen@helsinki.fi)
1
2

Abstract

Academic editor: Thorsten Lumbsch
Received: 19 March 2024
Accepted: 6 June 2024
Published: 5 July 2024
Citation: Kantelinen A, Svensson
M, Malíček J, Vondrak J, Thor G,
Palice Z, Svoboda S, Myllys L (2024)
A phylogenetic study of Micarea
melaeniza and similar-looking
species (Pilocarpaceae) unveils
hidden diversity and clarifies species
boundaries and reproduction modes.
MycoKeys 106: 327–354. https://doi.
org/10.3897/mycokeys.106.123484
Copyright: © Annina Kantelinen et al.
This is an open access article distributed underterms of the Creative Commons Attribution
License (Attribution 4.0 International – CC BY 4.0).

Micarea (Ascomycota, Pilocarpaceae) is a large cosmopolitan genus of crustose lichens.
We investigated molecular systematics and taxonomy of the poorly known Micarea
melaeniza group focussing on M. melaeniza, M. nigella and M. osloensis. A total of 54
new sequences were generated and using Bayesian and maximum likelihood analysisof two markers (nuITS and mtSSU), we discovered two previously unrecognized phylogenetic lineages, one of which is described here as Micarea eurasiatica Kantelinen & G.
Thor, sp. nov., morphologically characterized by pycnidia that are sessile to emergent, cylindrically shaped, with greenish-black K+ olive green, wall pigmentation and containing
large mesoconidia up to 6 µm in length. The species is known from Japan and Finland.
In addition, we show that the reproduction biology of M. osloensis has been poorly understood and that the species often occurs as an anamorph with stipitate pycnidia. We present a species synopsis and notes on pigments. Our research supports previous results ofasexuality being an important reproductive strategy of species growing on dead wood.
Key words: Biodiversity, DNA-barcoding, lichenized ascomycete, new species,
overlooked taxa, reproduction mode

Introduction
Species of the genus Micarea Fr. are lichenized ascomycetes belonging to
the family Pilocarpaceae. Currently, more than 140 species are known, andnew species are continually described (e.g., Czarnota 2007; Czarnota and Guzow-Krzemińska 2010; Sérusiaux et al. 2010; Guzow-Krzemińska et al. 2016,2019; van den Boom et al. 2017; Kantvilas and Coppins 2019; Launis and Myllys
2019; Launis et al. 2019a, b; van den Boom et al. 2020; Vondrák et al. 2022;
Index Fungorum 2023). Micarea species are globally distributed with representatives found on all continents. These species occur across a wide range327

Annina Kantelinen et al.: A phylogenetic study of Micarea melaeniza groupof habitats and grow on substrates such as bark, dead wood, rocks, soil andbryophytes. Some Micarea species are specialized and prefer specific habitats,such as dead wood in old-growth forests (Fig. 1). Their typical substrate has
acidic pH (e.g. Coppins 1983; Czarnota 2007).
Despite the diversity and global presence of Micarea species, they are often
overlooked and poorly understood. Several factors contribute to the challengeof identifying them. First, they are typically small. Second, Micarea exhibits awide range of sexual and asexual propagules, including ascospores, three typesof conidia (micro-, meso-, and macroconidia), and thallus fragments called goniocysts which likely serve as asexual propagules including both symbiotic partners. Some Micarea species are primarily sexual, while others often lack sexual
structures but form numerous pycnidia where asexual conidia are produced(e.g. Coppins 1983; Czarnota 2007; Kantelinen et al. 2022a). Third, some Micarea species display intraspecific colour variations, which depend on light exposure and corresponding pigment levels. For example, the Sedifolia-grey pigment,
commonly found in the apothecia of Micarea species, varies in concentrationfrom light grey in shaded situations to almost black in well-lit habitats (Coppins
1983; Czarnota 2007; Launis et al. 2019a, 2019b). Due to these challenges, identifying Micarea species typically requires careful examinations of microscopic
features, chemical testing (spot tests and TLC) and/or DNA sequencing.
Significant progress in the understanding of species boundaries and diversity
within Micarea has been achieved through the application of molecular methods(Czarnota and Guzow-Krzemińska 2010; Sérusiaux et al 2010; Guzow-Krzemińska et al. 2016; van den Boom et al. 2017; Launis et al. 2019 a, b). However, someinfra-generic groups in Micarea have been more studied than others. One groupthat remains poorly understood and has been sequenced only rarely is Micarea
melaeniza Hedl. and similar species, i.e. M. anterior (Nyl.) Hedl., M. botryoides(Nyl.) Coppins, M. deminuta Coppins, M. denigrata (Fr.) Hedl., M. melaeniza,
M. misella (Nyl.) Hedl., M. nigella Coppins, M. olivacea Coppins and M. osloensis (Th. Fr.) Hedl.. These species share morphological characteristics, such as athin or endosubstratal thallus, small (0.1–0.3 mm wide) dark apothecia and/or
dark stipitate mesopycnidia, as well as often simple to one septate ascospores.
Furthermore, M. botryoides, M. deminuta, M. melaeniza, M. nigella, M. olivacea
and M. osloensis are similar in having dark hypothecium and dimorphic paraphyses. The Cinereorufa-green (K+ green, HNO3+ purple) and Superba-brown(K–, HNO3–) pigments are often present, as well as sometimes Melaena-red(K+ green, HNO3+ purple-red). Most of these species are obligate or facultative
lignicoles (Coppins 1983, Czarnota 2007). Despite their morphological and ecological similarities, all the species are not necessarily closely related (Coppins
1983; Andersen and Ekman 2005; Sérusiaux et al. 2010).

Figure 1. Typical habitats for species in the M. melaeniza group in boreal and boreonemoral forests a Koli National Park, Eastern Finland (photo: Kantelinen) b Nikko National
Park, Central Honshu, Japan near the type locality of M. eurasiatica sp. nov. (photo: Thor).
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We aim to clarify the molecular systematics and morphology of the poorly known M. melaeniza and its similar-looking species, focusing especially on
M. melaeniza, M. nigella and M. osloensis, species that are morphologically challenging to identify. We use phenotypic characters and sequence datafrom two loci (nuITS and mtSSU). Additionally, we also address the species ́reproduction biology. Our study generates reliable sequences of several rarely
collected species and furthers understanding on lichen diversity in boreal, boreonemoral and hemiboreal forests especially on dead wood and conifer bark.

Materials and methods
A substantial portion of the sequenced specimens in this study were collectedfrom southern and central Finland as part of a research project that investigated lichen diversity on dead wood (years 2012–2014, Kantelinen et al. 2022b).
Logs and stumps of decaying Picea abies trees in decay stages 2, 3, 4 and 5
were inventoried (following Renvall 1995).
In addition, specimens were obtained from the Czech Republic, Japan, Russian Caucasus, Sweden and Ukraine. These specimens were collected fromdead wood and bark and sequenced when possible. Geo-coordinates are givenin the format WGS84. Relevant specimens were also looked for amongst fresh
Micarea collections from Australia, Brazil, Kenya, Rwanda and Tasmania withno success. Herbarium collections and type specimens from the herbaria FR,
H, PRA, RBGE, S and UPS were studied.

Species identification
Specimens were identified with a dissecting (Leica S4E) and compound (Leica
DM750) microscopes. Anatomical characters and ascospore dimensions were
measured in water and K. The number of measured ascospores and conidia depended on their availability, but usually 10–30 were measured and the rest were
examined superficially to ensure that they fell into the same size category. To
detect and determine the insoluble pigments present in the specimens chemical spot tests with 10% potassium hydroxide (K) and sodium hypochlorite (C)and nitric acid (HNO3) were used (Orange et al. 2010). Fresh material was often
not sufficient for thin-layer chromatography (TLC). The specimens are generally quite small and have no or thin thallus, meaning that a substantial partof the collection would need to be taken for TLC. No secondary substances
have previously been detected in M. melaeniza, M. nigella or M. osloensis, nor
in the similar-looking species M. anterior, M. misella and M. substipitata Palice& Vondrák (Coppins 1983; Czarnota 2007; Vondrák et al. 2022). Thus, TLC is oflimited practical value in the study of this species group.
Total genomic DNA was extracted from lichen structures (apothecia, pycnidia
and/or thallus) in labs in Finland and the Czech Republic. In Finland, extractions
were conducted using DNeasy® Blood & Tissue kit by Qiagen following the
manufacturer´s instructions with the following exceptions. Lichen structures ofapproximately 0.5–1 mm in diam. were ground with mini-pestles in 40 µl of lysis
buffer, after which 140 µl of the buffer was added by simultaneously flushinglichen fragments from the mini-pestle. The extracted DNA was eluted in 50 µl of
the eluation buffer. Samples were incubated for 5 minutes and centrifuged. After
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Annina Kantelinen et al.: A phylogenetic study of Micarea melaeniza groupthe first elution, a second elution was performed to increase sample availability
by adding another 50 µl of the elution buffer, incubated for 5 minutes and centrifuged. The two elutions were stored in the freezer in separate microcentrifuge
tubes. In the Czech Republic, extractions were conducted using ISOLATE II DNA
Plant Kit (Bioline) according to the manufacturer´s protocol using a cetyltrimethylammonium bromide (CTAB)-based protocol (Aras and Cansaran 2006).
In Finland, PCR reactions were prepared using PuReTaq Ready-To-Go PCRbeads (GE Healthcare). The 25 µl reaction volume contained 19 µl dH2O,
0.4 µM of each primer and 4 µL extracted DNA. The ITS and mtSSU regions wereused for species identification. PCR was run under the following conditions: initial denaturation for 10 min at 95 °C followed by six cycles of 1 min at 95 °C (denaturation), 1 min at 62 °C (annealing), and 1 min 45 s at 72 °C (extension); forthe remaining 35 cycles, the annealing temperature was decreased to 56 °C; thePCR program ended with a final extension of 10 min at 72 °C. The primers usedwere ITS1LM and ITS2KL (Myllys et al. 1999), and mrSSU1 and mrSSU3R (Zoller
et al. 1999) and they were used for PCR amplification and sequencing. In the
Czech Republic, PCR reactions for Malíček´s specimens were prepared following
the protocol in Malíček et al. (2023). For Vondrák´s and Palice´s specimens PCR
reactions were prepared as follows: Polymerase chain reactions were performed
in a reaction mixture containing 2.5 mmol/l MgCl2, 0.2 mmol/l of each dNTP,
0.3 µmol/l of each primer, 0.5 U Tag polymerase (TOP-Bio, Praha, Czech Republic) in the manufacturer’s reaction buffer, and sterile water to make up a final
volume of 10 µl. The ITS and mtSSU regions were used for species identification.
PCR was run under the following conditions: for ITS initial denaturation for 3 min
at 94 °C followed by 30 cycles of 30 sec at 94 °C (denaturation), 30 sec at 56 °C(annealing), and 2 min at 72 °C (extension); the PCR program ended with a final
extension of 7 min at 72 °C. For mtSSU initial denaturation for 10 min at 94 °C followed by 40 cycles of 30 sec at 94 °C (denaturation), 30 sec at 58 °C (annealing),
and 2 min at 72 °C (extension); the PCR program ended with a final extension of
7 min at 72 °C. The primers used were ITS1F (White et al. 1990) and ITS4 (Gardes
and Bruns 1993), and mrSSU1 (Zoller et al. 1999) and mrSSU7 (Zhou and Stanosz 2001), and they were used for PCR amplification and sequencing.

