Introduction
The Micarea prasina group has been studied by numerous researchers. Using only phenotypic characters, Hedlund (Reference Hedlund1892) considered different forms of M. prasina Fr. (now treated as separate species) as well as M. anterior (Nyl.) Hedl., M. subviridescens (Nyl.) Hedl. and M. globularis (Ach.) Hedl. to be related. Coppins (Reference Coppins1983) concluded that M. hedlundii Coppins and M. levicula (Nyl.) Coppins were related to M. prasina Fr., but he suggested that M. misella (Nyl.) Hedl., M. melanobola (Nyl.) Coppins and M. synotheoides (Nyl.) Coppins might also belong to this group. At that time M. prasina was treated in a wide sense and comprised three different chemotypes characterized by the presence of unidentified substances called prasina unknowns A, B and C (Coppins Reference Coppins1983), which were later identified as methoxymicareic, micareic and prasinic acids, respectively (Elix et al. Reference Elix, Jones, Lajide, Coppins and James1984; Coppins Reference Coppins1992). Subsequently, the taxonomy of M. prasina s. lat. was reorganized and species status was given to each chemical race; M. micrococca (Körb.) Gams ex Coppins for the methoxymicareic acid chemotype, M. prasina s. str. for the micareic acid chemotype and M. subviridescens (Nyl.) Hedl. for the prasinic acid chemotype (Coppins Reference Coppins2002). Additionally, M. xanthonica Coppins & Tønsberg with xanthones (thiophanic acid with satellites) and M. viridileprosa Coppins & van den Boom containing gyrophoric acid were recognized as members of this species complex (Coppins & Tønsberg Reference Coppins and Tønsberg2001; van den Boom & Coppins Reference van den Boom and Coppins2001).
Later Czarnota (Reference Czarnota2007) showed that micareic acid is not produced solely by M. prasina, but is also present in M. nowakii Czarnota & Coppins. The latter species, despite its non-granular thallus, and also M. tomentosa Czarnota & Coppins which lacks any lichen substances detectable by TLC, appeared to belong to the M. prasina group (Czarnota Reference Czarnota2007; Czarnota & Guzow-Krzemińska Reference Czarnota and Guzow-Krzemińska2010). In addition, Czarnota & Guzow-Krzemińska (Reference Czarnota and Guzow-Krzemińska2010), using morphological characters and phylogenetic approaches, segregated M. byssacea (Th. Fr.) Czarnota et al. from M. micrococca. These authors discovered an additional distinct evolutionary lineage also containing methoxymicareic acid, which appeared morphologically intermediate between both these species (but more similar to M. micrococca), and thus indeterminable without molecular data. Due to this, Barton & Lendemer (Reference Barton and Lendemer2014) refrained from separating M. byssacea from M. micrococca; they concluded that in the absence of more comprehensive molecular studies in Europe and North America, it is better to adopt a broad concept of M. micrococca.
Recently three new species, which probably also belong to the M. prasina group, have been described; two from Réunion, M. melanoprasina Brand et al. producing ‘unknown 1’, a substance probably related to micareic acid, and M. hyalinoxanthonica Brand et al. containing a xanthone (probably thiophanic acid) (Brand et al. Reference Brand, van den Boom and Sérusiaux2014), and one from Brazil, M. corallothallina M. Cáceres et al. which lacks lichen substances (Cáceres et al. Reference Cáceres, Mota, de Jesus and Aptroot2013). Czarnota & Guzow-Krzemińska (Reference Czarnota and Guzow-Krzemińska2010) and Brand et al. (Reference Brand, van den Boom and Sérusiaux2014) mentioned some problems within the M. prasina group which remain to be solved, for example the identity of some morphs of M. prasina from Réunion with a pigmented hypothecium. Thus several species are probably still unrecognized.
During our field studies we collected a very characteristic sorediate Micarea species in many localities, which we putatively determined as M. prasina due to the presence of micareic acid. Its morphology was, however, quite different as most samples were always sterile and with well-delimited soralia, at least in young stages of the thallus development. To clarify its identity we have analyzed mitochondrial rDNA sequences revealing that it is different from all known species in the M. prasina group. It is described in this paper as M. soralifera sp. nov.
