# Evolution of far-red light photoacclimation in cyanobacteria

Laura A Antonaru, Cecilia Rad-Menéndez, Susan Mbedi, Sarah Sarmann, Matthew Pope, Thomas Oliver, Shujie Wu, David H Green, Muriel Gugger, Dennis J Nürnberg

► To cite this version:

Laura A Antonaru, Cecilia Rad-Menéndez, Susan Mbedi, Sarah Sarmann, Matthew Pope, et al. Evolution of far-red light photoacclimation in cyanobacteria. Current Biology, In press, 10.1016/j.cub.2025.04.038. pasteur-05068924

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# Current Biology

## Evolution of far-red light photoacclimation in cyanobacteria

### Highlights

* Far-red light photoacclimation evolved in the early Paleoproterozoic

* The evolution of FaRLiP relies mainly on vertical descent, not HGT

* Sequence data mining provided resources for improved phylogenetics

* A conserved intergenic regulatory motif has been maintained for billions of years

### Authors

Laura A. Antonaru,
Cecilia Rad-Menéndez,
Susan Mbedi, ..., David H. Green,
Muriel Gugger, Dennis J. Nürnberg

### Correspondence

laura.antonaru@fu-berlin.de (L.A.A.),
dennis.nuernberg@fu-berlin.de (D.J.N.)

### In brief

Some cyanobacteria can thrive using only near-infrared/far-red light for oxygenic photosynthesis. Antonaru et al. show that this ability is an ancient adaptation inherited from a common ancestor in the early Paleoproterozoic. This hypothesis is supported by gene and genome trees and synteny within the far-red light photoacclimation gene cluster.
<figure><img src="image_2.png" /></figure>

Article

Evolution of far-red light photoacclimation
in cyanobacteria

Laura A. Antonaru,<sup>1,2,*</sup> Cecilia Rad-Menéndez,<sup>3</sup> Susan Mbedi,<sup>4,5</sup> Sarah Sparmann,<sup>4,6</sup> Matthew Pope,<sup>1,7</sup> Thomas Oliver,<sup>1,8</sup> Shujie Wu,<sup>2,9</sup> David H. Green,<sup>3</sup> Muriel Gugger,<sup>10</sup> and Dennis J. Nürnberg<sup>2,9,11,*</sup>

SUMMARY

Cyanobacteria oxygenated the atmosphere of early Earth and continue to be key players in global carbon and nitrogen cycles. A phylogenetically diverse subset of extant cyanobacteria can perform photosynthesis with far-red light through a process called far-red light photoacclimation, or FaRLiP. This phenotype is enabled by a cluster of ~20 genes and involves the synthesis of red-shifted chlorophylls *d* and *f*, together with paralogs of the ubiquitous photosynthetic machinery used in visible light. The FaRLiP gene cluster is present in diverse, environmentally important cyanobacterial groups, but its origin, evolutionary history, and connection to early biotic environments have remained unclear. This study takes advantage of the recent increase in (meta) genomic data to help clarify this issue: sequence data mining, metagenomic assembly, and phylogenetic tree networks were used to recover more than 600 new FaRLiP gene sequences, corresponding to 51 new gene clusters. These data enable high-resolution phylogenetics and—by relying on multiple gene trees, together with gene arrangement conservation—support FaRLiP appearing early in cyanobacterial evolution. Sampling information shows that considerable FaRLiP diversity can be observed in microbialites to the present day, and we hypothesize that the process was associated with the formation of microbial mats and stromatolites in the early Paleoproterozoic. The ancestral FaRLiP cluster was reconstructed, revealing features that have been maintained for billions of years. Overall, far-red-light-driven oxygenic photosynthesis may have played a significant role in Earth's early history.

INTRODUCTION

Cyanobacteria are important primary producers and played a
fundamental part in the Great Oxygenation Event that changed
Earth's atmosphere. <fnref n="1" />⁻<fnref n="3" /> The evidence can be seen in fossil stro-
matolites, some of the earliest evidence we have of life on
Earth. <fnref n="2" />,<fnref n="4" /> Molecular clock data combined with the fossil record
approximates their last common ancestor at 2.3⁻3.4 billion years
ago. <fnref n="5" />⁻<fnref n="8" /> Since then, cyanobacteria have diversified, colonizing a
wide variety of habitats from the low-nutrient open ocean to
the interior of rocks in hyperarid deserts, including regions inhos-
pitable to eukaryotic photosynthetic organisms. <fnref n="3" />,<fnref n="9" /> Some of this
environmental diversity is reflected in morphology, as cyanobac-
teria encompass lineages that are unicellular or filamentous,
including those with specialized nitrogen-fixing cells. <fnref n="7" />,<fnref n="10" /> Fila-
mentous growth forms have evolved multiple times, and this

morphology may have been acquired early on in cyanobacterial
evolution.<fnref n="7" />,<fnref n="11" />

Filamentous cyanobacteria are vital for understanding one of
the major early biological innovations on Earth: the development
of microbial mats. These structures would have protected
against ultraviolet radiation and, through calcification and sedi-
mentation, further led to the formation of stromatolites. Stromat-
olites were major biotic environments for a period of ca. 2 billion
years during the Proterozoic.<fnref n="7" />,¹² Based on data from extant
early-diverging lineages, the earliest cyanobacterial morpho-
types likely had small cell diameters (<2.5 µm).<fnref n="5" />,<fnref n="6" /> Despite their
evolutionary importance, few strains and genomes are available
for the early-diverging, thin-filamentous cyanobacteria.¹³ This
leaves gaps in the data when investigating, for example, the evo-
lution of ancient shading responses concomitant with the emer-
gence of stromatolites.

