2.1. Phylogenetic analyses
In the present study we generated 54 new ITS rDNA sequences, 29 sequences of chloroplast rbcL gene fragment, and 14 sequences of actin type I of Asterochloris algae. In addition, we obtained one ITS rDNA sequence belonging to the genus Vulcanochloris (Table S1). The Bayesian phylogenetic tree inferred the genus Asterochloris to be divided into two major clades with high support values (Fig. 2), similarly to the results of Vančurová et al. . The first major clade (PP=99, ML77/75) consists of three poorly supported subclades, although in Vančurová et al. only two subclades were found . The second clade (PP=99, ML=77/75) consists of two subclades. We recovered 56 lineages (Fig. 2), 44 of them being previously identified [4,5,9,13,14,15,18,19,25] and for those we applied the same nomenclature as in original papers. 28 Asterochloris lineages obtained from Bolivian lichen samples grouped within 11 already recognized clades on the tree, of which only two were ascribed to the species, i.e. A. mediterranea  and A. friedlii  (Fig. 2). Remaining 26 photobiont sequences formed 12 new lineages of Asterochloris, probably representing undescribed species.
Our data have shown that some Asterochloris photobiont species and lineages known to date may occur in a broader spectrum of climatic conditions (data summarized in Table S8 and in Fig. 2 in reference to altitude). Thus, A. mediterranea known so far only from Mediterranean and temperate Europe can also occur in Neotropics. Annual precipitation determined for this species is between 18–974 mm, and precipitation of driest quarter: 2–95 mm (Fig. 3). Asterochloris friedlii was known from temperate Europe and USA and tropical areas of China and Korea, and it was also found in Bolivian sample of a widespread Lepraria finkii. Similar situation was observed for A. sp. clades StA1 and StA5, which till date were found to associate with Stereocaulon spp. in temperate regions or subtropical continental climate. A. sp. clade 9 found in South America, India and Southern North America associated only with cosmopolitan, Neotropical or Pantropical Cladonia spp. This lineage shows the highest annual mean temperature range (14.1–24.9 °C) and is characterized by drought resistance and rehydration (annual precipitation: 591–4771 mm, precipitation of driest quarter: 8–156 mm, precipitation of wettest quarter: 361–3968 mm). The widespread lineage A. sp. clade 12 associates with Cladonia spp., Stereocaulon spp. and Diploschistes muscorum and is characterized by widest temperature annual range: -5.5–18.1°C and high drought resistance (precipitation of driest quarter: 10–165 mm), and represents the lowest rehydration resistance of all Asterochloris photobionts. Asterochloris sp. clade P2 was found to associate with different lichen species and genera (Cladia, Cladonia, Pilophorus and Stereocaulon) similarly to Asterochloris sp. clade L14 (Cladonia and Stereocaulon). Those lineages together with A. sp. clade L54 were found only in the Neotropics. So far, A. sp. clade L54 and clade A6 form a symbiosis exclusively with Lepraria spp. (Fig. 2).
Furthermore, in all 12 localities where lichen samples were collected in Bolivia, we found different Asterochloris species and lineages. In locality Chuquisaca 2 (N=7; for locality codes see Table S1), we found six different Asterochloris photobionts, of which four were detected for the first time. Lepraria harrisiana from this locality is associated with three different Asterochloris lineages, while Cladonia aff. ahtii collected at different altitudes contains only one photobiont type. In locality Cochabamba 1 (N=7) we identified 7 photobionts lineages: A. mediterranea, A. friedlii, A. sp. StA1, clade 12, clade P2, and clades Bol 3 and Bol 9 associated with different lichen species. Furthermore, we identified the same haplotype of A. sp. clade Bol 2 in Lepraria impossibilis in a range of altitudes from 1943 to 2149 m a.s.l. (Table S13). The most abundant lineages identified in Bolivia, i.e. A. sp. clade Bol 1, Bol 2, P2 and StA1, were found to occur in most of the sampled localities of Bolivia. Asterochloris sp. clade Bol 1 were recorded in localities Chuquisaca 2, La Paz 1, Santa Cruz 1, Tarija 1 and 2, within thalli of Neotropical (Cladonia aff. ahtii, C. ceratphyla) or Pantropical (Lepraria sipmaniana) lichens in altitudes from 980 to 2950 m a.s.l., whereas, Asterochloris sp. clade Bol 2 associates with Lepraria impossibilis from localities Tarija 1 and 2 and with L. harrisiana from locality Chuquisaca 2. Asterochloris sp. clade P2, till date detected in Pilophorus cf. cereolus from Costa Rica, was also found in four Bolivian localities in altitudes between 2450 and 3860 m a.s.l. in different genera and species of lichens (Fig. 2).
