Clade A

Clade A comprises eleven subclades A1 – A11 and 23 ITS-variants (FIG. 4.5). All ITS-variants except A8a (T. showmanii UTEX 2234) were found in the Physciaceae analyzed in this study (TABLE 4.4, FIGS. 4.4, 4.5). Although more subclades are differentiated in clade A than in the other clades, the average genetic distances within this clade are smaller than in the other clades. The average nucleotide substitution rate among subclades was 8 % (FIG. 4.2). The largest P-distance within a subclade of clade A was observed in subclade A1 with 8 %. Substitution rates within the other subclades were below 4 % (FIGS. 4.2, 4.4).

Relationships among the subclades of clade A are not well resolved in several instances. Some subclades or groups of subclades received significant support in bootstrap and Bayesian analyses or were characterized by unique sequence insertions. These characteristic insertions, however, could not be included in the phylogenetic analyses.

4.4.2.1 Sequence insertions and relationships among subclades

As a reference sequence for numbering the positions of sequence insertions and deletions, the sequence of T.

arboricola SAG 219-1a, (accession number Z68705, ITS-variant A2a) was chosen. The numbering of sequence positions starts with the first position of the ITS-1 region. All ITS-variants of clade A are characterized by the lack of an AG rich stretch of 4 – 9 bp in the ITS 1 region after positions 59 (appendix A.2, FIG. A.1). The single origin of subclade A1 and A2 was consistently supported in phylogenetic analyses as well as by an insertion which both subclades had in common. This insertion resided in the ITS-2 region, position 577 – 588 (appendix A.2, FIG. A.2). Subclade A2 was delimited from A1 by an additional insertion in the ITS 1 region (positions 146 – 177). Notably this insertion plus flanking positions was absent in ITS-variant A2c (appendix A.2, FIG. A.3). Among all distinguished subclades, subclades A1 and A2 were separated by the least p-distances (6% - 9%, FIG. 4.4). Such p-distances were occasionally found within single subclades, e.g. A1 and I4. However, an additional insertion only present in subclade A2 as well as different substrate preferences and an apparent discrimination between the two subclades by certain mycobionts (e.g. Anaptychia ciliaris, see chapter 5) were considered sufficient evidence to justify the delimitation of both subclades. Together with subclades A3 and A4 both subclades formed a consistently supported lineage. Subclades A9, (T. gigantea, A9a), A10 (T. incrustata, A10a) and the new ITS-variant A11a formed one lineage that was characterized by the presence of an insertion of 85 – 87 bp length at position 494 in the ITS-2 region (appendix A.2, FIG. A.4). However, their common origin was not significantly supported in bootstrap and Bayesian analyses. These three subclades were found to form a lineage together with T. showmanii (A8), which, received significant support in Bayesian analysis. Notably, T. showmanii was devoid of the sequence insertion that characterized subclades A9-A11. All other relationships among subclades of clade A appeared ambiguous.

Photobionts of the Physciaceae and the genus Trebouxia

4.4.2.2 Authentic strains, new ITS-variants, subclade support, internal distances

Subclade A1 contains the authentic strain of T. decolorans UTEX 901 (variant A1a). Four more ITS-variants were found in this subclade, which are not represented as cultures. The common origin of these five ITS-variants is well supported in phylogenetic analyses. Notably the maximal p-distances observed in this subclade is already in the range of p-distances found between subclades, e.g. distances between A7 and most other subclades of clade A. However, Anaptychia ciliaris was found associated with ITS-variants A1a and A1e only, and the phylogenetic delimitation of A1b from these two former ITS-variants was uncertain, because of which the whole group of ITS-variants was included into one subclade. However, the uniformity of this subclade might remain uncertain.

Subclade A2 contains the authentic strain and type species of Trebouxia, T. arboricola SAG 219-1a (ITS-variant A2a). This strain is synonymous with the authentic strain of T. aggregata UTEX 180, as revealed by the identity of both's nrITS sequences. One further authentic strain belongs to this subclade, T. crenulata CCAP 219/2, which represents ITS-variant A2c. Further two new ITS-variants were found to be closely related to these authentic strains (max. p-distance 3%). All four ITS-variants formed a highly supported subclade. Subclades A1 and A2 are the most closely related subclades in this study. The delimitation of both was recognized by different insertions in the ITS sequence as well as an apparent discrimination between the two subclades by e.g. Anaptychia ciliaris and Xanthoria parietina. Both lichens were exclusively associated with members of subclade A1.

The authentic strain of T. jamesii (UTEX 2233) represents ITS-variant A4a. Only one further ITS-variant in this subclade was detected, A4b. Both ITS-variants were separated by 3% nucleotide substitutions.

Two new ITS-variants were found that were closely related to T. asymmetrica (A7b). A common origin of these three ITS-variants was consistently found with different phylogenetic methods, but not significantly supported. However, all three genotaxa appeared to be the preferred photobionts of the closely related mycobionts Buellia elegans and B. zoharyi that were not found with other photobionts than these three ITS-variants. This was regarded as an indication for the unity of these ITS-variants into one subclade. Maximal p-distances were 4% in this subclade.

Subclades A8, A9, and A10 comprised only one ITS-variant each and were represented by the authentic strains of T. showmanii (UTEX 2234), T. gigantea (UTEX 2231), and T. incrustata (UTEX 784) respectively. All three subclades were separated by 7% to 10% nucleotide substitutions.