<footnote n="1">¹Department of Life Sciences, Imperial College London, London SW7 2AZ, UK</footnote>

<footnote n="2">²Institute for Experimental Physics, Freie Universität Berlin, 14195 Berlin, Germany</footnote>

<footnote n="3">³Culture Collection of Algae and Protozoa, Scottish Association for Marine Science, Oban PA37 1QA, UK</footnote>

<footnote n="4">⁴Berlin Center for Genomics in Biodiversity Research, 14195 Berlin, Germany</footnote>

<footnote n="5">⁵Museum für Naturkunde, Leibniz Institute for Evolution and Biodiversity Science, 10115 Berlin, Germany</footnote>

<footnote n="6">⁶Leibniz Institute for Freshwater Research and Inland Fisheries, 12587 Berlin, Germany</footnote>

<footnote n="7">⁷Esox Biologics, London W12 0BZ, UK</footnote>

<footnote n="8">⁸Department of Physics and Astronomy, Vrije Universiteit Amsterdam, 1081 HZ Amsterdam, the Netherlands</footnote>

<footnote n="9">⁹Dahlem Centre of Plant Sciences, Freie Universität Berlin, 14195 Berlin, Germany</footnote>

<footnote n="10">¹⁰Institut Pasteur, Université Paris Cité, Collection of Cyanobacteria, 75015 Paris, France</footnote>

<footnote n="11">¹¹Lead contact</footnote>

<footnote>*Correspondence: laura.antonaru@fu-berlin.de (L.A.A.), dennis.nuernberg@fu-berlin.de (D.J.N.)</footnote>

<footnote>https://doi.org/10.1016/j.cub.2025.04.038</footnote>
Photosynthetic organisms can adapt to different light conditions. Diverse mechanisms have evolved for photoacclimation, and organisms may change their photosynthetic systems in response to light quantity or quality (e.g., spectral niches).<sup>14-17</sup> Spectrum-based photoacclimation can help cyanobacteria thrive in various niches, from clear open ocean (enriched in blue light) to increasingly turbid seas and coastal waters (red light), as well as microbial mats and terrestrial environments (far-red light).<sup>15,18</sup> Associations between photoacclimation strategies and specific environments have been observed on a global scale, and some types of photoacclimation are present in many ancient lineages.<sup>19-21</sup>

One recently discovered spectral adaptation is far-red light photoacclimation (FaRLiP), but its evolution is still unclear. This process relies on red-shifted chlorophylls, chlorophyll *d* and chlorophyll *f*, to allow photosynthesis in the near-infrared/far-red (700-750 nm).<sup>22-25</sup> The distribution of FaRLiP cyanobacteria appears to favor shaded environments, which are enriched in far-red light.<sup>16</sup> These include microbial mats, the subsurface of rocks, and microbialites (sedimentary rock-like structures formed by microbes). Microbialites include present-day stromatolites, which are formed by layers of microbial mats iteratively growing and lithifying outward in a laminar matrix, and also thrombolites, which are similar but with a less organized structure.<sup>16,23,26-32</sup>

The basis of this adaptation is a cluster of around 20 genes encoding not only the enzyme synthesizing chlorophyll *f* (*chlF*) but also alternative components of photosystem I (*psa*), oxygen-evolving photosystem II (*psb*), phycobilisomes (*apc*), and a phytochrome signaling cascade (*rfp*).<sup>22,33-39</sup> Organisms that have been used to study FaRLiP include the unicellular *Synechococcus* sp. PCC 7335,<sup>40</sup> the aggregate-forming *Chroococcidiopsis thermalis* PCC 7203,<sup>22,41</sup> the filamentous *Halomiconema hongdechloris* CC206,<sup>42</sup> and the filamentous, heterocyst-forming *Chlorogloeopsis fritschii* PCC 9212.<sup>43</sup> However, FaRLiP is rare across the cyanobacterial species tree (approx. 4% based on previous data).<sup>20</sup> Because of this rarity, combined with morphological and phylogenetic diversity, it was originally hypothesized to have been largely transmitted by horizontal gene transfer (HGT).<sup>37</sup> However, recent work on ApcE2, the far-red phycobilisome-membrane linker, has emphasized the significance of vertical descent combined with gene loss in driving the evolution of this adaptation.<sup>26</sup> This hypothesis has been generally accepted.<sup>39,44-46</sup> Nevertheless, the data were insufficient to fully support vertical descent from the cluster's most recent common ancestor (MRCA).