Phylogenetic analyses
To examine the phylogenetic position of our study species within Micarea s.
lat., we ran a preliminary analysis of an mtSSU data matrix using Psora decipiens (Hedw.) Hoffm. from the family Psoraceae as an outgroup, based on
the studies by Andersen and Ekman (2005) and Sérusiaux et al. (2010). In thephylogeny (tree not shown) our new samples fall outside of Micarea s. str. (i.e.
the M. prasina group) and close to M. doliiformis (Coppins & P. James) Coppins& Sérus., M. paratropa (Nyl.) Alstrup, M. assimilata (Nyl.) Coppins and Leimonis
erratica (Körb.) R.C. Harris & Lendemer (see phylogeny in Sérusiaux et al. 2010).
The final phylogenies, including 29 newly generated mtSSU and 25 ITS sequences (Table 1), were first aligned with MUSCLE v.3.8.31 (Edgar 2004) using the European Molecular Biology Laboratory, European Bioinformatics Institute’s (EMBL-EBI) freely available web server (http://www.ebi.ac.uk/Tools/msa/muscle/).
Based on our previous studies (Launis et al. 2019 a, b) and our preliminary phylogenetic reconstruction of the genus, M. byssacea and M. prasina belonging to
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Table 1. List of Micarea specimens used in the phylogenetic analysis with locality, voucher information and GenBank
accession numbers.
Taxon

Locality

Voucher information, sequence ID

mtSSU

ITS

M. anterior

FinlandKantelinen 199 (H), A265

PP811702

PP811675

M. botryoides

NorwayAndersen 79b (BG)

AY567741

AY756471

M. byssacea

FinlandKantelinen (Launis) 289103 (H), A98

MG707768

MG521562

M. contexta

FinlandKantelinen 1914 (H), A569

PP811722

PP811692

M. deminuta

Japan

Thor 40245 (UPS), A926

PP811719

PP811689

M. deminuta

Czech Republic

Palice 6745 & Voříšková (PRA)

AY756446

AY756474
PP811691

M. denigrata

FinlandKantelinen 723 (H), A686

PP811721

M. doliiformis

UK, Wales

Orange, LG database 29 (LG)GU138666

M. doliiformis

UK

Andersen 178a (BG)

HQ650654

M. eurasiatica sp. nov.

FinlandKantelinen 2729 (H), A466

PP811712

PP811684

M. eurasiatica sp. nov.

Japan

Thor 40053 (UPS) A914, holotype

PP811720

PP811690

M. eximia

FinlandKantelinen 3785 (H), A785

MT982134

MT981600

M. globulosella

FinlandKantelinen (Launis) 67114 (H), A243

MG707744

MG521547

M. incrassata

FinlandKantelinen 90 (H), A90

MT982132

MT981598

Micarea sp.

FinlandKantelinen 2640 (H), A487

PP811703

PP811676

M. melaeniza

FinlandKantelinen 2430 (H), A772

PP811709

PP811682

M. melaeniza

UkraineVondrák 21921 (PRA)

PP811724

PP811694

M. melaeniza

RussiaVondrák 23358 (PRA)

PP811725

PP811695

Czech Republic

Palice 32013 (PRA)

OQ646318

M. misella

FinlandKantelinen (Launis) 108111 (H), A264

MG707742

MG521545

M. nigella

FinlandKantelinen 1971 (H), A589

PP811706

PP811679

M. nigella

FinlandKantelinen 1974 (H), A588

PP811714

PP811686

M. nigella

FinlandKantelinen 1921 (H), A572

PP811715

PP811687

M. nigella

Czech RepublicMalíček 16287

PP811726

PP811696

M. nigella

Czech RepublicMalíček 14664

PP811729

PP811698

M. osloensis

FinlandKantelinen 1685 (H), A574

PP811704

PP811677

M. osloensis

FinlandKantelinen 1865 (H), A575

PP811705

PP811678

M. osloensis

FinlandKantelinen 1909 (H), A583

PP811717

M. osloensis

FinlandKantelinen 2899 (H), A736

PP811716

PP811688

M. osloensis

FinlandKantelinen 1923 (H), A573

PP811713

PP811685

M. osloensis

FinlandKantelinen 1686 (H), A768

PP811708

PP811681

M. osloensis

FinlandKantelinen 2643 (H), A484

PP811710

PP811683

M. osloensis

FinlandKantelinen 2003 (H), A594

PP811707

PP811680

M. osloensis

FinlandKantelinen 2648 (H), A485

M. osloensis

Czech RepublicMalíček 16281

PP811730

M. osloensis (M. melaeniza in GenBank)

Czech Republic

Palice 30267 (PRA)

OQ646316

M. osloensis (M. nigella in GenBank)

Czech Republic

Palice 30266 (PRA)

OQ646320

M. osloensis

SwedenSvensson 4385 (UPS), C792

PP811718

M. osloensis

RussiaVondrák 22836 (PRA)

PP811723

M. osloensis (M. melaeniza in GenBank)

Czech Republic

Vondrak 25774 (PRA)

OQ646315

OQ717944

M. osloensis

Czech Republic

Vondrák 19007 (PRA)

PP811727

PP811697

M. osloensis

Czech Republic

Vondrák 22217 (PRA)

PP811728

M. prasina

FinlandKantelinen (Launis) 265101 (H), A92

MG707747

M. substipitata

FinlandKantelinen 2700 (H), A469

PP811711

M. melaeniza (M. nigella in GenBank)

MycoKeys 106: 327–354 (2024), DOI: 10.3897/mycokeys.106.123484

PP811693

OQ717948

MG521549

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Micarea s. str. were selected as outgroups. We ran separate single-marker analyses by using MrBayes3.2.7a and did not detect conflicting clades between
the analyses, although missing data was higher in the ITS matrix, and hence
decided to concatenate the data by using Mesquite v. 3.61 (Maddison andMaddison 2023). The two-locus data matrix from sequences of 43 specimens
included 1245 aligned nucleotide characters, with 707 positions in the mtSSU
and 537 positions in the ITS regions. The hypervariable region at the end of themtSSU was removed from the analyses. Micarea doliiformis is represented bysequences from two specimens that were combined as one, the other specimen represented by an mtSSU sequence and another by ITS sequence (seeTable 1). The data matrix was subjected to Bayesian inference using MrBayes(v. 3.2.7a) (Ronquist and Huelsenbeck 2003) and to maximum likelihood (ML)analysis using freely available IQ-Tree 1.6.12 (Trifinopoulos et al. 2016) web
server (http://iqtree.cibiv.univie.ac.at/). For the Bayesian analysis, substitution
models were selected by having the MCMC procedure sample across models (Huelsenbeck et al. 2004). The convergence of the four parallel runs waschecked after 2000000 generations using Tracer (v. 1.5) (Rambaut et al. 2018)
and graphed using FigTree (v. 1.4.4). For the ML analysis, model TIM2+F+I+G4
was chosen by having IQ-Tree run the best-fitting substitution model for our one
partition matrix, and branch lengths were assumed to be proportional acrosssubsets. Node support was estimated with 1000 bootstrap replicates using theultrafast bootstrap algorithm. The alignment is available from the Dryad Digital
Repository https://doi.org/10.5061/dryad.79cnp5j44.

Results
We present the first molecular phylogeny of M. melaeniza and similar-looking
species. We recorded 72 specimens of the M. melaeniza group of which 26
were sequenced successfully. In addition, we downloaded sequences from
GenBank (Table 1). The topologies of the Bayesian and ML analyses showed
no conflict and hence only the Bayesian tree is shown (Fig. 2).
The phylogenies are well-supported and resolved into 18 taxa. Micarea
misella, M. globulosella, M. eximia and M. botryoides form basal nodes for thephylogeny. The remaining taxa resolve into four clades that include the M. melaeniza group and its sister groups. The M. melaeniza group, delimited in thisstudy, includes six monophyletic taxa, i.e. M. deminuta, M. melaeniza, M. nigella,M. osloensis and two undescribed species, each represented by two specimens. We describe the first as M. eurasiatica Kantelinen & G. Thor, sp. nov., but
refrain from describing the other because of insufficient material and sequence
data (“Kantelinen 2640 and Kantelinen 1870”; sequences from the latter specimen are not of sufficient quality and therefore not included in the final analyses
even though it formed a monophyletic clade in preliminary analysis). Based on
our results the similar looking M. anterior, M. contexta and M. subtipitata are
not included in the M. melaeniza group. Instead, they form a sister clade to thisgroup, but the relationship receives no support and is based on limited taxon
sampling. Several outgroups, including M. incrassata, M. doliiformis, and Lecania cyrtella have been tested but the overall topology of the phylogeny has notchanged. The main distinguishing morphological characters are presented in
the species synopsis Table 2. Pigments are presented in Table 3.
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Micarea byssacea Kantelinen 289103
Micarea prasina Kantelinen 265101
Micarea incrassata Kantelinen 90
Micarea doliiformis Andersen 178a & Orange 29
Micarea contexta Kantelinen 1914

0.99/791

Micarea anterior Kantelinen 199
Micarea substipitata Kantelinen 2700
1

Micarea deminuta Palice 6745
Micarea deminuta Thor 40245

1

Micarea melaeniza Kantelinen 2430
Micarea melaeniza Palice 32013

1

Micarea melaniza Vondrák 21921
1

1

Micarea melaeniza Vondrák 23358
Micarea osloensis Kantelinen 1923
1

Micarea osloensis Kantelinen 2899
Micarea osloensis Kantelinen 1686
Micarea osloensis Palice 30266

