683 MycoKeys 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 Kantelinen'®, Mans Svensson, Jiri MaliGek?©, Jan Vondrak?“©, Goran Thor®®, Zdenék Palice?®, Stanislav Svoboda*®, Leena Myllys'® on FP wo NY — Botany and Mycology Unit, Finnish Museum of Natural History, University of Helsinki, PO. Box 7, Fl-00014 Helsinki, Finland Museum of Evolution, Uppsala University, Norbyvagen 16, SE-752 36 Uppsala, Sweden Czech Academy of Sciences, Institute of Botany, Zamek 1 252 43, Pruhonice, Czech Republic Department of Botany, Faculty of Science, University of South Bohemia, CZ-37005 Ceské Budéjovice, Czech Republic Department of Ecology, Swedish University of Agricultural Sciences, PO. Box 7044, SE-750 07 Uppsala, Sweden Corresponding author: Annina Kantelinen (annina.kantelinen@helsinki. fi) OPEN Qaceess Academic editor: Thorsten Lumbsch Received: 19 March 2024 Accepted: 6 June 2024 Published: 5 July 2024 Citation: Kantelinen A, Svensson M, MaliGek 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 under terms of the Creative Commons Attribution License (Attribution 4.0 International - CC BY 4.0). Abstract 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 analysis of two markers (nulTS and mtSSU), we discovered two previously unrecognized phylo- genetic lineages, one of which is described here as Micarea eurasiatica Kantelinen & G. Thor, sp. nov., morphologically characterized by pycnidia that are sessile to emergent, cy- lindrically shaped, with greenish-black K+ olive green, wall pigmentation and containing large mesoconidia up to 6 um in length. The species is known from Japan and Finland. In addition, we show that the reproduction biology of M. os/oensis has been poorly under- stood and that the species often occurs as an anamorph with stipitate pycnidia. We pres- ent a species synopsis and notes on pigments. Our research supports previous results of asexuality 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, and new species are continually described (e.g., Czarnota 2007; Czarnota and Gu- zow-Krzeminska 2010; Sérusiaux et al. 2010; Guzow-Krzeminska 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; Vondrak et al. 2022; Index Fungorum 2023). Micarea species are globally distributed with repre- sentatives found on all continents. These species occur across a wide range 327 Annina Kantelinen et al.: A phylogenetic study of Micarea melaeniza group of habitats and grow on substrates such as bark, dead wood, rocks, soil and bryophytes. 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 challenge of identifying them. First, they are typically small. Second, Micarea exhibits a wide range of sexual and asexual propagules, including ascospores, three types of conidia (micro-, meso-, and macroconidia), and thallus fragments called go- niocysts which likely serve as asexual propagules including both symbiotic part- ners. 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 Micar- ea species display intraspecific colour variations, which depend on light expo- sure and corresponding pigment levels. For example, the Sedifolia-grey pigment, commonly found in the apothecia of Micarea species, varies in concentration from 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, iden- tifying 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-Krzeminska 2010; Sérusiaux et al 2010; Guzow-Krzemins- ka et al. 2016; van den Boom et al. 2017; Launis et al. 2019 a, b). However, some infra-generic groups in Micarea have been more studied than others. One group that 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. osloen- sis (Th. Fr.) Hedl.. These species share morphological characteristics, such as a thin 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 para- physes. The Cinereorufa-green (K+ green, HNO,+ purple) and Superba-brown (K-, HNO,-) pigments are often present, as well as sometimes Melaena-red (K+ green, HNO,+ purple-red). Most of these species are obligate or facultative lignicoles (Coppins 1983, Czarnota 2007). Despite their morphological and eco- logical similarities, all the species are not necessarily closely related (Coppins 1983; Andersen and Ekman 2005; Sérusiaux et al. 2010). a = Figure 1. Typical habitats for species in the M. melaeniza group in boreal and boreon- emoral 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). MycoKeys 106: 327-354 (2024), DOI: 10.3897/mycokeys.106.123484 398 Annina Kantelinen et al.: A phylogenetic study of Micarea melaeniza group We aim to clarify the molecular systematics and morphology of the poor- ly known M. melaeniza and its similar-looking species, focusing especially on M. melaeniza, M. nigella and M. osloensis, species that are morphological- ly challenging to identify. We use phenotypic characters and sequence data from two loci (nulTS 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, bore- onemoral and hemiboreal forests especially on dead wood and conifer bark. Materials and methods A substantial portion of the sequenced specimens in this study were collected from southern and central Finland as part of a research project that investigat- ed 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, Rus- sian Caucasus, Sweden and Ukraine. These specimens were collected from dead wood and bark and sequenced when possible. Geo-coordinates are given in the format WGS84. Relevant specimens were also looked for amongst fresh Micarea collections from Australia, Brazil, Kenya, Rwanda and Tasmania with no 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 de- pended 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 chem- ical spot tests with 10% potassium hydroxide (K) and sodium hypochlorite (C) and nitric acid (HNO,) were used (Orange et al. 2010). Fresh material was often not sufficient for thin-layer chromatography (TLC). The specimens are gener- ally quite small and have no or thin thallus, meaning that a substantial part of 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 & Vondrak (Coppins 1983; Czarnota 2007; Vondrak et al. 2022). Thus, TLC is of limited 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 of approximately 0.5-1 mm in diam. were ground with mini-pestles in 40 ul of lysis buffer, after which 140 ul of the buffer was added by simultaneously flushing lichen fragments from the mini-pestle. The extracted DNA was eluted in 50 ul of the eluation buffer. Samples were incubated for 5 minutes and centrifuged. After MycoKeys 106: 327-354 (2024), DOI: 10.3897/mycokeys.106.123484 399 Annina Kantelinen et al.: A phylogenetic study of Micarea melaeniza group the first elution, a second elution was performed to increase sample availability by adding another 50 ul of the elution buffer, incubated for 5 minutes and cen- trifuged. 