Early HGT between distantly related cyanobacteria remained a possibility. Trees of different FaRLiP genes were not fully congruent,<sup>37,38</sup> and considerable variation existed in gene arrangement,<sup>24,47</sup> raising the possibility of extensive single-gene HGT. In particular, the HGT of the FaRLiP cluster to early-diverging filamentous lineages, after the acquisition of photosystem I genes, has been proposed.<sup>38</sup> The limited FaRLiP data from these lineages were a particularly large issue.<sup>26</sup> It left the evolutionary history of FaRLiP unclear.

This work aims to clarify the evolution of FaRLiP by investigating the present distribution, phylogenetics, and arrangement of FaRLiP genes, together with the phylogenetics of intergenic regions. Sequence data mining of genomes and metagenomes

ensured the recovery of many novel, highly diverse FaRLiP gene clusters for this purpose. The comprehensive phylogenetic analysis performed combined gene trees, genome trees, synteny, GC content analysis, and metatranscriptomics. We demonstrate that there is strong evidence for the cluster being vertically inherited since its MRCA, with losses in many cyanobacterial groups and the maintenance of a conserved regulatory motif since its origin.

## RESULTS

### FaRLiP is associated with microbialites and microbial mats

A total of 51 new complete or nearly complete FaRLiP gene clusters were recovered from a variety of sources, adding to previous work.<sup>38,39,48,49</sup> These include those from the NCBI database (14 clusters), additional cultured strains (16), raw data from the Sequence Read Archive (SRA) that were assembled in the course of this project (18), and enriched isolates from our laboratory (3) (Tables S1, S2, S3, and S4). Of these clusters, 32 had unique sequences, and together with the 50 unique previously known clusters, they formed the working dataset. The significance of metagenomic data as a resource for this project cannot be overstated. More than half of the clusters were sourced from metagenome-assembled genomes (MAGs) from species-rich environmental samples. Overall, common sampling locations included microbialites—both stromatolites and thrombolites (24%). FaRLiP was also present in non-lithifying microbial mats (23%), together with other types of environments (48%) and unknown sources (5%) (Figure 1A).

The microbialite sampling sites varied geographically and environmentally, encompassing five coastal and lake locations from three continents. Two of the stromatolite sites, notably Alchichica Lake (Mexico) and Cape Recife/Schoenmakerskop (South Africa), showed remarkable FaRLiP diversity. They contained ≥6 FaRLiP clusters (and MAGs) each, from four cyanobacterial orders, and approximately half of the cyanobacterial MAGs in these datasets had FaRLiP genes. Given the rarity of microbialites in the current Phanerozoic eon, the presence (and occasional abundance) of FaRLiP genes in so many of them suggests a significant role for FaRLiP in this habitat. The only other microenvironments with a similarly high diversity and abundance of FaRLiP genes were marine microbial mats on microplastics in the comparatively recent Pacific Garbage Patch. FaRLiP gene fragments have been reported from there before and were presumed to be associated with mat-forming, filamentous cyanobacteria inhabiting the plastics.<sup>26,51,52</sup>

Microplastics-associated sequences, together with microbialite sequences, were vital for recovering data from previously underrepresented, early-branching lineages of thin (cell diameter < 2.5 µm), filamentous cyanobacteria,<sup>6</sup> such as *Noodosilineales* and *Oculatellales* (Figure 1B), and helped to resolve early FaRLiP evolution. Overall, >600 new FaRLiP genes were recovered in addition to previous work,<sup>38,39,48,49</sup> in part through assembly of publicly available data complemented by motif-based strategies (Figure S1; Table S5), enabling for the first time more complex analyses of ancient FaRLiP evolution.
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /><figcaption>**Figure 1. The environmental and phylogenetic distribution of FaRLiP**</figcaption></figure>

(A) Microbialites, together with non-lithifying mats, are the source of about half of the FaRLiP gene clusters identified in this work. Environmental dataset size: 86 clusters.

(B) FaRLiP is present in a wide range of cyanobacterial orders, indicated by a blue-green box. The relationships between these lineages are shown on the left, and dashed lines mark alternative phylogenetic paths. The table shows the number of clusters associated with each order, already known (in blue) or newly identified, counted either as unique sequences (orange) or including duplicates (light orange). Tree schematic based on previous data.<sup>13,20,50</sup>

**The evolution of FaRLiP genes supports vertical descent**

The FaRLiP cluster consists of 19–24 genes. Out of them, 19 core genes are present in all known cyanobacteria with a complete FaRLiP cluster. They encode paralogous components of phycobilisomes (*apcB2/D2/D3/D5/E2*), photosystem I (*psaA2/B2/F2/I2/J2/L2*), photosystem II (*psbA3/B2/C2/D3* and *chlF*), and a phytochrome signaling cascade (*rfpA/B/C*). Their functions have been described in detail before.<sup>22,24,33,35,36</sup> Phylogenetic trees were inferred from all 19 core genes (Table S6). The tree for *apcE2*, the gene encoding the phycobilisome-photosystem linker, was especially highly supported. Sequences for *apcE2* fall into six groups, found in seven orders: *Nostocales* (I), *Chroococcidiopsidales* (II), *Coleofasciculales* (III), *Chroococcales* (IV), *Oculatellales* and *Leptolyngbyales* (V), and *Nodosiliinales* (VI) (Figure 2). Group V includes two orders that could not be reliably separated, as the branching of the FaRLiP sequences within them was not well resolved. Out of all groups, *Coleofasciculales* (III) is new to FaRLiP research. It is represented by a single strain, *Wilmottia* sp. PCC 9708. Overall, the *apcE2* tree also accurately matched the species tree (Figure 2). This provides strong evidence for vertical descent from a common ancestor and, due to the rarity of FaRLiP, implies that there were repeated losses of the gene cluster throughout evolutionary history.

