Research Article |
Corresponding author: Beata Guzow-Krzemińska ( beata.guzow@biol.ug.edu.pl ) Academic editor: Pradeep Divakar
© 2019 Beata Guzow-Krzemińska, Emmanuël Sérusiaux, Pieter P. G. van den Boom, A. Maarten Brand, Annina Launis, Anna Łubek, Martin Kukwa.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Guzow-Krzemińska B, Sérusiaux E, van den Boom PPG, Brand AM, Launis A, Łubek A, Kukwa M (2019) Understanding the evolution of phenotypical characters in the Micarea prasina group (Pilocarpaceae) and descriptions of six new species within the group. MycoKeys 57: 1-30. https://doi.org/10.3897/mycokeys.57.33267
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Six new Micarea species are described from Europe. Phylogenetic analyses, based on three loci, i.e. mtSSU rDNA, Mcm7 and ITS rDNA and ancestral state reconstructions, were used to evaluate infra-group divisions and the role of secondary metabolites and selected morphological characters on the taxonomy in the M. prasina group. Two main lineages were found within the group. The Micarea micrococca clade consists of twelve species, including the long-known M. micrococca and the newly described M. microsorediata, M. nigra and M. pauli. Within this clade, most species produce methoxymicareic acid, with the exceptions of M. levicula and M. viridileprosa producing gyrophoric acid. The M. prasina clade includes the newly described M. azorica closely related to M. prasina s.str., M. aeruginoprasina sp. nov. and M. isidioprasina sp. nov. The species within this clade are characterised by the production of micareic acid, with the exception of M. herbarum which lacks any detectable substances and M. subviridescens that produces prasinic acid. Based on our reconstructions, it was concluded that the ancestor of the M. prasina group probably had a thallus consisting of goniocysts, which were lost several times during evolution, while isidia and soredia evolved independently at multiple times. Our research supported the view that the ancestor of M. prasina group did not produce any secondary substances, but they were gained independently in different lineages, such as methoxymicareic acid which is restricted to M. micrococca and allied species or micareic acid present in the M. prasina clade.
Ancestral state reconstruction, lichenised fungi, morphology, mtSSU rDNA, secondary metabolites, taxonomy
Traditionally, morpho-anatomical characters, together with secondary metabolites, have played an important role in the lichen classification (e.g.
Secondary metabolites have been traditionally used in the taxonomy of lichens at different taxonomic levels, although their values have been questioned by many authors (
The genus Micarea Fr., comprising ca. 100 species, is a cosmopolitan group of lichens which has been extensively studied in Europe by
Species delimitation within Micarea has been especially difficult in the M. prasina group which was first characterised by
Moreover, several other new species likely to belong to the M. prasina group have been described. Two such species were described from Réunion, i.e. M. melanoprasina Brand, van den Boom & Sérus. producing a substance probably related to micareic acid and M. hyalinoxanthonica Brand, van den Boom & Sérus. containing a xanthone (probably thiophanic acid) (
This study is based on specimens from years of collection in Belgium, France, Germany, Portugal (including Madeira and the Azores), Poland, Romania and the Netherlands. Using these collections for a phylogenetic reconstruction, six new species, belonging to the M. prasina group, are described by means of morphological, anatomical, chemical and molecular data. Moreover, by reconstructing ancestral states, the evolution of diagnostic traits, that are traditionally used for the taxonomic classification of species belonging to the M. prasina group, were investigated. Infra-group divisions and the role of secondary metabolites for species taxonomy within the M. prasina group were also evaluated. The production of selected secondary metabolites is further analysed (i.e. gyrophoric, methoxymicareic, micareic, prasinic and thiophanic acids), as well as the presence of several pigments in the apothecia commonly used in lichen taxonomy (
Material of the new species, including samples used for DNA analyses, is deposited in KTC, UGDA and LG, with additional specimens stored in private herbaria of van den Boom and Brand.
Apothecial sections and squashed thallus preparations were studied in tap water with or without the addition of C (commercial bleach) and K (water solution of potassium hydroxide) (
A total of 63 new sequences were generated for this study (Suppl. material
DNA was extracted directly from pieces of thalli using a modified CTAB method (
Amplifications of mtSSU rDNA, employing mrSSU1 and mrSSU3R primers (
Amplifications of the Mcm7 region employing Mcm7_AL1r and Mcm7_AL2f primers (
Amplifications of the ITS region employed the following primer pairs: ITS1F (
The newly generated sequences (GenBank accession numbers are given in Suppl. material
Partition Finder 2 (
The data were analysed using a Bayesian approach (MCMC) in MrBayes 3.2 (
Maximum likelihood analyses were performed using RaxML HPC v.8 on XSEDE (
Morphological and chemical characters from taxa of the Micarea prasina group and selected outgroup taxa were obtained from herbarium material and complemented with data from literature. In order to reduce the number of missing data in our dataset, we did not include M. pycnidiophora, M. stipitata and M. synotheoides, which do not belong to the M. prasina group and for which mtSSU sequences were only available and Micarea sp. lineage A, which represents a single specimen that has not been formally described. The following secondary metabolites were analysed: gyrophoric, methoxymicareic, micareic, prasinic and thiophanic acids. The presence of apothecial pigmentation was also analysed and the following pigments were noted: Sedifolia-grey, Elachista-brown, Cinereorufa-green and Superba-brown. The presence of selected morphological characters was also analysed, i.e. goniocysts, isidia and soredia. The morphological and chemical characters were coded as a multistate data matrix (Suppl. material
The final DNA alignment consisted of sequences obtained from 119 individual specimens and three markers, i.e. mtSSU, Mcm7 and ITS rDNA, with a total of 1784 characters. Since the topologies from the maximum likelihood and Bayesian analyses did not show any strongly supported conflict, the maximum likelihood tree (RaxML Optimisation Likelihood was -14426.795913) is presented in Figure
The phylogenetic reconstruction (Fig.