0.72/61

Micarea osloensis Kantelinen 1865

1

Micarea osloensis Kantelinen 2643
Micarea osloensis Kantelinen 2003
0.51
0.99

Micarea osloensis Kantelinen 2648
Micarea osloensis Kantelinen 1909
Micarea osloensis Svensson 4385
Micarea osloensis Palice 30267

0.90/79

Micarea osloensis Vondrák 25774

10.68/74

M. melaeniza
group

Micarea osloensis Vondrák 22836
0.98
Micarea osloensis Vondrák 19007
Micarea osloensis Vondrák 22217
Micarea osloensis Malíček 16281

1

Micarea osloensis Kantelinen 1685
Micarea nigella Kantelinen 1974
0.80

Micarea nigella Malíček 16287

1

1

Micarea nigella Kantelinen 1971
Micarea nigella Malíček 14664
Micarea nigella Kantelinen 1921
Micarea eurasiatica sp. nov Thor 40053

10.70/63

Micarea eurasiatica sp. nov Kantelinen 2729
Micarea sp. Kantelinen 2640

Micarea denigrata Kantelinen 723

1

Micarea botryoides Andersen 79b
Micarea eximia Kantelinen 3785
Micarea misella Kantelinen 108111
Micarea globulosella Kantelinen 67114

0.03

Figure 2. Bayesian tree of the Micarea melaeniza -group and similar-looking species based on mtSSU and ITS sequences.
Bayesian posterior probabilities are always indicated near the branches together with bootstrap support when less than80 (e.g. 0.70/63).

In addition to our specimens, we downloaded M. melaeniza and M. nigella sequences from Genbank. Based on our phylogenetic analyses, all of themfall inside Micarea osloensis (OQ646318, OQ646320, OQ717948, OQ646315,
OQ717944, OQ646316, in addition OQ717947 and OQ646319 are identical withothers and excluded from our final analysis because of repetition). Three sequences from Genbank were left out from our analyses (AY756488, AY756484,
OQ717944), because they are substantially different compared to the othersequences in the M. melaeniza group and hence the alignment and phylogenetic analysis became unreliable. Based on blast searches AY756484 is a species of Lepraria, AY756488 perhaps Micarea melaena or M. nitschkeana, andOQ717944 an uncultured fungus.
Our data include a taxon with a high morphological resemblance to Micarea osloensis, a species found only twice before in years 1874 and 2007. Unfortunately, we were not successful in sequencing the old collections. Despite
the high morphological resemblance, our new specimens have some subtle
differences compared to the type. The type specimen of M. osloensis is a fertile specimen with apothecia, whereas our specimens are usually asexual withpycnidia. The specimens are mostly dimorphic, meaning that the existing sequenced specimens are usually either sexual or asexual, and rarely both. In
addition, some of the fresh material is with Cinereorufa green pigment which is
not present in the type. See taxonomy section for further info.
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7–11(–11.8) ×
4–5(–5.5) μm; (5.8–)7.7–10.1(–11.5) ×(3.2–)3.7–4.5(–4.8) μm(Coppins 1995)

7–13(–14) × 3–4.6 μm

8–12(–12.5) × 3–4(–
4.8) μm 0–1(–2) sept.

spores (simple, if notmentioned otherwise)

pycnidia, ap rare

ap and pycnidia

pycnidia, no ap

M. melaeniza(sequenced)

M. nigella (TYPE)

M. nigella(sequenced)

brownish-black, often
purple tingebrownish-black, often
purple tingebrownish-black, greenish, rarely purplish tone

ap and pycnidia brownish-black, greenish

M. melaeniza (TYPE)

none

6.5–12 × 2.5–4 μm

5–9.0 × 2.5–3.8 μm

5–9.0 × 2.5–3.8 μm

6–9(–10) × (2–)2.5–
3.5(–4) μm; 0(–1) sept.

olivaceous epihymeni- (7.2–)8.2–11.3(–11.8) ×
um (with dull brown to(4.1–)4.4–5.5(–6.1) μm
brown-black streaks inhymenium), brown-black
hypothecium

pycnidia, ap not dark olive-brown epihyalways present menium, hyaline hymenium and hypothecium

apothecia,
pycnidia rare

M. deminuta (sensu
Czarnota 2007)

dark-brown epihymenium, brown-black (notwarm) [dark ± reddish
brown / Coppins 1995]
hypothecium

dark green, purple(orange-/reddish-)brownanatomical coloration

M. misella

apothecia

apothecia,
pycnidia rare

M. contexta

M. deminuta (TYPE)

pycnidia, ap
quite rare

Micarea anterior

reproduction

none

two types: thick
2–3 μm; thinner c.
0.7–1 μm

two types: thickc. 2 μm; thinner c.
0.7–1 μm

two types: thickc. 2 μm; thinner c.
0.7–1 μm

one type: (0.8–)1–
1.2(–1.5) μm

two types: thick(1-)1.2–2.0 μm; thin
0.8–1.0 μm

two types: thick
1.5–2.0(–3.0) μm;
thin 0.8–1.0 μm

two types: : thick
1.5–2.0 μm; thin lessthan 1 μm

one type: 1–1.5 μm,
sometimes upper
part 2.5–3 μm

paraphyses (μmwide)

3.4–4.3 (–4.5) × 1.2–1.6(–1.8) μm

2.5–3.5 × 1.2–1.8 µm

3–3.5 × 1.75 μm

3.5–5(–6.5) × 1.2–1.4(–1.7) μm(4.9–)5.5–8(–8.1) × 1.5–1.8 um

not reported

4.2–5.0 × 1.2–1.5 μm

3.5–4.5 × 1.2–1.5 μm

mesoconidia

black, stipitate, walls brownish black or (3.5–)3.8–4.5 × 1.2–1.5(–1.8) μm
often purplish black, sometimes growing
from the same baseblack, stipitate, purplish-brown according
to the description, but the type specimen
is not clearly purplish, but walls are
instead brown-black to slightly greenish-black

black, stipitate up to 300 μm tall, upper
part of the walls greenish-brown andlower part reddish-brown, rarely purplish
tone, sometimes growing from thesame baseblack, stipitate up to 300 μm tall, upper
part of the walls greenish-brown andlower part reddish-brown sometimes
growing from the same basebrown to black, glossy, stipitate up to
300 μm tall, walls olive, olive-brown

emergent, often with gaping ostioles, black-brown with brown toolive-brown walls

no emergent/stalked pycnidia

sessile to emergent, c. 40 μm diam.

stipitate, brown from base and blackfrom the top, up to 250 μm tall (Finnish
specimens); sessile to stipitate, pallid,
often with reddish-brown blotches, up
to 250 μm tall (Coppins 1983); dilute
brownish (Czarnota 2007)

mesopycnidia

Table 2. Species synopsis representing key morphological characters based on our study and literature (Hedlund 1892; Coppins 1983; Czarnota 2007).

K+ greenK+ greenK+ green, or rarely K–

K+ green, or rarely K–

K+ violet

K- or K+ dull olive grey[=K+ green]K+ greenK+ green

no reaction or dulling

K-reaction

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ap, no pycnidia

pycnidia, ap few

M. osloensis (TYPE)

M. osloensis(sequenced)

pycnidia, ap few

pycnidia, ap notalways present

M. sp.
(Kantelinen 2640)

M. substipitata

M. eurasiatica sp. nov. pycnidia, ap few

ap and pycnidia

M. olivacea (TYPE)

reproduction

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pallid to whitish, hyalinebrownish-black, greenish-black, violet-black

greenish black, darkbrown

warm brown and olivaceous tones

warm brown tones

olivaceous, olive-brownanatomical coloration

7–10 (–11) × 2.2–3.5(–3.8) μm (0–) 1 sept.

none seen(6–)7–9 × 3–4 µm

7–10 × 3.0–3.5(–4.0) μm

6–9.5(–10) × 3–4 μm(7–)9–12.3 × 2.5–
3.5 μmspores (simple, if notmentioned otherwise)

two types: mostly
0.9–1.3 μm; rarely
thicker 1.5–2.0 μm

two types

two types: thick
2–3 μm sometimes
branched; thinner c.
0.7–1.5 µm

two types: thick
2–3 μm often
uneven in shape;
thinner c. 1–1.5 μm

two types: mostly
2–3 μm, unevenly
shaped, sometimes
branched; thinner
1.5–1.8 μm

two types: thick
2–3 μm; thinner c.
1–1.2 μm

paraphyses (μmwide)

sessile to shortly stipitate, white, up to
250 μm tallsessile (to emergent), c. 50 μm tall,
50 μm wide

sessile to emergent, 50–80 (–100) μm
tall, 30–45 μm wide

often numerous and crowded, simple
or branched from the base, emergent or
shortly stalked up to 180 μm, dark brown
to blackish, walls greenish black togreenish brown from the top with a warm
brown lower partnot seensordid green, numerous, inconspicuous,+/-immersed

mesopycnidia

2.5–3.5(–4) × 1.0–1.5 μm

4.2–5.0 × 1.2–1.5 μm

4.5–6.0 × 1.2–1.5(–1.8) μm

3.5–4.5(–5) × 1.2–1.5(–1.8) μm

not seen

3.4–4.3 × 1.2–1.6 μm

mesoconidia

No reaction

K+ greenK+ green

No reaction, or K+
green epihymenium

No reaction

K+ green

K-reaction

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Table 3. Pigments found in apothecia and pycnidia based on our study and literature (Hedlund 1892; Coppins 1983;
Czarnota 2007).
Cinereorufa-green [Pigment
A] K+ green intensifying,
HNO3+ purple

Melaena-red [Pigment
B] K+ dull sordid green,
HNO3+ purple-red

Melaenida-red [Pigment C]
K+ purplish-red, HNO3 -M. deminutax

M. melaeniza

x

(x)

(x)