All core FaRLiP gene trees were generally congruent with each other, as well as the species tree. This can be visualized by “overlapping” the trees into networks (Figure 3). The same six groups (I–VI) were often present. However, some discrepancies remain. In previous work, it was uncertain whether they were caused by biological factors (e.g., HGT) or by technical artifacts (e.g., different evolutionary rates and limited data).<sup>37,38</sup> This study lends strong support for technical artifacts as a reason. Increased data availability likely contributes to the fact that more similar gene trees were observed in this study.

The wide range of evolutionary rates and sizes between FaRLiP genes explains most of the differences remaining between the gene trees (Figure S2; Table S7). Large genes (>300 bp) that evolve moderately fast, such as the phycobilisome and photosystem II (PSII) genes, tended to also have

gene trees that agreed in fine detail and agreed with the species tree (Figures 3A and 3C). To a lesser extent, this agreement was also observed for the faster-evolving *rfp* genes of the phytochrome signaling cascade (Figure 3D). In contrast, photosystem I (PSI) gene trees showed considerable differences (Figure 3B).

It has been previously suggested that these mismatches indicate different phylogenetic histories, with PSI genes evolving later and then the cluster being transferred by HGT to early branches of the FaRLiP cyanobacterial tree.<sup>38</sup> This was based on the branching point of FaRLiP paralogs within trees of orthologous proteins.<sup>38</sup> However, the hypothesis discounts the influence of divergent evolutionary rates on phylogenetic reconstruction. A re-analysis of the initial large-scale PSI trees<sup>38</sup> revealed large differences from the species tree, even for major proteins like PsaA/B, which are expected to be little affected by HGT (Figure S3), raising doubts about the reliability of the branching points. This unreliability can be explained by the fact that PSI genes, FaRLiP and otherwise, include both very small, fast-evolving genes such as *psaI/J* and the very slow-evolving *psaA/B* (Table S7).<sup>53</sup> We further demonstrate this effect for FaRLiP genes by performing a minor artificial alteration of reverse-complementing DNA sequences prior to alignment and tree-building (the “heads or tails” method, or HoT).<sup>54</sup> This significantly changed the shape of the aforementioned FaRLiP PSI gene trees, due to unreliable alignments but not of the other core FaRLiP gene trees. Therefore, when evolutionary rates and likelihoods are taken into consideration, all core FaRLiP genes appear likely to share a common evolutionary trajectory, suggesting they have been inherited together since the cluster’s MRCA.

There was one significant dissimilarity that could not be explained by the genes’ different evolutionary rates, as gene trees agreed between themselves but not with the species tree (red cassettes in Figures 2 and 3). In the branch of interest, FaRLiP sequences of MAG origin were assigned to a different order (*Nodosiliinales*, VI) than the genome (*Oculatellales*, V). This may indicate HGT of the whole gene cluster. The mismatch observed is unlikely to be due to MAG contamination, as the three
<figure><img src="image_6.png" /><figcaption>**Figure 2.** The genome tree (left) resembles the tree of a FaRLiP marker gene (apcE2, right)</figcaption></figure>

This suggests that the FaRLIP cluster is an ancient innovation that mainly evolved by vertical descent. It is present in seven orders, listed on the right, organized into six groups (I–VI). Red boxes in the middle mark branches where FaRLiP gene phylogenies are different from the genome tree. *Gloeobacter* (purple) was used as outgroup. Genome tree was built with Orthofinder from single- and multi-copy protein orthologues. Bootstrap 100. On the left, in circles, the genome tree is cross-referenced with the different cluster orientations in Figure 5.
See also Figure S5 and Table S15.

genomes involved come from two separate geographic loca-
tions and sequencing projects. In unrooted networks, the branch
in question consistently diverges between the clusters for
groups V and VI (Figure 3). While HGT cannot be fully excluded,
it is more likely that this incongruity represents a long branch
attraction artifact instead. Between the aforementioned entries
and known homologs, there is low sequence similarity (AAI:
73%) at both the genome and the cluster level. Large evolu-
tionary distances make phylogenetic reconstruction more prone
to artifacts,⁵⁵ and as such, vertical descent remains the most
likely possibility. This *Oculatellales* branch will be discussed later
in the context of ancestral gene cluster composition.