Moreover, we observed dissimilarities between diversity of photobionts composition of four habitat types, i.e. lower montane cloud forest, upper montane cloud forest section 1, upper montane cloud forest section 2 and open high Andean vegetation (Table S1), e.g., in lower montane forest Asterochloris sp. clades Bol 1, Bol 2, Bol 3, Bol 6 and A14 were present, whereas in first section of upper montane cloud forest A. sp. clades 9, A14, L54, P2, S1 and Bol 1, 2, 5, 7, 12 and 13, A. mediterranea and A. friedlii occurred, whereas in second section of upper montane cloud forest Asterochloris sp. clades P2, S1, 12, A14, StA1, Bol 9. A. medirerranea, A. friedlii and Vulcanochloris sp. were also detected. In the open high Andean vegetation, Asterochloris sp. clades StA5, A6, Bol 8, 9, 10 and 11 were present. This shows that diverse photobiont types may occur within the gradient of altitude.
2.2. Statistical analyses
To identify the impact of selected factors on the distribution of Asterochloris photobionts, we performed variation partitioning analyses (Tables S5, S5, S7) that showed that selected variables explained 36-40% of the variation. 22-24% of the variation was explained by the species of the mycobiont (Table S6), which shows low correlation between photobiont distribution and mycobiont hosts. Remaining 14-16% was explained by environmental factors. 7% of the variability was shared between mycobiont host and climate in case of analyses when habitat was taken into account, and 6% of the variability was shared between mycobiont host, climate and geographical distance (when altitude was taken into account).
In the case of Stereocaulon 39% of the variation was explained by selected variables. The largest part of the variation in diversity of photobionts associated with Stereocaulon spp. was explained by mycobiont hosts (12%, Table S6). As independent factors, altitude explained 8% of the variation, while geographical distance - 4%. In Cladonia spp. 68% of the variability was explained by chosen variables (Table S6). 36% was explained by mycobiont species, whereas climate, altitude and geographical distance explained 1, 3 and 2%, respectively. However ecological factors shared 13% of the variability. In the case of Lepraria spp., altitude appeared to be insignificant and was excluded from analyses (Table S5). Results of PCoA analyses for Lepraria spp. show moderate correlation between photobiont distribution and mycobiont host (35% of the variation explained by mycobiont). Remaining factors did not reveal significant influence.
In addition, we selected twelve cosmopolitan species, seventeen Neotropical and ten Pantropical from our dataset and data available in GenBank (Table S4). In the case of cosmopolitan species, variation partitioning analyses revealed that between 0 to 13% of the variability was explained by mycobiont hosts (Table S7). Furthermore, the climate explained 15% of the variability. In further analyses, altitude and substrate, appeared to be insignificant (dbRDA analyses Table S5). 59% of the variability of Asterochloris distribution in Neotropical and Pantropical lichens were explained by mycobiont host. We performed PCA analyses to identify which composition of photobionts was represented by selected groups of lichens, depending on climatic factors. PCA ordination of all analyzed species resulted in 72.7% cumulative variance explained on the first 2 axes (first – 47.8%, second – 24.9% of the variance). Lichen groups and their photobionts respectively showed indifference relative to climatic parameters, being scattered across the hyperdimensional climatic space (Fig. S2). However, Cosmopolitan, Neotropical and Pantropical species as groups represent different range of climatic conditions. In case of cosmopolitan species, the total explained variation in the biplot was 68.4% by component 1 and 2 (Fig. S3). PCA ordination of Neotropical species resulted in 85% (Fig. S4) cumulative variance explained on the first 2 axes and Pantropical in 79.3 % (Fig. S5).
However, in cosmopolitan, Neotropical and Pantropical groups of lichens and between them, different genera show indifferences, dependent from diverse climatic conditions (potentially climatic preferences). Tropical species prefer higher temperature in warm and wet periods of the year, whereas the cosmopolitan species are acclimated to extremes in temperatures (BIO4, 7).
