Introduction

In lichen systematics, a “morphological” species concept (Du Rietz 1930; Mayr 1963) is broadly applied. It is generally accepted that those phenotypic differences are of relevance which correlate with variations in ecology, life-style, or geographic distribution. Without such correlations, delimitation of lichen species is difficult, and phenotypic variation is then controversially interpreted by lichenologists. Such delimitation problems affect many geographically widespread crustose lichens. Some of these can display an extraordinarily high morphological heterogeneity which is only partly attributable to different ecological conditions. These lichens represent taxonomically unresolved species complexes (Grube and Kroken 2000), which are often burdened with a complicate nomenclature.

The genus Tephromela was introduced by Choisy (1929), to allocate a single species of Lecanora with dark purple pigmented hymenium and straight conidiospores. Neglected by most authors before, the genus was resurrected by Hafellner (1984) and placed in a new family, Tephromelataceae, within the Lecanorales (Hafellner 1984). After the recent segregation of the phenotypically deviating T. armeniaca and T. aglaea in the genus Calvitimela (Hafellner and Türk 2001), the remaining “core” Tephromela species share diagnostic anatomical characters with the type species, Tephromela atra (Huds.) Hafellner & Kalb. The species belonging to Tephromela s.str., are distinguishable by the poorly developed true exciple, dark violaceous hymenium, Bacidia-type ascus with amyloid tholus, unbranched paraphyses, and straight, chain-forming conidiospores (Hafellner 1984; Purvis et al. 1992). According to recent phylogenetic studies, Tephromela is not closely related to Lecanoraceae, but rather forms a monophyletic group with Mycoblastus (Mycoblastaceae; Miadlikowska et al. 2006). With the exception of the peculiar shrubby Himalayan Tephromela siphulodes (Poelt and Grube 1993), all Tephromela species are crustose, and distinguished by more or less clear phenotypic characters or life-style (Rambold 1993; the relationships with Heppsora, a poorly understood squamulose relative requires further study; see also Poelt and Grube 1993).

The cosmopolitan Tephromela atra is morphologically and ecologically rather polymorphic, and a par excellence example for a lichen species complex with a controversial nomenclature. In the present contribution, we study the phylogenetic relationships among four entities of this taxonomically difficult group, which are characterised by distinct substrate preference or reproductive mode, and show slight morphological differences. Two of them occur on siliceous and intermediate rocks, and one on limestone and bark, respectively. The material was collected by the authors in southern Europe on a wide range of substrata from the eu-Mediterranean to the Alpine vegetation belts.

Materials and methods

The taxa

In Europe, fertile thalli without soredia occurring on siliceous rocks are traditionally assigned to Tephromela atra s.str., in agreement with the original collection of Hudson (1762). As currently understood, this holarctic taxon has a uniform composition of secondary metabolites, including atranorin, α-collatolic acid, alectoronic acid (Purvis 1992; Hesbacher et al. 1996), shows a wide ecological amplitude, and colonises different substrates, ranging from ultramafic rocks to sandstone and man-made materials such as bricks and cement. In the eu-Mediterranean belt it is restricted to sheltered situations, but elsewhere it preferentially occurs in sun-exposed habitats, although it can also occur in shaded ones.

Thalli occurring on limestone and dolomite are particularly common in open, sunny exposed habitats throughout the Mediterranean region, from sea level to the montane belt (Salvadori and Tretiach 2002). They are absent from the Alps (Nimis and Tretiach 1999), and become progressively rarer northwards, reaching southern Sweden (Fröberg 1989). The thalli occurring on carbonatic rocks are thick, cretaceous, with slightly effigurate margins, and contain two further unidentified secondary metabolites (Huneck et al. 1997). Taxonomically, this eco-morphotype has been interpreted differently, as some authors merged it in T. atra s.str. (Fröberg 1989; Wirth 1995; Egea and Alonso 1996; Galun and Mukhtar 1996; Seaward 1996; Llimona and Hladun 2001), or recognised it at form (T. atra f. pachythallina), variety (T. atra var. calcarea (Jatta) Clauzade & Cl.Roux: Clauzade and Cl.Roux 1985; Santesson 1993; Litterski and Mayrhofer 1998; Grube et al. 2001; Nimis and Martellos 2003), or at species (T. cypria (Körb.) Hafellner: Kalb and Hafellner 1992) level. It will be mentioned throughout the text as “T. calcarea”.