In addition to the 19 core genes that are present in all clusters, other putative FaRLiP paralogs may occur in specific lineages. Previous research has identified three through experimental data (*psbH2/O2/V2*)²²,⁴²,⁴³,⁵⁶,⁵⁷ and another through bioinformatics³⁹ (named here *psbH2*'). This study made it possible to recover two additional genes, *psbF2* (a minor PSII gene) and *rfpD* (a putative regulatory protein) (Table S8). Earlier work showed evidence of them being preferentially expressed under far-red light (Table S9). Although the small size of these genes severely limits the accuracy of phylogenetic trees, these FaRLiP-associated sequences cluster together (Figure 4), and their trees resemble the ones of core FaRLiP genes and the species tree (Table S10).
# Current Biology
Article

<figure><img src="image_7.png" /></figure>

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<figure><img src="image_8.png" /><figcaption>**B** Photosystem I
(psA/B/F/I/J/L)</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>**C** Photosystem II
(psB3/A4/B/C/D)</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>**D** Regulatory cascade
(rfpA/B/C)</figcaption></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

Overall, the evidence supports the co-inheritance of all genes in the FaRLiP cluster from a common ancestor. This MRCA would have existed prior to the split of the earliest FaRLiP-containing cyanobacterial lineage, *Nodosilineales* (VI), from the cyanobacterial species tree.

## FaRLiP cluster architecture reveals evolutionary patterns

FaRLiP gene arrangement (synteny) is conserved in a manner consistent with the genome tree, which further supports vertical descent of this phenotype. Additional evidence is provided by GC% values that are very similar between gene clusters and associated genomes (Table S11). Synteny was used to classify FaRLiP clusters, which also resolved the same six groups (I–VI) as sequence similarity. There is typically one major (most frequently encountered) syntenic variant of the FaRLiP cluster in each group (Figure 5A). All these variants share certain conserved characteristics in gene arrangement but also exhibit aspects that are group specific.

Gene arrangements that are (near-)universal were likely to be present in the ancestral FaRLiP cluster. This includes the order of *apc* genes, followed by *psbD/C/B*, which are common across all lineages (except IV) (Figure 5A). In some cases, *psbA3* may be present upstream (I, II, and V). This pattern suggests that co-transcription and associated co-regulation,

<caption>**Figure 3.** The phylogenetic tree reconstructions of core FaRLiP genes tend to match each other</caption>

This can be seen by combining trees into networks for genes that encode phycobilisomes (A), photosystem I (B), photosystem II (C), and regulatory cascade components (D). The same six groups (I–VI, color-coded and listed at the bottom) can be seen. Thin branches mark agreements between the trees, and thicker branches mark disagreements. Red boxes highlight a divergent lineage, probably *Oculatellales*, that could not be easily assigned. Evolutionary rates affect how well the trees match, with the photosystem I network (B) being noisy. Networks built with SplitsTree. Individual trees are listed in Table S6.

especially between phycobilisome and PSII genes, are important for FaRLiP. Linkage can also be seen between the PSI genes (*psaA/B/F/II/J/L*), which are always found together in the same orientation (apart from small components such as *psaF* and *psaJ*). Phytochrome signaling cascade genes (*rfpA/B/C*) are also always syntenically linked, with the exception of the optional downstream component *rfpD*.

Other gene arrangements are group specific and conserved in a manner consistent with vertical descent. This includes *psaF/J* being present at the opposite end of the cluster from other PSI genes (in group I) or even in another

area of the genome (group II); *chlF* immediately upstream of *apc* genes (III); a unique translocation of *psbC/psbD* (IV); and the order of *rfp*, *chlF*, then *psa* (V) or, by contrast, *chlF*, *psa*, then *rfp* (VI) (Figures 5A and 5B). Some groups (I and II) exhibit only minor variants, both within and between them (Figures S4 and S5). Others, especially early-diverging lineages (V and VI), show a remarkable diversity, with evidence for multiple events of cluster splitting, gene translocation, and inversion (clusters 26, 29, and 30 in Figure S4). This reinforces the likelihood that they also were the earliest lineages to have FaRLiP.

Groups V and VI represent the earliest FaRLiP lineages to branch out from the cyanobacterial species tree (Figure 5B), and aspects that they share may define ancestral FaRLiP traits. The genes *rfp*, *psa*, and *chlF* are grouped together, though typically in different orientations. Which orientation is the oldest can be revealed by also considering minor cluster variants. Two such variants from group V (clusters 24–25 in Figure S4; red boxes in Figures 2 and 3) are defined by the order of *chlF*, *psa*, and then *rfp*, which is normally characteristic of group VI. This orientation is therefore likely the one in the ancestral FaRLiP cluster. That the minor cluster variants in question belong to group V, and not VI, can be supported by the fact that they contain a small hypothetical protein that is conserved within the *Oculatellales/Leptolyngbyales* (V) FaRLiP
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Article

<figure><img src="image_13.png" /><figcaption>B psbH2'</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>C psbO</figcaption></figure>

<figure><img src="image_15.png" /><figcaption>D psbV</figcaption></figure>

<figure><img src="image_16.png" /><figcaption>E psbF</figcaption></figure>

<figure><img src="image_17.png" /><figcaption>F rfpD orthologues</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>Figure 4. The evolution of optional FaRLiP proteins</figcaption></figure>

