Introduction
Candelariella Müll. Arg. is a common lichen genus that includes corticolous, terricolous and saxicolous species. The identification of many corticolous species has proved to be problematic, with one reason being that apothecia are often absent in many species, and these contain diagnostic characteristics used to separate the species (e.g. 8-spored vs multispored asci; Lendemer & Westberg Reference Lendemer and Westberg2010). Apothecia are especially scarce in sorediate Candelariella species. For a long time, the name Candelariella reflexa (Nyl.) Lettau was used for all sorediate Candelariella species growing on bark in large parts of the world, for example North America (Lendemer & Westberg Reference Lendemer and Westberg2010) and Europe (e.g. van Herk & Aptroot Reference van Herk and Aptroot2004; Smith et al. Reference Smith, Aptroot, Coppins, Fletcher, Gilbert, James and Wolseley2009; Wirth et al. Reference Wirth, Hauck and Schultz2013). Lendemer & Westberg (Reference Lendemer and Westberg2010) showed that the name C. reflexa should be used for a European species with relatively large, thick areoles and coarse soredia, and that in North America the sorediate species on bark include Candelariella efflorescens R. C. Harris & W. R. Buck (with multispored asci) and C. xanthostigmoides (Müll. Arg.) R. W. Rogers (with 8-spored asci).
Recently, it has also been demonstrated that in Europe at least three distinct sorediate corticolous species occur (Bomble Reference Bomble2015), namely C. reflexa and two species with fine soredia. However, it remains unclear whether the names C. xanthostigmoides (described from Australia) and C. efflorescens (described from North America) should be applied to the material with fine soredia. Fertile specimens of C. efflorescens have been reported from several European countries, including Sweden (Arup & Ekman Reference Arup and Ekman1992), Slovakia (Guttová & Palice Reference Guttová and Palice2004), Poland (Kubiak & Westberg Reference Kubiak and Westberg2011), Switzerland (Westberg & Clerc Reference Westberg and Clerc2012), the Czech Republic (Steinová et al. Reference Steinová, Bouda, Halda, Kukwa, Malíček, Müller, Palice, Peksa, Schiefelbein and Svoboda2013) and Romania (Malíček et al. Reference Malíček, Bouda, Liška, Palice and Peksa2015), based on a sorediate thallus in combination with multispored asci. Candelariella xanthostigmoides has been reported from the Czech Republic, Germany and Switzerland (Westberg & Clerc Reference Westberg and Clerc2012; Malíček & Palice Reference Malíček and Palice2013; Bomble Reference Bomble2015), and the name is provisionally applied in the British Isles (Cannon et al. Reference Cannon, Coppins, Orange, Sanderson and Simkin2021). Sterile specimens of corticolous Candelariella with fine soredia in Europe have been putatively assigned to Candelariella efflorescens agg. in several countries (e.g. Westberg & Clerc Reference Westberg and Clerc2012; Bomble Reference Bomble2015).
Several other sorediate corticolous species have been described, most of them recently. Candelariella sorediosa Poelt & Reddi was described from high altitudes (≥ 3500 m) in the Himalaya in Nepal (Poelt & Reddi Reference Poelt and Reddi1969), but since then has rarely been recorded and remains poorly understood (Lendemer & Westberg Reference Lendemer and Westberg2010). In addition, Candelariella flavosorediata Kalb & Aptroot (Kalb & Aptroot Reference Kalb and Aptroot2021) and C. makarevichiae S. Y. Kondr. et al. (Kondratyuk et al. Reference Kondratyuk, Lőkös, Halda, Farkas, Upreti, Thell, Woo, S-O and Hur2018) are poorly known species, and their descriptions have not been compared to C. xanthostigmoides, to which they seem most similar. Both C. flavosorediata and C. makarevichiae resemble C. xanthostigmoides in forming yellow squamules from which yellow soralia with fine soredia develop, and in having 8-spored asci (apothecia were observed only in C. flavosorediata and not in C. makarevichiae, the latter being described from a sterile specimen). An additional species, Candelariella subsquamulosa D. Liu & Hur, was described in 2019 (Liu et al. Reference Liu, Wang, Wang and Hur2019) but was considered a synonym of C. makarevichiae based on matching ITS nrDNA sequences (Kondratyuk et al. Reference Kondratyuk, Lőkös, Jeong, Oh, Kondratiuk and Hur2020). Candelariella rubrisoli D. Liu & Hur was described from China in 2019 and was morphologically compared to C. xanthostigmoides. However, this species also remains somewhat poorly understood since DNA sequences of C. xanthostigmoides were not yet available and the type material of C. rubrisoli lacked apothecia (Liu et al. Reference Liu, Wang, Wang and Hur2019). Lastly, Candelariella magellanica Etayo was described from Nothofagus on Navarino Island in southern Chile, differing from C. xanthostigmoides mainly by the larger and predominantly 1-septate ascospores (Etayo et al. Reference Etayo, Sancho, Gómez-Bolea, Søchting, Aguirre and Rozzi2021).
In addition to the truly sorediate corticolous species, there are also several species that form a granular or blastidiate thallus, and these are sometimes difficult to distinguish from the sorediate species. These include Candelariella boleana Etayo et al., C. faginea Nimis et al. and C. xanthostigma (Pers. ex Ach.) Lettau. Additionally, Candelaria pacifica M. Westb. & Arup normally a lobate subfruticose species, can be dissolved into an almost continuous sorediate crust and then resembles Candelariella species (Westberg & Arup Reference Westberg and Arup2010, Reference Westberg and Arup2011). Candelaria pacifica is not closely related to C. concolor (Dicks.) Stein, the type species of the genus Candelaria, but is instead more closely related to Candelariella species (e.g. Liu & Hur Reference Liu and J-S2018).
DNA analysis has the potential to test the validity and application of species names for the sorediate corticolous Candelariella species. The first phylogenetic analysis of Candelariaceae was published by Westberg et al. (Reference Westberg, Arup and Kärnefelt2007) based on ITS sequence data. Several contributions with new ITS data have been added since, many of which used ITS data to support the description of a new species (e.g. Westberg et al. Reference Westberg, Frödén and Wedin2009, Reference Westberg, Morse and Wedin2011; Westberg & Arup Reference Westberg and Arup2011; Liu et al. Reference Liu, Wang, Wang and Hur2019; Kondratyuk et al. Reference Kondratyuk, Lőkös, Jeong, Oh, Kondratiuk and Hur2020; Haughland et al. Reference Haughland, Hood, Thauvette, Toni, Cao, Birch and Shier2022; Halıcı et al. Reference Halıcı, Kahraman Yiğit, Bölükbaşı and Güllü2023). In a recent genome-level analysis, the Candelariaceae were placed in the Lichinomycetes (Díaz-Escandón et al. Reference Díaz-Escandón, Tagirdzhanova, Vanderpool, Allen, Aptroot, Češka, Hawksworth, Huereca, Knudsen and Kocourková2022).
In this study, we aimed to disentangle 1) how many sorediate corticolous Candelariella species occur in Europe, and 2) which names should be applied for those species. For this purpose, we studied morphology and molecular data (ITS nrDNA) of corticolous species of Candelariella, mostly from European specimens but supplemented by specimens from other continents. We show that worldwide there are currently five well-documented corticolous sorediate Candelariella species that are also supported by molecular data, four of which occur in Europe, and that there are three other species for which molecular data are currently lacking. We provide a full description of all five species for which molecular data are available, including a complete description of Candelariella reflexa s. str., the first description of the apothecial characteristics of C. rubrisoli and a description of Candelariella pulchella sp. nov.
Methods
Material collection and morphological study
To achieve the set goals, fresh lichen specimens and herbarium material from different countries and habitats were examined. Fresh specimens with a sorediate or granulate thallus were collected in Europe, mainly from the Netherlands and the Czech Republic, and stored in the private herbaria of the first and third authors. We attempted to sample a wide variety of habitats and substrata (i.e. from both open habitats and woodlands, lower and higher elevations, and from trunks and thinner branches). Additionally, herbarium specimens from UPS, LD and NYS were studied. Specimens were studied with dissecting microscopes (×7–45 magnification) and light microscopes (×40–1000 magnification). Microscopic slides were mounted and measurements performed in water, unless stated otherwise. To compare soredium diameter among specimens, 20 soredia were measured from standardized images of 24 specimens (five specimens of Candelariella efflorescens, five of C. pulchella, five of C. reflexa, seven of C. rubrisoli and two of C. xanthostigmoides). Images were captured with a Canon 1200D camera through a Raynox DCR 150 tube lens with a ×4 microscope lens.
Molecular methods
DNA sequences from the internal transcribed spacer (nuITS) region were generated for 46 specimens and uploaded to the GenBank database (Table 1). Different methodologies were used to obtain the sequences. For 25 specimens (54%), DNA was extracted and ITS sequences were generated by Alvalab in Spain (www.alvalab.es). Total DNA was extracted from dry specimens employing a modified protocol based on Murray & Thompson (Reference Murray and Thompson1980). PCR amplification reactions (Mullis & Faloona Reference Mullis and Faloona1987) included 35 cycles with an annealing temperature of 54 °C. The primers ITS1F and ITS4 (White et al. Reference White, Bruns, Lee and Taylor1990; Gardes & Bruns Reference Gardes and Bruns1993) were used to amplify the ITS region. PCR products were checked in 1% agarose gels and positive reactions were sequenced with one or both PCR primers. Chromatograms were checked, searching for any putative reading errors which were then corrected.
