Infraspecific variation of the ITS rDNA region of some brown Parmeliae occurring in Poland is studied and compared with non-molecular characters. Haplotype networks are used to illustrate the variability within the species. Both newly-produced sequences from Central Europe and from all over the world, downloaded from the GenBank, are used.

The number of haplotypes found for each taxon ranged from five in Melanelia stygia to 12 in Melanelia hepatizon and Montanelia disjuncta; however, their numbers correlate with the number of specimens tested. New haplotypes for Melanelia agnata, M. hepatizon and Cetraria commixta are found. Based on our 169-sample dataset, we could not infer any geographical correlation, either locally or world-wide. Many of the analysed haplotypes were widely distributed and the same haplotype was often shared between temperate and polar populations. A comparison of molecular, morphological, anatomical and chemical characters also shows no correlation.

Cryptic species, haplotype, lichenised fungi, Parmeliaceae, phylogeny, taxonomy

The brown Parmeliae (Esslinger 1977) have been an object of numerous studies (Guzow-Krzemińska and Węgrzyn 2003; Blanco et al. 2005; Crespo et al. 2010, 2011; Nelsen et al. 2011; Divakar et al. 2012; Thell et al. 2012; Leavitt et al. 2014, 2015) and, due to this exceptional attention, they are one of the best-studied assemblages in the family Parmeliaceae. These lichens are a polyphyletic group possessing foliose, a dark to medium brown thallus and usually lacking atranorin or usnic acid in the cortex (Esslinger 1977; Blanco et al. 2004).

For many years, one of the largest genera within this group was Melanelia Essl., segregated from Parmelia Ach. by Esslinger (1978) to accommodate species with brown, foliose thalli and an N– cortex layer. However, during the following years, it has been demonstrated that the genus Melanelia s. lat. was polyphyletic and several new genera were distinguished within it, such as Melanelixia O. Blanco et al., Melanohalea O. Blanco et al. (Blanco et al. 2004) and Montanelia Divakar et al. (Divakar et al. 2012). In traditional terms, brown Parmeliae includes other genera, such as Allantoparmelia (Vain.) Essl., Pleurosticta Petr. and some species of Xanthoparmelia (Vain.) Hale. Moreover, due to the historical taxonomic approach (Thell 1995; Rico et al. 2005) and the similarity in the morphological and anatomical features of thalli, Cetraria commixta is also referred to this group.

Our studies have focused on the saxicolous species of Melanelia and Montanelia genera. According to Otte et al. (2005), species of these genera are arctic-alpine, circumpolar and occur on silicate rocks in the mountain areas of the Northern Hemisphere, including Arctic Regions (Divakar et al. 2012). Nowadays, Melanelia s. str. is restricted to a small clade of saxicolous, cetrarioid lichens and includes four species: M. agnata (Nyl.) A. Thell, M. hepatizon (Ach.) A. Thell, M. pseudoglabra (Essl.) Essl. and M. stygia (L.) Essl. According to Thell (1995), these species are characterised by broadly clavate asci with a small tholus and a broad axial body, a thick, paraplectenchymatous cortex and dumb-bell-shaped pycnoconidia. Montanelia, representing the parmelioid clade, includes eight species: M. disjuncta (Erichsen) Divakar, A. Crespo, Wedin & Essl., M. occultipanniformis S.D. Leav., Essl., Divakar, A. Crespo & Lumbsch, M. panniformis (Nyl.) Divakar, A. Crespo, Wedin & Essl., M. predisjuncta (Essl.) Divakar, A. Crespo, Wedin & Essl., M. saximontana (R.A. Anderson & W.A. Weber) S.D. Leav., Essl., Divakar, A. Crespo & Lumbsch, M. secwepemc S.D. Leav., Essl., Divakar, A. Crespo & Lumbsch, M. sorediata (Ach.) Divakar, A. Crespo, Wedin & Essl. and M. tominii (Oxner) Divakar, A. Crespo, Wedin & Essl. (Divakar et al. 2012; Leavitt et al. 2015; Leavitt et al. 2016). The characteristic features of the Montanelia genus are short and narrow lobes, with flat to convex lobe margins, a non-pored epicortex, cylindrical to fusiform conidia, a medulla containing orcinol depsides and flat, effigurate pseudocyphellae (absent only in M. sorediata; Divakar et al. 2012). Three of these species (M. disjuncta, M. panniformis and M. sorediata) have broad, intercontinental distributions, with no evidence of phylogeographic substructure (Leavitt et al. 2015).

The genera Melanelia and Montanelia have been the subject of a critical revision in Poland and data concerning their distribution, ecology and morphological, anatomical and chemical features are presented in previous papers (Szczepańska et al. 2015; Szczepańska and Kossowska 2017). However, recent molecular studies imply that both genera may include previously unrecognised species-level diversity (Divakar et al. 2012; Leavitt et al. 2014), especially within Icelandic populations of M. stygia (Xu et al. 2017).