These protein sequences are shown here in the context of related (orthologous) sequences, such as PsbH (A), PsbH2' (B), PsbO (C), PsbV (D), PsbF (E), and RfpD (F). These network representations were built from sequence alignments. They show that FaRLiP sequences (red) are more similar to each other than to other related sequences. Similarly, distinct branches may be seen for orthologues in eukaryotes as distantly related as land plants and algae (A, C, and E), divergent cyanobacterial lineages (the Syn-Pro clade in E), or for other paralogous lineages (D). The protein PsbH2' appears to be FaRLiP-specific, and no other related sequences could be found. For PsbF, FaRLiP sequences appear to form two neighboring clusters, not one; however, this can likely be explained as noise from the small size of this protein (approx. 45 aa). Overall, these data support optional FaRLiP genes representing distinct evolutionary lineages. Sequence similarity networks built with SplitsTree. Sequences available via STAR Methods.
See also Tables S8, S9, and S10.

clusters (Figures 5A and S6). Another potentially ancestral sim-
ilarity between these minor cluster variants from group V and
group VI is the presence of the optional FaRLiP gene psbO2.
The standard, visible-light version of this gene is known to sta-
bilize the manganese cluster of photosystem II.<sup>58,59</sup> We postu-
late that the presence of psbO2 may represent an ancestral
trait linked to FaRLiP-PSII function.

The cluster in the MRCA of known FaRLiP cyanobacteria
possibly had the following four elements. (1) It co-transcribed
chlF with downstream PSI genes, followed by independently
transcribed rfp genes. (2) Allophycocyanin genes were co-tran-
scribed with downstream PSII genes, including minor genes
psbH2 and psbH2'. (3) The FaRLiP D1 gene, psbA3, had its
own promoter. (4) The far-red psbO gene was present and,
tentatively, also far-red psbV and psbF, as seen in the earliest
lineage, VI (Figure 5).

The FaRLiP genes are co-regulated in operons through
an ancient motif

An important aspect of the evolution of FaRLiP is its regulation. The highly conserved nature of FaRLiP gene arrangements suggests that parts of the cluster could be co-transcribed as operons, with a conserved regulatory structure. This hypothesis is supported by metatranscriptomics data, by the presence of conserved syntenic variants, and by previous laboratory work, as addressed below. Analysis of 14 metatranscriptomes (downloaded from the IMG/MER database) shows that multi-gene mRNAs, which likely represent operons, are present across three FaRLiP groups (Figure S7; Table S12).

Analyzing both rare and common cluster variants also sug-
gests conserved patterns of transcription. For example, single-
gene translocations and inversions have only been observed
for core genes psbA3 and the chlorophyll f synthase (chlF).
<figure><img src="image_19.png" /></figure>

A

I. Nostocales

Fischerella (e.g. *Fischerella* sp. PCC 7521, NIES-592)

<figure><img src="image_20.png" /></figure>

Calothrix parasitica NIES-267

<figure><img src="image_21.png" /></figure>

II. *Chroococcidiopsidales*

*Chroococcidiopsis thermalis* PCC 7203

<figure><img src="image_22.png" /></figure>

III. *Coleofasciculales*

*Wilmottia* sp. PCC 9708

<figure><img src="image_23.png" /></figure>

IV. *Chroococcales*

*Hydrococcus rivularis* NIES-593

<figure><img src="image_24.png" /></figure>

V. *Oculatellales* + *Leptolyngbyales*

*Leptolyngbya* sp. JSC-1

<figure><img src="image_25.png" /></figure>

VI. *Nodosilineales*

MAG: *Romeria gracilis* LEGE 07310

<figure><img src="image_26.png" /></figure>

0. Ancestral FaRLiP cluster

<figure><img src="image_27.png" /></figure>

Scale: 1kB

B

<figure><img src="image_28.png" /></figure>

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This fact indicates that *chlF* and *psbA3* are likely to have their own promoters, which they might share with downstream genes. Other groups of genes (like *psa* genes) may share a promoter/ regulatory mechanism between them, as hinted at by partial clusters (Figure S8). In addition, this study showed that most large insertions, translocations, and/or split clusters were asso- ciated with the downstream area of the *rfp* genes. In order for far-red light to induce expression of the rest of the FaRLiP clus- ter, the phytochrome (*rfpA*) and the other components of the signaling cascade (*rfpB/C*) need to be already present. This has been shown by transcriptomic data and genetic studies.<sup>33,43,60</sup> Therefore, the *rfp* genes are likely to be regulated differently from the rest of the FaRLiP cluster. We hypothesized that a specific intergenic motif (defined as a conserved DNA sequence) would control the transcription of the FaRLiP operons under far-red light, except for *rfp* genes. This conserved region would likely interact with the DNA-binding residues in the regula- tory protein RfpB, a transcriptional activator,<sup>33</sup> to promote tran- scription, and it would be expected to commonly be present up- stream of *chlF* and *psbA3*.

A class of conserved, homologous, T-rich motifs was identified in FaRLiP intergenic regions (Figure 6A). These motifs were pre- sent within the first 440 bp upstream of particular genes in all known FaRLiP lineages. BLAST searches suggested that this type of motif was specific to FaRLiP species. No known role was identified when searching DNA motif databases with Tom- tom,<sup>61</sup> but we hypothesize that it is likely to be regulatory, as fol- lows. Previous work has shown that in some strains, the intergenic sequence upstream of *chlF* (which contains one such motif) can drive the expression of a fluorescent reporter when induced by far-red light in the presence of regulators RfpA/B/C.<sup>60,62</sup> This ap- pears to rely on a conserved region matching the aforementioned motif.<sup>62</sup> In this study, the homologous motifs identified could be classified into three groups, from high to low conservation: *chlF* motifs, “long” motifs, and “short” motifs.