Table 1. nuITS DNA sequences of Candelariella newly generated during this study with specimen details (voucher, country and substratum) and GenBank Accession number. The Method column indicates which method was used to generate the sequence: A = see Methods in this paper; B = Wedin et al. (Reference Wedin, Westberg, Crewe, Tehler and Purvis2009); C = Svensson & Fryday (Reference Svensson and Fryday2022); D = Westberg et al. (Reference Westberg, Arup and Kärnefelt2007). Clades are indicated for specimens of Candelariella rubrisoli (A or B) and C. xanthostigma (A, B or C), corresponding to clades identified in Figs 1 and 2, respectively.
For 17 (37%) specimens, the methodology described by Wedin et al. (Reference Wedin, Westberg, Crewe, Tehler and Purvis2009) was followed, using the primers ITS1F and LR3 to amplify the ITS region. For three (7%) and one (2%) specimen(s) the methodologies described by Svensson & Fryday (Reference Svensson and Fryday2022) and by Westberg et al. (Reference Westberg, Arup and Kärnefelt2007) were followed, respectively, with both methods using the primers ITS1F and ITS4 to amplify the ITS region. See Table 1 for which method was used on which specimen.
Not all sequences obtained were from sorediate corticolous specimens; some species with a granular-areolate thallus (e.g. C. xanthostigma and Candelariella lutella (Vain.) Räsänen), a minutely foliose thallus (Candelaria pacifica) and some terricolous or saxicolous species (Candelariella coralliza (Nyl.) H. Magn. and C. granuliformis M. Westb.; Table 1) were also included.
Specimens of other species examined
Candelariella spp. Argentina: Rio Negro: Dept Bariloche, W side of Lake Los Juncos, open grazing land by gravel road, 900 m alt., 41.05835°S, 71.00927°W, 2009, M. Svensson 1712 (UPS L-1075681; GenBank Accession no.: PQ932181).—Italy: South Tyrol: Bozen, Sulden, on bark of Salix in park surrounding lake, 1900 m alt., 46.5273°N, 10.5858°E, 2023, H. van der Kolk 3897 (hb. van der Kolk; GenBank Accession no.: PQ932152).—Switzerland: Valais: Zeneggen, Eich, S–SE exposed, rocky, steppe grassland with forest fragments (Quercus pubescens), on Quercus, 1010–1030 m alt., 46.27306°N, 7.87528°E, 2010, M. Westberg 10-188 (S F178464; GenBank Accession no.: PQ932180); Pfynwald, Preissen, on fruit trees at the edge of the forest, 600 m alt., 46.31028°N, 7.61889°E, 2010, M. Westberg 10-200 (S F178475; GenBank Accession no.: PQ932182).
Candelaria pacifica. The Netherlands: Gelderland: Ede, on bark of roadside Quercus robur in village, 52.0416°N, 5.6892°E, 2021, H. van der Kolk 2182 (hb. van der Kolk; GenBank Accession no.: PQ932162).
Candelariella coralliza. Austria: Vorarlberg: Gargellen, on granite in mountain heathland, 2000 m alt., 46.9516°N, 9.8956°E, 2022, H. van der Kolk 3179 (hb. van der Kolk; GenBank Accession no.: PQ932153).
Candelariella granuliformis. Sweden: Lule lappmark: Padjelanta National Park, 11 km WNW of Staloluokta, N of the River Duvggejåhkå (Tukijåkkå, Tokijokk), S slope of Mt Unna Duvgge (Unna Toki, Unna Tuki), bare mountain region, 790 m ESE of small cabin, just above the river on bryophytes and plant debris on the ground, 651 m alt., 67.343812°N, 16.465457°E, 2020, M. Westberg PAD251 (UPS L-1049792; GenBank Accession no.: PQ932183); Padjelanta National Park, along the Nordkalott trail, on bryophytes on rock, 690 m alt., 67.2544°N, 16.6527°E, 2004, M. Westberg 3031 (LD; GenBank Accession no.: PQ932184).
Candelariella lutella. Italy: South Tyrol: Bozen, Prad, Pradersand, on bark of Populus at forest edge, 900 m alt., 46.6327°N, 10.6089°E, 2023, H. van der Kolk 3886 (hb. van der Kolk; GenBank Accession no.: PQ932159); Bozen, Sulden, on bark of Salix in park surrounding lake, 1900 m alt., 46.5272°N, 10.5862°E, 2023, H. van der Kolk 3890 (hb. van der Kolk; GenBank Accession no.: PQ932158).
Candelariella vitellina. The Netherlands: Gelderland: Dodewaard, Boelenhamsestraat, on bark of roadside Quercus robur, 51.9230°N, 5.6749°E, 2022, H. van der Kolk 2745 (hb. van der Kolk); Malden, Maas-Waal kanaal, on bark of Quercus robur trees alongside canal, 51.7747°N, 5.8471°E, 2022, H. van der Kolk 2781 (hb. van der Kolk).
Candelariella xanthostigma s. lat. Czech Republic: Southern Bohemia: Český Krumlov Distr., Šumava Protected Landscape Area, Černá v Pošumaví, Bližná, on bark of Populus canadensis, 770 m alt., 48.7239°N, 14.0958°E, 2012, J. Malíček 5066 (hb. Malíček; GenBank Accession no.: OQ717357). Western Moravia: Žďár nad Sázavou Distr., Žďárské vrchy Protected Landscape Area, Cikháj, old trees along road 0.5 km N of village, on bark of Acer platanoides, 700 m alt., 49.6519°N, 15.9678°E, 2012, J. Malíček 5125 (hb. Malíček; GenBank Accession no.: OQ717358).—The Netherlands: Gelderland: Doorwerth, on bark of roadside Quercus robur, 51.9754°N, 5.7739°E, 2021, H. van der Kolk 2548 (hb. van der Kolk; GenBank Accession no.: PQ932166); Doorwerth, on bark of roadside Quercus robur, 51.9755°N, 5.7746°E, 2021, H. van der Kolk 2549 (hb. van der Kolk; GenBank Accession no.: PQ932165); Groot Wolfswinkel, on branch of Salix alongside fen, 52.0806°N, 5.5257°E, 2022, H. van der Kolk 3168 (hb. van der Kolk).—Sweden: Torne lappmark: Jukkasjärvi par., Tornehamn, W end of Lake Torneträsk, on dry birch branches at lakeshore, 333 m alt., 68.4302°N, 18.6758°E, 10 viii 2011, M. Westberg s. n. (UPS L-1027544; GenBank Accession no.: PQ932186). Lule lappmark: Padjelanta National Park, at Lake Virihaure, on wood at lake shoreline, 580 m alt., 67.4117°N, 16.6419°E, 2004, M. Westberg 3088 (LD; GenBank Accession no.: PQ932187); Padjelanta National Park, at Lake Virihaure, on juniper wood at lake shoreline, 570 m alt., 67.4099°N, 16.6430°E, 2004, M. Westberg 3076 (LD; GenBank Accession no.: PQ932188). Östergötland: Västra Ny par., c. 1.7 km NNW of Medevi Brunn, on bark of Acer platanoides in avenue with large old trees, 92 m alt., 58.6865°N, 14.9767°E, 2011, M. Westberg 11-033 (UPS L-1027543; GenBank Accession no.: PQ932185). Gotland: Fleringe par., c. 200 m SE of Utoje, on dead branches of Quercus in mixed deciduous forest, 13 m alt., 57.8580°N, 18.8755°E, 13 v 2013, M. Westberg s. n. (UPS L-1027548; GenBank Accession no.: PQ932190).
Phylogenetic analysis
A dataset with ITS sequences was obtained, using the generated sequences and ITS sequences from Candelariaceae available on GenBank. We included all sequences of species of Candelariella in the analysis (i.e. also terricolous and saxicolous species), and additionally of some species of Candelaria A. Massal. (i.e. Candelaria fibrosoides M. Westb. & Frödén, C. fruticans Poelt & Oberw. and C. pacifica) and Candelina Poelt (Candelina mexicana (B. de Lesd.) Poelt and C. submexicana (de Lesd.) Poelt; see Supplementary Material Table S1, available online, for the complete list of sequences obtained from GenBank). To simplify the phylogenetic analysis, sequences from Candelaria s. str. (Candelaria concolor-group) and from Placomaronea Räsänen were not included, since none of the sequences of these species were similar to sequences of corticolous sorediate Candelariella specimens. Sequences from Candelariella blastidiata Yakovchenko and C. subdeflexa (Nyl.) Lettau were used as outgroup, following previous studies that showed the more distant phylogenetic position of these species compared to other species of Candelariella (Westberg et al. Reference Westberg, Arup and Kärnefelt2007; Halıcı et al. Reference Halıcı, Kahraman Yiğit, Bölükbaşı and Güllü2023).
All sequences were aligned using MUSCLE (Edgar Reference Edgar2004). A maximum likelihood (ML) analysis was then performed using RAxML v. 8.2.12 (Stamatakis Reference Stamatakis2014) in the software raxmlGUI 2.0 (Edler et al. Reference Edler, Klein, Antonelli and Silvestro2021), with 1000 ML bootstrap iterations (ML + thorough bootstrap) and the GTRGAMMA model. The layout of the phylogenetic trees was edited in FigTree v. 1.4.4 (Rambaut Reference Rambaut2014) and Inkscape (https://inkscape.org/). Internodes with ML-BS ≥ 70 were considered to be significant. In the resulting phylogeny, we checked which well-supported clades (ML bootstrap value > 70) included sorediate corticolous specimens. In the phylogenetic tree (Figs 1 & 2), clades that did not include any sorediate corticolous specimens were collapsed at species or species-group level to ease interpretation of the results (details of which sequence was included in which clade are provided in Table 1 and Supplementary Material Table S1).
