Photobiont heterogeneity in a single specimen, species, or genus of the Physciaceae

The range of compatible algae found in a single specimen, species or genus of the Physciaceae varied considerably. Some foliose genera seemed to be restricted to a single Trebouxia ITS subclade, while in other cases, single thalli of some crustose species contained up to three distantly related Trebouxia ITS-variants (TABLES 5.3 and 5.4). On average, a higher percentage of crustose specimens was found to be lichenized with different Trebouxia subclades than foliose specimens. This might be partly explained by the sampling method. Due to the thinness of many crustose thalli, a larger area of the thallus, including several apothecia, was used for DNA extraction. In foliose species, only single apothecia or lobe tips were used, thus sampling only minute parts of the whole thallus. Since intrathalline photobiont heterogeneity was not the primary focus of this study, a systematic investigation cannot be presented here. Therefore, it might be suspected that photobiont heterogeneities were not detected in every instance in foliose specimens. Notably, Paulsrud (2001) investigated the aspect of intrathalline photobiont heterogeneity in the genera Peltigera and Nephroma. In single thalli of these cyanolichens he detected only single strains of Nostoc. This might support the suspicion that in most foliose specimens of the Physciaceae only one Trebouxia ITS-variant had been present. However, exceptions were found in Physcia caesia and Santessonia sorediata (TABLE 5.4). In single thalli of each of these two species, two closely related but distinct Trebouxia ITS-variants were found.

This contrasts to the photobiont heterogeneity found in many crustose thalli, where photobionts of different subclades or even different clades were detected (TABLE 5.4). In a single specimen of Buellia pulverulenta photobionts of subclades A4 and I1 were detected, in a single thallus of B. triphragmioides subclades A4 and I3, in Hafellia dissa subclades A7 and G6, in Rinodina atrocinerea subclades A6, A10 and A11, in Rinodina oxydata subclades A6, A10 and I5, and in Rinodinella controversa subclades A5 and A9 (TABLE 5.4). Since only single specimens were analyzed of each of these species it cannot be evaluated here, if multiple photobiont subclades are typical in these species or not. A couple of crustose species appeared to have a similar degree of selectivity than foliose taxa. Buellia elegans and B. zoharyi appeared selective for subclade A7. Specimens of Diplotomma venustum collected in Greece, Hungary, and Sweden were associated with ITS-variant A5a. In a fourth specimen from Arizona an ITS-variant of subclade S4 was found. This observation appeared atypical in two respects. First, an alga of this clade is not considered the typical photobiont of Diplotomma, and second members of Trebouxia clade S appear to prefer acid substrates, while the Diplotomma specimen from which Trebouxia subclade S4 was obtained grew on limestone.

The pattern of intrathalline photobiont heterogeneity correlated with growth habit rather than with phylogeny. Probably crustose forms which grow along the substrate surface have a higher chance to overgrow and incorporate new Trebouxia ITS-variants than foliose taxa. Foliose taxa elevate themselves above the substrate and are delimited by a cortex. This might prevent the incorporation of new photobionts after thallus establishment, even for compatible photobionts.

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TABLE 5.4: Physciaceae species in which single specimens were found to be associated with multiple Trebouxia subclades. A2b’: This ITS-variant deviated in less than 2% of its sequence positions from A2b (see chapter 4) but was distinct by an insertion in its nrSSU.

Lichen species Trebouxia ITS-variants crustose

Amandinea cacuminum A10a, A1a Buellia pharcidia A1c,d Buellia pulverulenta A4a, I1d Buellia triphragmioides A4b, I3b Hafellia dissa A7a, G6b Rinodina atrocinerea A6a, A10a, A11a Rinodina lecanorina I1g, I1p

Rinodina oxydata A10a, A6a, I5a Rinodina tunicata A5b, A9a Rinodinella controversa A5a, A9a foliose / fruticose

Physcia caesia (PiaCae2) I1s, I1t Santessonia sorediata A2b, A2b'