Maximum likelihood tree based on three-loci dataset. Bootstrap supports ≥ 70 for ML and posterior probabilities ≥ 0.95 (second value) for Bayesian methods are indicated near the branches. The highly supported clades with previously described species represented by numerous sequences are collapsed. Herbarium collection numbers for newly sequenced specimens precede the names of species and type specimens are marked. Newly described species are marked in bold. Micarea micrococca (orange) and M. prasina (blue) clades are indicated with shading. Ancestral states for selected characters reconstructed based on binary or multistate datasets using maximum parsimony (MP) or maximum likelihood methods (ML) are marked for the main clades: M. prasina group, M. micrococca clade and M. prasina clade using red (= present/significant) and white (= absent/insignificant) boxes or ? (= uncertain).
The Micarea micrococca clade in Figure
The Micarea prasina clade (93 BS and 1 PP) consists mostly of species containing micareic acid and accommodates the newly described M. aeruginoprasina, M. azorica and M. isidioprasina, as well as M. herbarum, M. meridionalis, M. nowakii, M. prasina, M. soralifera and M. subviridescens. Several highly supported lineages are further distinguished within this clade. The newly described M. azorica forms a highly supported group with the type species of M. prasina s.str. (100 BS and 1 PP), whereas specimens of M. prasina form a well-supported group (87/0.99). Furthermore, they are sister to M. nowakii and M. herbarum, which are the only species within the M. prasina group developing almost entirely an endosubstratal thallus with only a few areoles. With the exception of M. herbarum and M. nowakii, this lineage (97 BS and 1 PP) also includes a sequence which seems to be different from both species (EF453665) and may indicate the existence of an undescribed taxon. Specimens of the newly described M. isidioprasina form a highly supported group (100 BS and 1 PP) with a single sequence from North America originally assigned to M. prasina (AY756452; see
To investigate the diagnostic traits traditionally used for the taxonomic classification within the M. prasina group, we focused both on the M. micrococca and the M. prasina clades separately and the whole M. prasina together (Fig.
Most parsimonious ancestral character states for selected subclades of the M. prasina group. The results that differ between maximum likelihood and maximum parsimony methods for each dataset are marked with *.
Characters | M. prasina group | M. micrococca clade | M. prasina clade |
Morphological characters | goniocysts | goniocysts | goniocysts |
Secondary metabolites | lack of any substance* | methoxymicareic acid | micareic acid |
Presence of apothecial pigments | uncertain (lack of pigments OR Sedifolia-grey) | lack of pigments or unknown* | Sedifolia-grey |
Goniocysts | present | present | present |
Isidia | absent | absent | absent |
Soredia | absent | absent | absent |
Gyrophoric acid | absent | absent | absent |
Methoxymicareic acid | absent | present | absent |
Micareic acid | absent | absent | present |
Prasinic acid | absent | absent | absent |
Thiophanic acid | absent | absent | absent |
Cinereorufa-green | absent | absent | absent |
Elachista-brown | absent | absent | absent |
Sedifolia-grey | absent* | absent* | present* |
Superba-brown | absent | absent | absent |
The evolution of pigments, present in the apothecia, was also analysed, but some of the results remain uncertain in our analyses. Parsimony reconstructions, based on the binary dataset, suggest the lack of any pigment in the apothecia, while other analyses do not exclude the possibility that Sedifolia-grey pigment was present in the ancestor of M. prasina group (Fig.
Most likely ancestral character states in multistate analysis and their likelihoods for selected subclades of M. prasina group. Values for the most likely states are given in bold. The results that differ between maximum likelihood and maximum parsimony methods are marked with *
Characters | State | M. prasina group | M. micrococca clade | M. prasina clade |
Morphological characters | other or unknown | 0.03672198 | 0.00009398 | 0.00056073 |
goniocysts | 0.95494636 | 0.99972253 | 0.99844142 | |
soredia | 0.00411906 | 0.00009117 | 0.00008044 | |
isidia | 0.00421261 | 0.00009232 | 0.00091742 | |
Secondary metabolites | lack of any substances | 0.78962855 | 0.05876201 | 0.00749498 |
prasinic acid | 0.01031084 | 0.00338938 | 0.00052042 | |
micareic acid | 0.117972* | 0.01320396 | 0.98892236 | |
methoxymicareic acid | 0.04762568 | 0.90867382 | 0.00176536 | |
gyrophoric acid | 0.01528514 | 0.00349062 | 0.00050881 | |
thiophanic acid | 0.01917779 | 0.01248021 | 0.00078808 | |
Presence of apothecial pigments | lack of pigment or unknown | 0.39914052 | 0.73149364 | 0.03357191 |
Sedifolia-grey | 0.44528926 | 0.21519349* | 0.95640101 | |
Cinereorufa-green | 0.04803049 | 0.02251569 | 0.00293747 | |
Elachista-brown | 0.05745307 | 0.01546335 | 0.00302814 | |
Superba-brown | 0.05008665 | 0.01533383 | 0.00406148 |
Morphological characters, i.e. the presence of goniocysts observed in many species of the M. prasina group, soredia observed in M. microsorediata, M. soralifera and M. viridileprosa and isidia present in M. aeruginoprasina, M. isidioprasina, M. nigra and M. pauli were also evaluated (Fig.
Most likely ancestral character states based on analysis of binary dataset and their likelihoods for selected subclades of M. prasina group. Values for the most likely states are given in bold. The results that differ between maximum likelihood and maximum parsimony methods are marked with *
Characters | State | M. prasina group | M. micrococca clade | M. prasina clade |
Goniocysts | Present | 0.85180046 | 0.99078786 | 0.93297055 |
Absent | 0.14819954 | 0.00921214 | 0.06702945 | |
Isidia | Present | 0.00354307 | 0.00079891 | 0.01513079 |
Absent | 0.99645693 | 0.99920109 | 0.98486921 | |
Soredia | Present | 0.00099039 | 0.00020816 | 0.00004518 |
Absent | 0.99900961 | 0.99979184 | 0.99995482 | |
Gyrophoric acid | Present | 0.00499412 | 0.00058456 | 0.00011577 |
Absent | 0.99500588 | 0.99941544 | 0.99988423 | |
Methoxymicareic acid | Present | 0.0023099 | 0.85313833 | 0.00010697 |
Absent | 0.9986901 | 0.14686167 | 0.99989303 | |
Micareic acid | Present | 0.02913989 | 0.00055219 | 0.98044653 |
Absent | 0.97086011 | 0.99944781 | 0.01955347 | |
Prasinic acid | Present | 0.00002438 | 0.0000051 | 0.00000103 |
Absent | 0.99997562 | 0.9999949 | 0.99999897 | |
Thiophanic acid | Present | 0.0000248 | 0.00000102 | 0.00001393 |
Absent | 0.9999752 | 0.99999898 | 0.99998607 | |
Cinereorufa-green | Present | 0.00002408 | 0.0000016 | 0.00000099 |
Absent | 0.99997592 | 0.9999984 | 0.99999901 | |
Elachista-brown | Present | 0.00002503 | 0.0000052 | 0.00000102 |
Absent | 0.99997497 | 0.9999948 | 0.99999898 | |
Sedifolia-grey | Present | 0.49999914* | 0.49994168* | 0.50492012 |
Absent | 0.50000086 | 0.50005832 | 0.49507988* | |
Superba-brown | Present | 0.00050986 | 0.00004725 | 0.00000944 |
Absent | 0.99949014 | 0.99995275 | 0.99999056 |
Challenges in species delimitation within M. prasina group were already mentioned by
The evolution of new morphological characters involves multiple subsequent evolutionary steps. In our study, ancestral state reconstructions showed that the presence of goniocysts is the most parsimonious and most likely state for the ancestor of the M. prasina group (Fig.