Materials and Methods
Morphology and chemistry
The material of the new species is deposited in GPN, KTC and UGDA. Apothecial sections and squashed thallus preparations were studied on material mounted in tap water with and without the addition of C (commercial bleach), K (aqueous solution of potassium hydroxide) and N (nitric acid). Dimensions of all anatomical features were measured in water. Thin-layer chromatography (TLC) was used for the determination of lichen substances according to standard methods (Orange et al. Reference Orange, James and White2001). All samples were studied in solvent C.
Taxon sampling for DNA
Three specimens of Micarea soralifera, including the holotype, were collected in the Białowieża Primeval Forest (Białowieża National Park, forest section no. 256) in Poland and were further used for DNA analysis. Three mtSSU sequences were generated for this study and their GenBank accession numbers are provided for each corresponding sample in the list of specimens examined. Moreover, 33 sequences were obtained from GenBank, including all available species belonging to the M. prasina group. Micarea adnata Coppins and M. elachista (Körb.) Coppins & R. Sant. (Fig. 1) were chosen as outgroup taxa based on the study of Czarnota & Guzow-Krzemińska (Reference Czarnota and Guzow-Krzemińska2010). The holotype of M. soralifera was used to generate the ITS rDNA sequence, a marker which has been proposed as a universal DNA barcoding region for fungi (Schoch et al. Reference Schoch, Seifert, Huhndorf, Robert, Spouge, Levesque and Chen2012).
Maximum likelihood tree based on mtSSU rDNA data for the Micarea prasina group. Micarea adnata and M. elachista are the outgroup taxa. Bootstrap supports ≥70 for PhyML (first value) and MP (second value) methods and posterior probabilities ≥0·90 are indicated near the branches. GenBank accession numbers are supplied apart from the newly sequenced specimens of Micarea soralifera which are in bold and are followed by their herbarium collection number.
DNA extraction, PCR amplification and DNA sequencing
DNA was extracted directly from pieces of thalli using a modified CTAB method (Guzow-Krzemińska & Węgrzyn Reference Guzow-Krzemińska and Węgrzyn2000). DNA extracts were used for PCR amplification of mtSSU rDNA, employing mrSSU1 and mrSSU3R primers (Zoller et al. Reference Zoller, Scheidegger and Sperisen1999); the same primers were used for sequencing. The ITS rDNA marker was amplified and sequenced using ITS1F (Gardes & Bruns Reference Gardes and Bruns1993) and ITS4 primers (White et al. Reference White, Bruns, Lee and Taylor1990). The 25 μl of PCR mix contained 1U of Taq polymerase (Thermo Scientific), 0·2 mM of each of the four dNTPs, 0·5 μM of each primer and 10–50 ng of genomic DNA. PCR amplifications were performed using a Mastercycler (Eppendorf) with the following program for mitochondrial gene: initial denaturation at 95ºC for 10 min followed by 6 cycles at 95ºC for 1 min, 62ºC for 1 min and 72ºC for 105 s, and then 30 cycles at 95ºC for 1 min, 56ºC for 1 min and 72ºC for 1 min, with a final extension step at 72ºC for 10 min. The following conditions were used for amplification of the ITS rDNA marker: initial denaturation at 95ºC for 5 min, followed by 35 cycles at 95ºC for 40 s, 54ºC for 45 s and 72ºC for 1 min, with a final elongation step at 72ºC for 10 min. PCR products were visualized on agarose gels in order to determine DNA fragment lengths. Subsequently, 5 µl of PCR products were treated with 10 units of Exonuclease I and 1 unit of FastAP™ Thermosensitive Alkaline Phosphatase enzymes (Thermo Scientific) to degrade primers and dephosphorylate dNTPs. Treatment was carried out for 15 min at 37°C, followed by a 15 min incubation at 85°C to completely inactivate both enzymes. Sequencing of each PCR product was performed using Macrogen sequencing service (www.macrogen.com).