M. nigella

x

x

x

Superba-brown [within Pigment F]
No reaction in K or HNO3x

M. osloensis (type)xx

M. osloensis (new)(x)

M. eurasiatica sp. nov

x

x
(x)

x

Taxonomy
Micarea eurasiatica Kantelinen & G. Thor, sp. nov.
MycoBank No: 854181
Fig. 3A, B
Type. Japan, Honshu, Gunma Prefecture, Katashina-mura, Nikko National Park,
4.7 km E of Marunuma Kogen Ski Resort, 550 m S of the parking lot at the startof the trail up to the summit of Mt. Oku-Shirane, along the trail. Open forest withmainly deciduous trees. On Tsuga diversifolia log. 36.81573°N, 139.37823°E (±10 m), elevation 1791 m. 2019. Thor 40053 (Holotype UPS). DNA sample A914.
Description. Thallus endoxylic. Photobiont micareoid, 4–7 µm.
Apothecia few, immarginate, convex, black, matt, 0.1–0.2 mm in diam.
Hymenium 25–40 µm tall, hyaline or tinged green, K+ greenish when tinged,
sometimes with darker vertical streaks. Epihymenium black to blackish-green,K+ green, HNO3+ purple (Cinereorufa-green). Asci clavate, 28–38 × 11–14 µm.
Ascospores ellipsoid to ovoid, simple, (6–)7–9 × 3–4 µm. Paraphyses numerous, of two types: 1. evenly distributed, branched, thin, c. 0.7–1.5 µm wide, 2.
evenly distributed, stout, sometimes branched, 2–3 µm wide, not always coated in pigment. Hypothecium c. 35–45 µm tall, dark brown, K – (Superba-brown),
or sometimes with a slight purple tinge (Melaenida-red), hyphae coated withdark brown pigment. Excipulum not evident.
Mesopycnidia few to abundant, sessile to emergent, cylindrical in shape, 50–
80(–100) µm tall, 30–45 µm wide, black, walls greenish black, K+ green, HNO3+
purple (Cinereorufa-green), sometimes merged from base, usually extruding
white mass of conidia that sometimes merge with neighbouring conidial mass.
Mesoconidia ellipsoid-cylindrical, (4–)4.5–6.0 × 1.2–1.5(–1.8) µm. Micro- or
macropycnidia not seen.
Chemistry. Material not sufficient for TLC.
Crystalline granules not present in apothecia or pycnidia.
Habitat and distribution. M. eurasiatica is currently known from Finland andJapan. In Finland, the species was collected in a shaded and dense, Picea abies dominated managed forest. In Japan, the species occurred in a semiopen
forest with mainly deciduous trees. On both occasions, the species was foundgrowing on dead wood.
Notes. M. eurasiatica is currently known from two collections. The typecollection has abundant mesopycnidia, and additionally few small apothecia.
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Annina Kantelinen et al.: A phylogenetic study of Micarea melaeniza group

Figure 3. Morphological and anatomical features A, B Micarea eurasiatica sp. nov. (Thor 40053) A habit B apothecial section in water C, D Micarea melaeniza (Holotype) C habit, apothecia and pycnidia D apothecial section in water
E–G Micarea nigella (Holotype) E apothecial section in K F apothecial section in water G drawing of M. nigella pycnidia on
dead wood (Kantelinen 1974, H), Ga.) Mesopycnidia extruding mesoconidia, Gb.) Mesoconidia are cylindrical, ellipsoid,
sometimes biguttulate. Photos and drawing Kantelinen. Scale bars: Habit 0.5 mm (A, C); Apothecial sections 100 µm (B,C, E, F); Drawing Ga 100 µm, Gb 1 µm.

The other collection has only pycnidia. The most important diagnostic characters are the combination of sessile to emergent pycnidia that are cylindrical in shape, greenish-black pycnidial walls, large mesoconidia (up to 6 µm in
length) and a K+ olive green reaction (Cinereorufa-green). If apothecia are present, they are 0.1–0.2 mm wide, have greenish-black epihymenium (K+ green,
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Cinereorufa-green) and dark brown hypothecium (K– or sometimes slightly
K+ purple if Melaenida-red present). Micarea eurasiatica resembles other often asexual Micarea species on dead wood such as M. melaeniza, M. misella,
M. olivacea and M. osloensis. Micarea eurasiatica differs from M. melaeniza by
having shorter mesopycnidia, longer mesoconidia (M. melaeniza: (3–)3.5–4.5
× 1.2–1.8 µm, M. eurasiatica: (4–)4.5–6.0 × 1.2–1.5(–1.8) µm), and a more
greenish black wall colouration in the pycnidia with no brown tones. Micarea
misella has a K+ violet reaction (Sedifolia-grey) instead of K+ olive green andits mesopycnidia are brownish-black and taller (Coppins 1983; Czarnota 2007).
Micarea olivacea has rather similar short mesopycnidia that react K+ green, but
the wall of pycnidia is olive-brown and the mesoconidia are shorter than thoseof M. eurasiatica (M. olivacea: 3.4–4.3 × 1.2–1.6 µm, M. eurasiatica: (4–)4.5–
6.0 × 1.2–1.5(–1.8) µm) (Coppins 1983). Micarea osloensis, on the other hand,
has a warm brown wall colouration and usually no K reaction.
Additional specimen studied. Finland, Uusimaa, Tuusula, W of Korso, shaded and dense Picea abies dominated managed forest (plot 2), on wood of fallen Picea abies (decay stage 2), 60.3544°N, 25.0322°E, 2013, Kantelinen 2729
(DNA A466), H.

Micarea melaeniza Hedl.
MycoBank No: 368074
Fig. 3C, D
Type. Bih. Kongl. Svenska Vetensk.Akad. Hand. III, 18: 96 (1892). Type: Sweden, Helsinglandiæ [= Hälsingland], Jerfsö [= Järvsö], VIII 1891. J. T. Hedlund (S L1471! – lectotype, designated by Coppins 1983 [ICN Art. 9.10], further specified here [ICN Art. 9.17], S L1472!, UPS L-005556!, UPS L-171894!,
LD 1056591!, isolectotypes).
Description. Thallus endoxylic. Photobiont micareoid, 4–7 µm.
Apothecia absent to numerous (mostly rare), immarginate, subglobose, often
becoming tuberculate, black, matt, 0.1–0.3 mm in diam. Hymenium (25–)28–
42 µm tall, hyaline or tinged aeruginose green, olive, or rarely purplish brown, often with darker vertical streaks. Epihymenium irregularly pigmented aeruginose
green, or rarely sordid brown, sometimes with a purplish tinge. Epihymenium
and hymenium K+ olive green, HNO3 purple (Cinereorufa-green), or rarely K–.
Asci clavate, 22–35 × 10–12 µm. Ascospores ellipsoid to usually ovoid, simple,
5–9 × 2.5–4.0 µm. Paraphyses numerous, of two types: 1.) evenly distributed,
branched, thin, c. 0.7–1 µm wide, 2.) scattered or in small fascicles, stout, c.
2 µm wide, coated by greenish pigment. Hypothecium c. 60–120 µm tall, darkbrown (Superba-brown), sometimes greenish or with a reddish tinge, often K+
olive green in the upper part (reaction in the greenish Cinereorufa-green pigment), hyphae coated with dark brown pigment. Excipulum not evident.
Mesopycnidia always present, usually numerous, black, sessile or moreusually stalked and then 80–300 µm tall, 40–70 µm in diam., stalks simple orbranched from the base bearing up to four pycnidia, upper part of the walls
greenish-brown and lower part reddish-brown, K+ dull green (the greenish
pigment) or sometimes K–. Mesoconidia ellipsoid to short cylindrical 2.5–
3.5 × 1.2–1.8 µm. Micro- or macropycnidia not seen.
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Crystalline granules not present in apothecia, pycnidia or thallus.
Chemistry no substances detected by TLC (information based on Coppins
1983 and Czarnota 2007).
Typification. In his original description of Micarea melaeniza, Hedlund (1892)
cited material that he had collected in Järvsö in Hälsingland, but without giving
further specimen data. There are five specimens of M. melaeniza in S, LD and
UPS collected by Hedlund in Järvsö in August 1891 and which all are likely to bepart of the original material. Coppins (1983) cited a ‘holotype’ in S, which constitutes a lectotypification following ICN Art. 9.10. There is, however, an additional specimen in S (S L1472) with the same label data, and as Coppins (1983)
did not indicate which of these specimens he considered to be the holotype,the lectotypification effectively concerns both specimens. We therefore further
specify this by here designating the specimen S L1471 as the lectotype. This
specimen was likely the one referred to as holotype by Coppins, as annotation
slips from him are included in the envelope. It should be noted that all five typespecimens of M. melaeniza are homogeneous.
Habitat and distribution. M. melaeniza occurs on lignum of conifer stumps
and logs. Based on sequenced specimens and type, the species is currently
known from the Czech Republic, Finland, Sweden, Ukraine and the Russian Caucasus. In addition, M. melaeniza has been reported from Alaska (Spribille et al.
2020), Austria (Berger and Türk 1991, this study) and Mongolia (Palka and Śliwa 2006). Further, it might have been reported as M. nigella and could be foundafter revising specimens.
Notes. In his monograph of European Micarea species, Coppins (1983) accepted M. melaeniza, and in his interpretation, the species is characterized byhaving a hymenium with green pigmentation, a dark brown hypothecium without any reaction with K, and black stalked pycnidia containing comparatively
short conidia. In the same work, the new species M. nigella was described,
which should differ from M. melaeniza by having a purplish brown, K+ green
pigment in the hymenium, hypothecium and pycnidial tissues, and slightly larger mesoconidia (M. melaeniza: 2.3–3.6 × 1–1.3 µm vs. M. nigella: 3.4–4.3 ×
1.2–1.6 µm; Coppins 1983). Czarnota (2007) noted that the amount of purple, K+ green pigment varied considerably in Polish collections determined as
M. nigella, and suggested that M. melaeniza and M. nigella could be conspecific. He further noted that Hedlund’s original description could be interpreted asindicating the presence of another pigment, the purple, K+ purple pigment andsuggested that the differences between Hedlund’s and Coppins’ descriptions
could be due to the studied material having aged (Czarnota 2007).
In our interpretation, M. melaeniza is a species with mostly two pigments:(i) a blackish-green, usually K+ green intensifying pigment, mostly located tothe epihymenium but sometimes also in the hymenium and the upper part of
the hypothecium (Cinereorufa-green) and (ii) a dark brown, K– pigment in the
hypothecium (possibly Superba-brown). The description in Coppins (1983) fits
our interpretation quite well, except that the specimens have a K+ greenish
reaction due to Cinereorufa-green pigment that is not mentioned by Coppins(l.c.) but is, on the other hand, mentioned in the original description of the species by Hedlund (1892) and seen by us in the type specimen. Pigmentation of
M. melaeniza may be more complex, however. One specimen from the Czech
Republic (ZP32013) shows patchily purplish, K+ dark green pigment that might
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Annina Kantelinen et al.: A phylogenetic study of Micarea melaeniza groupbe the Melaena-red pigment (in apothecia concentrated mainly in hymenium
as darker streaks). This specimen was originally downloaded to GenBank as
M. nigella, but it is monophyletic with M. melaeniza in our phylogenetic analyses. More sequenced specimens are needed to understand better the pigment
profile of M. melaeniza.
We considered the possibility of M. melaeniza and M. nigella being synonymous. A careful study of the type specimens showed morphological differences,e.g. in the size of conidia and pigmentation of apothecia and pycnidia. A brownor purple-brown, K+ green pigment in the hymenium, hypothecium and pycnidia
walls (Melaena-red) of M. nigella is an important difference between M. melaeniza and M. nigella, although this is not true for all the studied specimens as was
mentioned above. The difference in pigmentation is also visible in nitric acid,i.e. in M. melaeniza the hypothecium is mostly HNO3– (rarely HNO3 intensifyingred), and in M. nigella HNO3+ purple-red. Compared to M. melaeniza, M. nigellaalso has longer conidia (3.4–4.5 × 1.2–1.6 µm), slightly shorter hymenium (upto 30 µm) and wider paraphyses (up to 3 µm), as also shown by Coppins (1983).
The molecular study supports the distinction of M. melaeniza and M. nigella(Fig. 2). Our sequenced specimens form two monophyletic clades, and these
specimens are morphologically similar with the type specimens (except for
ZP32013 discussed above).
In external appearance, M. melaeniza also resembles M. botryoides, M. eurasiatica, M. misella and M. osloensis. Micarea botryoides is usually not lignicolous, has longer ascospores (8–13(–16) × 2.3–3.7(–4) µm) that are often
septate, and longer mesoconidia (Coppins 1983). Micarea eurasiatica has similar pigmentation like M. melaeniza, but the shape of apothecia is different (adnate vs subglobose), its pycnidia are sessile and mesoconidia are longer (up to6 µm). Micarea misella, on the other hand, can be microscopically distinguishedfrom M. melaeniza by the olivaceous pigment that reacts violet instead of dull
green in K, and by its hyaline hypothecium (Coppins 1983). Micarea osloensis
is similar to M. melaeniza in many characters, and is a close relative basedon our phylogenetic study. However, M. osloensis has shorter pycnidia (max.
180 µm), longer mesoconidia 3.5–4.5(–5) × 1.2–1.5 (–1.8) µm, and apothecia and pycnidia are mostly K– (although a higher concentration of Cinereorufa-green pigment is known to occur in some of the C-European specimens).
Additional specimens studied. Austria, Niederösterreich, Ybbstaler Alpen, Wildnisgebiet Dürrenstein, Lunz am See Rothwald, Kleiner Urwald, primeval beech dominated forest on a crest above the valley of Moderbach brook,
47°46'31.0"N, 15°06'10.5"E, 1010 m, on wood of snag of Picea abies, 2022,
Malíček 16229, Berger, Palice & Vondrák, hb Malíček.
Czech Republic, S Bohemia, Šumava Mts, Volary, České Žleby: Radvanovický
hřbet - E foothill, managed spruce forest with left old beeches on E-NE-facing
slope, 48°54'02"N, 13°48'28"E, on decaying wood of (?)Picea stump, 820 m,
2021, Palice 32013, PRA (in GenBank as M. nigella: OQ646318); ibid., Horní
Vltavice, Zátoň: Jilmová skála Nature Monument, scree old-growth forest(150–200 years old) with maple, beech, sycamore, silver fir etc., 48°57'13"N,
13°47'48"E, 1000–1030 m, on decaying stump, 2014, Malíček 7322, hb Malíček.
Finland, Uusimaa, Vantaa, Herukkapuro nature reserve (plot 1), old-growthforest, on wood of a dead stump of Picea abies (decay stage 5), WGS84
60.3215°N, 24.7658°E, 2013, Kantelinen 2430 (DNA A772), H.
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Ukraine, Ukrainian Carpathians, Nadvirna, Bystrytsia, in valley of stream
Dzhurbzinets, c. 3 km south of village Maksymets, 48°28'30"N, 24°18'23"E,
1005 m, on soft wood of coniferous (Picea?) stump, 2019, Vondrák 21921, PRA.
Russia, Russian Western Caucasus, Adler, Krasnaya Polyana, primeval firbeech forest below timberline, 43°41'50"N, 40°21'25"E, 1690 m, on soft rotten
wood of Abies snag, 2019, Vondrák 23358, PRA.
Sweden, Ångermanland, Långsele par., VII. 1891. Hedlund, UPS (L-171893).