The most conserved motif version is present upstream of *chlF*. These *chlF* motifs are ~61 bp long and have conserved 5' and 3' regions, separated by a variable gap (Figure 6A). The second group of motifs has moderate conservation, especially in the 5' region, and these are ~59 bp “long” motifs preceding other genes (*apc*, *psa*, and some *psbA3* variants). The 5' region may be lost altogether in “short” motifs (~33 bp), such as those up- stream of some *psbA3* sequences, as well as minor genes *rfpD* and *psbO*. Any of these motifs is nearly always present in the intergenic region upstream of expected FaRLiP operons (Figure 6B; Table S13). Due to differences in gene arrangement, expected operons vary between lineages. Notably, no motif was detected upstream of *rfpA/B/C*, which is likely due to the different regulatory requirements of these genes.

Our phylogenetic analysis strongly suggests that a highly conserved *chlF* motif was the ancestral version, from which other motifs originated through repeated, independent duplications (Figures 6C and S9). The ancestral FaRLiP cluster carried a *chlF* motif. Duplications resulted in long motifs preceding *apc* genes in the *Nodosilineales* (VI) or the *psa* genes in the *Nosto- cales* (I)/*Chroococcidiopsidales* (II) and some *psbA3* genes. Short motifs likely originated from a long *chlF* motif too, possibly in an early event before the split of the earliest FaRLiP lineage, the *Nodosilineales* (VI), from the rest of the cyanobacteria. Motifs preceding *psbA3* may have a complex evolutionary history, including both long and short motifs, with ancient and recent transitions from the former to the latter. In general, all motifs, even short and divergent ones (e.g., upstream of *rfpD*), show evolutionary patterns overall supporting vertical descent (Figure S9). Motifs have been maintained across evolutionary time, some perhaps since the MRCA (2.2–2.5 billion years ago).<sup>5,6</sup>

The high conservation within the *chlF* motifs, combined with the high level of expression of the *chlF* gene within the cluster<sup>42,43,56</sup> and the evidence that this is the oldest FaRLiP gene,<sup>39,63</sup> underscores the vital role that the *chlF* synthase has in FaRLiP. However, the co-transcription of *chlF* and *psa* genes in early-diverging lineages (V and VI) also suggests that PSI genes may too have played an important role in early FaRLiP evolution prior to the cluster MRCA.

DISCUSSION

This study proves that FaRLiP is an evolutionarily early pheno-
type. It has been inherited vertically at least since its MRCA, prior
to the split of the order *Nodosilineales* (VI) from the cyanobacte-
rial species tree. This event was preceded by the split of *Acaryo-
chloris*, the only other far-red light photosynthesizing cyanobac-
terium, but the strategy of *Acaryochloris* (using a majority of
chlorophyll *d*) has remained constrained to one genus.<sup>16,64</sup> In
contrast, FaRLiP is spread throughout the cyanobacteria. The
evidence for vertical FaRLiP evolution can be seen in gene trees,
which generally agree with the species tree, as well as in synteny
and in supporting evidence such as GC% (Table S11). Prior to
the MRCA, different genes were suggested to have entered the
cluster at different times, with the earliest being the *chl f* syn-
thase, followed by *psbA3*.<sup>39,63</sup> Originally, PSI genes were sug-
gested to have emerged later, thereby requiring HGT to explain
gene presence in early-diverging lineages.<sup>38</sup> Our results confirm
the vital role of the *chl f* synthase in the history of the cluster;
however, our results do not support the HGT hypothesis. This
study shows that many FaRLiP PSI genes are characterized by
unusually slow/fast evolutionary rates, which make phylogenetic

<caption>Figure 5. Synteny and gene orientation are conserved within FaRLiP gene clusters in a manner consistent with vertical descent</caption>

(A) There are six cyanobacterial groups in which FaRLiP is present (I–VI). Each shows gene arrangements that are specific to itself (black boxes) as well as one feature that is conserved between most orders (*apc*, followed by *psb* genes, gray highlight). Only the most abundant syntenic variants are listed here. Based on both abundant and rare variants (especially with regard to *psbO*), the composition of the ancestral FaRLiP cluster (0) may be inferred.
(B) Syntenic features specific to each group (right) are conserved in a way consistent with vertical descent (species tree schematic shown to the left). The *psbF* gene is specific to group VI, though not present in the most common variant shown above. Color code: PSI (red), PSII (green), apart from *psbA3* (turquoise), *chlF* (yellow), phycobilisome (blue), regulatory signaling cascade (brown), non-FaRLiP hypothetical proteins (gray), and other proteins (pink). All variants, both abundant and rare, are listed in Figure S4.
See also Figure S6.
Current Biology

Article

<figure><img src="image_29.png" /></figure>

CellPress

OPEN ACCESS

<figure><img src="image_30.png" /><figcaption>Figure 6. The FaRLiP gene cluster contains repeats of intergenic motifs</figcaption></figure>

(A) Two distinct conserved regions, 5' (black box) and 3' (red box), separated by a variable gap of ~12 bp, have been observed within them. Based on the conservation of these regions, three types of motifs (1-3) can be defined. The chIF motif (1) represents the most highly conserved sequence, ~61 bp in length. It

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reconstructions difficult to build and challenging to interpret. Nevertheless, our evidence supports vertical descent as the principal mechanism explaining the distribution of FaRLiP genes in the genomes of the lineages studied.