Sequence alignment and phylogenetic analysis
The newly generated mtSSU rDNA sequences and ITS rDNA sequence (GenBank acc. no. KT119887) were compared to the sequences available in GenBank (http://www.ncbi.nlm.nih.gov/BLAST/) using BLASTN search (Altschul et al. Reference Altschul, Gish, Miller, Myers and Lipman1990) in order to confirm their identity. The mtSSU rDNA sequences were aligned with sequences of selected representatives of the genus Micarea obtained from GenBank (GenBank accession numbers are given in Fig. 1). Alignment was performed using Seaview software (Galtier et al. Reference Galtier, Gouy and Gautier1996; Gouy et al. Reference Gouy, Guindon and Gascuel2010) employing the clustalw2 option and followed by manual optimization. Portions of the alignment with ambiguous positions that might not have been homologous were excluded. The phylogenetic analyses were performed using PAUP* 4.0b10 (Swofford Reference Swofford2001) with maximum parsimony (MP) as the optimality criterion. Heuristic searches were performed with 1000 random sequence additions and TBR branch swapping. Gaps were treated as missing and the support for the branches was evaluated with a bootstrap method with 1000 pseudoreplicates (Felsenstein Reference Felsenstein1985).
Maximum likelihood (ML) analyses were performed with the fast likelihood software PhyML 3.0 (Guindon & Gascuel Reference Guindon and Gascuel2003; Guindon et al. Reference Guindon, Dufayard, Lefort, Anisimova, Hordijk and Gascuel2010), starting with a BioNJ tree. The GTR model of evolution was selected based on Hierarchical Likelihood Ratio Tests and the Akaike Information Criterion in Modeltest 3.5 (Posada & Crandall Reference Posada and Crandall1998) and used in the analysis. Non-parametric bootstrap analyses were performed with 1000 bootstrap replicates.
The data were also analyzed using a Bayesian approach (MCMC) in MrBayes 3.2 (Huelsenbeck & Ronquist Reference Huelsenbeck and Ronquist2001; Ronquist & Huelsenbeck Reference Ronquist and Huelsenbeck2003). The GTR+G+I model was selected based on analysis using MrModeltest 2.0 (Nylander Reference Nylander2004). A run with 2 000 000 generations employing 4 chains was selected and every 100th tree was saved. The initial 5000 trees were discarded as burn-in and the majority-rule consensus tree was calculated to obtain posterior probabilities (BA), of which values above 0·95 were considered to be significant supports.
The phylogenetic tree was drawn using TreeView (Page Reference Page1996). Bootstrap supports (in MP and PhyML) above or equal to 70% and posterior probabilities above or equal to 0·90 (in BA) were indicated near the branches.
Alignment and trees were deposited in Treebase as submission 18376.
Results and Discussion
The final alignment consisted of 36 mtSSU sequences with 946 characters. Ambiguous positions were excluded, and of the 182 variable characters, 44 were parsimony-uninformative and 138 were parsimony-informative.
Since trees of the same topology were obtained using maximum likelihood methods and a BA/MCMC approach, we present only the ML tree with bootstrap support above or equal to 70% for ML and MP methods and posterior probabilities above or equal to 0·90 for Bayesian analyses (BA) (Fig. 1). The only difference between the maximum parsimony tree (not shown) and the tree presented in this work (Fig. 1) was the placement of Micarea soralifera within the M. prasina s. str. and M. nowakii clade, but this grouping was not supported.
Phylogenetic trees (Fig. 1) obtained in this study confirmed the close relationship of Micarea viridileprosa with M. micrococca and M. byssacea. Although the latter two species form three distinct evolutionary lineages containing methoxymicareic acid, as shown in the previous study of Czarnota & Guzow-Krzemińska (Reference Czarnota and Guzow-Krzemińska2010), at the moment it would be better not to introduce any taxonomic innovation within the M. micrococca agg. until a more precise study in Europe and North America is performed. Barton & Lendemer (Reference Barton and Lendemer2014) found no correlation between ecology or morphology to separate M. micrococca and M. byssacea in eastern North America. However, based on the known molecular evidence in addition to other distinct phenetic differences, the separation of M. byssacea from the two lineages of M. micrococca agg. is correct, and the recognition of M. byssacea even in the field is surely possible (Czarnota & Guzow-Krzemińska Reference Czarnota and Guzow-Krzemińska2010). Material of M. byssacea differs from M. micrococca in the darker pigmented apothecia containing Sedifolia-grey pigment within an epihymenium and goniocysts. Some morphs of M. byssacea develop pale apothecia, but in such cases the granular thallus is always more olive and not as mealy as in M. micrococca, and apothecia are larger and adnate (Czarnota & Guzow-Krzemińska Reference Czarnota and Guzow-Krzemińska2010).