Figure 1. Phylogeny based on ITS sequences of Candelariella (with Candelariella blastidiata and C. subdeflexa as outgroup) obtained using a RAxML analysis (Stamatakis Reference Stamatakis2014). Values indicate ML-BS values and are displayed only for well-supported branches with ML-BS values ≥ 70. Highlighted are the three sorediate corticolous Candelariella species, as well as the Candelariella s. str. clade that is displayed in Figure 2. The scale bar is proportional to the substitution rate. In brackets are the three-letter ISO country codes (for a selection of specimens) or the number of sequences collapsed under that branch (see Table 1 and Supplementary Material Table S1 (available online), for details). GenBank accessions of newly generated sequences are highlighted in bold. In colour online.
Figure 2. Phylogeny of Candelariella s. str. based on ITS sequences obtained using a RAxML analysis (Stamatakis Reference Stamatakis2014). Values indicate ML-BS values and are displayed only for well-supported branches with ML-BS values ≥ 70. See Fig. 1 for the position of Candelariella s. str. within the phylogeny of Candelariella s. lat. Highlighted are the two sorediate species, and a well-supported clade of non-sorediate specimens (Candelariella cf. lutella) that requires further study. The scale bar is proportional to the substitution rate. In brackets are the three-letter ISO country codes (for a selection of specimens) or the number of sequences collapsed under that branch (see Table 1 and Supplementary Material Table S1 (available online), for details). GenBank Accessions of newly generated sequences are highlighted in bold. In colour online.
Results
The dataset with ITS sequences used for the maximum likelihood analysis contained 518 aligned sites of 327 sequences (46 newly generated sequences and 281 sequences extracted from GenBank). The resulting molecular phylogeny for Candelariella is presented in Figs 1 and 2. Five well-supported clades (ML bootstrap values ≥ 70) were identified that included sorediate corticolous Candelariella species, and whose distinction from each other is supported by morphological data. Three clades represent taxa with 8-spored asci:
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1) Candelariella reflexa s. str., a species with prominent, thick areoles and coarse soredia, forms a highly supported monophyletic clade with sequences from the Czech Republic, the Netherlands, Norway and Sweden (Fig. 1).
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2) Candelariella rubrisoli forms a highly supported monophyletic clade, with two highly supported sister clades within (Fig. 1). Candelariella rubrisoli clade A contains the sequence of the holotype from China (GenBank Accession no. MG694273), as well as sequences from Austria, the Czech Republic, Denmark, the Netherlands and Sweden. Candelariella rubrisoli clade B contains four sequences from the Netherlands. We did not observe obvious differences in morphology between the two clades.
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3) Sequences of Candelariella xanthostigmoides from Argentina, Australia and the USA, together with sequences of C. subsquamulosa (synonym of C. makarevichiae; Kondratyuk et al. Reference Kondratyuk, Lőkös, Jeong, Oh, Kondratiuk and Hur2020), including its type sequence (GenBank Accession no. MG694274), form a highly supported monophyletic clade (Fig. 1).
Two clades represented sorediate corticolous Candelariella species with multispored asci. Both clades were placed inside the Candelariella s. str. clade which also contains C. vitellina (Hoffm.) Müll. Arg., the type species of the genus Candelariella (Fig. 2). The two sorediate species within this group are:
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1) Candelariella efflorescens forms a well-supported clade with specimens from Austria, the Czech Republic, the Netherlands, Sweden, Pakistan and the USA (Fig. 2). The specimens observed in this study had generally fine soredia that initially arise from small areoles but quickly form a continuous crust.
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2) Candelariella pulchella forms a well-supported clade that includes sequences from Austria, the Czech Republic, Sweden and Canada (Fig. 2). The specimens had coarser soredia compared to C. efflorescens (Fig. 3), and also differed in several other morphological characteristics (see notes under C. pulchella).
Figure 3. Diameter of soredia from 24 Candelariella specimens. Black circles show the means of each measured specimen (from each specimen 20 soredia were measured). Grey dots show all 480 individual measurements. Measurements of Candelariella rubrisoli are split for specimens belonging to clades A and B. Letters show significant differences between groups (linear model using specimen means as response variable; significance levels computed with post hoc Tukey test).
Taxonomy
Candelariella efflorescens R. C. Harris & W. R. Buck
Michigan Bot. 17, 155 (1978); type: USA, Michigan, Mackinac Co., edge of swamp across highway from Island Point State Forest Campground (Hog Island State Forest Campground), on Populus balsamifera, 5 October 1974, W. R. Buck (MICH—holotype; H—isotype).
Figure 4. Candelariella efflorescens. A, van der Kolk 2185. B, van der Kolk 2550. C, van der Kolk 3102, note the two apothecia immersed in the soredia. D, M. Westberg s. n. (UPS L-1027545). E, van der Kolk 3797. F, van der Kolk 3102. Scales: A–D = 1 mm; E & F = 0.5 mm. In colour online.
Figure 5. Candelariella efflorescens (van der Kolk 3102), microscopical characteristics. A & B, multispored asci. C, paraphyses tips. D–G, ascospores. Scales: A–G = 10 μm. In colour online.
Thallus areolate. Areoles scattered, green-yellow to yellow, generally very small and soon inapparent when dissolved and obscured by soredia, but rarely also larger and apparent, then up to 250 μm diam., sometimes slightly incised, ±120 μm thick, with a thin (10–15 μm thick) cortex consisting of 1–3 layers of ±isodiametric cells. Soralia emerging from the areoles, 150–400 μm diam., first convex, solitary and well delimited, but soon becoming confluent in well-developed material, and frequently forming large continuous sorediate crusts. Soredia 20–55 μm diam., yellow, composed of 10–15 photobiont cells irregularly surrounded by slightly elongated or somewhat sigmoid mycobiont cells often with a yellow-brownish pigment. Photobiont cells chlorococcoid, 7–14 μm diam.
Apothecia lecanorine, infrequent, scattered, 0.2–0.4 mm diam.; disc flat to somewhat convex, yellow-orange, concolorous with the thallus; proper margin macroscopically indistinct; thalline margin thin, to 60 μm thick, irregular, sometimes sorediate, sometimes excluded in older apothecia. Apothecial anatomy: thalline margin with an indistinct pseudocortex, 4–9 um thick, of thin-walled non-gelatinized hyphae with isodiametric cells. Proper margin up to 45 μm in the top part, consisting of radiating hyphae with rectangular cells, 7–15 × 1–2 μm, similar throughout. Hypothecium colourless. Epihymenium reddish yellow to yellow-brown. Hymenium colourless, 70–75 μm tall. Paraphyses simple or sometimes forked, septate, 1.5–2 μm wide, tips mostly cylindrical, sometimes clavate and up to 3 μm wide. Asci clavate, 24–30-spored, 50–55 × 18–23 μm. Ascospores often not well developed, simple or rarely with a thin septum, oblong to narrowly ellipsoid, 10–15 × 3.5–5.0 μm.
Ecology
On well-lit bark of deciduous and coniferous trees, especially in areas with nitrogen pollution.
Distribution
Widespread in temperate parts of North America and Europe. Also in Asia (Pakistan; as Candelariella xanthostigmoides in Iqbal et al. (Reference Iqbal, Ashraf, Asghar and Khalid2024)).
Notes
Candelariella efflorescens forms tiny, rounded areoles that soon dissolve into fine soredia. The species was originally described from Michigan (USA; Harris & Buck Reference Harris and Buck1978). Descriptions of this species are provided in Harris & Buck (Reference Harris and Buck1978), Westberg (Reference Westberg2007a) and Lendemer & Westberg (Reference Lendemer and Westberg2010). Both Candelariella efflorescens and C. pulchella belong to Candelariella s. str. and are related to C. xanthostigma and C. vitellina, sharing the yellow areolate thallus and multispored asci. Candelariella efflorescens and C. pulchella differ from the other corticolous sorediate Candelariella species in their multispored asci (Harris & Buck Reference Harris and Buck1978; Westberg Reference Westberg2007a).
The European specimens of Candelariella efflorescens resemble the specimens from North America in all aspects, and also match well with previous descriptions of C. efflorescens (Harris & Buck Reference Harris and Buck1978; Westberg Reference Westberg2007a; Lendemer & Westberg Reference Lendemer and Westberg2010) and pictures of the holotype material at the University of Michigan Herbarium (MICH). The European and American specimens share the small areoles that are first apparent but later dissolve into soredia, the relatively fine soredia and the relatively small apothecia that lack a macroscopically visible proper margin and have thin paraphyses tips.
Compared to the other sorediate corticolous species, Candelariella efflorescens shows more variation in soredium size between specimens (Fig. 3). Specimens that differed considerably in soredium size were collected from the same country (the Netherlands) and in a similar habitat (i.e. well-lit tree trunks of roadside Quercus robur trees). Thus, there seems to be no obvious relationship between soredium size and geography or substratum. The soredium size of Candelariella efflorescens shows some overlap with that of C. pulchella, and therefore other characteristics should always be studied to reliably identify a specimen. See the notes under C. pulchella for a detailed description of the differences between the two species.
Candelariella efflorescens is a widespread and probably common species in large parts of North America and Europe. However, the species rarely develops apothecia, and then it is hard to distinguish from the 8-spored sorediate species C. xanthostigmoides and C. rubrisoli. In the latter two species, the areoles are often more dominant and the soredia have less of a tendency to form a continuous crust. Furthermore, the areoles of C. rubrisoli tend to be more greenish and more scattered. All of these characteristics are, however, rather variable and seem to partially depend on the growth site. Sterile specimens can therefore often not be identified to species level with certainty, and may then be recorded under the aggregate name Candelariella efflorescens agg.