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 cetyltrime- thylammonium bromide (CTAB)-based protocol (Aras and Cansaran 2006). In Finland, PCR reactions were prepared using PuReTaqg Ready-To-Go PCR beads (GE Healthcare). The 25 ul reaction volume contained 19 ul dH20, 0.4 uM of each primer and 4 uL extracted DNA. The ITS and mtSSU regions were used for species identification. PCR was run under the following conditions: ini- tial denaturation for 10 min at 95 °C followed by six cycles of 1 min at 95 °C (de- naturation), 1 min at 62 °C (annealing), and 1 min 45 s at 72 °C (extension); for the remaining 35 cycles, the annealing temperature was decreased to 56 °C; the PCR program ended with a final extension of 10 min at 72 °C. The primers used were 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 Malicek’s specimens were prepared following the protocol in Maliéek et al. (2023). For Vondrak’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 umol/I of each primer, 0.5 U Tag polymerase (TOP-Bio, Praha, Czech Repub- lic) in the manufacturer’s reaction buffer, and sterile water to make up a final volume of 10 ul. 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 fol- lowed 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 Stano- sz 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 de- cipiens (Hedw.) Hoffm. from the family Psoraceae as an outgroup, based on the studies by Andersen and Ekman (2005) and Sérusiaux et al. (2010). In the phylogeny (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 (K6rb.) R.C. Harris & Lendemer (see phylogeny in Sérusiaux et al. 2010). The final phylogenies, including 29 newly generated mtSSU and 25ITS sequenc- es (Table 1), were first aligned with MUSCLE v.3.8.31 (Edgar 2004) using the Eu- ropean Molecular Biology Laboratory, European Bioinformatics Institute’s (EM- BL-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 phy- logenetic reconstruction of the genus, M. byssacea and M. prasina belonging to MycoKeys 106: 327-354 (2024), DOI: 10.3897/mycokeys.106.123484 330 Annina Kantelinen et al.: A phylogenetic study of Micarea melaeniza group Table 1. List of Micarea specimens used in the phylogenetic analysis with locality, voucher information and GenBank accession numbers. Taxon M. anterior M. botryoides M. byssacea M. contexta M. deminuta M. deminuta M. denigrata M. doliiformis M. doliiformis M. eurasiatica sp. nov. M. eurasiatica sp. nov. M. eximia M. globulosella M. incrassata Micarea sp. M. melaeniza M. melaeniza M. melaeniza M. melaeniza (M. nigella in GenBank) M. misella M. nigella M. nigella M. nigella M. nigella M. nigella osloensis osloensis osloensis osloensis osloensis osloensis osloensis osloensis osloensis osloensis osloensis (M. melaeniza in GenBank) osloensis (M. nigella in GenBank) osloensis osloensis osloensis (M. melaeniza in GenBank) osloensis osloensis prasina S/S/S/S/S5/5]/5/5/5])/5/5)/5])/5/5])5/35/5]}5/8 substipitata Locality Finland Norway Finland Finland Japan Czech Republic Finland UK, Wales UK Finland Japan Finland Finland Finland Finland Finland Ukraine Russia Czech Republic Finland Finland Finland Finland Czech Republic Czech Republic Finland Finland Finland Finland Finland Finland Finland Finland Finland Czech Republic Czech Republic Czech Republic Sweden Russia Czech Republic Czech Republic Czech Republic Finland Finland Voucher information, sequence ID | Kantelinen 199 (H), A265 Andersen 79b (BG) Kantelinen (Launis) 289103 (H), A98 Kantelinen 1914 (H), A569 Thor 40245 (UPS), A926 Palice 6745 & VoriSkova (PRA) Kantelinen 723 (H), A686 Orange, LG database 29 (LG) | Andersen 178a (BG) | Kantelinen 2729 (H), A466 Thor 40053 (UPS) A914, holotype | Kantelinen 3785 (H), A785 Kantelinen (Launis) 67114 (H), A243 Kantelinen 90 (H), A90 Kantelinen 2640 (H), A487 Kantelinen 2430 (H), A772 Vondrdk 21921 (PRA) Vondrak 23358 (PRA) Palice 32013 (PRA) Kantelinen (Launis) 108111 (H), A264 Kantelinen 1971 (H), A589 Kantelinen 1974 (H), A588 Kantelinen 1921 (H), A572 Malicek 16287 Malicek 14664 Kantelinen 1685 (H), A574 Kantelinen 1865 (H), A575 Kantelinen 1909 (H), A583 Kantelinen 2899 (H), A736 Kantelinen 1923 (H), A573 Kantelinen 1686 (H), A768 Kantelinen 2643 (H), A484 Kantelinen 2003 (H), A594 Kantelinen 2648 (H), A485 Malicek 16281 Palice 30267 (PRA) Palice 30266 (PRA) Svensson 4385 (UPS), C792 Vondrak 22836 (PRA) Vondrak 25774 (PRA) Vondrak 19007 (PRA) Vondrdk 22217 (PRA) Kantelinen (Launis) 265101 (H), A92 Kantelinen 2700 (H), A469 MycoKeys 106: 327-354 (2024), DOI: 10.3897/mycokeys.106.123484 mtSSU PP811702 AY567741 MG707768 PP811722 PP811719 AY756446 PP811721 GU138666 PP811712 PP811720 MT982134 MG707744 MT982132 PP811703 PP811709 PP811724 PP811725 0Q646318 MG707742 PP811706 PP811714 PP811715 PP811726 PP811729 PP811704 PP811705 PP811717 PP811716 PP811713 PP811708 PP811710 PP811707 PP811730 0Q646316 0Q646320 PP811718 PP811723 0Q646315 PP811727 PP811728 MG707747 PP811711 ITS PP811675 AY756471 MG521562 PP811692 PP811689 AY756474 PP811691 HQ650654 PP811684 PP811690 MT981600 MG521547 MT981598 PP811676 PP811682 PP811694 PP811695 MG521545 PP811679 PP811686 PP811687 PP811696 PP811698 PP811677 PP811678 PP811688 PP811685 PP811681 PP811683 PP811680 PP811693 0Q717948 0Q717944 PP811697 MG521549 331 Annina Kantelinen et al.: A phylogenetic study of Micarea melaeniza group Micarea s. str. were selected as outgroups. We ran separate single-marker anal- yses 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 and Maddison 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 the mtSSU was removed from the analyses. Micarea doliiformis is represented by sequences from two specimens that were combined as one, the other spec- imen represented by an mtSSU sequence and another by ITS sequence (see Table 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 1Q-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 mod- els (Huelsenbeck et al. 2004). The convergence of the four parallel runs was checked 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 across subsets. Node support was estimated with 1000 bootstrap replicates using the ultrafast 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 the phylogeny. The remaining taxa resolve into four clades that include the M. me- laeniza group and its sister groups. The M. melaeniza group, delimited in this study, includes six monophyletic taxa, i.e. MW. deminuta, M. melaeniza, M. nigella, M. osloensis and two undescribed species, each represented by two speci- mens. 