Evolution of Phototrophy in the Chloroflexi Phylum Driven by Horizontal Gene Transfer

Abstract

The evolutionary mechanisms behind the extant distribution of photosynthesis is a point of substantial contention. Hypotheses range from the presence of phototrophy in the last universal common ancestor and massive gene loss in most lineages, to a later origin in Cyanobacteria followed by extensive horizontal gene transfer into the extant phototrophic clades, with intermediate scenarios that incorporate aspects of both end-members. Here, we report draft genomes of 11 Chloroflexi: the phototrophic Chloroflexia isolate Kouleothrix aurantiaca as well as 10 genome bins recovered from metagenomic sequencing of microbial mats found in Japanese hot springs. Two of these metagenome bins encode photrophic reaction centers and several of these bins form a metabolically diverse, monophyletic clade sister to the Anaerolineae class that we term Candidatus Thermofonsia. Comparisons of organismal (based on conserved ribosomal) and phototrophy (reaction center and bacteriochlorophyll synthesis) protein phylogenies throughout the Chloroflexi demonstrate that two new lineages acquired phototrophy independently via horizontal gene transfer (HGT) from different ancestral donors within the classically phototrophic Chloroflexia class. These results illustrate a complex history of phototrophy within this group, with metabolic innovation tied to HGT. These observations do not support simple hypotheses for the evolution of photosynthesis that require massive character loss from many clades; rather, HGT appears to be the defining mechanic for the distribution of phototrophy in many of the extant clades in which it appears.

Keywords: comparative genomics; lateral gene transfer; microbial diversity; microbial metabolism; phylogenetics.

Figure 1

Reference phylogeny of Chloroflexi based on RpoB protein sequences, with our newly sequenced strains indicated with daggers, phototrophic strains highlighted (pink for fused pufLM, green for unfused), and Candidatus Thermofonsia noted. Most phototrophic Chloroflexi occur within a monophyletic clade in the Chloroflexia class, yet two distinct lineages of phototrophs occur outside of this class, separated by many non-phototrophic lineages. This phylogeny is based on RpoB—a single organismal marker protein which was recovered in all Ca. Thermofonsia genome bins—and is primarily intended as a reference for the critical phylogenetic relationships presented here (e.g., divergence of Ca. Thermofonsia from Anaerolineae, separation of phototrophic Thermofonsia from phototrophic Chloroflexia). Potentially more robust organismal phylogenies (e.g., 16S or larger concatenated protein datasets) will be possible with higher completeness Ca. Thermofonsia genomes.

Figure 2

Phylogeny of Type 2 phototrophic reaction center proteins made from concatenated sequences of PufL and PufM. Lineages with a fused pufLM gene are highlighted in pink while lineages with unfused reaction center genes are shown in green. The phylogeny of reaction center proteins is incongruent with the organismal tree (Figure 1), suggesting a history of horizontal gene transfer. However, the monophyly of fused pufLM genes (pink) is consistent with a singular gene fusion event.

Figure 3

Cladogram of the Chloroflexi phylum based on RpoB protein sequences, illustrated with the simplest possible evolutionary history of phototrophy that honors the relationships between the reaction center proteins and organismal markers. Non-phototrophic lineages are shown in black, lineages with fused pufLM reaction center genes are highlighted in pink, and lineages with unfused reaction center genes are shown in green. Arrows mark the inferred horizontal gene transfers of phototrophy genes. The most parsimonious scenario for the evolution of phototrophy within the Chloroflexi requires two separate horizontal gene transfer events, and a single gene fusion of pufLM.