Photobionts of the Physciaceae and the genus Trebouxia

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The genetic distances of the new ITS-variant A11a to A10 and A9 were similar to the distance between A9 and A10. Therefore, the new ITS-variant was given subclade rank.

Subclades A3 and A6 were represented by one ITS-variant only and subclades A4 and A5 by two. All four subclades were well separated from each other and from subclades that included authentic strains and were therefore considered to be distinct.

sources of Trebouxia ITS variants

A1a T. decolorans UTEX 901 A1b ex Tornabea scutellifera 1 A1c ex Buellia pharcidia 1c20 A1d ex Buellia pharcidia 1c14 A1e ex Anaptychia ciliaris T A2a T. arboricola SAG 219-1a A2b ex Santessonia sorediata 1 A2c T. crenulata CCAP 219/2 A2d ex Diplotomma lutosum 1c39 A3a ex Anaptychia ulotrichoides 1 A4a T. jamesii UTEX 2233 A4b ex Buellia triphragmioides 53 A5a ex Diplotomma venustum 45 A5b ex Rinodina tunicata c53 A6a ex Rinodina oxydata 1grBd A7a ex Buellia elegans 1 A7b T. asymmetrica SAG 48.88 A7c ex Buellia elegans 2klBd A8a T. showmanii UTEX 2234 A9a T. gigantea UTEX 2231 A10a T. incrustata UTEX 784 A11a ex Buellia georgei 1

P-distances (%) among ITS-variants of clade A

A1a A1b A1c A1d A1e A2a A2b A2c A2d A3a A4a A4b A5a A5b A6a A7a A7b A7c A8a A9a A10a A1b 7

A1c 2 8 A1d 4 7 3 A1e 4 7 6 5 A2a 6 9 7 7 7 A2b 7 9 7 7 7 3 A2c 6 8 7 7 6 3 3 A2d 7 9 7 7 7 3 3 3 A3a 6 8 7 7 6 7 6 6 7 A4a 9 10 10 9 10 9 9 9 9 8 A4b 7 8 8 7 8 7 8 7 7 5 3 A5a 9 11 9 9 10 10 10 9 10 7 10 8 A5b 9 11 10 10 10 10 10 10 10 8 10 9 2 A6a 8 10 8 8 8 9 9 8 9 6 8 7 5 5 A7a 6 8 7 7 7 7 8 7 7 5 8 6 6 6 5 A7b 7 9 8 8 8 8 8 8 8 6 8 7 6 6 6 4 A7c 7 9 8 8 7 7 8 8 8 6 9 7 6 7 5 3 4 A8a 9 11 10 10 10 10 10 10 10 8 11 9 8 8 7 7 8 7 A9a 8 11 8 9 8 8 8 8 9 7 10 8 7 7 6 6 7 6 7 A10a 9 11 10 10 10 10 9 9 10 8 10 9 9 9 8 7 8 8 9 10 A11a 9 12 11 10 10 10 10 10 10 9 10 9 9 9 8 7 8 8 9 7 9

FIG.4.4: P-distances among subclades and ITS-variants of clade A. P-distances within subclades are shaded.

Photobionts of the Physciaceae and the genus Trebouxia

ML Phylogeny of Clade A

Support values:

PP/wMP/NJJC 0.1 subst./site

A1c ex Buellia pharcidia (1/-) A1d ex Buellia pharcidia (1/-) A1a T. decolorans UTEX 901 (11/11)

A1e ex Anaptychia ciliaris (2/-)

A1b ex Tornabea scutellifera (1/-) A2b ex Santessonia sorediata (2/-)

A2c T. crenulata CCAP 219/2 (1/1) A2a T. arboricola SAG 219-1a (2/1) A2d ex Diplotomma lutosum (1/-) A3a ex Anaptychia ulotrichoides (2/-)

A4a T. jamesii UTEX 2233 (7/6) A4b ex Buellia triphragmioides (1/-)

A7b T. asymmetrica SAG 48.88 (1/3)

A7c ex Buellia elegans (1/-) A7a ex Buellia elegans (5/-)

A10a T. incrustata UTEX 784 (4/1) A11a ex Buellia georgei (3/4) A9a T. gigantea UTEX 2231 (4/1)

A8a T. showmanii UTEX 2234 (-/1) A6a ex Rinodina oxydata (2/1)

A5a ex Diplotomma venustum (5/-) A5b ex Rinodina tunicata (2/-)

S3a T. simplex TW-1A2

FIG.4.5: Maximum Likelihood (ML) phylogeny of clade A. Branch supports obtained through Bayesian analysis (PP, 200 000 generations) and bootstrap analyses under the Maximum Parsimony (MP) criterion (500 replicates) and the Neighbor Joining method (NJJC, 1000 replicates). ITS-variants found in the Physciaceae are marked with a dot at the terminal nodes and ITS-variants obtained from cultures are underlined. Lichen names indicate the species from which the represented Trebouxia ITS-variant was obtained. Numbers in parentheses indicate the number of Physciaceae specimens (first number, bold) as well as the number of non-Physciacean specimens (second number) in which the respective ITS-variants were found. Blue subclade labels indicate subclades that include cultured strains, red labels indicate subclades from which no cultures are available. The same representative taxa as in F^IG.4.4 were used in this figure.

Photobionts of the Physciaceae and the genus Trebouxia

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