Thalli growing on bark are not rare in the chestnut and beech belts of southern European mountains, being rarer at lower altitudes (Nimis 1993). They show a clear preference for the first stages of bark colonisation, and are more frequent in humid sites. Taxonomically, they were alternatively segregated from T. atra s.str. as var. torulosa (Hafellner 1992; Kalb and Hafellner 1992; Suppan et al. 2000; Hafellner and Türk 2001; Nimis and Martellos 2003; Türk et al. 2004), recognised at species level (Motyka 1996), or considered a mere substratum variant of no taxonomic value, as the colonisation of bark would be induced by favourable climatic conditions (Clauzade and Roux 1985; Wei 1991; Purvis et al. 1992; Wirth 1995; Litterski and Mayrhofer 1998; Limona and Hladun 2001; Clerc 2004; Nash et al. 2004). This taxon will be mentioned throughout the text as “T. torulosa”.

The fourth taxon, closely related to Tephromela atra s.str., is T. grumosa (Pers.) Hafellner & Roux. Mainly a cool-temperate lichen, found on steeply inclined, acidic siliceous rocks, T. grumosa is morphologically and chemically clearly distinct, as the thallus is often completely sorediate. The species is almost always sterile, and differs also by the presence of lichesterinic acid (Purvis 1992), and the absence of the α-collatolic and alectoronic acid of T. atra s.str., apart from atranorin. Tephromela grumosa is well accepted as a species by the majority of the authors (Clauzade and Roux 1985; Purvis et al.1992; Santesson 1993; Brodo et al. 1994; Wirth 1995; Limona and Hladun 2001; Hafellner and Türk 2001; Nimis and Martellos 2003; Clerc 2004), having also its own specific lichenicolous fungus, Niesslia robusta (Tretiach 2002). Only a few authors retain it as a further variety of T. atra (Maheu and Gillet 1992; Jüriado 1997). This taxon will be mentioned throughout the text as “T. grumosa”.

A fifth taxon, T. pertusarioides (Degel.) Hafellner & Roux, could not be included in the analysis, as it was not recently collected in the survey area.

Sampling

For this study, two sets of samples were collected. The first consists of 20–30 thalli from four populations of T. atra s.str., T. calcarea, T. torulosa, and T. grumosa co-occurring in a limited area of 25 km2 (Mt. Amiata, Central Italy) (Fig. 1 insert a, sites 1–3), and therefore defined as “sympatric”. The second set consists in 3 to 10 thalli of each taxon eventually present in 24 further sites (Fig. 1, sites 4–27) scattered throughout Italy (sites 1–18), Greece (sites 19–23), and Austria (sites 24–27), from 300 to 2500 m altitude, i.e. from the Mediterranean to the Alpine vegetation belts, on a wide array of substrata (silicates, limestone, dolomite, bricks, sandstone, bark) (Table 1). Voucher material is deposited in the herbarium of the Department of Biology, University of Trieste (TSB).

Fig. 1
figure 1

Map of sampling sites of Tephromela atra s.str. (■), T. calcarea (●), T. torulosa (▲), T. grumosa (□). Insert a: sympatric populations of Mt. Amiata, central Italy

Full size image
Table 1 Geographic provenience of specimens. Number of collected and genetically analysed samples are reported for each taxon in each locality
Full size table

Molecular analysis: DNA extraction, PCR-amplification and sequencing

DNA extraction was performed on 20 thalli for each sympatric population of sites 1–3, and 3–10 for each taxon collected in sites 4–27. Saxicolous thalli were scraped off from an area of c. 0.5 cm2, while only the apothecia and the upper portions were taken from the corticolous thalli to avoid contaminants from the bark below. The DNA isolation protocol of Cubero et al. (1999) was applied for all samples. However, c. 70% of those occurring on limestone were not successfully amplified, notwithstanding several trials in which experimental conditions (DNA elution, MgCl2 concentration, pH of lyses buffer, etc.) were modified. In these cases, the DNAeasy Plant Mini Kit (QIAGEN, Vienna, Austria) and the GenElute™ Plant Genomic DNA Kit (SIGMA, Missouri, USA) was successfully applied to obtain sequence data from additional 10 specimens.

