Trebouxia – Asterochloris, one or two genera?

Morphological and especially genetic data suggest that the genus Trebouxia is a paraphyletic assemblage (Archibald 1975, Hildreth & Ahmadjian 1981, Tschermak-Woess 1989, Friedl & Zeltner 1994, Friedl &

Rokitta 1997). On the basis of different types of immobile reproductive cells (spores), a segregation of the genus into Trebouxia Puymaly and Pseudotrebouxia Archibald was first proposed by Archibald in 1975.

Gärtner (1985) and Tschermak-Woess (1989) also recognized differences in the formation of non-motile reproductive cells, but rejected Archibald's concept. Gärtner (1985a,b) interpreted all differences in non-motile reproductive cells as a suppression of zoospore formation at different ontogenetic stages and therefore rejected the division of the genus. In contrast, Tschermak-Woess (1989) assumed only one type of non-motile reproductive cells to originate from ontogenetic suppression (aplanospores) while she supposed the other type (autospores) to be a phylogenetically fixed mode of spore development. Therefore, she splitted Trebouxia into two subgenera, Trebouxia and Eleutherococcus. Eleutherococcus sensu Tschermak-Woess includes all species which do not develop autospores. Although referring to the same characteristics, her grouping was not congruent with that of Archibald. Friedl (1993) also recognized differences in autospore formation and distinguished two cell cycles A and B, resulting in groupings rather similar to Tschermak-Woess' subgenera. However, Friedl assigned T. flava and T. usneae to cell cycle B, corresponding to the

"Eleutherococcus-group" sensu Tschermak-Woess (TABLE 4.1). Furthermore, he rejected the split of Trebouxia based on this trait because of its variability even within one strain and its dependence on culture conditions. With the exceptions of T. gelatinosa and T. anticipata, which were regarded as synonymous species by Friedl (1989b), all taxa included in Trebouxia subgenus Eleutherococcus by Tschermak-Woess were found to be identical in more than 93% of their nrITS sequence positions in a recent study by Piercey-Normore & DePriest (2001). These authors also included the photobiont of Anzina carneonivea (Anzi) Scheid. (formerly Varicellaria carneonivea (Anzi) Erichs.). From this lichen species the green algal genus Asterochloris was originally isolated and described (Tschermak-Woess 1980) with the species A.

phycobiontica. Sequence comparison revealed that the Anzina photobiont was very closely related to the Eleutherococcus species of Tschermak-Woess (Tschermak-Woess 1989, Piercey-Normore & DePriest 2001). The affiliation of Asterochloris phycobiontica with the species of Eleutherococcus had been also

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recognized by Tschermak-Woess (1989) from phenotypic characters. Accordingly, she transferred Asterochloris phycobiontica to Trebouxia subgenus Eleutherococcus and suggested, however, the reestablishment of Asterochloris if the taxa lacking autospores should be given genus rank (Tschermak-Woess 1989). All Trebouxia species found to resemble Asterochloris phycobiontica can be distinguished from the remaining Trebouxia species in five traits: A uniform and shorter nrITS region which strongly deviates from those of the remaining Trebouxia species. Friedl & Zeltner (1994) and Friedl & Rokitta (1997) inferred from nrSSU and nrLSU sequence data that Trebouxia (Asterochloris) magna was more closely related to Myrmecia biatorellae Tschermak-Woess & Plessl than to Trebouxia s.str.. In contrast to Trebouxia s. str., the pyrenoid matrix in the Asterochloris/Eleutherococcus species is hard to distinguish from the chloroplast stroma or the pyrenoid has an irregular shape (Friedl 1989a, b). Further, the chloroplasts of species resembling Asterochloris may flatten and assume a parietal position prior to cell division as well as in zoo- and aplanospores, while chloroplasts of Trebouxia species remain lobed and at a more central position during division (Ahmadjian 1960, 1970, Hildreth & Ahmadjian 1981, Gärtner 1985b, Friedl &

Gärtner 1988). Also the different chloroplast types of the Trebouxia s. str. and "Asterochloris" species are mutually exclusive (Friedl 1989b). Interestingly, the delimitation of Asterochloris and Trebouxia s. str. is accurately reflected by the photobiont selection behavior of Cladonia cristatella Tuck. in resynthesis experiments reported by Ahmadjian & Jacobs (1981). In these experiments, C. cristatella formed squamules with all algal strains of the "Asterochloris-group" but parasitized and killed all other Trebouxia strains tested (TABLE 3.1). Rambold et al. (1998) assumed that all Asterochloris-like Trebouxia species would be the only compatible photobionts for the majority of the Cladoniaceae. This assumption was supported by Piercey-Normore & DePriest (2001). Compiling these findings, a delimitation of Trebouxia s.l. in Trebouxia (s.str.) and Asterochloris as noted in TABLE 4.1 is supported by these five independent traits which are considered strong evidence supporting the concept of two genera. Therefore, the genus Trebouxia is understood here in the strict sense, excluding the species which are assigned to Asterochloris here.

Photobionts of the Physciaceae and the genus Trebouxia

TABLE4.1: Traits that separate Trebouxia and Asterochloris. Clade and subclade affiliation as presented in this study;

pyrenoid type follows Friedl (1989a); chloroplast type follows Friedl (1989b); Chloroplast shape and position prior to division follows Ahmadjian (1960, 1970), Hildreth & Ahmadjian (1981), Gärtner (1985b), Friedl & Gärtner (1988): c = central and lobate, p = parietal and flattened; autospore presence as in Tschermak-Woess (1989), cell cycle A and B follows Friedl (1993); Compatibility with Cladonia cristatella follows Ahmadjian & Jakobs (1981). +: autospore present / compatible with C. cristatella, -: autospore absent / incompatible with C. cristatella, *: results published by Piercey-Normore & DePriest (2001), n.a.: not analyzed.

Trebouxia species Subclade affiliation Pyrenoid type Chloroplast type Chloroplast position before division Autospore presence / Cell cycle Compatibility with C. cristatella

T. decolorans A1 arboricola-type 4 c +/A -

T. arboricola A2 arboricola-type 3 c +/A n.a.

T. aggregata A2 arboricola-type 3 c +/A -

T. crenulata A2 arboricola-type 4 c +/A -

T. jamesii A4 impressa-type 2 c +/A -

T. asymmetrica A7 gigantea- type 6 c +/A n.a.

T. showmanii A8 gigantea- type 6 c +/A -

T. gigantea A9 gigantea- type 6 c +/A -

T. incrustata A10 gigantea- type 6 c +/A n.a.

T. flava I1 impressa-type 1 c +/B n.a.

T. impressa I1 impressa-type 1 c +/A -

T. potteri I1 impressa-type 5 c +/A -

T. gelatinosa I2 gelatinosa-type 7 c -/B -

T. anticipata I2 gelatinosa-type 7 c -/B -

T. usneae G1 corticola-type 8 c +/B -

T. corticola G1 corticola-type 9 c +/A -

T. galapagensis G2 corticola-type 9 c +/A -

T. higginsiae G2 corticola-type 9 c +/A -

T. simplex S3 impressa-type 2 c +/A n.a.

Trebouxia

T. irregularis Clade I* irregularis-type 10 p -/B n.a.

T. glomerata Clade I* irregularis-type 10 p -/B +

T. pyriformis Clade I* irregularis-type 10 p -/B +

T. italiana n.a. irregularis-type 10 p ?/B +

T. excentrica Clade II* irregularis-type 11 p -/B +

T. magna Clade II* magna-type 12 p ?/B +

T. erici Clade II* erici-type 10 p -/B +

Asterochloris

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