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 speci- men 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 this group, but the relationship receives no support and is based on limited taxon sampling. Several outgroups, including M. incrassata, M. doliiformis, and Leca- nia cyrtella have been tested but the overall topology of the phylogeny has not changed. The main distinguishing morphological characters are presented in the species synopsis Table 2. Pigments are presented in Table 3. MycoKeys 106: 327-354 (2024), DOI: 10.3897/mycokeys.106.123484 339 Annina Kantelinen et al.: A phylogenetic study of Micarea melaeniza group Micarea byssacea Kantelinen 289103 Micarea prasina Kantelinen 265101 Micarea incrassata Kantelinen 90 Micarea doliiformis Andersen 178a & Orange 29 0.99/79 Micarea contexta Kantelinen 1914 1 Micarea anterior Kantelinen 199 Micarea substipitata Kantelinen 2700 1 Micarea deminuta Palice 6745 Micarea deminuta Thor 40245 Micarea melaeniza Kantelinen 2430 1 Micarea melaeniza Palice 32013 Micarea melaniza Vondrak 21921 4 Micarea melaeniza Vondrak 23358 Micarea osloensis Kantelinen 1923 Micarea osloensis Kantelinen 2899 Micarea osloensis Kantelinen 1686 Micarea osloensis Palice 30266 = OF 2164) 1 Micarea osloensis Kantelinen 1865 Micarea osloensis Kantelinen 2643 Micarea osloensis Kantelinen 2003 O[5 Micarea osloensis Kantelinen 2648 Micarea osloensis Kantelinen 1909 0.99 Micarea osloensis Svensson 4385 0.90/79 Micarea osloensis Palice 30267 Micarea osloensis Vondrak 25774 4 : 0.68/74 Micarea osloensis Vondrak 22836 M. melaeniza 0.98 vicarea osloensis Vondrak 19007 group Micarea osloensis Vondrak 22217 Micarea osloensis Maliéek 16281 1 Micarea osloensis Kantelinen 1685 Micarea nigella Kantelinen 1974 0.80 Micarea nigella Kantelinen 1971 1 4 Micarea nigella Maliéek 16287 Micarea nigella Maliéek 14664 Micarea nigella Kantelinen 1921 4 Micarea eurasiatica sp. nov Thor 40053 Micarea eurasiatica sp. nov Kantelinen 2729 0.70/63 Micarea sp. Kantelinen 2640 4 Micarea denigrata Kantelinen 723 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 than 80 (e.g. 0.70/63). In addition to our specimens, we downloaded M. melaeniza and M. nigel- la sequences from Genbank. Based on our phylogenetic analyses, all of them fall inside Micarea osloensis (0Q646318, 0Q646320, 0Q717948, 0Q646315, 0Q717944, 0Q646316, in addition 0Q717947 and 0Q646319 are identical with others and excluded from our final analysis because of repetition). Three se- quences from Genbank were left out from our analyses (AY756488, AY756484, 0Q717944), because they are substantially different compared to the other sequences in the M. melaeniza group and hence the alignment and phyloge- netic analysis became unreliable. Based on blast searches AY756484 is a spe- cies of Lepraria, AY756488 perhaps Micarea melaena or M. nitschkeana, and 0Q717944 an uncultured fungus. Our data include a taxon with a high morphological resemblance to Micar- ea osloensis, a species found only twice before in years 1874 and 2007. Un- fortunately, 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 fer- tile specimen with apothecia, whereas our specimens are usually asexual with pycnidia. The specimens are mostly dimorphic, meaning that the existing se- quenced 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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-u9ei6 ‘yoeR|g-ysIuMolq (paouanbas) ajes de ‘elpiudAd eziuaejaw ‘VJ eipluohd puede | (4dAL) eziuaejaw ‘WV uunioayjodAy pue win juasaid skemje you de ‘elpiuoAd -Ayida UMOIG-aAI|O yep e]JOSIW uuinioayjyodAy yoe|q-umolg ‘(wuntuawAy ul Syeass yOeR|G-UMOIq 0} UMOJg |[NP YUM) win -luawAYida SnoadeAl|oO ase elpluoAd ‘eloaujode (Z00Z ejouezo nsuas) ejnulwap ‘Wy uunioayjyodAy [S66L Suiddog / umolq Usippes = yep] (eM OU) 4Oe;|G-UMOJ ‘uN -luawAYida UMO1g-yJep eloayjode (AdAL) e1NuIWwap ‘W ales elpluoAd a|dind ‘uaes6 yep ‘eloaujode 2)]Xa]U09 ‘WV oases ayinb umoiq(-ysippas/-a6uelo) de ‘elpiuoAd JOUajUe BaleOI| UOH}e10j/09 jeoIWO}eue uononpoidal 334 MycoKeys 106: 327-354 (2024), DOI: 10.3897/mycokeys.106.123484 Annina Kantelinen et al.: A phylogenetic study of Micarea melaeniza group uol10eaJ ON usel6 +4 usel6 +4 uuniuawAuidea uaas6 +> JO ‘uoloee1 ON uol]0eaJ ON usel6 +4 uonoeal-y win G*L-O'L x (v-)S°E-G'Z WH G'L-@'L x 0'S-ZY win (8° L-)S°L-@'L x 0°9-S'7 wil (8°L-)S"L-@'L x (G-)S'p-S'€ UdaS }0U WH O'L-@ LXE v-v'e eIpluodosaw yey Wil OSZ 0} dn ‘avy ‘ajyeldiys Ajjuoys 0} ajissas apim wi OS ‘Wye Wun Og *d ‘(jUaBsaWA 0O}) ajissas apim wun Gy—-0E ‘1123 wn (QO L—-) 08-0 ‘JuaHJawWa 0} a]ISsas qed Jamo] UMOJG WUEM e YUM do} ay} Woy UMOJG Ysiueet6 0} yOeI/q YsiuaaJB sjjem ‘ysyoe|q 0} UuMOJg yep ‘lu Og, 0} dn payjeys A}oys Jo juabiawa ‘aseg ay} Wl payoueig JO a|duuis ‘papmoso pue snoJauuNnu ualyjo UdaS }0U pasJauWUW-/+ ‘snonoidsuoou! 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Pigments found in apothecia and pycnidia based on our study and literature (Hedlund 1892; Coppins 1983; Czarnota 2007). M. deminuta M. melaeniza M. nigella M. osloensis (type) M. osloensis (new) M. eurasiatica sp. nov Cinereorufa-green [Pigment Melaena-red [Pigment A] K+ green intensifying, B] K+ dull sordid green, HNO,*+ purple HNO,*+ purple-red X (x) Melaenida-red [Pigment C] | Superba-brown [within Pigment F] K+ purplish-red, HNO, - No reaction in K or HNO, x (x) (x) x 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 start of the trail up to the summit of Mt. Oku-Shirane, along the trail. Open forest with mainly 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 um. Apothecia few, immarginate, convex, black, matt, 0.1-0.2 mm in diam. Hymenium 25-40 um tall, hyaline or tinged green, K+ greenish when tinged, sometimes with darker vertical streaks. Epihymenium black to blackish-green, K+ green, HNO,+ purple (Cinereorufa-green). Asci clavate, 28-38 x 11-14 ym. Ascospores ellipsoid to ovoid, simple, (6—)7—-9 x 3-4 um. Paraphyses numer- ous, of two types: 1. evenly distributed, branched, thin, c. 0.7-1.5 um wide, 2. evenly distributed, stout, sometimes branched, 2-3 um wide, not always coat- ed in pigment. Hypothecium c. 35-45 um tall, dark brown, K — (Superba-brown), or sometimes with a slight purple tinge (Melaenida-red), hyphae coated with dark brown pigment. Excipulum not evident. Mesopycnidia few to abundant, sessile to emergent, cylindrical in shape, 50- 80(-100) um tall, 30-45 ym wide, black, walls greenish black, K+ green, HNO,+ 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 x 1.2-1.5(-1.8) um. Micro- or macropycnidia not seen. Chemistry. Material not sufficient for TLC. Crystalline granules not present in apothecia or pycnidia. Habitat and distribution. WM. eurasiatica is currently known from Finland and Japan. In Finland, the species was collected in a shaded and dense, Picea ab- ies dominated managed forest. In Japan, the species occurred in a semiopen forest with mainly deciduous trees. On both occasions, the species was found growing on dead wood. Notes. MV. eurasiatica is currently known from two collections. The type collection has abundant mesopycnidia, and additionally few small apothecia. MycoKeys 106: 327-354 (2024), DOI: 10.3897/mycokeys.106.123484 336 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 apothe- cial 