The clade system and Trebouxia ITS-variant designation

Trebouxia nrITS sequencing revealed a much greater diversity in Trebouxia, than had been previously described (see below). A clade system was used to describe the phylogenetic hierarchy of the Trebouxia ITS-variants, as inferred by phylogenetic analyses of nrITS sequences. The lowest taxonomic rank was represented by ITS-variants. Monophyletic groups of closely related ITS-variants were united into subclades and monophyletic groups of subclades were combined to clades.

Clades are noted with an upper case letter, subclades by Arabic numbers and ITS-variants by a lower case letter (e.g. A2a). The clade that includes T. arboricola (authentic strain SAG 219-1a) was designated clade A, the clade including T. impressa (authentic strain UTEX 893) clade I, the clade with T. galapagensis (authentic strain UTEX 2230) clade G and the clade with T. simplex (authentic strain TW-1A2) clade S.

4.3.1.1 ITS-variants

An ITS-variant denoted a Trebouxia strain with a particular ITS sequence. In order to avoid an artificial inflation of the number of distinguished ITS-variants, due to sequencing errors or differential PCR amplification of within genome heterogeneities, a sequence variability of 2% within particular ITS-variants was admitted. In addition, particular ITS-variants had to be monophyletic in a neighbor-joining analysis employing the Jukes-Cantor model. As a consequence of the variability within ITS-variants, a number of ITS-variants comprized "short branch taxa" and "long branch taxa". Where "long branch taxa" of the different variants were separated by more than 2% substitutions, "short branch taxa" of the same ITS-variants could be separated by less than 2% nucleotide substitutions (FIG.4.1). Here, long branch taxa of monophyletic clusters of ITS sequences were used to delimit ITS-variants. As a consequence short branch taxa of distinct ITS-variants could be separated by less than 2% nucleotide substitutions. This situation, however, occurred only in subclade I1, where a large number of very closely related taxa was sampled.

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A

0.4 0.4

0.4 0.4

0.8 0.8

B C

D

ITS variant

a

ITS variant

b

d AB = 1.2 d CD = 1.2 d BC = 1.6

d AD = 2.4

d BD = 2.0 d CA = 2.0

4.3.1.2 Subclades

The level above ITS-variants was represented by “subclades”. This level was supposed to correspond to the taxonomic level of species. However, nrITS sequence data alone were not considered to provide sufficient information for a species definition (Grube & Kroken 2000, Taylor et al. 2000). Therefore, in addition to the monophyly criterion and p-distances as used for ITS-variant delimitation, mycobiont selection behavior was regarded as an important delimiter of subclades.

Ahmadjian & Jacobs (1981), demonstrated the usefulness of mycobiont selectivity in photobiont systematics in resynthesis experiments. The range of photobionts compatible with Cladonia cristatella and the delimitation of Asterochloris were found to be congruent (TABLE 4.1). According to Ahmadjian (1993) and Hawksworth & Honegger (1994) lichens can be interpreted as algal parasites. Eichler (1941a, b, 1948) proposed the hypothesis that parasites' taxonomies reflect their hosts' taxonomies (Fahrenholz's rule). This hypothesis has been followed in numerous instances (e.g. Mitter & Brooks 1983). Although it is now clear that Fahrenholz's rule is anything less than universal, it is generally accepted that most parasites are quite host specific (Mitter & Brooks 1983). Accordingly, mycobionts that appeared to discriminate between sister groups of ITS-variants were regarded as important indicators for the delimitation of subclades (TABLE 5.1, chapter 5). The applicability of this method could be shown in numerous instances where genetic delimitation was congruent with selection behavior of particular mycobionts (see chapter 5). Therefore, where p-distances did not allow an obvious delimitation of subclades, mycobiont selection behavior was used for subclade assignment (e.g. subclades A1, A7 and I4).

FIG. 4.1: As a consequence of the admission of intra-ITS variant variability (p-distances < 2%), conflicts between monophyly of ITS variants and the 2% limit of ITS variants variability occurred. Sequences B and C belong to different lineages but exhibit less than 2% sequence variability. In these instances, monophyly as revealed by a neighbor-joining analysis (using Jukes-Cantor model) was given priority above genetic distances in ITS variant assignment. Therefore, B and C were assigned to different ITS variants. Numbers on branches: p-distances (%), d = p-distances (%).

Photobionts of the Physciaceae and the genus Trebouxia

In order to approximate p-distances that suggest a delimitation of subclades, p-distances between the most closely related but morphologically clearly distinct species were determined. Genetic distances between T.

asymmetrica SAG 48.88 and all other authentic strains of clade A (T. arboricola SAG219-1a, T. jamesii UTEX 2233, T. incrustata UTEX 784, T. gigantea UTEX 2231, and T. showmanii UTEX 2234) ranged between 6 % and 8 % (FIG.4.4), p-distances between T. impressa UTEX 893 and T. gelatinosa UTEX 905 were 11 % (FIG.4.6), p-distances between T. simplex TW-1A2 and T. angustilobata AB97.027B3 were 8 % (FIG. 4.6). However, mutation rates apparently varied among clades (FIG. 4.2; for clade delimitation see below). Average genetic distances in clade A were only 8% while in clades I and G 12% and 17% were observed respectively (FIG. 4.2). This observation questioned the applicability of a fixed distance value across all clades to specify maximal boundaries for the inclusion of ITS-variants into single subclades. In general, a 6% threshold appeared appropriate for the delimitation of subclades, i.e. the inclusion of ITS-variants into one subclade. For example, in the most frequently sampled subclade I1 (113 sequences determined) maximal p-distances were 5% or less. However, this threshold was not considered mandatory.

When mycobiont selection behavior suggested a larger range of photobionts to be ecologically linked, larger p-distances within subclades were admitted (e.g. subclade I4 when considering Heterodermia leucomela including the conspecific H. boryi). When particular ITS-variants were found only once, and information about mycobiont selection behavior and potential intermediate sequences was not accessible, the 6%

threshold was relaxed to up to 10%. This conservative strategy of subclade delimitation was adopted, in order to avoid an artificial inflation of taxa. Although uncertainty may remain about the degree of mycobiont's selectivity as well as the taxonomic significance of p-distances, it is believed that including both types of information into a concept of subclade delimitation results in a more natural and ecologically meaningful subclade concept than relying on one of both only. Both aspects are strongly dependent on taxon sampling and subclade delimitation might therefore change in future, when additional information becomes available.

4.3.1.3 Clades

The highest level in this Trebouxia system was represented by clades. Also clades had to be monophyletic and previously published delimitations were adopted. According to Friedl et al. (2000), four clades were delimited, A, I, G, and S. Clade I includes clades 2 and 3 of Helms et al. (2001), which resulted in a more even range of genetic variability among clades.

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TABLE 4.4: List of Trebouxia nrITS sequences that were obtained in this study and those obtained from GenBank and student labs supervised by the author and numbers of available Trebouxia ITS-variants that originate from Physciacean specimens and Physciacean specimens. Note: Numbers of ITS-variants reported from Physciaceae and non-Physciaceae are not additive, since single ITS-variants were found in both groups.

Clade /

Photobionts of the Physciaceae and the genus Trebouxia