A previously published image of C. efflorescens from Poland (fig. 1 in Kubiak & Westberg (Reference Kubiak and Westberg2011)) is atypical in the presence of scattered and somewhat greenish areoles, and perhaps belongs to C. rubrisoli. Another, this time from Switzerland (fig. 4 in Westberg & Clerc (Reference Westberg and Clerc2012)), shows no visible areoles, no well-defined soralia and an apothecium with a clear proper margin, and therefore belongs to C. pulchella. Two sterile specimens reported as C. xanthostigmoides from Pakistan belong to C. efflorescens (GenBank Accession nos. OR133496 and OR133537; Iqbal et al. Reference Iqbal, Ashraf, Asghar and Khalid2024) based on the phylogenetic position of the ITS sequences.
The sequence with GenBank Accession number EF535203 was referred to as an 8-spored species by Westberg et al. (Reference Westberg, Arup and Kärnefelt2007), but its phylogenetic position indicates that it belongs to C. efflorescens, possibly as a result of a specimen mix-up or contamination. We are therefore not certain of the origin of this sequence.
Specimens examined
Austria: Steiermark: Graz, Schubertstrasse, on bark of roadside Aesculus in city, 380 m alt., 47.0809°N, 15.4568°E, 2022, H. van der Kolk 3102 (hb. van der Kolk; GenBank Accession no.: PQ932169).—Czech Republic: Southern Bohemia: Blanský les, Holubovské hadce Nature Reserve, S-facing slopes on serpentinite above Křemžský potok brook, on Frullania on serpentinite rock, 460–490 m alt., 48.8931°N, 14.3408°E, 2022, J. Malíček 16083 & H. Ghlimová (hb. Malíček; GenBank Accession no.: PQ499477).—The Netherlands: Gelderland: Doorwerth, on bark of roadside Quercus robur, 51.9755°N, 5.7743°E, 2021, H. van der Kolk 2550 (hb. van der Kolk; GenBank Accession no.: PQ932164); Epe, on bark of roadside Quercus robur, 52.3359°N, 5.9328°E, 2021, H. van der Kolk 2185 (hb. van der Kolk; GenBank Accession no.: PQ932161). Noord-Brabant: Uden, Karperdijk, on bark of roadside Quercus robur, 51.6636°N, 5.5874°E, 2023, H. van der Kolk 3797 (hb. van der Kolk; GenBank Accession no.: PQ932155).—Sweden: Östergötland: Vadstena par., Vadstena, churchyard, on bark of Ulmus glabra laying on the ground in churchyard, 102 m alt., 58.4512°N, 14.8931°E, 14 viii 2013, M. Westberg s. n. (UPS L-1027545; GenBank Accession no.: PQ932176).—USA: New York: Albany County, town of Chatham, Hand Hollow Preserve, on fallen Acer saccharum sapling in mixed hardwood forest, 289 m alt., 42.467222°N, 73.475833°W, 2024, J. C. Lendemer 83301 (NYS; GenBank Accession no.: PV230795); ibid., on fallen Pinus strobus branch in mixed hardwood forest, 289 m alt., 42.467222°N, 73.475833°W, 2024, J. C. Lendemer 83356 (NYS; GenBank Accession no.: PV230796).
Candelariella pulchella van der Kolk, M. Westb. & Malíček sp. nov.
MycoBank No.: MB 857949
Differing from Candelariella efflorescens in the smaller inapparent areoles, lack of well-delimited soralia, coarser soredia, larger apothecia with a persistent proper margin and broader paraphyses tips.
Type: Austria, Tirol, Heiterwang, Heiterwanger See, parking place at W edge of lake, on bark of Fraxinus excelsior, 980 m alt., 47.4550°N, 10.7589°E, 22 June 2014, J. Malíček 7004 (UPS L-1163134—holotype; PRA—isotype). GenBank Accession no.: PQ932160.
Figure 6. Candelariella pulchella sp. nov. A, E–K, holotype. B, Malíček 9130. C, Malíček 11867. D, M. Westberg s. n. (UPS L-1027546). E, apothecial margin. F & G, multispored asci. H & I, paraphyses tips. J & K, ascospores. Scales: A–D = 0.5 mm; E = 20 μm; F–K = 10 μm. In colour online.
(Fig. 6)
Thallus areolate. Areoles scattered, green-yellow to yellow, very small and almost immediately dissolved and obscured by soredia, up to 150 μm diam., ±75 μm thick. Soralia emerging from the areoles, not well delimited and soredia initially scattered, gradually becoming confluent in well-developed material, and sometimes eventually forming continuous sorediate crusts. Soredia 30–70 μm diam., yellow, composed of 10–20 photobiont cells irregularly surrounded by ellipsoid, slightly elongated or somewhat sigmoid mycobiont cells, outer cells covered with yellow-brown crystals. Photobiont cells chlorococcoid, 8–20 μm diam.
Apothecia lecanorine, scattered, 0.2–0.5 mm diam.; disc flat, yellow-orange, slightly darker than the thallus; thalline margin irregular, sometimes becoming sorediate, concolorous with the thallus, to 70 μm thick; proper margin often distinct, visible as a smooth, somewhat raised ring around the disc, later sometimes disappearing. Apothecial anatomy: thalline margin with an indistinct pseudocortex, 4–8 um thick. Proper margin up to 60 μm in the top part, consisting of radiating hyphae with rectangular cells, 7–12 × 2–3.5 μm, in the top part with ellipsoid cells, 5–7 × 3–4 μm. Hypothecium colourless. Epihymenium brown to yellow-brown, consisting of small crystals that do not dissolve in K. Hymenium colourless, 65–75 μm tall. Paraphyses simple or sometimes sparingly branched, septate, 2.0–2.5 μm wide, tips cylindrical to clavate, up to 4.5 μm wide. Asci clavate, 12–24(–32)-spored, 45–55 × 14–20 μm. Ascospores 1-celled, narrowly ellipsoid to ellipsoid, hyaline, straight or slightly curved, with rounded ends, often biguttulate, 10–14 × 4–5 μm, often smaller in asci with many ascospores.
Etymology
Referring to the beautiful appearance of the thallus and apothecia.
Ecology
So far known from the bark of deciduous solitary or roadside trees (e.g. Acer platanoides, Fraxinus, Populus) and from wood in the temperate and boreal zone. One specimen was collected in an old-growth scree forest. Candelariella pulchella is accompanied by Physcia sp., Phaeophyscia sp. and Physconia sp., suggesting that it prefers nutrient-rich substrata.
Distribution
Widespread in Europe, with confirmed records from Austria, the Czech Republic, Norway, Romania and Sweden. Also in Switzerland based on fig. 4 (as Candelariella efflorescens) in Westberg & Clerc (Reference Westberg and Clerc2012). Presumably also in North America (matching sequence of GenBank Accession no. ON682912; as Candelariella sp. in Haughland et al. (Reference Haughland, Hood, Thauvette, Toni, Cao, Birch and Shier2022)).
Notes
The new species Candelariella pulchella belongs to the Candelariella vitellina-group. The species in this group are characterized by a yellow areolate or sorediate thallus and multispored asci. Of the species within this group, C. pulchella is most similar to C. efflorescens. Candelariella pulchella differs from C. efflorescens most notably in the soralia that are not well defined and the relatively coarse soredia measuring 30–70 μm diam., the soralia in C. efflorescens (at least when young) being well defined and forming smaller soredia measuring 20–55 μm diam. Other characteristics of C. pulchella are the very small and almost always inapparent areoles (vs larger and sometimes more apparent areoles in C. efflorescens), the larger apothecia up to 0.5 mm diam. with a persistent proper margin (vs smaller apothecia up to 0.4 mm that often lack a visible proper margin in C. efflorescens) and the frequently clavate and broad paraphyses tips up to 4.5 μm (vs mostly cylindrical paraphyses tips up to 3.0 μm in C. efflorescens).
Other corticolous species in the Candelariella vitellina-group include C. boleana, C. faginea, C. lutella, C. vitellina and C. xanthostigma. All these species differ from C. pulchella by having a thallus that is areolate or granular and never truly sorediate. Candelariella vitellina is a species that grows on a wide variety of substrata, mostly on rocks but occasionally also on trees, especially on trunks in dusty places. It forms a more or less continuous crust of large areoles that have a more or less irregular outline. Candelariella lutella resembles C. vitellina but has smaller apothecia and smaller thallus areoles, and generally grows on twigs (Westberg & Sohrabi Reference Westberg and Sohrabi2012). Candelariella xanthostigma forms more or less rounded granules that are first scattered, but later form a continuous crust in which several granules can adhere to each other and form larger units. Candelariella boleana also has a thallus of scattered granules but differs from all other corticolous Candelariella species in having globose ascospores (Etayo et al. Reference Etayo, Palice and Spribille2009). Candelariella faginea forms areoles with an irregular outline that can eventually form granulose blastidia, and has asci with a variable (8–32) number of ascospores (Nimis et al. Reference Nimis, Poelt and Puntillo1989).
Sterile specimens of the 8-spored species Candelariella rubrisoli and C. xanthostigmoides differ from C. pulchella in the smaller soredia that are initially formed in discrete soralia and in the more prominent thallus areoles. Sterile specimens of C. reflexa differ from C. pulchella in the prominent, thick areoles and discrete, initially crateriform soralia.