The mycobiont genetic diversity was analysed with sequence data of the ITS region, beta tubulin fragment, and the ketoacyl synthase (KS) domain of a homologous polyketide synthase gene (PKS). The ITS region was amplified with the primers ITS1F (Gardes and Bruns 1993) and ITS4 (White et al. 1990), the beta tubulin with primers bt2a and bt2b (Glass and Donaldson 1995), and the KS domain with primers LC1 and LC2c (Bingle et al. 1999). In some cases PCR products of the KS domains were cloned in E. coli Xl-1blue using the pGEM-T Easy Vector System (Promega, Madison, USA) following the manufacturer’s instructions. The photobiont diversity was analysed using the ITS region by the Trebouxia-specific primers ITS1T and ITS4T (Kroken and Taylor 2000).

PCR reactions were prepared for a 30 μl final volume containing 4.05 μl double-distilled water, 3 μl 10× Taq polymerase reaction buffer (10 mM Tris pH 8.3), 1.8 μl MgCl2 (25 mM), 3 μl of dNTPs (2.5 mM), 0.15 μl Taq DNA polymerase, 1.5 μl for each of the 10 μM primers. PCR amplifications of both the mycobiont and photobiont ITS region were performed under the following conditions: one initial heating step of 2 min at 94°C linked to 30 cycles of 1 min at 94°C, 1 min at 53°C, 2 min at 72°C, and one final extension step of 7 min at 72°C after which the samples were kept at 4°C. The PCR amplification for the beta tubulin genes was performed with 58°C as annealing temperature, while the PCR amplification of the PKS KS domain required an initial heating step of 2 min at 94°C, linked to 36 cycles of 45 s at 94°C, 45 s annealing conditions alternative with 58–52°C touch-down during the first six cycles and 52°C for the remaining 30 cycles, 1 min 45 s at 72°C, and one final extension step of 7 min at 72°C, after which the samples were kept at 4°C. PCR products were purified using QIAGEN quick spin columns (Qiagen, Vienna, Austria) following the manufacturer’s instructions. Both complementary strands were sequenced, and sequences were either run by MWG Biotech-AG Company (Regensburg, Germany) or by ABI 310 (Applera, Vienna, Austria), and assembled in BioEdit (Hall 1999, http://jwbrown.mbio.ncsu.edu/BioEdit/bioedit.html). Specimen and DNA related data are listed with isolation, GenBank accession, and TSB herbarium collection numbers in Table 2.

Table 2 Samples of Tephromela with GenBank accessions, isolation, and herbarium numbers (top)
Full size table

Alignment and phylogenetic analysis

Four phylogenetic analyses were performed. The first two analyses focused on the ITS sequences belonging to the mycobionts (n = 46) and to the photobionts (n = 35) of samples belonging to the four sympatric populations of Mt. Amiata (sites 1–3). The sequences of Tephromela atra AY541279 and Trebouxia arboricola AF389915 were retrieved from GenBank and used as outgroups of fungal and algal datasets, respectively, in these analyses. The second two analyses focused on selected representatives of mycobionts (n = 72) and photobionts (n = 62) sequences originating from all the 27 sampling sites of Austria, Greece, and Italy. The mycobiont phylogeny was obtained by combining data of the three loci: ITS, beta tubulin, and PKS KS domain. Two new Lecanora cenisia ITS sequences (TSB 37464, TSB 37478), and ITS sequences of three representatives of the genus Lecanora, Mycoblastus sanguinarius (DQ782842), Calvitimela armeniaca (AY541278) and Tephromela atra (AY541279, DQ534487) were retrieved from GenBank and included in the analysis. Sequences of Lecanora, the genus from which Tephromela was once segregated, were selected as outgroup. PKS KS sequences were used in the combined analysis since several previous analyses (including lichenised and non-lichenised fungal KS domains) confirmed their homology (not shown). A supplementary haplotype analysis with the TCS program (Clement et al. 2000) was used to show the genetic heterogeneity of this paralog (see Fig. 4, below).