In our study, ancestral state reconstructions suggest that isidia evolved independently multiple times in this group of lichens resulting in the formation of almost entirely isidiate thalli in four species, i.e. M. aeruginoprasina, M. isidioprasina, M. nigra and M. pauli (Suppl. material
Secondary metabolites have been extensively used in the chemotaxonomy of lichens. The Micarea prasina group shows a high variation in chemistry even in closely related species (e.g.
Micareic and methoxymicareic acids are the most common secondary metabolites produced by species of the M. prasina group. They are structurally related diphenyl ethers (‘pseudodepsidones’) (
Reconstructions of the ancestral state for the whole M. prasina group suggest that the most recent common ancestor did not produce any secondary metabolites. This may suggest that the production of a wide range of secondary metabolites in this group of lichens could have resulted from independent gains of ability to biosynthesise various substances during evolution. The scenario, in which the ability to produce micareic acid in the ancestor of M. prasina clade or methoxymicareic acid in the ancestor of M. micrococca clade being gained only once during evolution, seems to be reasonable since losing an existing character could be expected to occur more rapidly and in fewer steps than gaining a new character (e.g.
To summarise, our study showed that phenotypical variation within the Micarea prasina group has been previously underestimated and, based on field work and laboratory studies, six new species within this group are described (see Taxonomy).
Species characterised by inconspicuous, pale brownish to moderately brownish, isidiate thallus, branched to coralloid isidia, emarginate, adnate to slightly convex apothecia measuring 0.1–0.5 mm in diam., which are pale cream to pale brown or aeruginose with pigment (Sedifolia-grey, K+ violet, C+ violet) present in hypothecium, (0–)1-septate ascospores measuring 9–14 × 4.5–5.5 µm and the production of micareic acid.
Portugal. Azores, Terceira, NW of Angra do Heroismo, W of Pico Gordo, Mistério dos Negros (N), trail from Lagoa do Negro to the West, 550 m alt., 38°44.15'N, 27°16.30'W, ± damp Juniperus brevifolia forest, with some young Vaccinium cylindraceum, on Juniperus brevifolia, 28 June 2014, P. & B. van den Boom 51445 (holotype LG; isotypes UGDA, hb v.d. Boom, mtSSU GenBank accession number: MK562024, Mcm7 GenBank accession number: MN105888).
Thallus indeterminate, inconspicuous, thin, endosubstratal to episubstratal in non-isidiate parts as a thin film over the substrate or minutely granular, pale to moderately brown, isidiate; prothallus not visible; granules vertically proliferating to form isidia; isidia branched to coralloid, crowded or separated, up to 250 μm tall and 25 μm wide, with a distinct and complete hyphal layer; apothecia abundant, adnate to slightly convex, emarginate, rounded to slightly irregular, pale cream to pale brown or aeruginose, often different colours in a single apothecium, 0.1–0.5 mm in diam.; excipulum sometimes paler, instinct; hymenium 40–50 µm high, hyaline; hypothecium hyaline to pale aeruginose brownish (Sedifolia-grey), K+ violet, C+ violet; paraphyses, sparse, branched, 1.0–1.2(–1.5) µm wide, tips not widened and not pigmented; asci cylindrical to clavate, 35–40 × 11–14 µm, 8–spored; ascospores ellipsoidal to ovoid, (0‒)1-septate, 9–14 × 4.5–5.5 µm; pycnidia not observed; crystalline granules (studied in polarised light) visible in hypothecium and in thallus, soluble in K.
Photobiont micareoid, cells thin walled, 6–9 µm in diam., clustered in compact groups.
Micareic acid detected by TLC. Sedifolia-grey in apothecia (hypothecium), its presence sometimes indistinct.
In the type locality Micarea aeruginoprasina grows abundantly on trunks of Juniperus brevifolia, in a subnatural degradated forest, dominated by J. brevifolia shrubs and trees. In other localities, it was found on Cryptomeria and Erica trunks, also in forested areas.
The new species is only known from the island Terceira in the Azores, where it is known from several localities.
The epithet refers to the often aeruginose colour of the apothecia and the resemblance in secondary chemistry to M. prasina.
Portugal. Azores, Terceira, NW of Angra do Heroismo, south edge of Reserva Florestal da Lagoa das Patas, area around a pond ‘Lagoa das Patas’, mature Cryptomeria trees and some Camellia shrubs, on Cryptomeria, 38°43.01'N, 27°17.32'W, 520 m alt., 28 June 2014, P. & B. van den Boom 51878 (hb v.d. Boom); NW of Angra do Heroismo, NNE of Santa Bárbara, Serra de Santa Bárbara, road to the summit, forests with mainly Cryptomeria trees, trees at edge of forest, on Cryptomeria, 38°43.49'N, 27°19.33'W, 800 m alt., 1 July 2014, P. & B. van den Boom 51622 (hb v.d. Boom); NE of Serreta, north trail to Lagoínha, forest with Cryptomeria japonica, Myrica faya, Erica, etc., on Erica, 38°45.28'N, 27°20.50'W, 500 m alt., 2 July 2014, P. & B. van den Boom 51691 (hb v.d. Boom).