Due to observed dissimilarities between diversity of photobionts composition of four habitat types in Bolivia, we performed PCA to visualize arrangement of photobionts of those habitat types depending on climatic factors (Fig. S6). This shows that on each habitat other conditions prevail, however, the ranges of photobionts from different habitats may overlap.
Due to differences in the impact of the mycobiont species on distribution of photobionts in selected genera of lichens and differences in tolerance for different climatic conditions we performed haplotype analyses to study relations between mycobiont hosts and photobionts in selected groups of lichens. In case of Asterochloris photobionts from 200 samples of Stereocaulon spp. (Table S9) we found low haplotype diversity (0.28) and detected 55 photobiont haplotypes (Tables S9, S15), of which 24 were identified in samples from tropical areas, 36 in samples from temperate zone and 5 were identified in both temperate and tropical regions. However, haplotype diversity for samples from tropical regions appeared to be higher (0.52) than in case of samples from temperate area (0.23) (Table S16). Asterochloris irregularis appeared to be the most common photobiont associating with Stereocaulon spp. in temperate climate (this haplotype found in 62 samples). In samples from Bolivia we found 5 distinct haplotypes:A. mediterranea, Asterochloris sp. clades P2, S1 and new lineages Bol 6 and 8.
In the case of Asterochloris associating with Cladonia spp., we found 144 ITS rDNA haplotypes (haplotype diversity = 0.36) based on 404 analyzed samples (Tables S10, S15). 65 of them originated from tropical region, 85 from temperate climate area and only 6 Asterochloris haplotypes were noted in both regions. Moreover, we observed differences in photobionts haplotype diversity in tropical and temperate samples associating with Cladonia spp. (tropical – 0.75, temperate – 0.27). The most common photobiont for Cladonia spp. from tropical areas seems to be Asterochloris clade 9 (found in 33 samples); furthermore this potentially new species is represented by fifteen haplotypes in our dataset. Also, A. sp. clade 9 was found in two lichen thalli of Cladonia spp. from temperate region. Samples from Bolivia are represented by fifteen haplotypes belonging to A. mediterranea, A. clades 9, 12, A14, S1, StA1, StA5, P2 and new lineages - Bol 1 and Bol 5.
Photobionts from Lepraria spp. (Tables S11, S15) represented by 44 haplotypes of Asterochloris showed high haplotype diversity (0.54). In tropical climate 14 haplotypes were found, while in temperate region we found 31 haplotypes. Moreover, haplotype diversity was higher in regions with tropical (0.70) than temperate (0.50) climate (Table S16.). Single haplotype of A. friedlii represented by fourteen sequences was found in both regions. In the case of groups representing cosmopolitan distribution patterns of lichen forming fungi, we observed 49 Asterochloris haplotypes (Table S12) with high haplotype diversity (0.65) (Table S15); together they represent 27 Asterochloris species or lineages. Samples from tropical climate were represented by 15 Asterochloris species or lineages, while specimens from temperate climate by 17. Some species of lichen forming fungi appeared to adopt different Asterochloris haplotypes in different climatic regions, e.g. Cladonia furcata associated with photobionts belonging to A. sp. clade 12 in temperate climate, while other haplotypes were found in tropical areas, i.e. A. sp. clades I1 and I2 in India and A. sp. clade P2 and clade Bol 5 in Bolivia. Moreover, Stereocaulon alpinum may associate with at least 10 different Asterochloris species or lineages. Asterochloris sp. clade Bol 8 was not noted in temperate climate, while A. sp. clade StA5 was found in Georgia, Austria (the same haplotype) and in Canada (different haplotype).
Seventeen Neotropical lichen species were analyzed; within them we identified nineteen haplotypes of Asterochloris belonging to 15 species or lineages (Table S13) with high haplotype diversity (0.76) (Table S15). In case of Pantropical group, ten lichen species associated with fourteen Asterochloris haplotypes belonging to 9 lineages (Table S14) and showed the highest haplotype diversity (0.85) (Table S15). Within Neotropical and Pantropical lichens, we found Asterochloris haplotypes, either restricted to single species or with potentially wide selectivity (i.e., occurring in different species in similar or diverse localities).