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 um (B, C, E, F); Drawing Ga 100 um, Gb 1 um. The other collection has only pycnidia. The most important diagnostic char- acters are the combination of sessile to emergent pycnidia that are cylindri- cal in shape, greenish-black pycnidial walls, large mesoconidia (up to 6 um in length) and a K+ olive green reaction (Cinereorufa-green). If apothecia are pres- ent, they are 0.1-0.2 mm wide, have greenish-black epihymenium (K+ green, MycoKeys 106: 327-354 (2024), DOI: 10.3897/mycokeys.106.123484 397 Annina Kantelinen et al.: A phylogenetic study of Micarea melaeniza group Cinereorufa-green) and dark brown hypothecium (K- or sometimes slightly K+ purple if Melaenida-red present). Micarea eurasiatica resembles other of- ten 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 x 1.2-1.8 um, M. eurasiatica: (4—)4.5-6.0 x 1.2-1.5(-1.8) ym), 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 and its 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 those of M. eurasiatica (M. olivacea: 3.4—4.3 x 1.2-1.6 um, M. eurasiatica: (4-)4.5- 6.0 x 1.2-1.5(—1.8) um) (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, shad- ed and dense Picea abies dominated managed forest (plot 2), on wood of fall- en Picea abies (decay stage 2), 60.3544°N, 25.0322°E, 2013, Kantelinen 2729 (DNA A466), H. Micarea melaeniza Hed. MycoBank No: 368074 Fig..3C,.D Type. Bih. Kongl. Svenska Vetensk.Akad. Hand. Ill, 18: 96 (1892). Type: Swe- den, Helsinglandize [= Halsingland], Jerfs6 [= Jarvs6], VIIl 1891. J. T. Hed- lund (S L1471! — lectotype, designated by Coppins 1983 [ICN Art. 9.10], fur- ther specified here [ICN Art. 9.17], S L1472!, UPS L-005556!, UPS L-171894I, LD 1056591!, isolectotypes). Description. Thallus endoxylic. Photobiont micareoid, 4-7 um. Apothecia absent to numerous (mostly rare), immarginate, subglobose, often becoming tuberculate, black, matt, 0.1-0.3 mm in diam. Hymenium (25-)28- 42 um tall, hyaline or tinged aeruginose green, olive, or rarely purplish brown, of- ten with darker vertical streaks. Epihymenium irregularly pigmented aeruginose green, or rarely sordid brown, sometimes with a purplish tinge. Epihymenium and hymenium K+ olive green, HNO, purple (Cinereorufa-green), or rarely K-. Asci clavate, 22-35 x 10-12 um. Ascospores ellipsoid to usually ovoid, simple, 5-9 x 2.5-4.0 um. Paraphyses numerous, of two types: 1.) evenly distributed, branched, thin, c. 0.7-1 um wide, 2.) scattered or in small fascicles, stout, c. 2 um wide, coated by greenish pigment. Hypothecium c. 60-120 um tall, dark brown (Superba-brown), sometimes greenish or with a reddish tinge, often K+ olive green in the upper part (reaction in the greenish Cinereorufa-green pig- ment), hyphae coated with dark brown pigment. Excipulum not evident. Mesopycnidia always present, usually numerous, black, sessile or more usually stalked and then 80-300 um tall, 40-70 um in diam., stalks simple or branched 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 x 1.2-1.8 um. Micro- or macropycnidia not seen. MycoKeys 106: 327-354 (2024), DOI: 10.3897/mycokeys.106.123484 338 Annina Kantelinen et al.: A phylogenetic study of Micarea melaeniza group Crystalline granules not present in apothecia, pycnidia or thallus. Chemistry no substances detected by TLC (information based on Coppins 1983 and Czarnota 2007). Typification. |n his original description of Micarea melaeniza, Hedlund (1892) cited material that he had collected in Jarvs6 in Halsingland, but without giving further specimen data. There are five specimens of M. melaeniza in S, LD and UPS collected by Hedlund in Jarvso in August 1891 and which all are likely to be part of the original material. Coppins (1983) cited a ‘holotype’ in S, which con- stitutes a lectotypification following ICN Art. 9.10. There is, however, an addi- tional 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 type specimens 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 Cau- casus. In addition, M. melaeniza has been reported from Alaska (Spribille et al. 2020), Austria (Berger and Tiirk 1991, this study) and Mongolia (Palka and Sli- wa 2006). Further, it might have been reported as M. nigella and could be found after revising specimens. Notes. In his monograph of European Micarea species, Coppins (1983) ac- cepted M. melaeniza, and in his interpretation, the species is characterized by having a hymenium with green pigmentation, a dark brown hypothecium with- out 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 larg- er mesoconidia (M. melaeniza: 2.3-3.6 x 1-1.3 um vs. M. nigella: 3.4-4.3 x 1.2-1.6 um; Coppins 1983). Czarnota (2007) noted that the amount of pur- ple, K+ green pigment varied considerably in Polish collections determined as M. nigella, and suggested that M. melaeniza and M. nigella could be conspecif- ic. He further noted that Hedlund’s original description could be interpreted as indicating the presence of another pigment, the purple, K+ purple pigment and suggested 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 to the 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 (I.c.) but is, on the other hand, mentioned in the original description of the spe- cies 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 MycoKeys 106: 327-354 (2024), DOI: 10.3897/mycokeys.106.123484 339 Annina Kantelinen et al.: A phylogenetic study of Micarea melaeniza group be 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 analy- ses. 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 synony- mous. A careful study of the type specimens showed morphological differences, e.g. in the size of conidia and pigmentation of apothecia and pycnidia. A brown or purple-brown, K+ green pigment in the hymenium, hypothecium and pycnidia walls (Melaena-red) of M. nigella is an important difference between M. melae- niza 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 HNO,- (rarely HNO, intensifying red), and in M. nigella HNO,+ purple-red. Compared to M. melaeniza, M. nigella also has longer conidia (3.4—4.5 x 1.2-1.6 um), slightly shorter hymenium (up to 30 um) and wider paraphyses (up to 3 um), 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. eur- asiatica, M. misella and M. osloensis. Micarea botryoides is usually not ligni- colous, has longer ascospores (8-13(-16) x 2.3-3.7(-4) um) that are often septate, and longer mesoconidia (Coppins 1983). Micarea eurasiatica has sim- ilar pigmentation like M. melaeniza, but the shape of apothecia is different (ad- nate vs subglobose), its pycnidia are sessile and mesoconidia are longer (up to 6 um). Micarea misella, on the other hand, can be microscopically distinguished from 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 based on our phylogenetic study. However, M. osloensis has shorter pycnidia (max. 180 um), longer mesoconidia 