Micarea prasina and M. nowakii form two closely related subclades, of which only M. prasina is highly supported (Fig. 1). However, the sequence of M. prasina with the GenBank accession number AY756452 included in our analyses was obtained from an American specimen that strongly differs from sequences of European specimens of M. prasina and M. nowakii. In fact it may represent a separate species, but its recognition is beyond the scope of this study.
The specimens of the new species, M. soralifera, form a single highly supported clade within the M. prasina group, but our analyses did not determine the position of the new species within the group. Micarea soralifera appears as closely related to the prasinic acid-containing M. subviridescens, but with low bootstrap support in MP and ML methods (79 and 76, respectively), and a posterior probability of 0·96 in the BA analysis (Fig. 1). Micarea prasina s. str. and M. nowakii both produce micareic acid (Czarnota & Guzow-Krzemińska Reference Czarnota and Guzow-Krzemińska2010), but in spite of their chemical similarity to M. soralifera, they are not the closest relatives of the new species. Maximum likelihood and Bayesian approaches placed M. soralifera outside the M. prasina s. str. and M. nowakii clade (Fig. 1), but the clade is poorly supported. Moreover, all these species were recovered as a single group in maximum parsimony analysis, but this grouping was not supported.
The Micarea prasina group shows high variation in secondary metabolite production within the genus Micarea Fr. (a detailed description of the chemistry within this group is presented in Czarnota (Reference Czarnota2007) and Czarnota & Guzow-Krzemińska (Reference Czarnota and Guzow-Krzemińska2010)). Species belonging to this group produce micareic, methoxymicareic, prasinic and gyrophoric acids as well as xanthones (Elix et al. Reference Elix, Jones, Lajide, Coppins and James1984; van den Boom & Coppins Reference van den Boom and Coppins2001; Coppins & Tønsberg Reference Coppins and Tønsberg2001). Within this group, the newly described M. soralifera produces micareic acid, while its closest relative M. subviridescens contains prasinic acid. Our study shows that a close phylogenetic relationship does not necessarily have to correspond with the chemical similarities, as it was previously presented in molecular studies for some lichens (e.g. Buschbom & Mueller Reference Buschbom and Mueller2006; Nelsen & Gargas Reference Nelsen and Gargas2008, Reference Nelsen and Gargas2009).
Taxonomy
Micarea soralifera Guzow-Krzemińska, Czarnota, Łubek & Kukwa sp. nov.
MycoBank No.: MB 814837
Similar to Micarea prasina s. str. due to the presence of micareic acid and green thallus, but differing in the thallus developing well-delimited soralia, which are absent in M. prasina s. str.
Type: Poland, Równina Bielska, Białowieża Primeval Forest, Białowieża National Park, forest section no. 256, Circeo-Alnetum, on log, October 2014, M. Kukwa 13001 & A. Łubek (UGDA—holotype; KTC—isotype). ITS GenBank Acc. No. KT119887, mtSSU GenBank Acc. No. KT119886.
(Fig. 2)
Micarea soralifera. A & B, habit; C, apothecial section in water; D, apothecial section with a K+ violet reaction of Sedifolia-grey pigment; E, branched and anastomosed paraphyses; F & G, ascospores. A, C, E–G, holotype; B & D, Czarnota 4659. Scales: A & B=1 mm; C & D=50 µm; E–G = 10 µm. In colour online.
Thallus crustose, indeterminate, endosubstratal to episubstratal in non-sorediate parts as a thin greyish green to dull green film over the substratum or minutely areolate, sorediate; areoles flat to convex, up to 0·1(–0·2) mm diam., grey greenish, soon bursting apically to form soralia. Prothallus not evident. Soralia green, often with a bluish grey or brownish tinge due to the pigmentation of external soredia, developing directly from the endosubstratal thallus or from thallus areolae, more or less rounded, up to 0·3 mm diam., convex or slightly concave, mostly discrete but in older parts of the thallus more or less fused and sometimes appearing to form a continuous leprose crust (but individual soralia still visible). Soredia farinose, bright to pale green, in exposed parts with Sedifolia-grey pigment (K+ violet, C+ violet) confined to the gel matrix, 10–25 μm diam., simple or in consoredia up to 35 μm diam. Photobiont chlorococcoid, micareoid, cells globose to ellipsoidal, 4–7 μm diam.