Additional specimens examined
Czech Republic: Northern Moravia: Jeseníky Protected Landscape Area, Bělá pod Pradědem, on bark of roadside Acer platanoides, 620 m alt., 50.1361°N, 17.2111°E, 2012, J. Malíček 5144 (hb. Malíček; GenBank Accession no.: PQ932175). Southern Bohemia: Českomoravská vysočina highlands, Kamenice nad Lipou, group of trees before castle, on bark of Acer, 560 m alt., 49.3021°N, 15.0785°E, 2016, J. Malíček 9130 (hb. Malíček; GenBank Accession no.: OQ717775); Šumava Protected Landscape Area, Lenora, Kaplice, Zátoňská hora Nature Reserve, on bark of Acer platanoides in old-growth scree and mixed deciduous forest, 1034 m alt., 48.9447°N, 13.8297°E, 2018, J. Malíček 11867 (GenBank Accession no.: OQ717354).—Norway: Telemark: Vinje, Haukeli, rural area on S exp. slope above village, on Populus, alt. 650 m, 59.74°N, 7.56°E, 2002, L. B. Sparrius 7021 (hb. Sparrius).—Romania: Central Romania: Fagaras Mts, Sibiu, pasture in valley of Porumbacu River 3.5 km SE of Porumbacu de Sus, on bark, alt. 520 m, 45.6888°N, 24.5075°E, 2013, J. Malíček 6586, F. Bouda & L. Syrovátková (hb. Malíček, PRC).—Sweden: Dalarna: Svärdsjö par., Nysjö fäbodar, old pasture by Lake Nysjön, on old stump, 394 m alt., 60.9439°N, 15.9656°E, 21 iv 2012, M. Westberg s. n. (UPS L-1027547; GenBank Accession no.: PQ932172). Jämtland: Alsen par., Ede, 2.5 km SSE of Alsen church, on bark of Populus in garden, 346 m alt., 63.3642°N, 13.9579°E, 6 vi 2007, M. Westberg s. n. (UPS L-1027546; GenBank Accession no.: PQ932189).
Candelariella reflexa (Nyl.) Lettau
Hedwigia 52(3–4), 196 (1912).—Lecanora vitellina var. reflexa Nyl., Bull. Soc. Bot. France 13, 241 (1866).—Candelaria reflexa (Nyl.) Arnold, Flora, Regensburg 62, 397 (1879).—Caloplaca reflexa (Nyl.) Flagey, Revue Mycol., (Toulouse) 19(39), 130 (1888).—Lecanora reflexa (Nyl.) Nyl., Lich. Envir. Paris, 51 (1896).—Gyalolechia reflexa (Nyl.) Dalla Torre & Sarnth., Fl. Tirol. Vorarlberg, Liechstenstein 4, 200 (1902); type: France, Ile-de-France, Dep. Seine-et Marne, Fontainebleau, ad Robiniam, 1854, W. Nylander s. n. (H-NYL 29179—lectotype, designated (as ‘holotype’) by Hakulinen (Reference Hakulinen1954), p. 53).
Figure 7. Candelariella reflexa. A & B, van der Kolk 2536. C, van der Kolk 2976. D, van der Kolk 2759. E, van der Kolk 2779. Scales: A–D = 1 mm; E = 0.5 mm. In colour online.
Figure 8. Candelariella reflexa, apothecium characteristics. A, Malíček 11534. B, Malíček 16240. C, Malíček 11534, apothecial margin, note that the yellow-brown crystals do not belong inside the excipulum, but are a contamination originating from the excipulum edge. D, Malíček 16240, ascus with ascospores. E, Malíček 11534, paraphyses tips. Scales: A & B = 0.5 mm; C = 20 μm; D & E = 10 μm. In colour online.
Thallus areolate. Areoles abundant, well developed, aggregated, sometimes forming distinct rosettes, convex, often incised, sessile, greenish to yellow, up to 600 μm diam., ±130 μm thick, in section with a thin (5–15 μm thick) cortex consisting of 1–3 layers of ±isodiametric cells (4–8 μm), an algal layer (40–75 μm) with 3–5 layers of photobiont cells and a spongy medulla of a very loose layer of elongated hyphae (2.5–3.5 μm wide). Soralia forming in the middle of the areoles, 200–450 μm diam., initially crateriform, later convex, sometimes becoming confluent but almost never forming large sorediate crusts. Soredia 40–70 μm diam., green-yellow to yellow, composed of photobiont cells enclosed by 1–2 layers of mycobiont cells (3–6 μm thick) with some yellow-brownish pigment. Photobiont cells chlorococcoid, 10–20 μm diam. in the areoles, 5–8 μm diam. in the soredia.
Apothecia rare, clearly lecanorine only when young, later appearing biatorine, 0.3–1.3 mm diam.; disc first flat, soon convex and eventually undulate, yellow-orange to orange, often clearly darker coloured than the thallus; proper margin macroscopically inapparent; thalline margin green-yellow to yellow, concolorous with the thallus, irregular, incomplete, soon excluded. Apothecial anatomy: thalline margin ±100 μm thick, with a cortex of ±isodiametric cells. Proper margin up to 120 μm in the top part, consisting of radiating hyphae with rectangular cells, 4–16 × 2.5–4 μm. Hypothecium colourless, with oil droplets. Epihymenium a layer of yellow-brown granules, the granules sometimes interspersing downwards in between the paraphyses, not soluble in KOH. Hymenium colourless, 60–100 μm tall. Paraphyses simple or sometimes branched, septate, 2.2–3.5 μm wide, tips not or only slightly swollen. Asci clavate, 8-spored, 45–65 × 12–20 μm. Ascospores narrowly ellipsoid, simple or sometimes 1-septate, sometimes narrowing at one end, uniguttulate, sometimes biguttulate or with multiple small guttules, 12–18 × 4.5–6 μm.
Ecology
On well-lit or shaded tree trunks of deciduous trees (e.g. Populus, Quercus, Fraxinus and Fagus), rarely on coniferous trees (Pinus).
Distribution
Widespread in Europe (Austria, the Czech Republic, France, Germany, the Netherlands, Norway, Poland, Spain, Sweden, Ukraine).
Notes
This is the most distinct sorediate corticolous Candelariella species, and can be recognized by the large areoles, sometimes forming rosette-like thalli, the large-sized soredia and if present, large biatorine apothecia. The colour of the thallus is variable but often more greenish compared to the other sorediate species. This species is widespread in large parts of Europe on roadside trees and deciduous tree trunks in parks, but it is clearly rarer than C. efflorescens and C. rubrisoli. It is also known from more shaded habitats, such as beech or pine forests.
Although the species has a distinct morphology, the name has been widely misapplied in the past. Candelariella reflexa was first published by Nylander (Reference Nylander1866) and referred to as Lecanora vitellina var. reflexa, later Lecanora reflexa (Nylander Reference Nylander1896). The taxon was first placed in the genus Candelariella by Lettau (Reference Lettau1912). Later descriptions were incomplete or probably partially based on other sorediate species (e.g. Hakulinen Reference Hakulinen1954). The description and illustrations of the first record of Candelariella reflexa in Great Britain (James Reference James1971) and of early records in central Europe (Kalb Reference Kalb1976) seem to be based on material of C. rubrisoli. Subsequently, in many lichen floras the name Candelariella reflexa was misapplied to specimens with fine soredia. The confusion over C. reflexa was not surprising, since in many areas in Europe C. reflexa s. str. is much rarer than the species with small-sized soredia. Although the name Candelariella xanthostigmoides was available at the time, this species was described from Australia and therefore possibly not considered for European material. Other names including Candelariella rubrisoli were only published later. Additionally, early publications focused on distinguishing C. reflexa from C. xanthostigma and Candelaria concolor (James Reference James1971; Kalb Reference Kalb1976), and not on disentangling different sorediate Candelariella species. Also, in North America, early records of 8-spored sorediate specimens were first referred to as Candelariella reflexa (e.g. Ladd et al. Reference Ladd, Wilhelm and Harris1994) but later shown to belong to C. xanthostigmoides (Lendemer & Westberg Reference Lendemer and Westberg2010). The type material of Candelariella reflexa was studied by Westberg (Reference Westberg2007b), in which C. reflexa in the strict sense was shown to be a species with relatively large light yellow sometimes lobate areoles and relatively coarse soredia forming in often crateriform soralia, and having apothecia with 8-spored asci (Westberg Reference Westberg2007a), thereby showing that the name had thus been widely misapplied in America and Europe. Since then, several other publication have correctly separated C. reflexa from the other sorediate species (e.g. Bomble Reference Bomble2015; Kubiak Reference Kubiak2023).