One of the goals of this study was to assess the intraspecific internal transcribed spacer (ITS) rDNA variability in brown Parmeliae species. Investigations of genetic variation in lichen-forming symbionts have advanced considerably in recent years and resulted in interesting conclusions (Palice and Printzen 2004; Lindblom and Ekman 2006; Domaschke et al. 2012; Starosta and Svoboda 2020). Although brown Parmeliae appear to be well studied in taxonomic terms, there are insufficient molecular data to estimate their genetic variation. Most of the available data concern samples collected in a few regions of the world, such as Europe and North America. The North American species of this group were studied in Greenland and Canada (Leavitt et al. 2014; Leavitt et al. 2015), while samples from Europe originated mainly from the north – Iceland, Finland, Norway and Sweden (Blanco et al. 2004; Divakar et al. 2012; Xu et al. 2017). Therefore, we decided to fill in the gap in sampling and focused our study on samples collected in Central Europe. We have used phylogenetic trees and haplotype networks to investigate the extent of molecular differences between newly-generated sequences from samples collected in Central Europe (Austria, Czech Republic, Germany, Poland and Slovakia) and others originating from different geographical regions. Due to additional samplings from previously unexplored areas, it was possible to evaluate and compare the genetic variability of the studied specimens in Central Europe with samples from other regions of the world and to identify areas with the greatest haplotype diversity. In addition, we analysed morphological, anatomical and chemical characters of collected specimens to find a potential correlation between phenotypic characters and genetic variation of the studied taxa. By analysing genetic diversity and geographical distribution of identified haplotypes, as well as phenotypic characters of collected samples, we tried to better define and designate the species boundaries within analysed taxa. Special emphasis was placed on analysis of European, Greenlandic and Icelandic samples of M. agnata and M. stygia to revise the hypothesis assuming a semi-cryptic or cryptic nature of their potential species-level diversity.

Although several studies focused on the phylogeny of brown Parmeliae, in the analysed datasets, there was an evident lack of molecular data concerning this group from Central Europe. The available data included only North America (mainly Greenland), Northern Europe (Scandinavian countries) and single sequences from specimens collected in Western Europe (Spain, Italy) and Asia (India, Russia). Having the opportunity to collect data from Poland, we focused on taxa occurring in this country, such as Cetraria commixta, Melanelia agnata, M. hepatizon, M. stygia, Montanelia disjuncta and M. sorediata. Additionally, in analyses, we also included newly-generated sequences from samples collected in Austria, Czech Republic, Germany and Slovakia. By supplementing the dataset with new sequences from a previously-unexplored area, we wanted to study the intraspecific internal transcribed spacer (ITS) rDNA variability of mentioned species and analyse distribution patterns of individual haplotypes. Previously, Leavitt et al. (2014) reported mean genetic distance (given as the number of nucleotide substitutions per site) in brown Parmeliae and found higher values in the case of Melanelia agnata and M. hepatizon (0.013) in contrast to Cetraria commixta and M. stygia (0.002 and 0.007, respectively). In this study, we found the highest nucleotide diversity in Melanelia agnata and M. hepatizon (0.01552 and 0.01421, respectively), but also in M. stygia (0.01418) as a result of additional sampling.

In our study, the haplotype networks illustrated that single-locus haplotypes and clades have no geographic clustering and cannot be useful in defining the species boundaries within brown Parmeliae. Haplotypes are dispersed amongst the sites and clades do not show apparent association with spatial location, as reported in literature data (Werth and Sork 2008; Starosta and Svoboda 2020). In addition, many of the analysed haplotypes of brown Parmeliae are widely distributed and, in many cases, the same haplotypes are shared between temperate and polar populations. What is more, all taxa, except Melanelia stygia, seem to be monophyletic and newly-sequenced specimens cluster together with other representatives of the species downloaded from GenBank. The extremely wide geographical distribution of mycobiont haplotypes has been observed in some other species, such as Cavernularia hultenii (Printzen et al. 2003), Cetraria aculeata (Fernández-Mendoza et al. 2011) and Cladonia subcervicornis (Printzen and Ekman 2003). In the first two cases, this phenomenon is assigned to lichens characterised by vegetative propagation and interpreted as evidence for ancestral polymorphisms and slow genetic drift (Printzen et al. 2003). This finding conforms well with the results of our study on Parmeliaceae, which are usually sterile species, reproducing by soredia (Montanelia) and conidia (Cetraria, Melanelia).