This species is unique within the group due to the presence of the Sedifolia-grey pigment in hypothecium. It is similar to M. prasina because of its production of micareic acid, but the latter has Sedifolia-grey pigment in the epihymenium and its thallus consists of goniocysts (isidiate in M. aeruginoprasina). However, it is not closely related to M. prasina, being resolved as basal in the M. prasina clade and the sequences of their molecular markers are very different. In the Azores archipelago, the most widespread prasina-like species is M. azorica, newly described in this paper, which, however, is not isidiate and contains Superba-brown in the apothecia. Micarea aeruginoprasina resembles M. byssacea, which can have somewhat the same coloured and adnate apothecia; however, M. byssacea is not isidiate, contains methoxymicareic acid and the apothecial pigment is absent in hypothecium (
Micarea isidioprasina, M. nigra and M. pauli also have isidiate thalli, but only M. aeruginoprasina has pale cream to pale brown or aeruginose apothecia. Micarea isidioprasina and M. pauli are often sterile and, to date, M. aeruginoprasina and M. nigra have always been found with apothecia, but, based only on the thallus characters, M. nigra and M. pauli can be distinguished due to the production of methoxymicareic acid and M. isidioprasina has green isidia (shades of brown in M. aeruginoprasina).
Species characterised by pale to moderately brownish thallus consisting of goniocysts, convex to subglobose, emarginate, pale greyish-brown to dark brown (with Superba-brown pigment) apothecia measuring 0.1–0.3 mm in diam., (0–)1-septate, narrowly ellipsoidal to ovoid ascospores measuring 9–11 × (2.5–)3–4 µm, sessile to slightly stalked, pale to moderately brown mesopycnidia, bacillar mesoconidia measuring 6.5–8 × 0.9–1.1 µm and the production of micareic acid.
Portugal. Azores, Terceira, NW of Angra do Heroismo, south edge of Reserva Florestal da Lagoa das Patas, area around a pond ‘Lagoa das Patas’, 520 m alt., 38°43.01'N, 27°17.32'W, mature Cryptomeria trees and Camellia shrubs, on Cryptomeria japonica, 28 June 2014, P. & B. van den Boom 51468 (holotype LG; isotypes UGDA, hb v.d.Boom, mtSSU GenBank accession number: MK562026, Mcm7 GenBank accession number: MN105891).
Thallus inconspicuous, thinly scurfy to somewhat farinose-granular, pale to moderately brownish and consisting of goniocysts; prothallus not seen; apothecia abundant, convex to subglobose, emarginate, pale greyish-brown to dark brown, often unevenly coloured in a single apothecium (partly dark, partly pale), 0.1‒0.3 mm in diam.; hymenium ca. 32‒40 µm tall; epihymenium with grey-brown pigment, K‒, C‒ (Superba-brown); hypothecium hyaline; paraphyses, abundant, branched, ca. 1.0–1.5(–1.8) µm wide, tips not widened and not pigmented; asci 25–35 × 11–14 µm, 8-spored; ascospores narrowly ellipsoidal to ovoid, (0–)1-septate, 9–11 × (2.5–)3–4 µm; mesopycnidia occasionally abundant, sessile to slightly stalked, 40–60 µm in diam., pale to moderately brown, the ostiole sometimes gaping; mesoconidia bacillar, simple, 6.5‒8 × 0.9‒1.1 µm; crystalline granules (studied in polarised light) visible in epithecium and in thallus, soluble in K.
Photobiont micareoid, cells thin-walled, 4–10 µm in diam., clustered in compact groups.
Micareic acid detected by TLC. Superba-brown in apothecia (epihymenium).
To date, known only from the Azores archipelago (Terceira island) from three localities where it was found on bark of trees.
The name refers to the archipelago of the Azores, where the species occurs.
Portugal. Azores, Terceira, NW of Angra do Heroismo, Reserva Florestal Viveira da Falca, 460 m alt., 38°42.90'N, 27°16.78'W, picnic area with many mature Cryptomeria trees, some Acer trees and Camellia, on Cryptomeria, 28 June 2014, P. & B. van den Boom 51330 (hb. v.d. Boom); N of Serreta, Reserva Florestal da Serreta, 80 m alt., 38°46.27'N, 27°21.42'W, picnic area in open forest with mixed trees and shrubs, on tree, 2 July 2014, P. & B. van den Boom 51733 (hb. v.d. Boom).
Notes. The new species is resolved as sister to M. prasina s.str. with strong support, being morphologically and chemically similar to that species, but differing in the absence of the Sedifolia-grey pigment, responsible for the typical reaction K+ violet in M. prasina s.str (
The identity of M. prasina s.str. has been recently solved by
Micarea azorica resembles M. lithinella (Nyl.) Hedl. due to its brownish, convex to subglobose small apothecia, but the latter is mainly a saxicolous species, has smaller conidia, 4‒5.5 × 0.5‒1 µm and does not contain secondary metabolites (
Species characterised by granular-isidiate thallus, pale grey to grey-beige apothecia, 0–1-septate, ovoid, ellipsoidal or oblong ascospores measuring 7–13 × 3.5–4.5 μm and the presence of micareic acid.
POLAND. Równina Bielska, Białowieża Primeval Forest, Białowieża National Park, forest section no 256, Pino-Quercetum, on wood of log, 21 Aug 2015, M. Kukwa 17367a, A. Łubek (holotype UGDA; isotype KTC, ITS GenBank accession number: MN095789, mtSSU GenBank accession number: MK562016, Mcm7 GenBank accession number: MN105897).