3.5—4.5(-5) x 1.2-1.5 (-1.8) um, and apothe- cia and pycnidia are mostly K- (although a higher concentration of Cinereor- ufa-green pigment is known to occur in some of the C-European specimens). Additional specimens studied. AUSTRIA, Niederdsterreich, Ybbstaler Al- pen, Wildnisgebiet Dirrenstein, Lunz am See Rothwald, Kleiner Urwald, prime- val 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, Malicek 16229, Berger, Palice & Vondrak, hb Malicek. CZECH REPUBLIC, S Bohemia, Sumava Mts, Volary, Ceské Zleby: Radvanovicky hrbet - 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: 0Q646318); ibid., Horni Vitavice, Zaton: Jilmova skala 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, Malicek 7322, hb Malicek. FINLAND, Uusimaa, Vantaa, Herukkapuro nature reserve (plot 1), old-growth forest, 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. MycoKeys 106: 327-354 (2024), DOI: 10.3897/mycokeys.106.123484 340 Annina Kantelinen et al.: A phylogenetic study of Micarea melaeniza group 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, Vondrak 21921, PRA. RussiA, Russian Western Caucasus, Adler, Krasnaya Polyana, primeval fir- beech forest below timberline, 43°41'50"N, 40°21'25"E, 1690 m, on soft rotten wood of Abies snag, 2019, Vondrak 23358, PRA. SweDEN, Angermanland, Langsele par., Vil. 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: Den- mark, 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. Pho- tobiont micareoid, 4-7 um. Apothecia absent to numerous (mostly rare), immarginate, subglobose, of- ten becoming tuberculate, black, matt, 0.1-0.3 mm in diam. Hymenium 25- 30 um tall, hyaline or tinged dull brown or purplish brown, K+ sordid green, HNO,+ purple-red (Melaena-red), often with darker vertical streaks. Epihyme- nium irregularly pigmented brown to purplish-brown (Melaena-red), sometimes dark greenish, K+ olive green, HNO,+ purple (Cinereorufa-green). Asci clavate, 22-30 x 10-12 um. Ascospores ellipsoid to usually ovoid, simple, 6.5-12 x 2.5-4.0 um. Paraphyses of two types: 1.) evenly distributed, branched, thin, c. 0.7-1 um wide, 2.) scattered or in small fascicles, stout, 2-3 um wide, coated by dark pigment. Hypothecium c. 70-120(-160) um tall, dark brown with vari- able amount of purplish tone, K+ olive green, HNO,+ purple-red (Melaena-red), hyphae coated with a dark brown pigment. Excipulum not evident. Mesopycnidia always present, usually numerous, black, sessile or more usually stalked and then 80-300 um tall, 40-80 um in diam., stalks simple or branched from the base bearing up to four pycnidia, walls brownish black to purplish black, sometimes olivaceous from the top, K+ dull green and HNO,+ purple-red especially in the brown parts (Melaena-red). Mesoconidia ellipsoid or short cylindrical 3.5—4.5(—5) x 1.2-1.8 um. 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), Fin- land and Sweden. In addition, M. nigella has previously been reported from bo- real and temperate forests in north-western, central and eastern Europe (e.g. Czarnota 2007). Notes. In external appearance, M. nigella resembles M. melaeniza. The dif- ferences between these two species are discussed in detail under M. melaeni- za and Coppins (1983). The species also resembles M. botryoides, M. misella MycoKeys 106: 327-354 (2024), DOI: 10.3897/mycokeys.106.123484 341 Annina Kantelinen et al.: A phylogenetic study of Micarea melaeniza group and M. osloensis. Micarea botryoides is usually not lignicolous and prefers rain-sheltered microhabitats on various substrata, it has slightly taller pycnidia (up to 400 um) and longer ascospores (8-13(-—16) x 2.3—4 um) that are often septate (e.g. Coppins 1983). Microscopically, M. misella can be distinguished by 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 pig- ment (K+ green, HNO,+ purple-red) in the hymenium, hypothecium and pycnid- ia. 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. nigella has the Melaena-red pigment, that looks brown with a purplish tinge, but of our three 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 can be 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+ pur- ple). 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 Melaeni- da-red pigments can be intermixed and appear in varying concentrations. Additional specimens studied. CZECH REPUBLIC, Central Bohemia, Brdy Pro- tected Landscape Area, MiSov, Na Skalach Nature Reserve, old-growth beech forest, small scree with rock outcrops and sparse spruce forest in NW part of the protected area, 49°36'20"N, 13°45'56"E, 715-740 m, on stump, 2023, Maliéek 16287, hb Malicek. Western Bohemia, DomazZlice, Cesky les Protected Landscape Area, Pec, Bystrice 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, Maliéek 8029 (hb Malicek); ibid., Tachov Lesna: managed spruce forest 1 km NE of Knizeci strom Hill (829 m), 49°46'13.0"N, 12°29'28.3"E, 750 m, on stump of Picea abies, 2019, Malicek 13161 & Rydlo, hb Malicek. Southern Bohemia, Prachatice, Sumava National Park, Nova Pec old-growth beech-spruce forest on N-facing slope of Mt Hraniénik (1281 m), 48°45'13"N, 13°54'17"E, 1170 m, on decaying wood, 2017, Maliéek 11296, hb Maliéek. Eastern Bohemia, Zd‘ar nad Sazavou, Zdarské vrchy Protected Landscape Area, Svratka managed beech-spruce forest on NW-facing slope of Bubnovany kopec Hill (780 m), 49°42'41.6"N, 16°05'13.7"E, 775 m, on stump of Picea abies, 2020, Malicek 13865 & Sejfova, hb Malicek. Svratka forest mosaic 0.2 km SSE of Spaleny kopec Hill (766 m), 49°43'28.0"N, 16°06'23.3"E, 755 m, on stump of Picea ab- ies, 2020, Maliéek 13868 & Sejfova, hb Maliéek. Svitavy, Ceska Trebova, Psi kuchyne Nature Reserve, old-growth beech forest 0.1 km NW of Psi kuchyne Hill (526 m), 49°50'38.3"N, 16°26'47.5"E, 505 m, on lying wood of Fagus syl- vatica, 2020, Malic¢ek 13975 & Rydlo, hb Malicek. Silesia, Beskydy Protected Landscape Area, Horni Lomna, Velky Polom Nature Reserve, valley of a brook MycoKeys 106: 327-354 (2024), DOI: 10.3897/mycokeys.106.123484 349 Annina Kantelinen et al.: A phylogenetic study of Micarea melaeniza group with 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, Malicek 14664 & Sejfova, hb. Malicek; ibid., gorge on NW-facing slope of Mt Velky po- lom (1067 m), c. 49°30'25"N, 18°40'04"E, 950-1000 m, on stump, 2021, Malicek 14649, Hlisnikovsky & Sejfova, hb Malicek; ibid., Karolinka, Maly Javornik Na- ture Reserve, old-growth spruce-beech forest, 49°18'20.9"N, 18°17'18.5"E, 880-970 m, on wood of stump, 2023, Malicek 16392, hb Malicek; ibid., Va- lasska Bystrice, Kutany 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, Malicek 14246 & Konecna, hb Malicek. Jeseniky Protected Landscape Area, Karlova Studanka, old-growth spruce forest on SE-facing slope in valley of Bila Opava Brook, along tourist path 1.1 km NE of Ovéarna, 50°04'37"N, 17°15'06"E, 1170-1200 m, on roots of Picea abies, 2015, Malicek 8547, hb. Malicek. 