Specimens examined
Austria: Niederösterreich: Ybbstaler Alpen Mts, Wildnisgebiet Dürrenstein, Lunz am See, Rothwald, Kleiner Urwald, on bryophytes on Fagus sylvatica in primeval beech-dominated forest, 1010 m alt., 47.7753°N, 15.1029°E, 2022, J. Malíček 16240 (hb. Malíček); ibid., Langau, Maierhöfen, few trees along road to Rothschildhaus, near building of Forstverwaltung Langau, on bark of Pyrus communis, 680 m alt., 47.8603°N, 15.1128°E, 2015, J. Malíček 8424, F. Berger, O. Breuss & R. Türk (hb. Malíček). Steiermark: Graz, Stadtpark, on bark of Robinia in park, 360 m alt., 47.0707°N, 15.4469°E, 2022, H. van der Kolk 3103.—Czech Republic: Eastern Moravia: Beskydy Mts, Malé Karlovice, Fraxinus at road in the village, on bark of Fraxinus excelsior, 605 m alt., 49.35016°N, 18.32692°E, 2020, J. Malíček 14206 & E. Konečná (hb. Malíček). Southern Bohemia: Blanský les, Holubovské hadce Nature Reserve, S-facing slopes on serpentinite above Křemžský potok brook, on Frullania on serpentinite rock, 465 m alt., 48.89249°N, 14.34318°E, 2022, J. Malíček 15669 (hb. Malíček).—The Netherlands: Gelderland: Zeddam, N335, on bark of roadside Quercus robur, 51.8977°N, 6.2934°E, 2022, H. van der Kolk 2976 (hb. van der Kolk); Gendringen, on bark of roadside Quercus robur, 51.8726°N, 6.3567°E, 2021, H. van der Kolk 2536 (hb. van der Kolk; GenBank Accession no.: PQ932167).; De Horst, Plak, on bark of roadside Quercus robur, 51.7688°N, 5.9871°E, 2022, H. van der Kolk 2779 (hb. van der Kolk); Groesbeek, on bark of roadside Quercus robur, 51.7856°N, 5.9317°E, 2022, H. van der Kolk 2759 (hb. van der Kolk). Noord-Holland: Purmerend, on bark of Populus in park, 52.5044°N, 4.9535°E, 2015, L. B. Sparrius 9182 (hb. Sparrius; GenBank Accession no.: PQ932168).—Spain: Andalusia: Sierra de las Nieves National Park, Ronda, tourist path near Refugio Los Quejigales, on bark of Abies pinsapo in old-growth forest with Abies pinsapo, 1410 m alt., 36.6928°N, 5.0369°W, 2017, J. Malíček 11534 (hb. Malíček).—Ukraine: Zakarpattia: East Carpathians, Khust, Velyka Uhol’ka, on Fraxinus excelsior in mixed deciduous old-growth forest, 820 m alt., 48.255833°N, 23.661111°E, 2015, J. Malíček 8193 (hb. Malíček).
Candelariella rubrisoli D. Liu & Hur
Mycobiology 47, 41 (2019).—Opeltiella rubrisoli (D. Liu & Hur) S. Y. Kondr., Acta Bot. Hung. 62, 304 (2020); type: China, Yunnan Province, Kunming City, Dongchuan District, Huagou Village, on Pinus armandi, 25.92306°N, 103.08417°E, alt. 2403 m, 3 August 2017, J.-S. Hur & D. Liu CH170039 (KoLRI—holotype).
Figure 9. Candelariella rubrisoli. A, van der Kolk 2914 (clade B). B, van der Kolk 3314 (clade unknown), with the pink lichenicolous fungus Illosporiopsis christiansenii. C, van der Kolk 3314 (clade unknown). D, van der Kolk 2707 (clade B). E, van der Kolk 3547 (clade B). F, van der Kolk 3569 (clade A). G, van der Kolk 3100 (clade A). Scales: A, B & D = 1 mm; C, E–G = 0.5 mm. In colour online.
Figure 10. Candelariella rubrisoli, microscopical characteristics. A, van der Kolk 2914 (clade A), section of apothecium. B, van der Kolk 3314, asci with ascospores (clade unknown). C, van der Kolk 2914 (clade A), close-up of paraphyses tips and ascus with ascospores. D, van der Kolk 2707 (clade A), soredia. Scales: A, B & D = 20 μm; C = 10 μm. In colour online.
Thallus areolate. Areoles often abundant, scattered or sometimes aggregated, often well developed and somewhat lobate, green to green-yellow, often somewhat ascending (subsquamulose), up to 350 μm diam., with a thin (around 10 μm thick) cortex consisting of 1–2 layers of ±isodiametric cells. Soralia emerging from the areoles, 150–350 μm diam., first convex, solitary and well delimited, becoming confluent in older well-developed material, but generally not forming large continuous sorediate crusts. Soredia 20–50 μm diam., yellow, composed of 5–15 photobiont cells surrounded by slightly elongated or somewhat sigmoid mycobiont cells often with a yellow-brownish pigment. Photobiont cells chlorococcoid, 6–15 μm diam.
Apothecia lecanorine, infrequent, scattered, 0.3–0.5 mm diam.; disc flat, yellow-orange, darker coloured than the thallus; proper margin smooth, entire, yellow; thalline margin thin, green-yellow to yellow, irregular, excluded in older apothecia. Apothecial anatomy: thalline margin ±60 μm thick, with an indistinct cortex of ±isodiametric cells. Proper margin generally well developed, 40–60 μm in the top part, consisting of radiating hyphae with rectangular cells in the bottom part, 6–12 × 2–3 μm, and with ±rounded cells in the upper part. Hypothecium colourless. Epihymenium yellow-brown. Hymenium colourless, 65–75 μm tall. Paraphyses simple or sometimes branched, septate, 2.0–3.0 μm wide, tips not or only slightly swollen. Asci clavate, (4–)8-spored, 50–65 × 13–20 μm. Ascospores simple or rarely with a thin septum, multiguttulate, narrowly ellipsoid, sometimes narrowing at one end, 11–18 × 5.5–7.0 μm.
Ecology
On trunks and twigs of a wide range of tree species in open landscapes and forests. Well-developed fertile specimens were all found on half-shaded tree bark (most often on Salix) in humid conditions in deciduous forests. Sterile specimens (confirmed by DNA sequences) were also collected from more open habitats.
Distribution
Asia (China), Europe (Austria, the Czech Republic, Denmark, Germany, the Netherlands, Sweden, probably also Switzerland and Ukraine).
Notes
Candelariella rubrisoli was described from China as a species forming greenish or green-yellowish subsquamulose areoles that dissolve into soredia (Liu et al. Reference Liu, Wang, Wang and Hur2019). The original description of Liu et al. (Reference Liu, Wang, Wang and Hur2019) is complemented here with a description of the apothecia, since those were lacking in the type material.
In Europe, Candelariella rubrisoli is a widespread species for which the name C. xanthostigmoides has been used in the past. At the moment, we do not have any evidence that C. xanthostigmoides occurs in Europe, and propose to henceforth use the name C. rubrisoli for European sorediate corticolous Candelariella species with 8-spored asci and fine soredia. In support, pictures of C. xanthostigmoides from Germany (Bomble Reference Bomble2015) show typical thalli of C. rubrisoli with abundant green-yellow areoles. Additionally, records of this species from the Czech Republic (e.g. Malíček & Palice Reference Malíček and Palice2013) belong to C. rubrisoli based on the phylogenetic analysis. The records of C. xanthostigmoides in Switzerland (Westberg & Clerc Reference Westberg and Clerc2012) and Ukraine (Khodosovtsev & Darmostuk Reference Khodosovtsev and Darmostuk2020) probably also belong to C. rubrisoli. Lastly, the image of C. efflorescens in Kubiak & Westberg (Reference Kubiak and Westberg2011) also resembles C. rubrisoli.
The morphology of the specimens that we have studied matches the description of C. rubrisoli provided by Liu et al. (Reference Liu, Wang, Wang and Hur2019), except for the size of the soredia which we found to be smaller than in the protologue (20–50 μm vs 40–70 μm in Liu et al. (Reference Liu, Wang, Wang and Hur2019)). All specimens that we measured, from both phylogenetic clades of C. rubrisoli, had an average soredium diameter of under 40 μm (Fig. 3).
Fertile specimens of this species are almost always found in somewhat shaded and humid habitats, as is the case in the Netherlands, Switzerland (Westberg & Clerc Reference Westberg and Clerc2012) and Germany (Bomble Reference Bomble2015). However, the phylogenetic analysis revealed that well-developed sterile specimens can also grow on more open and exposed tree bark. Candelariella rubrisoli is variable in the colour, size and abundance of areoles. Specimens from exposed tree bark typically tend to be more richly covered with soredia and have a brighter yellow colour (e.g. compare Fig. 9E & G from open bark vs Fig. 9C & D from shaded bark). Sterile specimens in Europe from shaded habitats with abundant green areoles can be recognized in the field, but sterile specimens with less conspicuous areoles from more open habitats cannot always be reliably separated from C. efflorescens.
The phylogenetic analysis shows that the specimens sequenced are divided into at least two distinct clades. Clade A represents Candelariella rubrisoli s. str. and contains sequences from a variety of countries as well as the sequence obtained from the holotype, whereas clade B contains only four sequences from the Netherlands. The sequence from the holotype (GenBank Accession MG694273) is 97.7–98.7% similar to the sequences of other specimens currently known to belong to clade A, and 93.3–94.6% to the four sequences in clade B. Such relatively large differences between the ITS sequences suggests that multiple cryptic species are possibly involved. However, the abovementioned variation in morphological characteristics was observed in both clades and there were no obvious differences in morphology or ecology between specimens belonging to the different clades (e.g. compare specimens of clade A in Fig. 9F & G with specimens of clade B in Fig. 9D & E). Given this lack of morphological distinction and given that all sequences form a monophyletic group, we prefer to adopt a broad concept of the species Candelariella rubrisoli. We recommend that future studies of this species include more specimens from outside Europe and aim to sequence multiple DNA markers to obtain a better understanding of whether multiple species could be involved.
