Next-generation sequencing reveals sterile crustose lichen phylogeny

I recently had a paper published that showed the utility of next-generation sequencing and a microbiome-based approach for basic lichen-forming fungal systematics. DNA sequences derived from the species known as Lepraria moroziana had for many years either been too messy to read or had seemingly been derived from disparate fungi, presumably because the species contains many endo-lichenic associates and/or it often grows in close proximity to other lichen-forming fungi. However, when using 454 sequencing to examine the whole pool of LSU (nuclear ribosomal large sub-unit) sequences from ten samples, it became clear that the dominant fungus in each was from the class Arthoniomycetes. This provided a breakthrough for the group, which was previously classified in the class Lecanoromycetes. After further investigations, it became clear that the two species Lepraria moroziana and Lepraria obtusatica belonged in their own genus, Andreiomyces, which itself we put in its own family, Andreiomycetaceae.

Relative abundances of the various putative classes represented by LSU sequences in each of the ten samples from the species of interest.

Interestingly, there were some sequences found in some of the amplicon pools derived from the "real" genus Lepraria (Lepraria s. str. in the class Lecanoromycetes). Had we gotten these sequences through standard Sanger sequencing, we may have been led astray, making it more difficult to discern the fact that the main lichen-forming fungus belongs to a previously unknown lineage in the class Arthoniomycetes!

I anticipate that this type of methodology will be advantageous for a lot of problematic fungi, especially those that remain uncultured. Here is the full abstract for the associated paper:

"The rapid phylogenetic placement and molecular barcoding of fungi is often hampered in organisms that cannot easily be grown in axenic culture or manually teased apart from their associated microbial communities. A high-throughput procedure is outlined here for this purpose, and its effectiveness is demonstrated on a representative species from an especially problematic group of fungi, the sterile crustose lichens. Sequence data of the LSU and ITS regions were generated from samples of a sterile crustose lichen species, Lepraria moroziana, using next-generation sequencing. DNA fragments most likely to represent the primary lichen-forming fungus were bioinformatically teased out using a specialized data processing pipeline. Phylogenetic analyses of the LSU region revealed that the lichen-forming fungus L. moroziana was previously placed in the incorrect class of fungi (Lecanoromycetes), and actually belongs to the class Arthoniomycetes, in the order Arthoniales. It is here treated as a member of a new family (Andreiomycetaceae Hodkinson & Lendemer fam. nov.) and genus (Andreiomyces Hodkinson & Lendemer gen. nov.). Additionally, Lepraria obtusatica Tønsberg is placed in the newly-defined genus based on its morphological, chemical, and ITS-based molecular similarity to L. moroziana. The procedure outlined here is projected to be especially useful for resolving the dispositions of diverse problematic fungi that remain unnamed, incertae sedis, or have taxonomic positions that are not expected to reflect their true phylogeny."

- Brendan

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Reference

Hodkinson, B. P., and J. C. Lendemer. 2013. Next-generation sequencing reveals sterile crustose lichen phylogeny. Mycosphere 4(6): 1028-1039.
Download publication (PDF file)
Download data and sequence-processing scripts (ZIP archive)
Download Ascomycota LSU alignment and analysis files (ZIP archive)
Download Arthoniales LSU alignment and analysis files (ZIP archive)

Leprocaulales

This year I published a paper with James Lendemer (Lendemer & Hodkinson 2013) in which we established a new order of fungi, Leprocaulales, for a group of sterile crustose lichens.  We first found that the species in the genera Lepraria and Leprocaulon were all shuffled up (they got this way due to their similar appearance), but we were able to sort them properly using molecular data.  It then became apparent, after further analyses, that the Leprocaulon clade is actually quite distantly related to other known groups of fungi (and is not even close to Lepraria), so we gave it a new family (Leprocaulaceae) and new order (Leprocaulales).  One really interesting aspect of this work is that it revealed to us that both groups (Lepraria and Leprocaulon) had crustose and fruticose growth forms within them.  This shows that growth form is more plastic than most of us had probably suspected, and that an entirely new growth form can evolve in fungi on a very short time scale!

Figure 1 from Lendemer & Hodkinson (2013): This tree shows the placement of Lepraria s.l. species (including the species of Leprocaulon) in multiple groups within four families of Ascomycetes. Newly generated sequences of Lepraria s.l. are mapped to the topology of the Schmull et al. (2011) Lecanoromycetidae phylogeny. All taxa with a "leprarioid" growth form are in red.

- Brendan

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Reference:

Lendemer, J. C., and B. P. Hodkinson. 2013. A radical shift in the taxonomy of Lepraria s.l.: molecular and morphological studies shed new light on the evolution of asexuality and lichen growth form diversification. Mycologia 105: 994-1018.
Download publication (PDF file)
View data and analysis file web-portal (website)