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 (DNA A588), H; ibid., Kantelinen 1921 (DNA A572), H. Etela-Hame, Hameenlinna, 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., Nova Sedlica, protected area Stuzica, NNE-facing slope of Temny vrSok Mt. (838 m), old-growth beech forest, 49°04'11"N, 22°32'26"E, 750-820 m, on stump of Abies alba, 2013, Malicek 6511 & Vondrak, hb Malicek. Micarea osloensis (Th. Fr.) Hed. MycoBank No: 627666 Fig. 4A, E Type. Bih. Kongl. Svenska Vetensk.Akad. Hand. Ill, 18: 97 (1892). Lecidea os- loensis 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 dark green-grey layer on top of substrate. Photobiont micareoid, 4-7 um in diam. Apothecia infrequent or rare, absent or numerous, immarginate, convex to hemispherical, dark brown to black, matt, simple, 0.1-0.2(—0.3) mm in diam. Hymenium 30-50 um tall (Coppins c. 30 um tall), hyaline, sometimes oliva- ceaous, often with warm brown vertical streaks, K-, HNO,- (Superba-brown). Epihymenium warm dark brown to blackish (Coppins: red-brown), rarely green- ish, mostly K- but sometimes K+ green, HNO,- or HNO,+ purplish (Cinereoru- fa-green). Paraphyses of two type: 1.) hyaline or coated with a brown pigment, thick, 2-3 um wide, simple or branched, sometimes wider from apices, often uneven in shape, abundant, sometimes concentrated into fascicles, 2.) thin- ner, c. 1-1.5 um wide, rarely branched, rare. Asci cylindrical to cylindrical-clav- ate, 30-40 x 10-12 um (Coppins: 26-30 x 11-13 um). Ascospores 7-10 x (2.5-)3.0-3.5 (-4.0) um (Coppins: 6-9.5 x 3-4 um), ellipsoid, cylindrical or sometimes roughly shaped, 0(-1) sept. Hypothecium warm dark brown (Cop- pins: red-brown), composed of hyaline hyphae 1-2 um wide surrounded by MycoKeys 106: 327-354 (2024), DOI: 10.3897/mycokeys.106.123484 343 Annina Kantelinen et al.: A phylogenetic study of Micarea melaeniza group brown pigment giving it an unevenly coloured/randomly spotted appearance, K-, HNO,— (Superba-brown), c. 85 um tall. Mesopycnidia often numerous and crowded, sometimes absent, simple or branched from the base, emergent or shortly stalked ca. 50-180 um tall, 40- 70 um 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, HNO,- or HNO,+ purplish (Cinereorufa-green), usually extruding white mass of conidia that may merge with neighbouring conidia. Mesoconidia ellipsoid or short cylindrical 3.5-4.5(-5) x 1.2-1.5 (-1.8) um. 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. Another morphologically identical specimen collected in 2007 occurs on bark of decay- ing trunk (Palice 11684). Our newer specimens occur on bark, dead wood and dead mosses. The type specimen was collected from Norway from a woodland clearing on the site of an old bonfire, and the newer specimens are from the Czech Republic, Finland, Sweden and Ukraine. In the Czech Republic, M. os/oen- sis occurs commonly from middle to montane elevations. It appears to be toxi- tolerant 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 of Fagus 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. os/oensis specimens, including the type, have not been sequenced, although an unsuccessful sequencing attempt of 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. osloen- sis 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, some specimens have a K+ greenish, HNO,+ 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 Cinereor- ufa-green pigmentation appear to be more frequent in the Czech Republic. The Finnish specimens have sometimes olivaceous tones that are K- but slightly HNO,*+ purple. Another difference between the type of M. osloensis and our newly se- quenced 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 speci- mens represent either sexual (rare) or asexual reproduction modes which are monophyletic 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 possi- bility that they are conspecific. On the other hand, we also cannot exclude the possibility that the new specimens represent a yet undescribed taxon in the M. melaeniza group. MycoKeys 106: 327-354 (2024), DOI: 10.3897/mycokeys.106.123484 344 Annina Kantelinen et al.: A phylogenetic study of Micarea melaeniza group o © os a & 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 (Kante- linen 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 um (B, D). M. osloensis resembles M. eurasiatica, M. melaeniza, M. misella and M. ni- gella. The most important characters of M. osloensis are the combination of sessile to shortly stalked pycnidia, mesoconidia of the size 3.5—4.5(—5) x 1.2- 1.5 (-1.8) um, warm-brown, sometimes olivaceous colouration in apothecia MycoKeys 106: 327-354 (2024), DOI: 10.3897/mycokeys.106.123484 345 Annina Kantelinen et al.: A phylogenetic study of Micarea melaeniza group and pycnidia (Coppins: red-brown), and often a K— reaction. Micarea eurasiat- ica sp. nov. has mostly sessile to emergent pycnidia and bigger mesoconidia ((4-)4.5-6.0 x 1.2-1.5(-1.8) um). 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 sis- ter species. They have morphological similarities including pigmentation and spore 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, HNO,+ purple) appears to vary in both taxa, but especially in M. osloensis. Micarea melaeniza is mostly K+ green in epi- hymenium, 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, Jizerske hory Mts, Joseftv Dul: valley of Jedlovy potok, protected zone of nature reserve Jedlovy dul, 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: 0Q646320); ibid., Hejnice, Jizerskohorské buciny National Nature Reserve, valley of Velky Stolpich brook, S of OfeSnik Mt. (800 m), ca 50°51'13'N, 15°11'13"E, 660 m, on base of Fagus sylvatica, 2013, Malic¢ek 6020, hb Malicek. Luzické hory Protected Landscape Area, Marenice, Horni Svetla: managed spruce-beech forest on E-facing slope, 0.3 km NNW of Kopfrivnice 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, Malicek 14019, 14020 & Rydlo, hb Malicek. Central Bohemia, Brdy Pro- tected Landscape Area, MiSov, Na Skalach Nature Reserve, old-growth beech forest, small scree with rock outcrops and sparse spruce forest in NW part of the protected area, 49°36'20"N, 13°45'56"E, 715-740 m, at base of Fagus sylvatica, 2023, Malicek 16281, hb Malicek. Brdy Hills, Rozmital pod Tremsi- nem, 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, Malicek 12007 & Vondrak, hb Malicek; ibid., StraSice, managed mixed forest 3 km E of village, 49°43'34'N, 13°47'56"E, 610 m, at base of Larix decidua, 2018, Malicek 12000 & Vondrak, hb. Malicek. Pribram, Brdy Hills, Jince managed coniferous forest 0.5 km SSE of Velci pond, 49°45'17"N, 13°56'34"E, 600 m, at base of Larix decidua, 2018, Malicek 12016 & Vondrak, hb Malicek. Eastern Bohemia, Rychnov n. Knéznou, Orlické hory Protect. Lands. Area, Rokytnice