Apothecia rarely developed (only in 6 of 32 specimens examined), white, pale beige-white, pale greyish brown or greyish, 0·1–0·3 mm diam., immarginate, convex. Excipulum absent. Hymenium up to 40 μm tall, hyaline or in upper part of darker morphs olive-grey due to the presence of Sedifolia-grey pigment (K+ violet, C+ violet) confined to the gel matrix. Hypothecium hyaline or very pale straw-coloured. Paraphyses of one type, 1·0–1·5 μm thick, sparse, mostly apically branched and anastomosed, hyaline throughout. Asci cylindrical-clavate, 35–40×10–12 μm, Micarea-type. Ascospores 8 per ascus, 0–1(–2)-septate, ovoid, ellipsoid or oblong, 6–12 × 3·5–4·5 μm.
Pycnidia not seen.
Chemistry. Micareic acid with traces of unidentified substances. Sedifolia-grey pigment in hymenium and pigmented soredia.
Notes. Micarea soralifera is characterized by the thallus developing distinct, mostly delimited green soralia, the presence of micareic acid and Sedifolia-grey pigment in darker apothecia and soredia. These features make the species very similar to M. prasina s. str., which also has a usually green thallus containing the same secondary metabolite; however, its thallus does not form soralia and consists of goniocysts (Czarnota Reference Czarnota2007).
Micareic acid is also produced by M. nowakii, but this species differs from M. soralifera in the lack of soralia and in the presence of black apothecia, as well as barrel-like to shortly stalked, emergent pycnidia producing mesoconidia (Czarnota Reference Czarnota2007).
The development of delimited soralia is a rare character within the genus and previously known only in four species: M. alectorialica Brand et al., M. coppinsii Tønsberg, M. pseudocoppinsii Brand et al. and M. viridileprosa. They all differ predominantly in the secondary chemistry, as M. alectorialica contains alectorialic acid, M. pseudocoppinsii and M. viridileprosa produce gyrophoric acid, whereas M. coppinsii has 5-O-methylhiascic acid (with a trace of gyrophoric acid). In addition, the ascospores of M. alectorialica, M. coppinsii and M. pseudocoppinsii are 3-septate and soralia of M. viridileprosa are usually not clearly delimited and give the thallus a leprose appearance (Tønsberg Reference Tønsberg1992; van den Boom & Coppins Reference van den Boom and Coppins2001; Brand et al. Reference Brand, van den Boom and Sérusiaux2014). Moreover, M. alectorialica and M. pseudocoppinsii have so far been found only on the Indian Ocean island of Réunion (Brand et al. Reference Brand, van den Boom and Sérusiaux2014).
Micarea soralifera can also be confused in the field with diminutive, greenish morphs of Trapeliopsis flexuosa (Fr.) Coppins & P. James. The latter, however, usually have at least some esorediate and clearly visible flat areoles and contain gyrophoric acid (soralia and thallus C+ red) (Tønsberg Reference Tønsberg1992). Also, Biatora chrysantha (Zahlbr.) Printzen and Trapelia corticola Coppins & P. James may morphologically resemble the newly described species due to the distinct green soralia. Both species differ, however, in the chemistry since they produce gyrophoric acid reacting C+ red (Tønsberg Reference Tønsberg1992).
The new species is also similar to Catillaria croatica Zahlbr., a corticolous species with delimited soralia, which is very common in Białowieża National Park and often grows in the same localities as M. soralifera (Kukwa et al. Reference Kukwa, Łubek, Szymczyk and Zalewska2012; M. Kukwa & A. Łubek, pers. comm.). Catillaria croatica does not, however, produce secondary metabolites (Harris & Lendemer Reference Harris and Lendemer2010; Kukwa et al. Reference Kukwa, Łubek, Szymczyk and Zalewska2012).