The general analysis of the photobiont ITS included sequences of different Trebouxia species retrieved from GenBank (their name and accession number are reported in the phylogeny of Fig. 5, below). The tree was rooted arbitrarily with Trebouxia higginsiae AJ249574.

The respective alignments were produced automatically with ClustalW (Hall 1999, http://jwbrown.mbio.ncsu.edu/BioEdit/bioedit.html) as implemented in BioEdit 5.0.6 (Hall 1999) and then manually adjusted. The phylogenetic hypotheses for the four analyses were established using a Bayesian approach as implemented in the program MrBayes 3.1.2 (Huelsenbeck and Ronquist 2003; Ronquist et al. 2005). The General Time Reversible substitution model (Rodriguez et al. 1990) with estimation of invariant sites and assuming a gamma distribution with four categories (GTR+I+G) was used for likelihood calculations. The optimal nucleotide substitution model was found before with the program MrModeltest 3.7 (written by J.A.A. Nylander and available at http://morphobank.ebc.uu.se/mrbayes/; Posada and Crandall 1998). For other parameters the default setting were used. For all the four phylogenetic analyses the Markov Chain Monte Carlo (MCMC) algorithm was run for 2 million generations, with 6 chains starting from a random tree and using the default temperature of 0.2. Every 100th trees were sampled while the first 200,000 generations were discarded as burn-in. The burn-in period was determined after testing for stationarity of likelihood values (i.e. by plotting the numbers of generation vs. the log probability and checking for the convergent diagnostic PSRF approaching 1; Ronquist et al. 2005; MrBayes 3.1 Manual). The consensus phylograms based on the mean branch lengths were calculated with the command sumt in MrBayes (see MrBayes 3.1 Manual; Ronquist et al. 2005). The phylogenetic trees were drawn using the program TreeView (Page 1996).

In each phylogenetic analysis the ambiguously aligned position were excluded. The MCMC parameters resulted for each Bayesian calculation and the numbers of included, variable and informative nucleotide positions are not reported but are available under request.

Results

Analysis of the sympatric populations: mycobionts

This analysis was performed with the alignment of 46 sequences obtained from 9 to 13 thalli of each substratum-defined taxon collected in the Mt. Amiata area (Fig. 1 insert a). The taxa segregate in two fully supported (100% PP) branches (Fig. 2a): one representing T. grumosa (clade I), and the other representing clades of the three ecotypic taxa of T. atra (clade II) occurring, respectively, on bark (IIa), limestone (IIb), and trachyandesite (IIc). Three samples from bark (TSB 38467), trachyandesite (TSB 37084), and calcareous rock (TSB 37936), respectively, do not cluster with the other samples of their corresponding ecotype. The first two are basal in the clade II, while the third is nested in the clade IIa of the epiphytic taxon.

Fig. 2
figure 2

Phylogenies of the sympatric populations of Mt. Amiata: a mycobionts, b photobionts. 50% majority-rule consensus trees of ITS sequences data from a B/MCMC sampling procedure. Posterior probabilities are indicated by the thickness of the branches (<90%, 90–94%, >95%), taxa are marked as in Fig. 1

Full size image

Analysis of the sympatric populations: photobionts

The analysis was performed with an alignment of the photobiont ITS sequences obtained from principally the same thalli that were also included in the mycobiont ITS analysis (photobiont sequences could not be obtained for nine thalli). Two highly supported clades are identified, I and II (Fig. 2b). Clade I is composed by two sister clades (one highly supported) in which photobiont from the calcicolous and silicicolous taxa group together. Clade II groups, in a highly supported (99% PP) unresolved branch, photobiont sequences of T. grumosa, the remaining silicicolous T. atra, and the epiphytic T. torulosa, which form a smaller, moderately supported (87% PP) subclade (clade IIa of Fig. 2b). The phylogenetic hypothesis of the photobionts does not correlate with the substratum-defined taxa of the mycobionts, but the well-defined segregation of the two main clusters suggests the presence of two different photobiont species.