v Orlickych horach, Cerny dial Na- ture 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, Maliéek 4536 et al., hb Maliéek. Zd’ar nad Sazavou, Zd’arské vrchy Protected Landscape Area, Svratka fragment of old beech predominated forest 0.6 km SW of Spaleny kopec Hill (766 m), 49°43'18.2"N, 16°06'11.4"E, 750 m, on stump of Picea abies, 2020, Malic¢ek 13864 & Sejfova, hb Malicek; ibid., Svratka, Pus- ta Rybna: spruce-beech forest on S-facing slope of KaStanktv kopec (753 m), 49°43'03.9"N, 16°06'46.4"E, 740 m, on stump of Picea abies, 2020, Malicek 13953, hb MaliGek. Svitavy, Ceska Tfebovaé 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, Maliéek 13970 & SAdmalova, hb Maliéek. Western Bohemia, Cesky les Protected MycoKeys 106: 327-354 (2024), DOI: 10.3897/mycokeys.106.123484 346 Annina Kantelinen et al.: A phylogenetic study of Micarea melaeniza group Landscape Area, Tachov, Lesna: young beech forest 0.8 km E of Knizeci strom Hill (829 m), 49°45'54.2"N, 12°29'18.5"E, 780 m, on stump of Picea abies, 2020, Malicek 13784 & Rydlo, hb Malicek; ibid., spruce-beech forest 1 km NE of Knizeci strom Hill (829 m), 49°46'17.3"N, 12°29'18.1"E, 770 m., at base of Picea abies, 2019, Malicek 13167 & Rydlo,hb Malicek; ibid., Pec, Bystrice Nature Reserve, natural mixed forest up to 150 years old, 49°22'56'"N, 12°48'39"E, 650 m, at base of Picea abies, 2015, Malicéek 8028, hb Malicek. Kdyné, Mezholesy: managed mixed forest 0.3 km SE of Korab Hill (773 m), 49°23'37.7"N, 13°04'44.1"E, 750 m, at base of Picea abies, 2019, Malicek 13371 & Rydlo, hb Malicek. Southern Bo- hemia, Novohradské hory Mts, Horni Stropnice, NPP Hojna Voda, fragment of primeval forest predominated by beech, 48°42'20'N, 14°45'08"E, 840-870 m, on snag, 15 October 2019, MaliGek 13500, herb. Maliéek. Sumava Mts., Vo- lary, Nova Pec, 740 m, 48°49'11.2"N, 13°56'12.151"E, on bark of Pinus sylves- tris at base of trunk, 2017, Vondrak 19007, PRA. Prachatice, Sumava Protected Landscape Area, Kubova Hut, Boubinsky prales National Nature Reserve, man- aged spruce forest c. 120 years old, 0.4 km NNE of top of Mt Boubin (1362 m), 48°59'42.2"N, 13°49'05.7"E, 1275 m, on decaying stump 2015, Malicek 8349 & Palice, hb Maliéek. Jindrfichtv Hradec, Javoricka vrchovina Hills, Straz nad Nezarkou, Sedlo: managed coniferous forest SSE of Otinsky kopec Hill (538 m), 49°02'44.0"N, 14°59'45.7"E, 530 m, on stump, 2020, Malicek 13817, hb Malicek; ibid., Lasenice: mixed forest S of Sembursky rybnik, 49°03'34.6'N, 14°59'55.2"E, 520 m, on stump, 2020, Malicek 13821, hb. Malicek; ibid., managed conifer- ous forest between Nova Ves and Sedlo, 49°03'36.1"N, 15°00'55.3"E, 560 m, at base of Pinus sylvestris, 2020, Malicek 13824, hb Malicek. Tabor Chynov, young beech forest SSE of Blanicka, 49°28'00.6'N, 14°50'34.9"E, 690 m, on fall- en wood, 2020, Malicek 13834 & Rydlo, hb Malicek; ibid., mixed forest SE of Bat- kovy Hill (724 m), 49°27'43.3"N, 14°50'00.9"E, 700 m, at base of Larix decidua, 2020, Malicek 13838 & Rydlo, hb Malicek. Northern Moravia, Jesenik, Jeseniky Protected Landscape Area, Bela pod Pradédem, Vysoky vodopad, Nature Re- serve, valley of Studeny p. brook, ca. 50°06'57"N, 17°12'10"E, 900-1000 m, on base of Picea abies, 2012, Maliéek 5102, hb Maliéek. Sumperk, Kralicky Snéznik Mts, Staré Mésto: Mt. Kralicky Snéznik, c. 100 years old spruce forest on S-fac- ing slope 0.1 km SE of FrantiSkova chata, 50°12'05.2"N, 16°51'28.3"E, 1210 m, at base of Picea abies, 2015, Malicek 8381, Kocourkova & Vondrak, hb Malicek. Eastern Moravia, Beskydy Protected Landscape Area, Bila, Salajka: beech dom- inated 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, Mali¢ek 13344 & Rydlo, hb Malicek; ibid., Frenstat pod Radhostém, Knéhyné-Certiv mlyn National Nature Reserve, W-fac- ing slope of Knéhyné 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, Maliéek 6090 & Vondrak, hb Maliéek. Western Moravia, Zd’ar nad Saza- vou, Zd'arské vrchy Protect. Landsc. Area, Cikhaj, Zakova hora National Nature Reserve, beech virgin forest, 49°39'18"N, 15°59'35"E, 750-800 m, on stump of Fagus, 2012, Malicek 5110 & Syrovatkova, hb Malicek. Silesia, Bruntal, Jeseniky Protected Landscape Area, Karlova Studanka, managed spruce forest (c. 100 years old) on N-facing slope in valley of Bila Opava Brook, 1.8 km ENE of Ovéar- na, 50°04'35'"N, 17°15'53"E, 1170-1180 m, on bark of Picea abies, 2015, Malicek 8490, Kocourkova, Vondrak & Zemanova, hb Malicek; ibid., Pradéd National Na- ture Reserve, old-growth spruce forest c. 200 years old on E-facing slope of Mt MycoKeys 106: 327-354 (2024), DOI: 10.3897/mycokeys.106.123484 347 Annina Kantelinen et al.: A phylogenetic study of Micarea melaeniza group Vysoka hole (1464 m), 0.2 km WNW of EustaSka hut, 50°03'35"N, 17°15'12’E, 1220 m, at base of Picea abies, 2015, Malicek 8563, Kocourkova, Palice & Von- drak, hb Malicek. Frydek-Mistek, Beskydy Protected Landscape Area, Ostravice, Mazak National Nature Reserve, old-growth spruce forest with intermixed syc- amores on W-facing slope of Mt Lysa hora (1323 m), 49°32'41"N, 18°26'43’E, 1200 m, on bark of Picea abies, 2016, Malicek 9743 & Palice, hb Malicek. 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 (DNA A583), H. Ibid., on wood of dead standing Picea abies (decay stage 3), Kan- telinen 1685 (DNA A574), H. Ibid., on wood of fallen Picea abies (decay stage 5), 2013, Kantelinen 2003 (DNA A594), H. Ibid., on wood of fallen Picea abies (decay stage 3), 2013, Kantelinen 1923 (DNA A573), H. Etela-Hame, Hameen- linna, 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 and Betula, 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 and dense Picea abies dominated managed forest (plot 2), on wood of a Picea ab- ies stump (decay stage 5), 60.3544°N, 25.0322°E, 2013, Kantelinen 2643 (DNA A484), H. SWEDEN, Jamiland, Kall par., about 850 m NW of the northwestern tip of Lake Spjuttjarnen, S side of stream Kondsan, on stump of Betula pubescens in old- growth 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, Vondrak 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 um 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 an- other, putatively new species, marked Micarea sp. in the phylogeny that we re- frain from describing because of insufficient morphological data and few avail- able collections. This putative new species has sessile to emergent, brownish black pycnidia with a purple tinge and a K+ green reaction, but apothecia are few (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 identi- fy because they are small, have relatively few morphological characters and because the current literature is not up to date particularly in relation to pig- mentation (Coppins 1983; Czarnota 2007). The challenges while identifying MycoKeys 106: 327-354 (2024), DOI: 10.3897/mycokeys.106.123484 348 Annina Kantelinen et