Specimens examined
Clade A: Austria: Steiermark: Judenburg, Seetaler Alpen, on bark of Salix at forest edge, 1800 m alt., 47.0943°N, 14.5679°E, 2022, H. van der Kolk 3100 (hb. van der Kolk; GenBank Accession no.: PQ932154).—Czech Republic: Eastern Bohemia: Železné hory Protected Landscape Area, Trhová Kamenice, 1 km W of Bystřice, on bark of Acer pseudoplatanus in a fragment of mixed forest, 560 m alt., 49.8155°N, 15.8602°E, 2020, J. Malíček 13948 (hb. Malíček; GenBank Accession no.: OQ717778). Western Bohemia: Kdyně, Smržovice, E slope of Zadní kopec hill, on bark of Fagus sylvatica, 650 m alt., 49.3805°N, 13.0939°E, 2010, J. Malíček 2937 (hb. Malíček; GenBank Accession no.: PQ932173). Central Bohemia: Sedlčany region, Týnčany, on bark of Salix caprea, 510 m alt., 49.5850°N, 14.3394°E, 2011, J. Malíček 4014 (hb. Malíček; GenBank Accession no.: PQ932174).—Denmark: Fyn: Svendborg, Bregninge Bakker, on a branch of a large oak, 55.0233°N, 10.6091°E, 13 iv 2024, M. Westberg s. n. (UPS L-1131173; GenBank Accession no.: PQ932192).—The Netherlands: Gelderland: Wageningen, Wageningse Bovenpolder, on bark of Salix at willow forest edge, 51.9600°N, 5.6633°E, 2021, H. van der Kolk 2551 (hb. van der Kolk; GenBank Accession no.: PQ932163); Ede, Noord Ginkel, on bark of Betula at forest edge, 52.0451°N, 5.7529°E, 2023, H. van der Kolk 3569 (hb. van der Kolk; GenBank Accession no.: PQ932151).—Sweden: Skåne: Lund, along Trollebergsvägen, c. 70 m SW of St Peter’s Priory church, on bark of young, planted Acer platanoides, 40 m alt., 55.7038°N, 13.1850°E, 28 xi 2023, M. Westberg s. n. (UPS L-1101103; GenBank Accession no.: PQ932191).
Clade B: The Netherlands: Zuid-Holland: Gorinchem, Linge, on bark of Salix in willow forest, 51.849°N, 4.999°E, 2022, H. van der Kolk 2707 (hb. van der Kolk; GenBank Accession no.: PQ932171); Veermansplaat, on bark of Salix in thickets with willows and sea-buckthorns, 51.7526°N, 3.9819°E, 2022, H. van der Kolk 2914 (hb. van der Kolk; GenBank Accession no.: PQ932170). Gelderland: Hoge Veluwe, on bark of Pinus in heathland, 52.0687°N, 5.8624°E, 2023, H. van der Kolk 3462 (hb. van der Kolk; GenBank Accession no.: PQ932156); Ede, Zuid Ginkel, on bark of Betula in birch forest, 52.0229°N, 5.7430°E, 2023, H. van der Kolk 3547 (hb. van der Kolk; GenBank Accession no.: PQ932157).
Clade unknown: The Netherlands: Limburg: Tegelen, Holtmühle, on shaded horizontal branch of Salix at the edge of mixed forest, 51.3234°N, 6.1595°E, 2022, H. van der Kolk 3314 (hb. van der Kolk).
Candelariella xanthostigmoides (Müll. Arg.) R. W. Rogers
Muelleria 5(1), 32 (1982).—Lecanora xanthostigmoides Müll. Arg., Flora, Regensburg 65, 484 (1882).—Candelaria xanthostigmoides (Müll. Arg.) Müll. Arg., Bull. Herb. Boissier 1(2), 33 (1893).—Placodium xanthostigmoides (Müll. Arg.) Gyeln., Rev. Bryol. Lichénol. 6, 205 (1933); type: Australia, New South Wales, Parramatta, W. Woolls (G 00290997—lectotype, designated by Rogers (Reference Rogers1982), p. 32).
Candelariella makarevichiae S. Y. Kondr., L. Lőkös & J.-S. Hur, in Kondratyuk et al., Abstr. Bot. (Budapest) 60, 134 (2018).—Candelinella makarevichiae (S. Y. Kondr., L. Lőkös & J.-S. Hur) S. Y. Kondr., in Kondratyuk et al., Acta Bot. Hung. 62, 304 (2020); type: Republic of Korea, Gangwon-do, Sacheon-myeon, Gadunji-gill, small pine plantation along the seashore road (Halanro) at Sacheon Beach, on bark of Pinus densiflora and P. thunbergii, 37°49′43.21″N, 128°52′41.50″E, alt. c. 5 m, 9 July 2015, S. Y. Kondratyuk & L. Lőkös 150828 (KoLRI 034061/A—holotype; KoLRI 034061/B, KoLRI 034062—isotypes).
Candelariella subsquamulosa D. Liu & J.-S. Hur, in Liu et al., Mycobiology 47, 44 (2019); type: South Korea, Jeollanam-do, Suncheon-si, Jungang-ro 225, Sunchon National University, on Cerasus sp., 34°58′01″N, 127°28′48″E, alt. 47 m, 26 September 2017, D. Liu 171419 (KoLRI—holotype).
Figure 11. Candelariella xanthostigmoides. A, J. C. Lendemer 83066 (NYS). B, F. B. Bungartz & F. Ziemmeck 3002 (ASU). C, Svensson 1803 (UPS L-1075676). D, F. B. Bungartz & F. Ziemmeck 3002 (ASU). Scales: A–D = 0.5 mm. In colour online.
(Fig. 11)
Thallus areolate. Areoles scattered or sometimes aggregated, larger areoles often somewhat incised, green-yellow to yellow, sometimes slightly ascending, up to 300 μm. Soralia emerging from the areoles, 150–350 μm diam., first convex, solitary and well delimited, becoming confluent in older well-developed material, but generally not forming large continuous sorediate crusts. Soredia 25–45 μm diam., yellow, composed of photobiont cells surrounded by slightly elongated and somewhat irregular mycobiont cells with often a yellow-brownish pigment. Photobiont cells chlorococcoid, 8–13 μm diam.
Apothecia lecanorine, infrequent, scattered, 0.2–0.4 mm; disc flat, yellow-orange, darker coloured than the thallus; proper margin smooth, thin, entire, yellow; thalline margin thin, yellow, sometimes irregular, sometimes excluded in older apothecia. Apothecial anatomy not studied and incompletely reported in the literature. Hymenium colourless, up to 70 μm tall. Asci 8-spored, 30–35 × 12.5–15 μm. Ascospores simple, long-ellipsoid, straight or curved, sometimes narrowing at one end, 11–16 × 4–6 μm.
Ecology
Most frequent on bark and branches of deciduous trees, but sometimes also on coniferous trees, wood or stone (Lendemer & Westberg Reference Lendemer and Westberg2010).
Distribution
Australia, Asia (Japan, South Korea), North America and South America (Argentina).
Notes
Candelariella xanthostigmoides is a species with 8-spored asci which was described from tree bark in Australia (Müller Reference Müller1882; Rogers Reference Rogers1982). It is the most common sorediate corticolous species in Australia (Filson Reference Filson, George, Grgurinovic, Thompson and Mowatt1992). Many old records of Candelariella reflexa in North America belong to this species (Lendemer & Westberg Reference Lendemer and Westberg2010). Most, if not all, European citations of C. xanthostigmoides refer to C. rubrisoli (see notes under that species).
Typical forms of C. xanthostigmoides have deep yellow areoles that dissolve into bright yellow soredia (Filson Reference Filson, George, Grgurinovic, Thompson and Mowatt1992; Lendemer & Westberg Reference Lendemer and Westberg2010). This is apparent in descriptions of American (fig. 4 in Lendemer & Westberg (Reference Lendemer and Westberg2010)) and Asian material (Kondratyuk et al. Reference Kondratyuk, Lőkös, Halda, Farkas, Upreti, Thell, Woo, S-O and Hur2018; Yakovchenko et al. Reference Yakovchenko, Davydov and Ohmura2022). However, as in the other species, C. xanthostigmoides also shows variability in the colour (sometimes being greenish) and size (sometimes being small and quickly obscured by soredia) of the areoles. There is some variation in the ITS sequence between different specimens of C. xanthostigmoides and C. subsquamulosa, which may reflect the geographical distribution over different continents (Fig. 1). However, given the lack of morphological characteristics between specimens from different continents, and since all sequences form a monophyletic group, we prefer to adopt a broad concept of C. xanthostigmoides. We therefore consider C. makarevichiae and C. subsquamulosa synonyms of C. xanthostigmoides. Candelariella makarevichiae was described based on sterile specimens from South Korea. It forms scattered yellowish areoles that dissolve into soredia, and was shown to be phylogenetically distant from C. reflexa and C. efflorescens; however, it was not compared to C. xanthostigmoides in the protologue (Kondratyuk et al. Reference Kondratyuk, Lőkös, Halda, Farkas, Upreti, Thell, Woo, S-O and Hur2018). Almost at the same time, C. subsquamulosa was described, also from sterile specimens from South Korea. It was described as differing from C. xanthostigmoides in the thallus areoles not dissolving into soredia and the soredia randomly spreading, whereas in C. xanthostigmoides soredia generally start at the margin of areoles and spread inwards (Liu et al. Reference Liu, Wang, Wang and Hur2019). However, the description of C. subsquamulosa fits within the morphological variation of C. xanthostigmoides. Candelariella subsquamulosa was previously synonymized with C. makarevichiae based on ITS sequences by Kondratyuk et al. (Reference Kondratyuk, Lőkös, Jeong, Oh, Kondratiuk and Hur2020). Unfortunately, the ITS sequences of C. makarevichiae have not been uploaded to GenBank, and therefore only sequences of C. subsquamulosa were included in our phylogenetic analysis. We recommend that future studies of this species include a larger number of specimens and analyze sequence data for multiple loci.