Micarea nigella Coppins
Fig. 3E, F, G
Micarea nigella Coppins. Bull. Brit. Mus. Nat. Hist. 11(2): 163 (1983). Type: Denmark, Jylland, c. 16 km N of Hobro, Rold Skov, Torstedlund Skov, on conifer
stump, lignum, 1979, Coppins 4429 (RBGE! – holotype).
Description. Thallus endoxylic or thin green-grey layer on top of substrate. Photobiont micareoid, 4–7 µm.
Apothecia absent to numerous (mostly rare), immarginate, subglobose, often becoming tuberculate, black, matt, 0.1–0.3 mm in diam. Hymenium 25–
30 µm tall, hyaline or tinged dull brown or purplish brown, K+ sordid green,
HNO3+ purple-red (Melaena-red), often with darker vertical streaks. Epihymenium irregularly pigmented brown to purplish-brown (Melaena-red), sometimes
dark greenish, K+ olive green, HNO3+ purple (Cinereorufa-green). Asci clavate,
22–30 × 10–12 µm. Ascospores ellipsoid to usually ovoid, simple, 6.5–12 ×
2.5–4.0 µm. Paraphyses of two types: 1.) evenly distributed, branched, thin, c.
0.7–1 µm wide, 2.) scattered or in small fascicles, stout, 2–3 µm wide, coatedby dark pigment. Hypothecium c. 70–120(–160) µm tall, dark brown with variable amount of purplish tone, K+ olive green, HNO3+ purple-red (Melaena-red),hyphae coated with a dark brown pigment. Excipulum not evident.
Mesopycnidia always present, usually numerous, black, sessile or moreusually stalked and then 80–300 µm tall, 40–80 µm in diam., stalks simple orbranched from the base bearing up to four pycnidia, walls brownish black to
purplish black, sometimes olivaceous from the top, K+ dull green and HNO3+
purple-red especially in the brown parts (Melaena-red). Mesoconidia ellipsoid or
short cylindrical 3.5–4.5(–5) × 1.2–1.8 µm. Micro- or macropycnidia not seen.
Crystalline granules not present in apothecia, pycnidia or thallus.
Chemistry no substances detected by TLC (information based on Coppins
1983 and Czarnota 2007).
Habitat and distribution. Micarea nigella occurs mainly on lignum of conifer
stumps or fallen trunks, sometimes spreading from wood to dead bryophytes.
Based on sequenced specimens and the type material, the species is known
from the Czech Republic, Denmark (holotype), Great Britain (paratypes), Finland and Sweden. In addition, M. nigella has previously been reported from boreal and temperate forests in north-western, central and eastern Europe (e.g.
Czarnota 2007).
Notes. In external appearance, M. nigella resembles M. melaeniza. The differences between these two species are discussed in detail under M. melaeniza and Coppins (1983). The species also resembles M. botryoides, M. misella
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and M. osloensis. Micarea botryoides is usually not lignicolous and prefersrain-sheltered microhabitats on various substrata, it has slightly taller pycnidia(up to 400 µm) and longer ascospores (8–13(–16) × 2.3–4 µm) that are often
septate (e.g. Coppins 1983). Microscopically, M. misella can be distinguishedby the olivaceous pigment that reacts violet instead of dull green in K, and by
its hyaline hypothecium (Coppins 1983; Czarnota 2007). Micarea osloensis, on
the other hand, is usually K– and its pycnidia are shorter. However, our study
includes specimens that are difficult to identify by morphological characters,
especially between M. melaeniza, M. nigella and M. osloensis.
One of the distinguishing characteristics of M. nigella is the Melaena-red pigment (K+ green, HNO3+ purple-red) in the hymenium, hypothecium and pycnidia. In the literature, the pigment is described as `purple` (Coppins 1983; Meyer
and Printzen 2000; Czarnota 2007). However, based on our study, the pigment
is mostly brown, sometimes with a purplish tinge. The holotype of M. nigellahas the Melaena-red pigment, that looks brown with a purplish tinge, but of ourthree sequenced specimens (Fig. 2, Table 1), one (collection Kantelinen 1971)
has no purplish tone, whereas the other two collections (Kantelinen 1974,
1921) have easily detected amounts of purple. Interpreting the colouration canbe difficult and confusing, but maybe a helpful hint is that the pigment is always
K+ green, even if it looks brown in water. According to our study, the K+ green
reaction mostly disappears in 30 minutes.
Occasionally, M. nigella also has a third pigment, the Melaenida-red (K+ purple). This pigment was not found in the Finnish specimens but is sometimes
seen in the Central European specimens included in this study and mentioned
also by Coppins (1983) and Czarnota (2007). The Melaena-red and Melaenida-red pigments can be intermixed and appear in varying concentrations.
Additional specimens studied. Czech Republic, Central Bohemia, Brdy Protected Landscape Area, Míšov, Na Skalách Nature Reserve, old-growth beech
forest, small scree with rock outcrops and sparse spruce forest in NW partof the protected area, 49°36'20"N, 13°45'56"E, 715–740 m, on stump, 2023,
Malíček 16287, hb Malíček. Western Bohemia, Domažlice, Český les Protected
Landscape Area, Pec, Bystřice Nature Reserve, natural mixed forest up to 150
years old, 49°22'56"N, 12°48'39"E, 650–750 m, on lying decaying trunk, 2015,
Malíček 8029 (hb Malíček); ibid., Tachov Lesná: managed spruce forest 1 km
NE of Knížecí strom Hill (829 m), 49°46'13.0"N, 12°29'28.3"E, 750 m, on stumpof Picea abies, 2019, Malíček 13161 & Rydlo, hb Malíček. Southern Bohemia,
Prachatice, Šumava National Park, Nová Pec old-growth beech-spruce forest
on N-facing slope of Mt Hraničník (1281 m), 48°45'13"N, 13°54'17"E, 1170 m,
on decaying wood, 2017, Malíček 11296, hb Malíček. Eastern Bohemia, Žďárnad Sázavou, Žďárské vrchy Protected Landscape Area, Svratka managed
beech-spruce forest on NW-facing slope of Bubnovaný kopec Hill (780 m),
49°42'41.6"N, 16°05'13.7"E, 775 m, on stump of Picea abies, 2020, Malíček
13865 & Sejfová, hb Malíček. Svratka forest mosaic 0.2 km SSE of Spálený
kopec Hill (766 m), 49°43'28.0"N, 16°06'23.3"E, 755 m, on stump of Picea abies, 2020, Malíček 13868 & Sejfová, hb Malíček. Svitavy, Česká Třebová, Psí
kuchyně Nature Reserve, old-growth beech forest 0.1 km NW of Psí kuchyně
Hill (526 m), 49°50'38.3"N, 16°26'47.5"E, 505 m, on lying wood of Fagus sylvatica, 2020, Malíček 13975 & Rydlo, hb Malíček. Silesia, Beskydy Protected
Landscape Area, Horní Lomná, Velký Polom Nature Reserve, valley of a brook
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Annina Kantelinen et al.: A phylogenetic study of Micarea melaeniza groupwith old-growth, beech predominated forest in the E part of the protected area,
49°30'36"N, 18°41'00"E, 860–910 m, on stump of Picea abies, 2021, Malíček
14664 & Sejfová, hb. Malíček; ibid., gorge on NW-facing slope of Mt Velký polom (1067 m), c. 49°30'25"N, 18°40'04"E, 950–1000 m, on stump, 2021, Malíček
14649, Hlisnikovský & Sejfová, hb Malíček; ibid., Karolinka, Malý Javorník Nature Reserve, old-growth spruce-beech forest, 49°18'20.9"N, 18°17'18.5"E,
880–970 m, on wood of stump, 2023, Malíček 16392, hb Malíček; ibid., Valašská Bystřice, Kutaný Nature Reserve, old-growth beech-silver fir forest,
49°22'15.6"N, 18°6'0.4"E, 610–770 m, on fallen wood, 2020, Malíček 14246 &Konečná, hb Malíček. Jeseníky Protected Landscape Area, Karlova Studánka,old-growth spruce forest on SE-facing slope in valley of Bílá Opava Brook, along
tourist path 1.1 km NE of Ovčárna, 50°04'37"N, 17°15'06"E, 1170–1200 m, onroots of Picea abies, 2015, Malíček 8547, hb. Malíček.
Finland, Pohjois-Karjala, Lieksa, Koli National Park (plot 9), East side,
old-growth forest, on wood of fallen Picea abies (decay stage 2), 63.1033°N,
29.8140°E, 2013, Kantelinen 1971 (DNA A589), H; ibid., Kantelinen 1974 (DNAA588), H; ibid., Kantelinen 1921 (DNA A572), H. Etelä-Häme, Hämeenlinna, Evo(plot 8), protected old managed forest, on wood of a Picea abies stump (decay
stage 5), 61.2088°N, 25.1363°E, 2013, Kantelinen 2851 (DNA A769), H; ibid.,
Kantelinen 2881 (decay stage 4, DNA A764), H.
Slovakia, Eastern Slovakia, Bukovské vrchy Mts., Nová Sedlica, protectedarea Stužica, NNE-facing slope of Temný vršok Mt. (838 m), old-growth beech
forest, 49°04'11"N, 22°32'26"E, 750–820 m, on stump of Abies alba, 2013,
Malíček 6511 & Vondrák, hb Malíček.

