A phylogenetic study of Micarea melaeniza and similar-looking species (Pilocarpaceae) unveils hidden diversity and clarifies species boundaries and reproduction modes Author Kantelinen, Annina 0000-0001-8664-7662 Botany and Mycology Unit, Finnish Museum of Natural History, University of Helsinki, P. O. Box 7, FI- 00014 Helsinki, Finland Author Svensson, Måns 0000-0003-1664-8226 Museum of Evolution, Uppsala University, Norbyvägen 16, SE- 752 36 Uppsala, Sweden Author Malíček, Jiří 0000-0002-3119-8967 Czech Academy of Sciences, Institute of Botany, Zámek 1 252 43, Průhonice, Czech Republic Author Vondrák, Jan 0000-0001-7568-6711 Czech Academy of Sciences, Institute of Botany, Zámek 1 252 43, Průhonice, Czech Republic & Department of Botany, Faculty of Science, University of South Bohemia, CZ- 37005 České Budějovice, Czech Republic Author Thor, Göran 0000-0003-1166-6898 Department of Ecology, Swedish University of Agricultural Sciences, P. O. Box 7044, SE- 750 07 Uppsala, Sweden Author Palice, Zdeněk 0000-0003-4984-8654 Czech Academy of Sciences, Institute of Botany, Zámek 1 252 43, Průhonice, Czech Republic Author Svoboda, Stanislav 0000-0001-9797-4984 Czech Academy of Sciences, Institute of Botany, Zámek 1 252 43, Průhonice, Czech Republic Author Myllys, Leena 0000-0002-9566-9473 Botany and Mycology Unit, Finnish Museum of Natural History, University of Helsinki, P. O. Box 7, FI- 00014 Helsinki, Finland text MycoKeys 2024 2024-07-05 106 327 353 journal article 299630 10.3897/mycokeys.106.123484 b3c16d01-35d2-44e8-99b2-797f2a00d2ba Micarea melaeniza Hedl. Fig. 3 C, D Type. Bih. Kongl. Svenska Vetensk. Akad. Hand. III, 18: 96 (1892) . Type: Sweden , Helsinglandiæ [= Hälsingland], Jerfsö [= Järvsö], VIII 1891 . J. T. Hedlund (S L 1471! – lectotype , designated by Coppins 1983 [ICN Art. 9.10], further specified here [ICN Art. 9.17], S L 1472 !, 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, HNO 3 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, 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 pigment), hyphae coated with dark brown pigment. Excipulum not evident. Mesopycnidia always present, usually numerous, black, sessile or more usually stalked and then 80–300 µm tall, 40–70 µm 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 × 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 ). 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 be part 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 L 1472) 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 L 1471 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 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 found after revising specimens. Notes. In his monograph of European Micarea species , Coppins (1983) accepted M. melaeniza , and in his interpretation, the species is characterized by having 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 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 (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 (ZP 32013) shows patchily purplish, K + dark green pigment that might 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 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 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. 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 HNO 3 – (rarely HNO 3 intensifying red), and in M. nigella HNO 3 + purple-red. Compared to M. melaeniza , M. nigella also has longer conidia (3.4–4.5 × 1.2–1.6 µm), slightly shorter hymenium (up to 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 ZP 32013 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 to 6 µm). 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 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 : OQ 646318 ); 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-growth forest, on wood of a dead stump of Picea abies (decay stage 5), WGS 84 60.3215 ° N , 24.7658 ° E , 2013, Kantelinen 2430 (DNA A 772), H. 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 fir-beech 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).