General phylogeny of the mycobionts

The general analysis combining ITS, β-tubulin and PKS KS domain loci includes 16 representatives of the 4 sympatric populations of Fig. 1 (insert), and 56 mycobionts of the same four taxa collected in the 24 other localities of Austria, Greece, and Italy. One to three samples are present for each taxon collected in each locality. With the exception of two single samples (TSB 37901 from the Adamello Massif, and TSB 33382 from the Ligurian Alps), the samples from the Southern Alps group together (clade I Fig. 3); five of them constitute a fully supported but unresolved subclade. The Alpine T. atra samples clearly segregate from the T. grumosa clade (II in Fig. 3), and from all the other samples, mostly of Mediterranean origin. The rest of the samples are grouped into three major sub branches: the epiphytic specimens split into two fully supported clades (clades III and IV of Fig. 3), while calcicolous and silicicolous specimens, as well as those found on intermediate substrata (sandstone, brick, etc.), cluster together intermixed in the unresolved and moderately supported third one (clade V of Fig. 3). Two samples of T. atra from siliceous rocks from insular Greece and Italy (respectively, TSB 37928 from Crete, and TSB 37486 from Sardinia) group together in a branch close to T. grumosa, while one sample of T. calcarea is nested in the clade IV of T. torulosa.

Fig. 3
figure 3

Phylogeny of the mycobionts: combined analysis of ITS, β-tubulin and polyketide synthase KS domain. For further information, see legend to Fig. 2

Full size image

Haplotype analysis of the polyketide synthase genes

Sequences of the ketoacyl synthase domain were obtained for 55 representative mycobionts from almost all the localities of Fig. 1. Preliminary phylogenetic analyses (not shown) including type I PKS genes from other lichenised and not lichenised fungi, and the sequencing of some cloned PCR products, revealed that a single homolog was amplified with the primers used. All the KS sequences differ only in few sites in the coding regions but show more variation in their spliceosomal intron sequences. Six specimens of Tephromela grumosa show the same intron sequence, and two further sequences differ only in two nucleotides. Three different intron allels are found in the epiphytic taxon: one differs in eight nucleotides from the other two, which have only a difference of two nucleotides among each other. Intron sequences of the calcicolous and the silicicolous taxa can be divided into four groups, which differ from each other only in one nucleotide. Only the intron sequence of T. atra TSB 37094 is clearly different from all others.

The complete KS DNA sequences -including coding and intron parts- were considered in the haplotype analysis. In the haplotype network (Fig. 4), KS sequences of T. grumosa group in a well distinguished branch, quite distant from the intermixed KS sequences of the silicicolous and calcicolous taxa. KS domain of the epiphytic taxon segregate in two independent smaller networks which do not correlate with the geographical provenience of the samples.

Fig. 4
figure 4

Haplotype analysis of the polyketide synthase KS domain of the mycobionts: 95% probability networks of epilithic (a), and epiphytic (b, c) taxa. The size of the circles are proportional to the number of sampled sequences of the haplotype. Small dots represent not sampled haplotypes, the line between two haplotype is one mutational step. Full circles of (a) belong to Tephromela atra s.str and T. calcarea, empty circles belong to T. grumosa; (b, c): T. torulosa

Full size image

General phylogeny of the photobionts

The general analysis of the photobionts included 19 representative sequences of the four sympatric mycobiont populations of Mt. Amiata, 43 sequences of samples from the remaining 24 localities of Austria, Greece, and Italy, 2 sequences generated for Lecanora cenisia, and 38 sequences of Trebouxia species retrieved from GenBank, as nothing was known about the phylogenetic position of the photobiont(s) of Tephromela species. Six highly supported clades can be identified (Fig. 5). The photobionts of the alpine Tephromela atra form a highly supported subclade (clade Ia of Fig. 5) nested with samples of Trebouxia simplex, T. jamesii and further Trebouxia sp. photobionts from different Lecanora species. We named this clade (clade I) as T. simplex sensu lato, because it refers to a group of taxa, most of which have not been described yet (Hauck et al. 2007). Clades II, III and IV (Fig. 5) represent T. impressa, T. incrustata and T. arboricola/ decolorans respectively, and do not include any photobiont of our Tephromela, which cluster in two further highly supported clades (V, VI of Fig. 5). Clade V is sister of T. arboricola, while clade VI is well segregated from the others five ones: it groups, in an unresolved topology, the majority of our sequences. Clades V and VI also include selected sequences of the photobionts identified by Blaha et al. (2006) which represent new, undescribed Trebouxia species (namely, Trebouxia sp. 1: clade V; Trebouxia sp. 2: clade VI).