Micarea osloensis (Th. Fr.) Hedl.
MycoBank No: 627666
Fig. 4A, E
Type. Bih. Kongl. Svenska Vetensk.Akad. Hand. III, 18: 97 (1892). Lecidea osloensis Th. Fr. Lich. Scand. 2: 524 (1874). Type: Norway, Oslo, In cacumine
Ryenbjerget, 10 July 1874. N.G. Moe (UPS L-153276! – holotype).
Description. Thallus endoxylic or visible as a thin pale greenish-grey to darkgreen-grey layer on top of substrate. Photobiont micareoid, 4–7 µm in diam.
Apothecia infrequent or rare, absent or numerous, immarginate, convex tohemispherical, dark brown to black, matt, simple, 0.1–0.2(–0.3) mm in diam.
Hymenium 30–50 µm tall (Coppins c. 30 µm tall), hyaline, sometimes olivaceaous, often with warm brown vertical streaks, K–, HNO3– (Superba-brown).
Epihymenium warm dark brown to blackish (Coppins: red-brown), rarely greenish, mostly K– but sometimes K+ green, HNO3– or HNO3+ purplish (Cinereorufa-green). Paraphyses of two type: 1.) hyaline or coated with a brown pigment,
thick, 2–3 µm wide, simple or branched, sometimes wider from apices, often
uneven in shape, abundant, sometimes concentrated into fascicles, 2.) thinner, c. 1–1.5 µm wide, rarely branched, rare. Asci cylindrical to cylindrical-clavate, 30–40 × 10–12 µm (Coppins: 26–30 × 11–13 µm). Ascospores 7–10 ×(2.5–)3.0–3.5 (–4.0) µm (Coppins: 6–9.5 × 3–4 µm), ellipsoid, cylindrical orsometimes roughly shaped, 0(–1) sept. Hypothecium warm dark brown (Coppins: red-brown), composed of hyaline hyphae 1–2 µm wide surrounded by
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Annina Kantelinen et al.: A phylogenetic study of Micarea melaeniza groupbrown pigment giving it an unevenly coloured/randomly spotted appearance,
K–, HNO3– (Superba-brown), c. 85 µm tall.
Mesopycnidia often numerous and crowded, sometimes absent, simple orbranched from the base, emergent or shortly stalked ca. 50–180 µm tall, 40–
70 µm in diam., dark brown to blackish, walls brown, greenish brown from the
top with a warm brown lower part, K– (Superba-brown) or sometimes K+ green,
HNO3– or HNO3+ purplish (Cinereorufa-green), usually extruding white mass ofconidia that may merge with neighbouring conidia. Mesoconidia ellipsoid or shortcylindrical 3.5–4.5(–5) × 1.2–1.5 (–1.8) µm. Micro- or macropycnidia not seen.
Chemistry no substances detected by TLC (Coppins 1983).
Crystalline granules not present in apothecia, pycnidia or thallus.
Habitat and distribution. The type of M. osloensis occurs on soil. Anothermorphologically identical specimen collected in 2007 occurs on bark of decaying trunk (Palice 11684). Our newer specimens occur on bark, dead wood anddead mosses. The type specimen was collected from Norway from a woodlandclearing on the site of an old bonfire, and the newer specimens are from theCzech Republic, Finland, Sweden and Ukraine. In the Czech Republic, M. osloensis occurs commonly from middle to montane elevations. It appears to be toxitolerant and is known in areas with higher levels of air pollution in the past (i.e.
acidification by acid rain). The typical habitats are bark on bases and roots ofFagus sylvatica, Larix decidua, Picea abies, Pinus sylvestris. It is abundant also
on dead wood and dead bark on stumps, fallen trunks and snags. In Finland,M. osloensis is likely relatively common but overlooked in coniferous forests
on bark, dead wood and dead mosses. In both countries, M. osloensis is known
from managed and old-growth forests.
Notes. The two previously known M. osloensis specimens, including thetype, have not been sequenced, although an unsuccessful sequencing attemptof a specimen collected by Palice (11684) was made by Kantelinen in 2011,
and therefore we cannot compare our new specimens to the type of M. osloensis using DNA. Subtle morphological features differentiate the type from new
specimens, i.e. taller hymenium and asci. Most of the new specimens are K–
and have only the Superba-brown pigment, similar to the type. However, somespecimens have a K+ greenish, HNO3+ purple reaction in the epihymenium
and pycnidial walls suggesting the presence of the Cinereorufa-green pigment
which is not known from the type of M. osloensis. Specimens with the Cinereorufa-green pigmentation appear to be more frequent in the Czech Republic. The
Finnish specimens have sometimes olivaceous tones that are K– but slightly
HNO3+ purple.
Another difference between the type of M. osloensis and our newly sequenced specimens is reproduction. The type specimen has apothecia and no
pycnidia. The new specimens, on the other hand, often have shortly stipitate
pycnidia. Our specimens appear to be dimorphic, however, so that the specimens represent either sexual (rare) or asexual reproduction modes which aremonophyletic in DNA level.
Because of overlapping variation in reproduction and pigmentation between
the type of M. osloensis and our new specimens, we cannot exclude the possibility that they are conspecific. On the other hand, we also cannot exclude thepossibility that the new specimens represent a yet undescribed taxon in the
M. melaeniza group.
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Figure 4. Morphological and anatomical features of old and new Micarea osloensis collections A, B old Micarea osloensis
A habit, apothecia on soil (Holotype) B apothecial section in water (Palice 11684, H) C–E new Micarea osloensis (Kantelinen 2648, H) C mesopycnidia extruding mesoconidia as a white drop D apothecial section in water E mesopycnidia on
dead wood and mosses. Scale bars: Habit 0.5 mm (A, C, E); Apothecial sections 100 µm (B, D).