Specimens examined
Argentina: Buenos Aires: Tornquist partido, 5 km W of Villa Ventana, Tornquist Provincial Park, on lignum of Pinus halepensis in open plantation, 500 m alt., 38.05609°S, 61.97729°W, 2009, Svensson 1803 (UPS L-1075676; GenBank Accession no.: PQ932179).—Australia: Western Australia: Perth Area, SW of Northam, along Highway 94, 60 km NE of Perth, on roadside trees of Eucalyptus, near pipeline, 31.68611°S, 116.59528°E, 1997, Kärnefelt 971704 (LD 1490610; GenBank Accession no.: PQ932177).—USA: Connecticut: Windham County, Mansfield Hollow State Park, on bark of Quercus velutina in young deciduous forest along stream, 41.7569°N, 72.1808°W, 2002, F. B. Bungartz & F. Ziemmeck 3002 (ASU; GenBank Accession no.: PQ932178). New Jersey: Monmouth County, Middletown Township, Gateway National Recreation Area, on Juniperus branch in maritime hardwood forest, 40.429328°N, 73.989115°W, 2024, J. C. Lendemer 83027 (NYS; GenBank Accession no.: PV230797); ibid., on Prunus maritima in disturbed dunes with forests and thickets, 40.471413°N, 74.004489°W, 2024, J. C. Lendemer 83066 (NYS; GenBank Accession no.: PV230798).
Species without molecular data
Candelariella flavosorediata Kalb & Aptroot
Archive for Lichenology 28, 2 (2021); type: Réunion, Piton Maïdo, just below summit, on tree bark in scrub, 21°03′S, 55°23′E, alt. c. 2100 m, 30 August 1991, K. Kalb & A. Kalb 26141 (B—holotype).
Notes
Candelariella flavosorediata was described from tree bark on Réunion, an island in the Indian Ocean. It forms yellow areoles (100–300 μm) from which yellow soredia of ±25 μm diam. develop, and has 8-spored asci with relatively large ascospores (Kalb & Aptroot Reference Kalb and Aptroot2021). Molecular data are not available. The species was not compared with C. xanthostigmoides, but it may differ from that species in having larger ascospores. Currently C. flavosorediata has been reported only from the type locality and we have not seen the material.
Candelariella magellanica Etayo
Anales del Instituto de la Patagonia 49, 29 (2021); type: Chile, Navarino, Ukika, track from Bulnes to the south, fertile thalli on Nothofagus pumilio bark, alt. c. 50 m, 21 January 2008, J. Etayo 24541 & A. Gómez-Bolea (MAF-Lich—holotype; hb. Etayo—isotype).
Notes
Candelariella magellanica resembles C. xanthostigmoides, but differs mainly in having larger ascospores that are predominantly 1-septate. Molecular data are not available. The species is currently known only from Navarino Island in southern Chile (Etayo et al. Reference Etayo, Sancho, Gómez-Bolea, Søchting, Aguirre and Rozzi2021).
Candelariella sorediosa Poelt & Reddi
Khumbu Himal 6, 10 (1969); type: Nepal, Khumbu, east of Khumzung against the Dudh-Kosi-Tal, on Juniperus recurva in open Abies-Rhododendron forest, alt. 3700-3800 m, Poelt L434 (M—holotype).
Notes
Candelariella sorediosa was described from high altitudes (≥ 3500 m) in the Himalayas in Nepal (Poelt & Reddi Reference Poelt and Reddi1969). It forms tiny, yellow, sometimes subsquamulose areoles (200–500 μm) with a sorediate margin and apothecia with 8-spored asci (Poelt & Reddi Reference Poelt and Reddi1969). Unfortunately, the type material is sparse and molecular data are not available (Lendemer & Westberg Reference Lendemer and Westberg2010). Poelt & Vězda (Reference Poelt and Vězda1977) included C. sorediosa in their identification key for European lichens (referring to the occurrence of the species in the Alps). The name was also used for sorediate specimens from Asia (Aptroot & Sparrius Reference Aptroot and Sparrius2003) and South America (Sipman Reference Sipman, Balslev and Luteyn1992). We have not seen any material identified as C. sorediosa, and it remains unclear if and how this species differs from C. xanthostigmoides and C. rubrisoli.
Identification key to the sorediate corticolous Candelariella species
The key presented here is based on typical specimens and focuses on sterile characteristics, since apothecia are rare in most species. However, note that sterile specimens can often not be identified with certainty; in particular, sterile specimens of Candelariella efflorescens, C. rubrisoli and C. xanthostigmoides are often hard to distinguish. All species are variable in the abundance, aggregation and colour of the areoles. Typically, the colour of the areoles is more greenish in C. rubrisoli and more yellowish in C. xanthostigmoides, but this characteristic is not completely reliable since the pigmentation seems to vary with the amount of shade. However, the two species are as far as we know now largely geographically separated, except in Asia where their distribution overlaps. In all species there is also variability in the soredium size, causing some overlap even between C. efflorescens and C. pulchella (Fig. 3); nevertheless, soredium size is one of the most useful characteristics to separate sterile specimens based on morphological characteristics. Figure 12 presents a same-scale comparison of all treated species. The key includes only truly sorediate species. Species with a blastidiate or granular thallus (e.g. C. xanthostigma) that could also resemble the sorediate species are not treated in the key, but some are depicted in Fig. 12.
Figure 12. Same-scale comparison of thalli of specimens of various granular or sorediate corticolous species. A, Candelariella vitellina (van der Kolk 2781). B, Candelariella vitellina (van der Kolk 2745). C, Candelariella xanthostigma s. lat. (clade ‘Candelariella xanthostigma s. lat. B’ in Fig. 2) (van der Kolk 2549). D, Candelariella xanthostigma s. lat. (clade ‘Candelariella xanthostigma s. lat. B’ in Fig. 2) (van der Kolk 3168). E, Candelariella xanthostigma s. lat. (clade ‘Candelariella xanthostigma s. lat. A’ in Fig. 2) (M. Westberg 3076; LD), intermediate morph between C. pulchella and typical C. xanthostigma that requires further study. F, Candelariella reflexa (van der Kolk 3103). G, Candelariella pulchella (M. Westberg s. n.; UPS L-1027546). H, Candelariella pulchella (M. Westberg s. n.; UPS L-1027547). I, Candelariella efflorescens (van der Kolk 2550). J, Candelariella xanthostigmoides (F. B. Bungartz & F. Ziemmeck 3002; ASU). K, Candelariella rubrisoli (van der Kolk 3314). In colour online.
Discussion
In this study, we have presented an overview of the sorediate corticolous Candelariella species and recognized five species that are widespread and currently well supported by morphological and molecular data, four of which occur in Europe. We adopted a rather conservative species concept. For example, since we found no consistent morphological differences among specimens from the two clades within Candelariella rubrisoli, it is in our opinion not justified to treat them as different species without further studies. Similarly, although there is some phylogenetic distance between the sequences of Candelariella subsquamulosa and the other sequences of C. xanthostigmoides, the lack of morphological differentiation also does not warrant a treatment as separate species without further studies.
Unfortunately, the large majority of sorediate corticolous Candelariella specimens lack apothecia. Crucial diagnostic characteristics to identify a specimen to species level can therefore not be observed, most notably the number of ascospores per ascus. Candelariella pulchella and C. reflexa are distinct species that can usually be reliably identified based on morphological characteristics, even when sterile. However, it is often not possible to reliably identify sterile specimens of the species with small-sized soredia, viz. Candelariella efflorescens, C. rubrisoli and C. xanthostigmoides. In Europe, a practical approach is to identify specimens with scattered greenish prominent areoles and discrete yellow soralia as Candelariella rubrisoli, especially those growing on twigs or those on stems in humid habitats. Specimens in which areoles are inconspicuous and that grow on well-lit dry habitats can be recorded under an aggregate name, viz. Candelariella efflorescens agg., as they are already in several European countries (e.g. Westberg & Clerc Reference Westberg and Clerc2012; Bomble Reference Bomble2015, Malíček et al. Reference Malíček, Palice, Bouda, Knudsen, Šoun, Vondrák and Novotný2025).
Future studies could attempt to sequence material of Candelariella flavosorediata, C. magellanica and C. sorediosa, and sequence sorediate Candelariella species from New South Wales in Australia (the type location of C. xanthostigmoides), all of which we were unfortunately not able to obtain fresh material of in the present study. Further studies are also needed for the corticolous species with an areolate or granular thallus, that is most species within the Candelariella vitellina-group, including C. boleana, C. faginea, C. lutella and C. xanthostigma. Our phylogenetic analysis shows that sequences identified as C. lutella and C. xanthostigma are currently present in multiple clades, and more detailed studies are needed to improve our understanding of these taxa.
Supplementary Material
The Supplementary Material for this article can be found at http://doi.org/10.1017/S0024282925101059.
Acknowledgements
We are grateful to James Lendemer (New York State Museum Herbarium), Laurens Sparrius and Jan Vondrák for loans and discussion on sorediate Candelariella species. Most of the ITS sequences newly published in this study were generated by ALVALAB (www.alvalab.es). Three specimens were sequenced by Måns Svensson (UPS), for which we are very grateful. We thank Tiina Randlane and two anonymous reviewers for their helpful comments which improved the manuscript. JM has been supported by the long-term research development project RVO 67985939.
Author ORCIDs
Henk-Jan van der Kolk, 0000-0002-8023-379X; Martin Westberg, 0000-0002-8346-0322; Jiří Malíček, 0000-0002-3119-8967.
Competing Interests
The authors declare none.
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