Fig. 5
figure 5

Phylogeny of the photobionts. Samples selected from the GenBank are reported with their accession number. For further information, see legend to Fig. 2

Full size image

Discussion

The systematic identity of the taxa of Tephromela atra occurring on bark, limestone and siliceous rocks, respectively, has for long been a much debated question. Due to the lack of chemical differences (Purvis et al. 1992; Hesbacher et al. 1996), they are regarded as representing the same species by most authors, whereas others distinguished them at different infrageneric level owing to their different ecological requirements and slight morphological differences. On the other hand, there is clear evidence that T. grumosa can be considered a morphologically and chemically distinct species: the thallus is almost always sterile and completely sorediate, and contains atranorin and lichesterinic acid as secondary metabolites. Samples assigned to T. grumosa group together in the phylogenetic tree, which also contradicts the possibility of sporadic loss of sexuality in a broader concept of T. atra. There is little doubt that T. grumosa is a reproductively isolated lineage distinct from Tephromela atra s.lat. The interpretation of the remaining three taxonomic entities (T. atra s.str., T. calcarea and T. torulosa), which was mainly based on the different substrates, is more difficult.

The restricted ITS analysis of the sympatric populations of the M. Amiata sites identified four well-distinguished clades, yet not all of them fully supported, corresponding to each substratum-characterised taxon. It would be of future interest to investigate whether such supposed local genetic differentiation would also occur in other sympatric populations. However, evidence for an ongoing genetic differentiation is obscured when the sampling is extended to a wider geographic area. Further sampling inevitably introduces a greater genetic variation in the dataset, which could supersede the distinctiveness of clades detected in the sympatric analyses. The four lineages are in fact not resolved as monophyletic lineages agreeing with the substrate type in the multilocus tree of Fig. 3. In this multilocus analysis, T. atra from the Alps appears as a basal lineage of the entire complex, whereas T. torulosa splits into two lineages, and no resolution is found in T. calcarea and T. atra s.str. from Mediterranean habitats.

The presence of two distinct groups in T. torulosa could indicate that this taxon underwent genetic separation from the other Mediterranean ecotypes. However, it remains unclear whether the two clades formed by T. torulosa denote two separate lineages. Motyka (1996) in his posthumous monographic treatment of the genus described 22 species of Tephromela, of which 3 represented new epiphytic taxa: T. depressula, T. hibernica, T. persordida. The former differs from T. torulosa by a rimose-areolate thallus and the hymenium covered by a hyaline layer. Tephromela hibernica and T. persordida are mainly distinguished by anatomical characters, such as the large cells of the hyphae of the hypothecium and the irregularly septate paraphyses, respectively. Although Motyka (1996) was recently added to the list of opera utrique oppressa (McNeill et al. 2006), the validity of the above mentioned characters for the segregation of infrageneric entities within T. torulosa should still be critically re-evaluated on the basis of Fig. 3.

Two possible hypotheses may explain the low resolution between the Mediterranean T. calcarea and T. atra s.str. First, this pattern could indicate that no genetic adaptation to the different ecological circumstances is present, and the morphological differences between the two taxa are simply induced by the accumulation of calcium oxalates in limestone occurring thalli, as suggested by Salvadori and Tretiach (2002). Alternatively, the specialisation to the different substrates has not yet led to reciprocally isolated alleles in our investigated loci. Incomplete lineage sorting was demonstrated in Cavernularia (Printzen et al. 2003), and in closely related neuropogonoid Usnea species (Wirtz et al. 2007), where haplotypes might even be shared among morphologically separable species. The process of lineage sorting may take exceptionally long in lichens (Printzen et al. 2003), which indicates that the results of population genetic studies in these organisms have to be interpreted with great caution, especially when few specimens are analysed. However, the lack of resolution with the mycobiont sequence data and the shared photobionts, the mere morphological and ecological differences between T. calcarea and T. atra s.str. from Mediterranean habitats, does not support the distinction of species. Therefore, the form level seems to be appropriate for the taxonomic recognition of the calcicolous ecotype at the present stage, and the correct name is thus T. atra f. pachythallina Th.Fr. On the other hand, the unique alleles of T. torulosa and the distinctiveness of T. atra s.str. from the Alps indicate that further, genetically isolated, lineages are likely present in the Tephromela atra complex in southern Europe. As no further characters have not been studied in detail so far, at this moment we hesitate to recognise them from the taxonomic point of view.