Thallus crustose, granular-isidiate, indeterminate, endosubstratal to rarely episubstratal in non-isidiate parts and then as a thin greenish film over the substrate or minutely areolate, isidiate; prothallus not seen; areoles up to 0.05 mm in diam., green, soon developing isidia; isidia abundantly branched and coralloid, crowded and forming an almost continuous layer locally over the substrate, but in younger parts of thalli separated, green to olive green (Sedifolia-grey, K+ violet), up to 250 μm tall and 25 μm wide, with a distinct and complete hyphal layer; apothecia rarely developed, white to beige, some patchily grey, up to 0.45 mm in diam., convex; excipulum poorly developed, as a narrow, hyaline zone, hyphae radiating, branched and anastomosing; hymenium up to 50 μm tall, hyaline; epihymenium and hypothecium hyaline; paraphyses of one type, 1–1.5 μm thick, sparse, mostly apically branched and anastomosed, hyaline throughout; asci cylindrical-clavate, 30–45 × 12–15 μm, 8-spored; ascospores, 0–1-septate, ovoid, ellipsoidal or oblong, 11–14 × 3.5–4.5 μm; pycnidia not seen; crystalline granules (studied in polarised light) present rather sparsely in hymenium (as strands between asci and paraphyses) and abundantly in isidia, soluble in K.
Photobiont chlorococcoid, micareoid, cells globose to ellipsoidal, 4–7 μm in diam.
Micareic acid detected by TLC. Sedifolia-grey pigment present in outermost parts of some isidia.
The species grows on wood (decomposing logs) and acidic bark of trees in various forest communities in well preserved forest.
To date, it is known from Belgium, Germany, France, Poland and Romania.
The name of the new species refers to the presence of isidia and the chemistry of M. prasina.
Belgium. Herbeumont, forest by the Semois river, 265 m alt., 49°45'N, 05°13'E, on Quercus tree in forest, 2013, E. Sérusiaux 3609 (LG). France. Vosges, Dépt. Haut-Rhin, Hohneck, Frankenthal nature preserve, 48°02'N, 07°01'E, 1100 m alt., on dead Fagus in forest, 2013, E. Sérusiaux LG DNA 3437 (LG). Germany. Niedersachsen, S of Goslar, Rammelsberg, 360 m alt., 51°53.01'N, 10°25.23'E, trail along Picea forest and brooklet with Acer, Alnus and Betula trees, 12 May 2015, P. & B. van den Boom 53248 (hb. v.d. Boom). Poland. Roztocze Środkowe, Roztoczański National Park, S of Zwierzyniec village, Bukowa Góra nature reserve, 50°35'47"N, 22°57'48"E, ca. 280 m alt., beech forest, on wood of log, 15 Sept 2015, M. Kukwa 17493 (UGDA); Równina Bielska, Białowieża Primeval Forest, Białowieża National Park, forest section no 256, Carici elongatae-Alnetum, on wood of logs, bark Picea abies and Alnus glutinosa, Aug 2014, M. Kukwa 14030, 14038, 14107, 14112, A. Łubek (KTC, UGDA); ibidem, Circaeo-Alnetum, on wood of log, Aug 2014, M. Kukwa 13299, A. Łubek (KTC, UGDA); ibidem, Tilio-Carpinetum, on wood of log, Aug & Oct 2014, M. Kukwa 13418, 14358, A. Łubek (KTC, UGDA); ibidem, Circaeo-Alnetum, on wood of snag, Oct 2014, M. Kukwa 14243, A. Łubek (KTC, UGDA). Romania. W of Brasov, S of Zarnesti, Praia Craiului National Park, 1350 m alt., 45°31'N, 25°16'E, on Fagus inside forest, 2016, E. Sérusiaux LG DNA 6260 & 6265 (LG).
Micarea isidioprasina is an isidiate species of the M. prasina group containing micareic acid as the main secondary metabolite. It is usually sterile and in Poland often grows in similar habitats with M. pauli, a species described in this paper, from which it can be separated with certainty by analyses of secondary metabolites, as the latter contains methoxymicareic acid.
Micarea aeruginoprasina and M. nigra also develop similar isidiate thalli, but M. aeruginoprasina has pale cream to pale brown or aeruginose apothecia (often mottled with all colours in the same apothecium) and M. nigra develops dark greyish to black apothecia. When sterile, all three species may be more difficult to separate, especially M. aeruginoprasina which also produces micareic acid (M. nigra contains methoxymicareic acid), but that species has pale brown isidia. Additionally, the so far known distributions of all three species do not overlap and M. aeruginoprasina and M. nigra are known from the Azores and continental Portugal, respectively.
Micareic acid is also the main secondary metabolite in the somewhat morphologically similar M. prasina, but the latter is not isidiate, often richly fertile and its thallus consists of goniocysts (
Species morphologically similar to Micarea viridileprosa, characterised by sorediate thallus, delimited or diffuse and confluent soralia with green or locally bluish soredia produced from the thallus areoles, white and immarginate when mature apothecia, 0.2–0.3 mm in diam., cylindrical to ellipsoidal (0–)1-septate ascospores measuring 9.5–13 × 2.8–3.5 µm and the presence of methoxymicareic acid.
Poland. Wysoczyzna Żarnowiecka, Pużyckie Łęgi nature reserve, 54°38'N 17°51'E, Circaeo-Alnetum, on wood of log, 12 Aug 2015, M. Kukwa 17053 (holotype UGDA, ITS GenBank accession number: MN095791, mtSSU GenBank accession number: MK562012, Mcm7 GenBank accession number: MN105906).
Thallus diffuse, up to 10 cm wide, consisting of finely granular soredia, often with a powdery appearance, vivid green or green, sometimes with bluish tinge; prothallus not seen; areoles up to 25 µm in diam., green, soon bursting to produce soredia; soralia at first delimited, produced from small, convex areoles, soon fused and confluent, sometimes forming a sorediate continuous layer; soredia simple, up to 20 µm in diam., sometimes slightly elongated or in more or less rounded consoredia up 35 µm in diam. apothecia rarely present, adnate, first with indistinct margin, then immarginate, 0.2–0.3 mm in diam., white or slightly brownish; excipulum in young apothecia present, 15–25 µm wide, of thin irregular hyphae; hymenium ca. 30‒42 µm tall; epihymenium and hypothecium hyaline; paraphyses thick (in K), branched and anastomosing, ca. 1.2‒1.5 µm wide; asci 29–35 × 7–10 µm, 8-spored; ascospores cylindrical to ellipsoidal, 9.5–13 × 2.8–3.5 µm, (0–)1-septate; micropycnidia present in some specimens, ca. 60 µm in diam., with dark brown tops (K–); microconidia narrow fusiform to bacilliform, 7 × 0.8 µm; mesopycnidia, mesoconidia 3.8 × 1.4 µm; crystalline granules (studied in polarised light) visible in hymenium and in thallus, soluble in K.