M. osloensis resembles M. eurasiatica, M. melaeniza, M. misella and M. nigella. The most important characters of M. osloensis are the combination ofsessile to shortly stalked pycnidia, mesoconidia of the size 3.5–4.5(–5) × 1.2–
1.5 (–1.8) µm, warm-brown, sometimes olivaceous colouration in apothecia
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Annina Kantelinen et al.: A phylogenetic study of Micarea melaeniza groupand pycnidia (Coppins: red-brown), and often a K– reaction. Micarea eurasiatica sp. nov. has mostly sessile to emergent pycnidia and bigger mesoconidia((4–)4.5–6.0 × 1.2–1.5(–1.8) µm). Micarea misella, on the other hand, has a
K+ violet reaction in the epihymenium and pycnidia (Coppins 1983; Czarnota
2007). Micarea nigella has similar mesoconidia and spore size, but its pycnidia
are usually taller and it has the brown/purple-brown Melaena-red pigment (K+green) in hypothecium and pycnidia.
Based on our phylogenetic analyses, M. osloensis and M. melaeniza are sister species. They have morphological similarities including pigmentation andspore size. However, M. osloensis has slightly larger mesoconidia, shorter
pycnidia and wider often roughly shaped paraphyses. The concentration of the
Cinereorufa-green pigment (K+ green, HNO3+ purple) appears to vary in bothtaxa, but especially in M. osloensis. Micarea melaeniza is mostly K+ green in epihymenium, hymenium, upper hypothecium and pycnidia. Micarea osloensis, on
the other hand, is rarely K+ green and then from the epihymenium and pycnidia.
Additional specimens studied. Czech Republic, Northern Bohemia, Jizerskéhory Mts, Josefův Důl: valley of Jedlový potok, protected zone of nature reserve
Jedlový důl, fragment of fir-beech old-growth forest, 50°47'45"N, 15°14'47.5"E,on dry wood of old conifer stump, 780 m, 2020, Palice 30266, PRA (in GenBank
as M. nigella: OQ646320); ibid., Hejnice, Jizerskohorské bučiny National Nature
Reserve, valley of Velký Štolpich brook, S of Ořešník Mt. (800 m), ca 50°51'13"N,
15°11'13"E, 660 m, on base of Fagus sylvatica, 2013, Malíček 6020, hb Malíček.
Lužické hory Protected Landscape Area, Mařenice, Horní Světlá: managed
spruce-beech forest on E-facing slope, 0.3 km NNW of Kopřivnice Hill (638 m),
50°50'13.6"N, 14°37'56.6"E, 600 m, at base of Picea abies, on stump of Picea,
2020, Malíček 14019, 14020 & Rydlo, hb Malíček. Central Bohemia, Brdy Protected Landscape Area, Míšov, Na Skalách Nature Reserve, old-growth beech
forest, small scree with rock outcrops and sparse spruce forest in NW partof the protected area, 49°36'20"N, 13°45'56"E, 715–740 m, at base of Fagus
sylvatica, 2023, Malíček 16281, hb Malíček. Brdy Hills, Rožmitál pod Třemšínem, Nepomuk: managed forest 0.4 km N of Praha Hill (862 m), 49°39'48"N,
13°49'06"E, 825 m, at base of Larix decidua, 2018, Malíček 12007 & Vondrák,
hb Malíček; ibid., Strašice, managed mixed forest 3 km E of village, 49°43'34"N,
13°47'56"E, 610 m, at base of Larix decidua, 2018, Malíček 12000 & Vondrák,
hb. Malíček. Příbram, Brdy Hills, Jince managed coniferous forest 0.5 km SSE
of Velcí pond, 49°45'17"N, 13°56'34"E, 600 m, at base of Larix decidua, 2018,
Malíček 12016 & Vondrák, hb Malíček. Eastern Bohemia, Rychnov n. Kněžnou,Orlické hory Protect. Lands. Area, Rokytnice v Orlických horách, Černý důl Nature Reserve, fragment of old-growth beech-spruce-silver fir forest, along brook,
50°12'01.4"N, 16°31'18.2"E, 800–810 m, on bark of stump of Picea abies, 2012,
Malíček 4536 et al., hb Malíček. Žďár nad Sázavou, Žďárské vrchy Protected
Landscape Area, Svratka fragment of old beech predominated forest 0.6 km
SW of Spálený kopec Hill (766 m), 49°43'18.2"N, 16°06'11.4"E, 750 m, on stumpof Picea abies, 2020, Malíček 13864 & Sejfová, hb Malíček; ibid., Svratka, Pustá Rybná: spruce-beech forest on S-facing slope of Kaštánkův kopec (753 m),
49°43'03.9"N, 16°06'46.4"E, 740 m, on stump of Picea abies, 2020, Malíček
13953, hb Malíček. Svitavy, Česká Třebová managed spruce forest 3 km W
of Opatov, 49°49'47.6"N, 16°27'40.0"E, 445 m, on stump of Picea abies, 2020,
Malíček 13970 & Šámalová, hb Malíček. Western Bohemia, Český les Protected
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Landscape Area, Tachov, Lesná: young beech forest 0.8 km E of Knížecí strom
Hill (829 m), 49°45'54.2"N, 12°29'18.5"E, 780 m, on stump of Picea abies, 2020,
Malíček 13784 & Rydlo, hb Malíček; ibid., spruce-beech forest 1 km NE of Knížecí
strom Hill (829 m), 49°46'17.3"N, 12°29'18.1"E, 770 m., at base of Picea abies,
2019, Malíček 13167 & Rydlo,hb Malíček; ibid., Pec, Bystřice Nature Reserve,
natural mixed forest up to 150 years old, 49°22'56"N, 12°48'39"E, 650 m, at baseof Picea abies, 2015, Malíček 8028, hb Malíček. Kdyně, Mezholesy: managed
mixed forest 0.3 km SE of Koráb Hill (773 m), 49°23'37.7"N, 13°04'44.1"E, 750 m,
at base of Picea abies, 2019, Malíček 13371 & Rydlo, hb Malíček. Southern Bohemia, Novohradské hory Mts, Horní Stropnice, NPP Hojná Voda, fragment ofprimeval forest predominated by beech, 48°42'20"N, 14°45'08"E, 840–870 m,on snag, 15 October 2019, Malíček 13500, herb. Malíček. Šumava Mts., Volary, Nová Pec, 740 m, 48°49'11.2"N, 13°56'12.151"E, on bark of Pinus sylvestris at base of trunk, 2017, Vondrák 19007, PRA. Prachatice, Šumava Protected
Landscape Area, Kubova Huť, Boubínský prales National Nature Reserve, managed spruce forest c. 120 years old, 0.4 km NNE of top of Mt Boubín (1362 m),
48°59'42.2"N, 13°49'05.7"E, 1275 m, on decaying stump 2015, Malíček 8349
& Palice, hb Malíček. Jindřichův Hradec, Javořická vrchovina Hills, Stráž nadNežárkou, Sedlo: managed coniferous forest SSE of Otínský kopec Hill (538 m),
49°02'44.0"N, 14°59'45.7"E, 530 m, on stump, 2020, Malíček 13817, hb Malíček;ibid., Lásenice: mixed forest S of Šemburský rybník, 49°03'34.6"N, 14°59'55.2"E,
520 m, on stump, 2020, Malíček 13821, hb. Malíček; ibid., managed coniferous forest between Nová Ves and Sedlo, 49°03'36.1"N, 15°00'55.3"E, 560 m,
at base of Pinus sylvestris, 2020, Malíček 13824, hb Malíček. Tábor Chýnov,young beech forest SSE of Blanička, 49°28'00.6"N, 14°50'34.9"E, 690 m, on fallen wood, 2020, Malíček 13834 & Rydlo, hb Malíček; ibid., mixed forest SE of Batkovy Hill (724 m), 49°27'43.3"N, 14°50'00.9"E, 700 m, at base of Larix decidua,
2020, Malíček 13838 & Rydlo, hb Malíček. Northern Moravia, Jeseník, Jeseníky
Protected Landscape Area, Bělá pod Pradědem, Vysoký vodopád, Nature Reserve, valley of Studený p. brook, ca. 50°06'57"N, 17°12'10"E, 900–1000 m, onbase of Picea abies, 2012, Malíček 5102, hb Malíček. Šumperk, Králický Sněžník
Mts, Staré Město: Mt. Králický Sněžník, c. 100 years old spruce forest on S-facing slope 0.1 km SE of Františkova chata, 50°12'05.2"N, 16°51'28.3"E, 1210 m,
at base of Picea abies, 2015, Malíček 8381, Kocourková & Vondrák, hb Malíček.
Eastern Moravia, Beskydy Protected Landscape Area, Bílá, Salajka: beech dominated forest 0.6 km SE of gamekeeper’s house, 49°24'37.9"N, 18°25'39.5"E,
730 m, on decaying stump, 2019, Malíček 13344 & Rydlo, hb Malíček; ibid.,
Frenštát pod Radhoštěm, Kněhyně-Čertův mlýn National Nature Reserve, W-facing slope of Kněhyně Mt. (1257 m), old-growth spruce forest above red-marked
tourist path, 49°29'57"N, 18°18'38"E, 1080–1100 m, on stump of Picea abies,
2013, Malíček 6090 & Vondrák, hb Malíček. Western Moravia, Žďár nad Sázavou, Žďárské vrchy Protect. Landsc. Area, Cikháj, Žákova hora National Nature
Reserve, beech virgin forest, 49°39'18"N, 15°59'35"E, 750–800 m, on stump of
Fagus, 2012, Malíček 5110 & Syrovátková, hb Malíček. Silesia, Bruntál, Jeseníky
Protected Landscape Area, Karlova Studánka, managed spruce forest (c. 100
years old) on N-facing slope in valley of Bílá Opava Brook, 1.8 km ENE of Ovčárna, 50°04'35"N, 17°15'53"E, 1170–1180 m, on bark of Picea abies, 2015, Malíček
8490, Kocourková, Vondrák & Zemanová, hb Malíček; ibid., Praděd National Nature Reserve, old-growth spruce forest c. 200 years old on E-facing slope of Mt
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Vysoká hole (1464 m), 0.2 km WNW of Eustaška hut, 50°03'35"N, 17°15'12"E,
1220 m, at base of Picea abies, 2015, Malíček 8563, Kocourková, Palice & Vondrák, hb Malíček. Frýdek-Místek, Beskydy Protected Landscape Area, Ostravice,
Mazák National Nature Reserve, old-growth spruce forest with intermixed sycamores on W-facing slope of Mt Lysá hora (1323 m), 49°32'41"N, 18°26'43"E,
1200 m, on bark of Picea abies, 2016, Malíček 9743 & Palice, hb Malíček.
Finland, Pohjois-Karjala, Lieksa, Koli National Park (plot 9), East side,
old-growth forest, on bark of fallen Picea abies (decay stage 3), 63.1033°N,
29.8140°E, 2013, Kantelinen 1865 (DNA A575, apothecia and pycnidia), H. Ibid.,
on wood of fallen Picea abies (decay stage 4), 2013, Kantelinen 1909 (DNAA583), H. Ibid., on wood of dead standing Picea abies (decay stage 3), Kantelinen 1685 (DNA A574), H. Ibid., on wood of fallen Picea abies (decay stage5), 2013, Kantelinen 2003 (DNA A594), H. Ibid., on wood of fallen Picea abies(decay stage 3), 2013, Kantelinen 1923 (DNA A573), H. Etelä-Häme, Hämeenlinna, Evo (plot 8), protected old managed forest, on wood/mosses of a Picea
abies stump (decay stage 5), 61.2088°N, 25.1363°E, 2013, Kantelinen 2899
(DNA A736). Ibid., on wood of Picea abies, 2013, Kantelinen 1686 (DNA A768),
H. Ibid., Rajakallio, boreal forest on a bouldery slope, forest of Picea, Pinus andBetula, 61°15.27'N, 025°06.43'E, on bark of rotten wood among boulders, 2007,
Palice 11684, conf. Coppins, H. Uusimaa, Tuusula, west of Korso, shaded anddense Picea abies dominated managed forest (plot 2), on wood of a Picea abies stump (decay stage 5), 60.3544°N, 25.0322°E, 2013, Kantelinen 2643 (DNAA484), H.
Sweden, Jämtland, Kall par., about 850 m NW of the northwestern tip of Lake
Spjuttjärnen, S side of stream Konäsån, on stump of Betula pubescens in oldgrowth Picea abies forest, 63°34'30"N, 13°04'05"E, elev. 440 m, 2022, Svensson
4335, UPS L-1091180.
Ukraine. Eastern Carpathians, Nadvirna, Bysrytsia, N of hill Skali verkhni,
48°27'48.492"N, 24°18'35.46"E, 1233 m, on bark of Picea abies, 2019, Vondrák
22217, PRA.