Although representatives of brown Parmeliae are known from both Hemispheres (Otte et al. 2005), all species studied in this paper represent circumpolar distribution and occur only on northern continents. The specimens used for the analyses originated mainly from mountain areas of Poland, both the Carpathians and the Sudetes; however, the range of sampling seems to be representative for this part of Europe. The number of analysed haplotypes representing different geographical regions was comparable for each taxon; nevertheless, the number of Melanelia agnata and Montanelia sorediata samples remain very small. Due to the newly-generated molecular data covering Central Europe, we were able to compare the haplotype distribution in this area with other parts of the world. Unfortunately, the data available for discussed lichens taxa include, almost exclusively, specimens from North America and Northern Europe; the data concerning Asia and Southern Europe are not sufficient to make a reliable comparison possible. In almost all analysed taxa, stronger genetic differentiation was found amongst North American populations, with a few haplotypes unique for this part of the world, especially for Greenland. Specimens occurring in Central Europe have lower haplotype diversification and many of these haplotypes have wide geographical distribution (Table 2). Nevertheless, it seems that the number of analysed sequences is still insufficient to indicate high diversity areas (hotspots), species speciation centres or glacial refugia. Although the numbers of haplotypes correlated with the number of specimens tested, two species occurring in Poland (Melanelia agnata and M. stygia) clearly indicate a very low level of genetic diversity. Both species are rare in Poland and their distribution is limited to the high mountain regions (Szczepańska and Kossowska 2017). Low genetic diversity and limited occurrence suggest considering both taxa as critically endangered in Poland.

In recent years, it has been proved that cryptic species-level lineages are very common amongst lichen-forming fungi (Crespo and Pérez-Ortega 2009; Crespo and Lumbsch 2010; Lumbsch and Leavitt 2011). At the same time, it has been shown that phenotypic variation is not always ‘sensitive’ enough for delimitation and description of new taxa. Modern methods of genetic analysis are recommended as an additional tool for this purpose (Molina et al. 2011; de Paz et al. 2012; Leavitt et al. 2013; Renner 2016). At the same time, it is necessary to include other evidence, such as chemistry, ecology, geography and morphology, for the proper delimitation of lichenised fungi species (Hawksworth 1976; Dayrat 2005; Crespo and Pérez-Ortega 2009). Such careful and versatile analysis of distinct phylogenetic lineages may lead to recognising some previously-overlooked characteristics (Kroken and Taylor 2001; del Prado et al. 2007; Frolov et al. 2016; Leavitt et al. 2016; Szczepańska et al. 2020). In the recent review paper, Lücking et al. (2021) proposed a detailed protocol for consistent taxonomy of lichen-forming fungi. The integrative taxonomy employing phylogeny, reproductive biology and phenotype should be used to delimit species (Lücking et al. 2020). Aime et al. (2021) recommended circumscription of new taxa, based on an appropriate sampling of multiple representatives from different collections for which multi-loci analyses should be performed. They also noted that description of a new species, based on single-locus phylogenetic analyses, could only be done in exceptional cases. The errors caused by contaminant sequences, laboratory mix-ups and chimeric sequences should be avoided for proper establishment of novel taxa, based on molecular data only (Lücking et al. 2021). Therefore, it is crucial to employ unlinked loci from different parts of the genome, even though the ITS rDNA marker is widely used in DNA barcoding of fungal taxa.

We analysed phenotypic diversity of samples representing individual haplotypes in our studies. However, in morphological, anatomical and chemical analyses, we observed that phenotypic characters of individuals representing different haplotypes are homogeneous and no visible distinctive features for samples with different geographic distribution were recognised. Recent molecular studies of one of the analysed genus – Melanelia, suggested previously unrecognised species-level diversity within this taxon (Divakar et al. 2012; Leavitt et al. 2014; Xu et al. 2017). However, the authors based their assumptions primarily on phylogenetic analyses without considering phenotypic features. Therefore, we have decided to analyse differences in morphology, anatomy and chemistry of M. stygia and M. agnata specimens originating from different geographic regions (Greenland, Iceland and Central Europe).

Melanelia agnata is a rare lichen recorded in North America and some European countries, such as Austria, Iceland, Norway, Poland, Russia, Sweden, Switzerland and Slovakia (Westberg et al. 2004; Hawksworth et al. 2008; Szczepańska and Kossowska 2017). The analysed holotype of Melanelia agnata is characterised by small (ca. 3.0 cm in diam.), foliose, olive-brown to dark-brown thallus, composed of flat, shiny, 0.25–2 mm broad, smooth lobes with thicker margins (Fig. 8A). Its lower surface is pale brown with single, dark rhizines. Polish (Figs 8G and H) and Greenlandic (Fig. 8E and F) specimens comply with the type. However, Icelandic material differs in a larger thallus size (up to 10 cm in diam.) and the appearance of the lobes, which are more convex than flat, 1–5 mm broad and distinctly wrinkled (Fig. 8C). Thell (1995) made an interesting taxonomic description of M. agnata, in which he noted that its thallus could reach up to 10 cm diam. However, in his research, Thell (1995) analysed only a few specimens, including one from Iceland (Kristinsson 14781, GZU, LD) and treated them all as a single taxon. A similar situation applies to conidia, reaching 5–7.5 µm in M. agnata, according to Thell (1995). Pycnidia observed in Icelandic specimens are usually marginal (Fig. 8D), very often double and produce bifusiform conidia, 4.5–6 × 1 µm, in contrast to the type specimen, which pycnidia are simple, marginal to laminal (Fig. 8B) with smaller conidia, at 3.5–5 × 1 µm. Pseudocyphellae are always whitish, rounded or irregular, marginal and laminal in all analysed material; they are much more abundant in specimens from Iceland (Fig. 8D). None of the Icelandic specimens had apothecia, so their anatomical analysis was impossible. All material was chemically homogeneous and no secondary metabolites were detected by thin-layer chromatography (TLC), which is consistent with other descriptions (Thell 1995; Xu et al. 2017).