Photobiont micareoid, cells thin-walled, 4–8(–9) µm in diam.
Methoxymicareic acid detected by TLC. Soredia in exposed habitats with Sedifolia-grey pigment, K+ violet.
The new species occurs on acidic bark of various trees such as Alnus, Betula, Fagus and Quercus, usually in humid forests, also on decaying wood (logs and stumps) and rarely on terrestrial decaying mosses in, for example, steep slopes in heath and dunes. It is a very common species in the south of the Netherlands and some areas in Poland and is mostly found on microhabitats where only few other lichens species co-occur. On several occasions, Normandina pulchella (Borrer) Nyl. and squamules of Cladonia spp. are the only accompanying lichens.
To date, the species has been found in Belgium, Germany, the Netherlands, Poland and Portugal.
One specimen of Micarea microsorediata was invaded by Nectriopsis micareae Diederich, van den Boom & Ernst (see below additional specimens examined).
The epithet refers to the production of soredia and the similarity to M. micrococca due to the same secondary chemistry.
Belgium. Limburg, N of Achel, Rozendaal, 51°17.0'N, 5°29.9'E, 35 m alt., Pinus forest with Betula and Quercus trees, on wood of fallen decaying trunk, 28 Dec. 2018, P. & B. van den Boom 58046 (hb v.d. Boom); NE of Achel, near Tomp, 51°16.10'N, 5°29.8'E, 35 m alt., along small road, Pinus forest with Betula and Quercus trees, on Betula, 28 Dec. 2018, P. & B. van den Boom 58052 (hb v.d. Boom); NE of Lommel, Kolonie, E of ‘Afwateringskanaal’, 51°14.40'N, 5°23.6'E, 50 m alt., Pinus forest, on Prunus, 28 Dec. 2018, P. & B. van den Boom 58054 (hb v.d. Boom); ENE of Lommel, E of Kolonie, 51°15.50'N, 5°24.35'E, 40 m alt., between edge of Pinus forest and edge of reserve Hageven, on Betula and Quercus robur, 28 Dec. 2018, P. & B. van den Boom 58055, 58056 (hb v.d. Boom). Germany. Nedersaksen, N of Bentheim, NE of Wengsel, Isterberg, 52°21.4'N, 7°9.0'E, Pinus forest with some sandstone outcrops, on Quercus rubra, 26 Aug. 2015, M. & D. Brand, P. & B. van den Boom 53633 (hb v.d. Boom). Poland. Bory Tucholskie, Kręgi Kamienne nature reserve, close to Wda river, NW from Odry, 53°53'59.50"N, 17°59'42.07"E, black alders close to river, on Alnus glutinosa, 19 June 2018, M. Kukwa 19991 (UGDA); Równina Bielska, Białowieża Forest, Białowieża National Park, forest section no 256, Peucedano-Pinetum, on twig of Picea abies, Quercus robur and wood of log, May 2014, M. Kukwa 13462, 13660, 13675, 13778, A. Łubek (KTC); ibidem, Pino-Quercetum, on Quercus robur, Aug. 2014, M. Kukwa, 13321, A. Łubek (KTC, UGDA); ibidem, Tilio-Carpinetum, on Quercus robur, Aug. 2014, M. Kukwa 13420a, 13392 (as admixture in a specimen of Biatora ligni-mollis T. Sprib. & Printzen), A. Łubek (KTC, UGDA); ibidem, Carici elongatae-Alnetum, on bark of log, Alnus glutinosa and Picea abies, Aug. 2014, M. Kukwa, 14000, 14002, 14012, 14045, 14109, A. Łubek (KTC, UGDA); ibidem, Circaeo-Alnetum, on wood of snag, Oct. 2014, M. Kukwa, 14139, A. Łubek (KTC); ibidem, Tilio-Carpinetum, on wood of branch of the log, Oct. 2014, M. Kukwa, 14350, A. Łubek (KTC, UGDA); ibidem, Peucedano-Pinetum, on Alnus glutinosa, March 2015, M. Kukwa, 13308, A. Łubek (KTC, UGDA); ibidem, Carici elongatae-Alnetum, on wood of stump, May 2015, M. Kukwa, 15789, A. Łubek (KTC, UGDA); ibidem, Circaeo-Alnetum, on Alnus glutinosa, 29 Sept. 2015, M. Kukwa, 17546, A Łubek (KTC, UGDA); ibidem, Querco-Piceetum, on Carpinus betulus, Aug 2015, M. Kukwa, 17379, 17436, A. Łubek (UGDA; KTC); ibidem, Circaeo-Alnetum, on Alnus glutinosa, 1 Oct. 2015, M. Kukwa, 17592, A Łubek (KTC, UGDA); ibidem, Pino-Quercetum, on wood of log, 4 Oct. 2015, M. Kukwa, 17641, A. Łubek (KTC, UGDA); Wybrzeże Słowińskie, Białogóra nature reserve, forest section no. 14, 54°49'29"N, 17°57'21"E, Empetro nigri-Pinetum, on Pinus sylvestris, 21 April 2010, M. Kukwa 7721 (UGDA); ibidem, forest section no. 22, 54°49'27"N, 17°57'52"E, open area with few trees and Myrica gale, on Betula pendula, 22 Sept. 2010, M. Kukwa, 8280, A. Jabłońska, M. Oset (UGDA); Wysoczyzna Polanowska, Skotawskie Łąki nature reserve, 54°16'03"N, 17°33'37"E, large group of black alders, on Alnus glutinosa, 11 April 2017, M. Kukwa, 19212, 19219 (UGDA); ibidem, by NE part of Lipieniec lake, 54°15'44"N, 17°33'26"E, group of black alder trees on meadow, on Alnus glutinosa, 27 June 2017, M. Kukwa, 19800a, 19801, 19806 (UGDA); ibidem, by unnamed lake (N of Lipieniec lake), 54°15'46"N, 17°33'03"E, black alder forest, by lake, on Alnus glutinosa, Pinus sylvestris and Sambucus nigra, 27 June 2017, M. Kukwa, 19839, 19845, 19849, 19850 (UGDA); Torfowisko Potoczek nature reserve, 54°09'49"N, 16°57'11"E, Vaccinio uliginosi-Betuletum pubescentis, on wood of log and Picea abies, 11 Aug. 2015, M. Kukwa, 16994, 17001a (UGDA); Wysoczyzna Żarnowiecka, Pużyckie Łęgi nature reserve, 54°38'N 17°51'E, Circaeo-Alnetum, on Alnus glutinosa and wood of log, 12 Aug. 2015, M. Kukwa, 