Photobiont selectivity leads to ecological tolerance and evolutionary divergence in a polymorphic complex of lichenized fungi

Abstract

Background and aims: The integrity and evolution of lichen symbioses depend on a fine-tuned combination of algal and fungal genotypes. Geographically widespread species complexes of lichenized fungi can occur in habitats with slightly varying ecological conditions, and it remains unclear how this variation correlates with symbiont selectivity patterns in lichens. In an attempt to address this question, >300 samples were taken of the globally distributed and ecologically variable lichen-forming species complex Tephromela atra, together with closely allied species, in order to study genetic diversity and the selectivity patterns of their photobionts.

Methods: Lichen thalli of T. atra and of closely related species T. grumosa, T. nashii and T. atrocaesia were collected from six continents, across 24 countries and 62 localities representing a wide range of habitats. Analyses of genetic diversity and phylogenetic relationships were carried out both for photobionts amplified directly from the lichen thalli and from those isolated in axenic cultures. Morphological and anatomical traits were studied with light and transmission electron microscopy in the isolated algal strains.

Key results: Tephromela fungal species were found to associate with 12 lineages of Trebouxia. Five new clades demonstrate the still-unrecognized genetic diversity of lichen algae. Culturable, undescribed lineages were also characterized by phenotypic traits. Strong selectivity of the mycobionts for the photobionts was observed in six monophyletic Tephromela clades. Seven Trebouxia lineages were detected in the poorly resolved lineage T. atra sensu lato, where co-occurrence of multiple photobiont lineages in single thalli was repeatedly observed.

Conclusions: Low selectivity apparently allows widespread lichen-forming fungi to establish successful symbioses with locally adapted photobionts in a broader range of habitats. This flexibility might correlate with both lower phylogenetic resolution and evolutionary divergence in species complexes of crustose lichen-forming fungi.

Keywords: Adaptation; Lecanoromycetes; Tephromela atra; Trebouxia; algal culture; crustose lichen; lichenized fungi; morphology; mycobiont; photobiont; phylogeny.

Fig. 1.

Phylogenetic hypothesis of the ITS locus of Trebouxia species: the 50 % majority rule consensus tree of the Bayesian analysis is presented; branch support (Bayesian PP >95 %/ML bootstrap values >70 %) is reported above or beside branches; branches and DNA extraction numbers of our new Trebouxia sequences (Supplementary Data, Table S1) are highlighted in bold. Incongruences (*1–8) of photobiont identities recovered between culture isolates and their original lichen thalli are colour-coded. DNA extraction numbers from cultures are in italics. Sequences retrieved from GenBank are reported in Supplementary Data Table S2. The growth substrate of lichens from which Trebouxia sequences were obtained is colour-coded beside the clades: green, bark, grey, rock (calcareous and siliceous), brown, soil.

Fig. 1.

Phylogenetic hypothesis of the ITS locus of Trebouxia species: the 50 % majority rule consensus tree of the Bayesian analysis is presented; branch support (Bayesian PP >95 %/ML bootstrap values >70 %) is reported above or beside branches; branches and DNA extraction numbers of our new Trebouxia sequences (Supplementary Data, Table S1) are highlighted in bold. Incongruences (*1–8) of photobiont identities recovered between culture isolates and their original lichen thalli are colour-coded. DNA extraction numbers from cultures are in italics. Sequences retrieved from GenBank are reported in Supplementary Data Table S2. The growth substrate of lichens from which Trebouxia sequences were obtained is colour-coded beside the clades: green, bark, grey, rock (calcareous and siliceous), brown, soil.

Fig. 2.

Comparison of Tephromela mycobiont (left) and Trebouxia photobiont (right) phylogenies. A high specificity of mycobiont–photobiont association is indicated by letters (A–F); low specificity between mycobionts and photobionts is highlighted by a dashed line. Mycobiont phylogeny is retrieved from Muggia et al. (2014).

Fig. 3.

Single strand conformation polymorphism (SSCP) analysis of ITS1 fragments of Trebouxia photobionts. Selected samples of Tephromela spp. and cultured photobionts were analysed. Bands excised from the gel are marked by letters (A–D); successfully sequenced bands are additionally encircled. Black bars above the numbers join original lichen samples to their corresponding photobiont isolates. The affiliations of the photobiont samples – identified in the Trebouxia phylogeny of Fig. 1 – are reported above the sample numbers and grouped by white lines. The position of SSCP bands corresponding to Trebouxia ‘sp.1’ and Trebouxia ‘TR1’ sequences (as they are the most frequent bands) is shown by black dashed lines.

Fig. 4.

Morphology and anatomy of Trebouxia species isolated in axenic culture. The cultures are named according to the DNA extraction numbers (Supplementary Data Tables S1 and S3); the name of the corresponding Trebouxia lineage of Fig. 1 is reported in parentheses. (A, C, E–G, I, L, P) Algal colonies on TM medium. (B, D, H, J, M, N, Q) Algal cells observed by light microscopy; arrows indicate the lobes of the chloroplast (central green body) and the nucleus; (N) Autospore; 12 daughter cells are visible. (K, O, R) Transmission electron microscopy (TEM) microphotographs of algal cells: nucleus (n), pyrenoid (p) – in detail in (K) – and thylakoid (t); the presence of two pyrenoids in (O) and (R) is probably due to the first duplication that chloroplasts undergo before the first cell division in generating autospores. (A, B) L1831 (clade I); (C, D) L1380 (T. impressa); (E) L1382 (T. arboricola); (F) L1828 (T. incrustata); (G, H) L1661, (clade IV); (I–K, O) L1535 (clade V); (L–N, R) L1379 (Trebouxia ‘sp.1’), (P, Q) L1654 (Trebouxia ‘TR1’). Scale bars: P = 0·5 mm; C = 1 mm, A, F, G, L = 2 mm; E, I = 3 mm; D, J = 25 μm; M, N, Q = 10 μm; B, H = 4 μm; K, O, R = 2 μm.