Distribution and habitat. Micarea soralifera is so far known from numerous localities in Poland, where it is especially abundant in the Białowieża Primeval Forest, and one locality in the Czech Republic. The species has been most often found in deciduous forests, rarely in coniferous plantations, but always in humid and often shaded situations. It usually grows on decaying logs, rarely on tree stumps; one sample was also found on the bark of dead oak and one on the bark of black alder. The common accompanying species include Absconditella lignicola Vězda & Pišut and Placynthiella icmalea (Ach.) Coppins & P. James, and rarely Amandinea punctata (Hoffm.) Coppins & Scheid., Parmelia sulcata Taylor, Micarea misella (Nyl.) Hedl. and M. peliocarpa (Anzi) Coppins & R. Sant.
Additional material examined. Czech Republic: Jihomoravský kraj: S of Lanžhot, Cahnov-Soutok National Nature Reserve, 48°39'22"N, 16°56'27"E, 152 m, 2014, Kukwa 12473 (UGDA).—Poland: Western Beskids: Babia Góra range, NE slope of Polica Mt., 49°38'03"N, 19°38'43"E, 900 m, 2004, Czarnota 3928 (GPN); Gorce Mts, Lubań range, valley of Kudowski stream, c. 700 m, 2004, Czarnota 4008 & Wojnarowicz (GPN); Gorce National Park, by Olszowy Potok stream, 49°33'82"N, 20°05'40"E, c. 770 m, 2003, Czarnota 3513 (GPN); Turbacz nature reserve, by Olszowy Potok stream, 820 m, 1996, Czarnota 1148/94 (GPN). Karkonosze Mts: Karkonoski National Park, Dolina Łomniczki valley, 50°45'N, 15°45'E, c. 700 m, 2003, Czarnota 3537 (GPN). Pogórze Przemyskie: Krępak nature reserve, 49°42'09"N, 22°31°55"E, 390 m, 2005, Czarnota 4502 (GPN). Pojezierze Iławskie: between Mątki and Ryjewo villages, 2002, Kukwa 1504 (UGDA). Pojezierze Kaszubskie: Staniszewskie Błoto nature reserve, on bark of Quercus sp., 2006, Kukwa 5444 (UGDA). Pojezierze Mrągowskie: by NW part of Lake Kiersztanowskie, 53°57'03"N, 21°13'47"E, 2006, Kukwa 5257a (UGDA). Pojezierze Wielkopolskie: Wielkopolski National Park, Wiry forest district, 52°17'54"N, 16°49'36"E, 2004, Czarnota 3912 (GPN). Puszcza Kampinoska: Sieraków nature reserve, 52°20'27"N, 20°47'42"E, on bark of Alnus glutinosa, 2004, Czarnota 3940 (GPN). Równina Bielska: Białowieża Primeval Forest, Białowieża National Park, forest section no 256, 2014, Kukwa 12722 (mtSSU GenBank Acc. No. KT119884), 12797, 12863, 12939, 12949, 12969, 12976, 12999 (mtSSU GenBank Acc. No. KT119885), 13000, 13221, 13270 & Łubek (KTC, UGDA); forest section no 285A, 2015, Kukwa 15624 & Łubek (KTC, UGDA); forest section no 255D, 2015, Kukwa 15625 & Łubek (KTC, UGDA); forest section no 342B, 2010, Łubek (KTC), forest section no 314C, 2010, Łubek (KTC); forest section 340D, 2012, Łubek (KTC); forest section no 225, on bark of Alnus glutinosa, 2010, Łubek (KTC). Równina Łukowska: near Żdżary Village, 51°57'11"N, 22°11'53"E, 2005, Czarnota 4659 (GPN). Tatry Wschodnie Mts: Tatra National Park, Dolina Roztoki valley, 49°13'N, 20°04'E, 2003, Czarnota 3339 (GPN). Wysoczyzna Żarnowiecka: Pużyckie Łęgi nature reserve, 54°38'N, 17°51'E, 2015, Kukwa 17054 (UGDA). Wyżyna Lubelska: between Urzędów and Dzierzkowice villages, c. 200 m, 2003, Czarnota 4192 (GPN).
This research was supported by funding from the Polish-Norwegian Research Programme operated by the National Centre for Research and Development, under the Norwegian Financial Mechanism 2009-2014, Project Contract No Pol-Nor/196829/87/2013. This research was also partially supported by the University of Gdańsk task grant no. 530-L140-D242-14 and by the University of Rzeszów task grant no. WBR/KA/DS/5/2015. We are also very grateful to the reviewers for their very helpful comments and suggestions.