17032, 17040, 17053 (UGDA). PORTUGAL. Estremadura, W of Lisbon, WSW of Sintra, WSW of Linhó, along road to Malveira da Serra, nature park Quinta do Pisão, trail in forest with mainly Pinus trees, on Pinus, 38°45.50'N, 9°24.60'W, 160 m alt., 18 Oct. 2015, P. & B. van den Boom 53789 (hb v.d. Boom); W of Lisbon, S of Sintra, garden of National Palace de la Pena, mixed (mature) trees and shrubs, on tree-fern, 38°47.23'N, 9°23.42'W, 490 m alt., 20 Oct. 2015, P. & B. van den Boom 53907 (hb v.d. Boom). The Netherlands. Noord-Brabant, NE of Oirschot, Woekensesteeg, grid-ref. 51.23.23, trail in mixed forest, on wood of fallen trunk, 4 Oct. 2014, P. & B. van den Boom 51991 (hb v.d. Boom, hb Brand 67113); N of Oirschot, De Mortelen, grid-ref. 51.23.12, trail in damp mixed forest, on Fagus sylvatica, 5 June 2017, P. & B. van den Boom 56372 (hb v.d. Boom); E of Best, S side of Wilhelmina channel, grid-ref. 51.24.53, trail in Pinus forest, on Quercus rubra, 22 July 2018, P. & B. van den Boom 57647 (hb v.d. Boom); ENE of Oostelbeers, Oostelbeerse Heide, grid-ref. 51.32.34, forest, on Pseudotsuga, 26 May 2016, P. & B. van den Boom 55028 (hb v.d. Boom); NNW of Wintelre, S side of Straatsche Heide, grid-ref. 51.33.51, Pinus forest at edge of Calluna heathland with some Quercus robur trees, on Quercus robur, 14 April 2016, P. & B. van den Boom 54996 (hb v.d. Boom); W of Son, E of Nieuwe Heide, grid-ref. 51.24.45, E side of trail in Pinus forest, on Betula, 22 June 2014, P. & B. van den Boom 51315 (hb v.d. Boom); S of Best, Aarlesche Heide, S of high-way, grid-ref. 51.33.25, in Pinus forest, on Quercus rubra, 1 Nov. 2014, P. & B. van den Boom 52515 (hb v.d. Boom); S of Best, Aarlesche Heide, S of highway, grid ref. 51.34.21, grassy Calluna heathland, with scattered trees, on Quercus robur, 24 Jan. 2014, P. & B. van den Boom 50279 (hb v.d. Boom).
The Netherlands. Noord-Brabant, S of Best, Aarlesche Heide, S of highway, grid ref. 51.34.21, grassy Calluna heathland, with scattered trees, on Micarea microsorediata growing on Quercus robur, 24 Jan. 2014, P. & B. van den Boom 50278 (hb v.d. Boom).
The new species is morphologically similar to M. viridileprosa and M. soralifera, but those species differ in their contents of secondary lichen metabolites: M. viridileprosa contains gyrophoric acid, whereas M. soralifera produces micareic acid (
Micarea microsorediata produces methoxymicareic acid, a substance present in M. byssacea, M. nigra, M. pauli and other species of the M. micrococca clade (Fig.
Species characterised by the production of branched isidia, dark greyish to almost black apothecia containing Cinereorufa-green pigment and measuring 0.15–0.5 mm in diam., (0–)1-septate, narrowly ellipsoidal to clavate ascospores measuring 7.5–12 × (2.5–)3–4.5 µm and the production of methoxymicareic acid.
Portugal. Estremadura, W of Lisbon, W of Sintra, Park de la Monserrate, 200 m alt., 38°47.30'N, 9°25.07'W, parkland with mixed (mature) trees and shrubs, on fern tree, 15 Oct. 2015, P. & B. van den Boom 53726 (holotype LG; isotypes UGDA, hb v.d. Boom, mtSSU GenBank accession number: MK562029).
Thallus inconspicuous, thin, consisting of often branched and vertically proliferating fine isidia; prothallus not seen; areoles up to 0.1 mm in diam.; isidia developing from small areoles, vertically branched and coralloid, in some parts crowded and forming almost a continuous layer, but separated in younger parts of thalli, brownish-green, up to 500 μm tall and 30 μm wide, with a distinct and complete hyphal layer; apothecia abundant, adnate, flat to moderately convex, emarginate, 0.15–0.5 mm in diam., dark greyish to almost black, sometimes with a pale greyish rim; hymenium greenish, with pale brownish streaks, K–, C‒, 30–40 µm tall; epihymenium aeruginose greenish, with Cinereorufa-green pigment, K+ green intensifying; hypothecium hyaline; paraphyses sparse, branched, tips not widened and not pigmented, ca. 1.0‒1.5 µm wide; asci cylindrical to clavate, 24–28 × 9–12 µm, 8-spored; ascospores narrowly ellipsoid to clavate, 7.5–12 × (2.5–)3–4.5 µm, (0–)1-septate; micropycnidia inconspicuous, rare, 30–60 µm in diam., with dark brown top (K–, C‒); microconidia bacilliform, sometimes slightly curved, aseptate, 7–10 × 0.5–0.9 µm; crystalline granules (studied in polarised light) not visible in apothecium, but detected in isidia (sometimes isidia very abundant and sometimes very few), insoluble in K.
Photobiont micareoid, cells thin-walled, 4–8 µm in diam., clustered in compact masses.
Methoxymicareic acid detected by TLC. Cinereorufa-green in apothecia (epihymenium).
Abundantly present on a trunk of a fern tree in a parkland where many tropical and exotic fern and tree species have been introduced.
To date, it is only known from the type locality in Portugal (Sintra).
The epithet chosen for this species refers to its very dark appearance, the thallus being dark greenish and the apothecia mostly blackish.