Discussion
Our aim was to clarify systematics and species boundaries among Micarea
melaeniza and similar-looking species. We propose the new species Micarea
eurasiatica, characterized by the combination of cylindrically shaped, sessile
to emergent pycnidia with greenish-black walls, long mesoconidia (up to 6 µm
in length), a K+ olive green reaction and by mostly occurring in the anamorphic
stage. The species is known from Japan and Finland. We also discovered another, putatively new species, marked Micarea sp. in the phylogeny that we refrain from describing because of insufficient morphological data and few available collections. This putative new species has sessile to emergent, brownish
black pycnidia with a purple tinge and a K+ green reaction, but apothecia arefew (see Table 2). Despite our efforts, we did not find or sequence M. olivacea,
a species that could be related to the M. melaeniza group.
Generally, the species in the M. melaeniza group are challenging to identify because they are small, have relatively few morphological characters andbecause the current literature is not up to date particularly in relation to pigmentation (Coppins 1983; Czarnota 2007). The challenges while identifying
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our fresh specimens underline this issue, and are discussed in the notes under
M. melaeniza, M. nigella and M. osloensis. According to our study, the species
in the M. melaeniza group are characterized by having a thin or endosubstratal
thallus and dark, sessile to stipitate mesopycnidia that often extrude a whitemass of conidia at their top. Apothecia are often absent, but when present, they
are 0.1–0.3 mm wide and brown to black in colour. They have hyaline or slightly
coloured hymenium, dimorphic paraphyses, simple spores and dark hypothecium where hyphae are surrounded by brown pigment giving them an unevenly
coloured appearance, also noted by Coppins (1983). Based on results of thisstudy, the size of pycnidia and mesoconidia, and to some extent also the general pigmentation in K and HNO3 are the most useful morphological characters inseparating the species, however in some cases DNA sequencing is the only reliable way for identification. Micarea eurasiatica, and M. olivacea develop sessile
to emergent pycnidia, M. osloensis emergent to shortly stipitate pycnidia, andM. melaeniza and M. nigella develop stipitate pycnidia. Micarea deminuta does
not develop distinctive stalked pycnidia, at least according to current knowledge. The Cinereorufa-green pigment (K+ green, HNO3+ purple) is present in
pycnidia and apothecia of M. eurasiatica, M. deminuta, M. melaeniza, M. nigella,
M. olivacea (according to Coppins 1983) and occasionally in M. osloensis invarying concentration, accompanied by the Superba-brown pigment (K–). The
brown or purple-brown Melaena-red pigment (K+ dull sordid green) is present
in M. nigella (and possibly in one specimen of M. melaeniza) and is sometimes
intermixed with the Melaenida-red pigment (K+ purplish-red) (Table 3).
Our study indicates that the pigmentation of some species may correlatewith geography. In the central European specimens some pigments are encountered more often or higher concentrations than in Fennoscandia. For example, in the Fennoscandian specimens of M. osloensis, the Cinereorufa-green
pigment is barely visible even when using HNO3, and the Melaenida-red pigment in M. nigella was not found at all. In the central European specimens,
however, both pigments were found, and sometimes in relatively high concentrations. The central European material also includes samples that appear to
be morphologically “intermediate” between species, for example M. melaeniza(ZP32013) may have Melaena-red pigment like M. nigella, although its mesoconidia size and DNA profile is similar to M. melaeniza. Some of the “intermediate” specimens have not been sequenced and therefore we do not know
their identity or whether they are undescribed species. Obviously, more work isneeded to understand the pigments and species in this group.
Because of these morphological challenges, we even considered that the species in the M. melaeniza group are conspecific, i.e. variation of just one species.
However, we excluded this possibility because of several reasons 1) molecular differences, 2) existing morphological differences, even though they may be
hard to interpret, 3) the type specimens studied are not morphologically conspecific, 4) the types correspond in morphology to most of our specimens. Basedon morphology, one might suggest that M. melaeniza, M. nigella and M. osloensis are variation of one species, but according to our phylogenies, they are
not monophyletic without M. deminuta, M. eurasiatica and Micarea sp. Even between M. melaeniza and M. nigella, the pigmentation is mostly different (Superba brown vs. Melaena-red), and although these pigments may look quite similar
to the human eye, they may have different ontologies and evolutionary paths.
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According to morphological studies by Coppins (1983), M. botryoides andM. melaeniza are relatives (group G) and possibly close to M. contexta, M. eximia, M. nigella and M. olivacea (group H). Our Bayesian and ML phylogenies
conclude that groups G and H are intermixed, however, based on DNA sequencing M. eximia is likely not a close relative of the M. melaeniza group (unpublished data) and molecular relationships of M. olivacea are still unknown. Our
phylogenetic analyses also show that M. melaeniza and M. osloensis are sisters, a relationship that has not been noted in previous publications, but thelatter clade is not supported (0.68 / 74). A closer look at the sequences andalignment shows that there are nucleotide differences in the mtSSU region
between M. melaeniza and M. osloensis (ca. 1–2% between specimens Kantelinen 2430 and 1923), but the ITS regions are nearly identical with only two
nucleotide differences. At least three conclusions could be drawn from theseresults. 1) The two clades are, in fact, two species as we suggest in our study.
This conclusion is supported by molecular and morphological data, to someextent at least, 2) The two clades are conspecific and represent morphologicaland molecular variation of M. melaeniza, 3) Our new specimens of M. osloensis
are not conspecific to the type of M. osloensis or M. melaeniza, but instead ascientifically undescribed species. Any conclusion we make suffers from theuncertainty caused by the M. melaeniza and M. osloensis type specimens nothaving sequenced, which means that we cannot compare our sequences to thetypes and the connections between fresh and old specimens are based solely
on morphology. This is also the reason why we refrain from describing our new
specimens of M. osloensis as a new species to science. The type specimens
are over 100 years old and hence likely beyond successful DNA sequencing –based on our experience, sequences are usually difficult to get from Micarea
specimens just over 3 years old, and nearly impossible when over 6 years old.
All species in the M. melaeniza group are either obligate or facultative lignicoles. According to a previous study, the wood-inhabiting lifestyle of Micarea
species influences their reproductive biology: obligate lignicoles primarily reproduce asexually, likely due to the transient nature of decaying wood, which
imposes a time constraint on the species occupying it (Kantelinen et al. 2022a).
Asexual reproduction via mesoconidia is likely a more rapid and efficient strategy than sexual reproduction. In the here studied M. melaeniza group and relatives, several species are found mostly asexual, viz. M. melaeniza, M. nigella,M. osloensis, M. eurasiatica, M. anterior and M. substipitata.
Based on our field experience as well as previous works by Coppins (1983)
and Czarnota (2007), the species in the M. melaeniza group are probably common in boreal and hemiboreal forests, both in natural and managed forests. In
spite of this, their small size and often anamorphic lifestyle make them easily
overlooked, resulting in rare mentions in ecological studies or species inventories. We hope that the morphological and molecular features presented in thisstudy will pave the way for future research endeavors.

Acknowledgements
We warmly thank prof. Teuvo Ahti, emeritus curator Arto Kurtto and Dr. Juha
Pykälä for their valuable help with this manuscript. Laura Häkkinen, Emilia Piki andEmelie Winquist are thanked for the DNA lab work. Permission for collection of
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Annina Kantelinen et al.: A phylogenetic study of Micarea melaeniza grouplichens on Mt. Oku-Shirane (Mt. Nikko-Shirane), Nikko National Park to GT in 2019was kindly granted by the Nikko National Park office, the Ministry of Environment(no. 1908283, 400), the Nikko District Forest Office of the Forest Agency and The
Nikko District Forest Office of Nihon Paper Company in Gunma Prefecture. ZP, JM
and JV acknowledge the long-term research development project RVO 67985939.

Additional information
Conflict of interest
The authors have declared that no competing interests exist.

Ethical statement
No ethical statement was reported.

Funding
This research was financially supported by the Finnish Ministry of Environment as apart of the research programme on data deficient and threatened forest species (grant
YTB067 to LM), a postdoctoral fellowship of the Finnish Museum of Natural History
and Societas pro Fauna et Flo-ra Fennica (personal grants for AK), and the ResearchCouncil of Finland (Grant 323711 to LM). Research by MS was financially supported by
the Swedish Taxonomy Initiative (grant no. 2016-206 4.3). Open access is funded by
Helsinki University Library.

Author contributions
Conceptualization: AK, Data curation/collection: AK, JV, JM, SS, MS, ZP, GT, Formal Analysis: AK, Funding acquisition: AK, LM, Investigation: AK, MS, JM, JV, ZP, Project administration: AK, LM, Visualization: AK, Writing – original draft: AK, MS, Writing – review &editing: AK, MS, ZP, JM, JV, GT, LM.

Author ORCIDs
Annina KantelinenMåns SvenssonJiří Malíček

https://orcid.org/0000-0001-8664-7662
https://orcid.org/0000-0003-1664-8226

https://orcid.org/0000-0002-3119-8967

Jan Vondrak

https://orcid.org/0000-0001-7568-6711

Göran Thor

https://orcid.org/0000-0003-1166-6898

Zdeněk Palice

https://orcid.org/0000-0003-4984-8654

Stanislav Svoboda
Leena Myllys

https://orcid.org/0000-0001-9797-4984

https://orcid.org/0000-0002-9566-9473

Data availability
All of the data that support the findings of this study are available in the main text.

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