al.: A phylogenetic study of Micarea melaeniza group 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 white mass 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 hypothe- cium where hyphae are surrounded by brown pigment giving them an unevenly coloured appearance, also noted by Coppins (1983). Based on results of this study, the size of pycnidia and mesoconidia, and to some extent also the gener- al pigmentation in K and HNO, are the most useful morphological characters in separating the species, however in some cases DNA sequencing is the only reli- able way for identification. Micarea eurasiatica, and M. olivacea develop sessile to emergent pycnidia, M. osloensis emergent to shortly stipitate pycnidia, and M. melaeniza and M. nigella develop stipitate pycnidia. Micarea deminuta does not develop distinctive stalked pycnidia, at least according to current knowl- edge. The Cinereorufa-green pigment (K+ green, HNO,+ 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 in varying 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 correlate with geography. In the central European specimens some pigments are en- countered more often or higher concentrations than in Fennoscandia. For ex- ample, in the Fennoscandian specimens of M. osloensis, the Cinereorufa-green pigment is barely visible even when using HNO,, and the Melaenida-red pig- ment in M. nigella was not found at all. In the central European specimens, however, both pigments were found, and sometimes in relatively high concen- trations. 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 meso- conidia size and DNA profile is similar to M. melaeniza. Some of the “inter- mediate” specimens have not been sequenced and therefore we do not know their identity or whether they are undescribed species. Obviously, more work is needed to understand the pigments and species in this group. Because of these morphological challenges, we even considered that the spe- cies in the M. melaeniza group are conspecific, i.e. variation of just one species. However, we excluded this possibility because of several reasons 1) molecu- lar differences, 2) existing morphological differences, even though they may be hard to interpret, 3) the type specimens studied are not morphologically conspe- cific, 4) the types correspond in morphology to most of our specimens. Based on morphology, one might suggest that M. melaeniza, M. nigella and M. oslo- ensis are variation of one species, but according to our phylogenies, they are not monophyletic without M. deminuta, M. eurasiatica and Micarea sp. Even be- tween M. melaeniza and M. nigella, the pigmentation is mostly different (Super- ba brown vs. Melaena-red), and although these pigments may look quite similar to the human eye, they may have different ontologies and evolutionary paths. MycoKeys 106: 327-354 (2024), DOI: 10.3897/mycokeys.106.123484 349 Annina Kantelinen et al.: A phylogenetic study of Micarea melaeniza group According to morphological studies by Coppins (1983), M. botryoides and M. melaeniza are relatives (group G) and possibly close to M. contexta, M. ex- imia, M. nigella and M. olivacea (group H). Our Bayesian and ML phylogenies conclude that groups G and H are intermixed, however, based on DNA sequenc- ing M. eximia is likely not a close relative of the M. melaeniza group (unpub- lished data) and molecular relationships of M. olivacea are still unknown. Our phylogenetic analyses also show that M. melaeniza and M. osloensis are sis- ters, a relationship that has not been noted in previous publications, but the latter clade is not supported (0.68 / 74). A closer look at the sequences and alignment shows that there are nucleotide differences in the mtSSU region between M. melaeniza and M. osloensis (ca. 1-2% between specimens Kan- telinen 2430 and 1923), but the ITS regions are nearly identical with only two nucleotide differences. At least three conclusions could be drawn from these results. 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 some extent at least, 2) The two clades are conspecific and represent morphological and 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 a scientifically undescribed species. Any conclusion we make suffers from the uncertainty caused by the M. melaeniza and M. osloensis type specimens not having sequenced, which means that we cannot compare our sequences to the types 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 ligni- coles. According to a previous study, the wood-inhabiting lifestyle of Micarea species influences their reproductive biology: obligate lignicoles primarily re- produce 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 strat- egy than sexual reproduction. In the here studied M. melaeniza group and rel- atives, 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 com- mon 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 invento- ries. We hope that the morphological and molecular features presented in this study will pave the way for future research endeavors. Acknowledgements We warmly thank prof. Teuvo Ahti, emeritus curator Arto Kurtto and Dr. Juha Pykala for their valuable help with this manuscript. Laura Hakkinen, Emilia Piki and Emelie Winquist are thanked for the DNA lab work. Permission for collection of MycoKeys 106: 327-354 (2024), DOI: 10.3897/mycokeys.106.123484 350 Annina Kantelinen et al.: A phylogenetic study of Micarea melaeniza group lichens on Mt. Oku-Shirane (Mt. Nikko-Shirane), Nikko National Park to GT in 2019 was 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 a part 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 Research Council 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 Anal- ysis: AK, Funding acquisition: AK, LM, Investigation: AK, MS, JM, JV, ZP, Project admin- istration: AK, LM, Visualization: AK, Writing — original draft: AK, MS, Writing - review & editing: AK, MS, ZP, JM, JV, GT, LM. Author ORCIDs Annina Kantelinen © https://orcid.org/0000-0001-8664-7662 Mans Svensson © https://orcid.org/0000-0003-1664-8226 Jifi Malicek © https://orcid.org/0000-0002-31 19-8967 Jan Vondrak © https://orcid.org/0000-0001-7568-6711 Goran Thor © https://orcid.org/0000-0003-1 166-6898 Zdenék Palice © https://orcid.org/0000-0003-4984-8654 Stanislav Svoboda © https://orcid.org/0000-0001-9797-4984 Leena Myllys © 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. References Andersen HL, Ekman S (2005) Disintegration of the Micareaceae (lichenized Ascomy- cota): A molecular phylogeny based on mitochondrial rDNA sequences. Mycological Research 109(1): 21-30. https://doi.org/10.1017/S0953756204001625 Aras S, Cansaran D (2006) Isolation of DNA for sequence analysis from herbarium ma- terial of some lichen specimens. Turkish Journal of Botany 30: 449-453. 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