This species is resolved in the M. micrococca group (Fig.
Micarea nigra resembles M. aeruginoprasina, M. isidioprasina and M. pauli. Micarea aeruginoprasina and M. isidioprasina differ in the presence of micareic acid instead of methoxymicareic acid and paler apothecia. In addition, M. aeruginoprasina produces different pigment in the apothecia (Sedifolia-grey). Micarea pauli differs in the production of methoxymicareic acid, Sedifolia-grey pigment in the apothecia and different distribution (see under that species).
Some morphs of M. prasina can also have dark apothecia, but this species contains micareic acid and Sedifolia-grey in the apothecia (
Species characterised by isidiate thallus, pale grey to grey beige apothecia with Sedifolia-grey pigment, 0–1-septate, ovoid, ellipsoidal or oblong ascospores measuring 7–13 × 3.5–4.5 μm and the presence of methoxymicareic acid.
Poland. Równina Bielska, Białowieża Forest, Białowieża National Park, forest section no 256, Carici elongatae-Alnetum, on Alnus glutinosa, 17 Aug 2015, M. Kukwa & 17240, A. Łubek (holotype UGDA; isotype KTC, mtSSU GenBank accession number: MK562014, Mcm7 GenBank accession number: MN105912).
Thallus crustose, indeterminate, endosubstratal to rarely episubstratal in non-isidiate parts and then as a thin greenish film over the substrate or minutely areolate, isidiate; prothallus not evident; areoles up to 0.1 mm in diam., green, soon developing isidia; isidia branched and coralloid, crowded and forming almost a continuous layer over the substrate, but separated in younger parts of thalli, green to olive green, up to 0.5 mm tall and 30 μm wide, with a distinct and complete hyphal layer; apothecia rarely developed (in 2 specimens only), beige with spots of grey pigment, pale grey to grey-beige, up to 0.5 mm in diam., irregular in shape, convex, with a white rim; excipulum as a narrow, hyaline zone, hyphae radiating, branched and anastomosing; hymenium up to 45 μm tall; epihymenium partly olive-grey due to the presence of Sedifolia-grey pigment (K+ violet, C+ violet) confined to the gel matrix; hypothecium hyaline to pale straw coloured in upper part; paraphyses 1–1.5 μm thick, sparse, mostly apically branched and anastomosing, hyaline throughout; asci cylindrical-clavate, 30–35 × 9–12 μm, 8-spored; ascospores, 0–1-septate, ovoid, ellipsoidal or oblong, 7–13 × 3.5–4.5 μm; pycnidia not seen; crystalline granules (studied in polarised light) abundant in hymenium and isidia, soluble in K.
Photobiont chlorococcoid, micareoid, cells globose to ellipsoidal, 4–7 μm in diam.
Methoxymicareic acid detected by TLC. Sedifolia-grey in apothecia (epihymenium).
This species is so far known only in Poland from Białowieża Forest, where it grows in deciduous forests on bark of Alnus glutinosa (5 specimens), Tilia cordata (1 specimen) and on wood (2 specimens).
The species is named after our friend, Paweł Czarnota, specialist in the genus who monographed it in Poland.
Poland. Równina Bielska, Białowieża Forest; Białowieża National Park, forest section no 256, Carici elongatae-Alnetum, on Alnus glutinosa and Picea abies, Aug. 2014, M. Kukwa 13194, 13330, 13345, A. Łubek (KTC, UGDA); ibidem, Tilio-Carpinetum, on Tilia cordata, Aug. 2014, M. Kukwa 14101, A. Łubek (KTC, UGDA); ibidem, Carici elongatae-Alnetum, on Alnus glutinosa, 17 Aug. 2015, M. Kukwa, 17227, A. Łubek (KTC, UGDA, hb v.d. Boom); ibidem, Querco-Piceetum, on Alnus glutinosa, 29 Sept. 2015, M. Kukwa 17544, A. Łubek (KTC, UGDA); ibidem, Peucedano-Pinetum, on Alnus glutinosa, March 2015, M. Kukwa 13308 (KTC, UGDA); Pino-Quercetum, on wood of snag, 1 Oct. 2015, M. Kukwa 17582a, A. Łubek (KTC); Pino-Quercetum, on wood of log, 2 Oct. 2015, M. Kukwa 17619, A. Łubek (KTC, UGDA); Carici elongatae-Alnetum, on Alnus glutinosa, 3 Oct. 2015, M. Kukwa 17621, A. Łubek (KTC, UGDA).
Micarea pauli is an isidiate species with Sedifolia-grey pigment in its apothecia. It can be separated from the similar M. isidioprasina, with which it grows in Białowieża Forest, by the presence of methoxymicareic acid.
Micarea aeruginoprasina and M. nigra are also similar in thallus morphology, but they differ in the pigmentation of apothecia. Micarea aeruginoprasina develops pale cream to pale brown or aeruginose apothecia, which are often mottled in colour in one apothecium, whereas in M. nigra the apothecia are dark greyish to black. Without apothecia, they can be difficult to separate from M. pauli, especially M. nigra which also contains methoxymicareic acid (M. aeruginoprasina produces micareic acid), but so far, M. aeruginoprasina and M. nigra are only known from the Azores and continental Portugal, respectively.
Methoxymicareic acid is the main secondary metabolite, also found in M. byssacea, M. micrococca and other species in the M. micrococca clade (Fig.
We thank Dr Jacek Pokrzywnicki (University of Gdańsk) for the help with the Latin name of Micarea pauli. Prof. Mark R.D. Seaward is acknowledged for language correction of the final version of the manuscript. We are grateful to Heidi Lee Andersen and the anonymous reviewer for their constructive comments that improved the manuscript. Martin Kukwa and Anna Łubek received funding for the research from the Polish-Norwegian Research Programme operated by the National Centre for Research and Development under the Norwegian Financial Mechanism 2009–2014 in the frame of Project Contract No Pol-Nor/196829/87/2013.
Conflict of interest: The authors declare that they have no conflict of interest.
Figures S1–S15 presenting ancestral character state reconstructions
Data type: PDF file
Tables S1–S4
Data type: PDF file
Explanation note: Additional tables with list of specimens used in this study, data matrices for ancestral character state reconstructions and probabilities of reconstructions with model Mk1.