Figure 8. 

Melanelia agnata specimens treated A Melanelia agnata H-NYL 36086 (holotype) B Melanelia agnata, H-NYL 36086 (holotype) C M. agnata, AMNH 27562 (Iceland) D M. agnata, AMNH 30974 (Iceland) E M. agnata, C 19019 (Greenland) F M. agnata, C 19019 (Greenland) G M. agnata, Szczepańska 1050, WRSL (Poland) H M. agnata, Szczepańska 1050, WRSL (Poland). Scale bars: 0.5 cm (A, C, E, G); 0.5 mm (B, D, F); 1 mm (H).

Melanelia stygia is a much more common species than M. agnata. In Europe, it was recorded in the upper mountain areas of Austria, the Czech Republic, Germany, Great Britain, Poland, Romania, Russia, Slovakia, Switzerland and Ukraine (Hawksworth et al. 2008).

After phenotypic studies, we have concluded that all material is homogeneous and none of the analysed morphological and anatomical features coincides with geographically-distinct M. stygia populations (Fig. 9A–F). However, some differences may be observed in the secondary chemistry. In his paper, Esslinger (1977) recognised six chemical races within M. stygia. He stated that some of them are broadly distributed and others are more frequent in particular regions. All the currently-examined samples originating from Greenland and Central Europe belong to Race 1, containing fumaroprotocetraric and protocetraric acids. Specimens from Iceland represent Race 6, without secondary metabolites. Both races are known to occur in Japan, North America and Europe; however, there is a possibility that Race 6 is the only chemical Race occurring in Iceland. Production of some secondary metabolites may be induced by environmental factors (Culberson 1986; Leavitt et al. 2011) and does not always correspond with molecular data. Moreover, chemical differences can be observed within some recognised haplotype groups and even in the same haplotype (Matteucci et al. 2017). At the same time, chemical characters may be successfully used to support delimitation of lichen taxa, but in any case, they cannot be treated as an exclusive diagnostic trait (Elix et al. 2009; Spribille et al. 2011; Leavitt et al. 2013; Onut-Brännström et al. 2018; Mark et al. 2019,).

Figure 9. 

Melanelia stygia specimens treated A M. stygia, AMNH 28243 (Iceland) B M. stygia, AMNH 16894 (Iceland) C M. stygia, C 19893 (Greenland) D M. stygia, C 19893 (Greenland) E M. stygia, Szczepańska 1160, WRSL (Poland) F M. stygia, Szczepańska 737, WRSL (Austria). Scale bars: 0.5 cm (A, C, E);1 mm (B, D); 0.5 mm (F).

In conclusion, we can state that all of the potential species lineages within Melanelia agnata and M. stygia are cryptic, with very slight morphological, anatomical and chemical variation. We were unable to distinguish any distinctive feature that could be considered diagnostic and useful for the delimitation of new species, except molecular variation. The phenotypic differences mentioned above may reflect environmental or climate conditions, such as temperature, light, humidity or substrate and may not be connected with genetic differences. However, this study was limited to a small number of samples and one genetic marker, ITS; therefore, we refrain from describing new species because further study is pending. We suggest that an extended phylogeographic study is necessary and an increase in the number of herbarium specimens would probably give additional information. Even though our analyses complement the knowledge on lichens in Central Europe, many areas remain insufficiently explored. Additional sampling from Asia and Southern Europe may bring new data on the phylogenetic and phenotypic diversity of species from the brown Parmeliae group.

The curators of AMNH, C and H, are gratefully acknowledged for the loan of specimens. The authors are also very grateful to the reviewers for their valuable comments and improvements to the manuscript. The publication is financed under the Leading Research Groups support project from the subsidy increased for the period 2020–2025 in the amount of 2% of the subsidy referred to Art. 387 (3) of the Law of 20 July 2018 on Higher Education and Science, obtained in 2019.