Photobiont variation among different thalli might be at least as large as within a single thallus. Finding only one photobiont variant in a particular thallus, however, does not indicate that all thalli of one species are associated with just one particular photobiont. Therefore, photobionts of multiple specimens of the same species were investigated in a number of instances. Emphasis was put on the analysis of specimens from as remote of locations as possible, in order to maximize the probability that associations were established independently and also under deviating environmental conditions (TABLE 5.1). Diversity of collection sites was considered to be of higher significance with respect to assessing the degree of selectivity in single species than analyzing numerous specimens of adjacent localities. With this strategy, photobiont heterogeneity within species was observed in most cases where multiple specimens of the same species were analyzed. In most foliose species of the Physciaceae that were sampled multiple times, different Trebouxia ITS-variants were observed that belonged to only one ITS subclade. A particularly high degree of selectivity was found in Anaptychia runcinata. Four specimens of A. runcinata which were collected in Spain, Italy, and Scotland were found with ITS-variant A4a (T. jamesii) only. Locations in Italy and Scotland were on the immediate coast, while two adjacent locations in Spain were 20 km inland, one on a sun exposed rock, the other on a shaded mossy rock beneath an oak tree (see voucher information given in the appendix A.1). The large distances and environmental differences between these habitats in combination with a photobiont constancy on the level of ITS-variant might suggest an exceptionally high degree of mycobiont selectivity.

However, this constancy might be alternatively explained by an exceptional low variability within subclade A4. Only one other ITS-variant in this subclade was detected only once in a specimen of Buellia

Ecological and Evolutionary Dependence in Associations of Physciaceae and Trebouxia

pulverulenta (FIG. 4.5). However, also instances were observed, where single, foliose Physciaceae species were found to be associated with representatives of different subclades at different locations. Different thalli of Physcia semipinnata were associated with members of subclades I1 and I2, and different thalli of Physcia tribacia were found lichenized with members of subclades I2 and I4 (TABLE 5.3).

At the level of subclades, photobiont steadiness not only prevailed in most thalli or species analyzed, but was also observed in whole genera. Twenty-six specimens of the genus Physconia, representing six species, which were collected in Greenland, central Europe, Scandinavia, and Spain, were exclusively lichenized with representatives of subclade I1. Eleven specimens of the genus Heterodermia, representing seven species, which were collected in Bolivia, El Salvador, Venezuela, the Canary Islands, South Africa, Tanzania, and the Philippines, were exclusively lichenized with representatives of subclade I4. Further lineages of closely related Physciaceae species were found with representatives of the same subclade. For example, photobionts of Dimelaena oreina, and D. tenuis, as well as Phaeorrhiza nimbosa and P. sareptana were all found to be selective for subclade I1. In other genera such as Anaptychia and Phaeophyscia, different species were selective for different photobiont subclades of the same clade. Phaeophyscia orbicularis was associated with algae of subclade I1 while Phaeophyscia endophoenicea and P. kairamoi were found with photobionts of subclade I3. Physcia, certainly the largest and most heterogeneous foliose genus of the Physciaceae, was found associated with photobionts belonging to two different clades, I and G. This was paralleled by the geographic range of this genus, which is distributed over temperate as well as tropical climates. None of the other foliose genera is distributed over such a wide range of different climates. All European species of Physcia were associated with algae of subclade I1 or, as exceptions, photobionts of subclades I2 or I4.

Tropical Physcia species did not contain algae of subclade I1 or I2. They were lichenized either with algae of subclade I4 or with subclades G4, G7, and G9.

In summary, the degree of selectivity is a quite variable trait in the Physciaceae. Most foliose species seem quite selective, while many crustose species seem to be compatible with a variety of Trebouxia lineages.

Fourteen of the 18 species of which three or more specimens were analyzed were associated with algae of only one subclade (TABLE 5.1). In all 77 species analyzed, nine species were found that were associated with photobionts of different subclades of the same clade and four species were found to be associated with Trebouxia ITS-variants from different clades (FIG. 5.2).

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