The phylogenetic analyses of the ITS region of photobionts of Tephromela atra s.l. revealed two well-resolved clades which likely represent hitherto undescribed photobiont species. Trebouxia sp. 1 and T. sp. 2, respectively, have already been found by Helms et al. (2001) in representatives of the Physciaceace, and by Blaha et al. (2006) in Lecanora rupicola. Blaha et al. (2006) identified the new photobiont species from crustose thalli growing on acid to sub-neutral to base rich rocks in the Mediterranean region. In our study, they were also identified in thalli occurring on limestone, dolomite, bricks, sandstone, and bark. Trebouxia sp. 1 seems to be restricted to saxicolous thalli, and it was recently also identified in a group of calcicolous endolithic Caloplaca species (Muggia et al. 2008), but interestingly, it has never been found as photobiont of T. grumosa. Trebouxia sp. 2 does not display specificity for the substratum, nor for the fungal partner, and was found as photobiont of foliose lichens as well (Helms et al. 2001). The mycobionts of our saxicolous Mediterranean Tephromela do not exhibit a high specificity for their photobionts, in agreement with the findings of Blaha and Grube (2006). In some cases, individual thalli of the same population form symbioses with either Trebouxia sp. 1 or sp. 2. Nevertheless, in the Mediterranean region a higher selectivity for the photosynthetic partner was found in Fulgensia fulgida, which associates strictly with Trebouxia asymmetrica (Beck et al. 2002).

A single algal lineage has been detected in T. grumosa. This can be justified either by its dominant vegetative propagation mode or by a high specificity for a certain photosynthetic partner. The second hypothesis accords well with the fact that, even though the dispersion by soredia (Fig. 6h) ensure T. grumosa the propagation of both symbionts together, this lichen reproduces sporadically by sexual means (Fig. 6f), which would allow reassociation with another photobiont lineage. A similar photobiont specificity could also explain the exclusively presence of Trebouxia sp. 2 in the epiphytic, esorediate taxon, but it should also be considered that Trebouxia sp. 1 may not be available on bark for unknown reasons.

The occurrence of photobionts belonging to T. simplex s.l. in alpine T. atra correlates with the genetic distinctiveness of the mycobiont sequence data in a separate basal lineage of the T. atra complex (Fig. 3). We therefore assume that the Alpine Tephromela atra samples are genetically isolated from Mediterranean ones, and that the isolation might be enforced by ongoing association with a climatically adapted photobiont.

Fig. 6
figure 6

Habitus of Tephromela atra s.str. (a, c: TSB 38666, bar = 3 mm, 2.5 mm), T. calcarea (b: TSB 38684, bar = 6 mm; d: TSB 38677, bar = 4 mm), T. torulosa (e: TSB 38699; bar = 2 mm; g: TSB 37128, bar = 2.5 mm), and T. grumosa (f: TSB 33882, bar = 1.5 mm; h: TSB 34232, bar = 1.0 mm)

Full size image

Tephromela atra s.l. is a subcosmopolitan species and its morphological heterogeneity is not constrained to the Mediterranean region. This species complex also shows a high morphological diversity in SW North America, SW Australia and Southern Africa, from where new species (Rambold 1989; Elix and Kalb 2006; Wirth 2007) have been described (and will be described; K. Kalb, personal communication). It remains to be studied whether taxa from these parts represent lineages that are genetically distinct from the southern European representatives of the T. atra complex. The T. atra species complex represents an interesting case, where the joint efforts of molecular and phenotypic studies will reveal new insights in the diversification of lichens.