Gene persistence may be affected by the presence of other genes, with which they form functional modules. The FaRLiP cluster MRCA likely co-transcribed allophycocyanin genes with most PSII genes, while *chlF* was co-transcribed with PSI genes. The cluster may have also included a far-red *psbO*. It is not possible to tell from the synteny data whether PSI or non-*chlF* PSII genes were the earliest. The fact that partial FaRLiP clusters exist, lacking either PSI or PSII genes, supports either option (Figure S8). The FaRLiP cluster from *Chroococcidiopsis* sp. CCMEE010, for example, lacks PSI genes but was shown to be functional.⁶⁵,⁶⁶ As a counterpoint, in another experiment, when the *chlF* gene was introduced into non-FaRLiP *Synechococcus* sp. PCC 7002, *chl f* was incorporated into standard PSI.⁶⁷,⁶⁸ It is feasible to imagine that selective pressure in an early cyanobacterium may have led to a duplication of PSI, leading to a photosystem that could more advantageously incorporate the new chlorophyll. However, it should be noted that without a similarly red-shifted PSII, the system might not be photosynthetic (fixing carbon) but only phototropic (producing ATP). Alternatives can be seen in modern plants, which rely on both visible and far-red light photons in response to shading, and in algae, which can use solely far-red light through mechanisms yet to be fully understood.⁶⁹⁻⁷¹

Based on this study, the FaRLiP cluster is characterized by regulatory simplicity. One class of highly conserved, homologous intergenic motifs may be responsible for much of its regulation. In addition, no functional FaRLiP cluster has yet to be observed to be broken into more than two pieces, and reunifications are also apparent. This stands in contrast to the complexity seen for visible-light paralogs, which are commonly scattered throughout the genome and transcribed and regulated independently.⁷²,⁷³ Co-localization typically is limited to a few gene pairs, such as *psaA/B* or *psbC/D*, or rarely more genes, as in the phycobilisome operon *cpcB/A/C/C/D*.⁷⁴,⁷⁵ A simpler and more symmetric system with fewer regulatory regions is likely to be more robust and easier to maintain when not in use for long periods of time. Robustness has mostly been described for essential genes ("the persistence model"), but recent work has also extended it to genes under weak selection by considering transposable elements.⁷⁶,⁷⁷

Co-regulation, as revealed by synteny and metatranscriptomic analysis, appears to be a vital characteristic of the FaRLiP cluster. However, it has remained unknown why genes are preferentially found in certain conserved orientations, for example, *psbD*, *psbC*, and *psbB*. One hypothesis is that for PSII, this may be

connected to photosystem component replacement rates. The most easily damaged protein in visible-light PSII is *psbA*,⁷⁸ whose FaRLiP version has its own promoter. Then, in order from second-most to least easily damaged, there are *psbD*, *psbC*, and *psbB*.⁷⁸ It might be that by simply relying on gene order and incomplete transcripts, the FaRLiP cluster can ensure part of the regulation necessary for timely replacement. This would be consistent with transcriptional rates observed in far-red-light-adapted *Chlorogloeopsis fritschii* PCC 9212.⁴³ However, to what point transcriptional (versus translational) regulation influences the amount of end product in bacteria is still debated.⁷⁹⁻⁸¹

The FaRLiP cluster was likely to be present in the common ancestor of early-diverging lineages (*Leptolyngbya*-like morphotypes, now known as *Nodosilineales/Oculatellales/Leptolyngbyales*), before their split from the rest of the cyanobacteria. In contrast with even earlier ("basal") branches, these cyanobacteria have been associated with evolutionary innovations in microbial mat formation, such as increased ability to tolerate salinity, a filamentous growth form and nitrogen fixation, and sometimes larger cell sizes (>2.5 µm), enabling the growth of cyanobacteria in thicker layers.⁵⁻⁷ The ability to photosynthesize using far-red light would likely have been beneficial in these shaded environments. Several molecular clock studies place this ancestor in the early Paleoproterozoic (or Siderian), concomitant with the rise in stromatolite diversity.⁴⁻⁶,¹² The presence and comparative abundance of FaRLiP species in modern microbialites, together with the presence of FaRLiP transcripts (Table S12), suggest that FaRLiP could have been highly influential in Paleoproterozoic stromatolite formation. During this time, and after the stromatolites' decline in the present Phanerozoic eon,¹² FaRLiP cyanobacteria continued to diversify. They are present on all continents, including Antarctica, though rarely in hot and cold deserts, from ocean microplastics to forest canopies and the inside of caves,²⁶,²⁸,³¹,⁴⁸ but especially in various microbial mats and modern microbialites.

## RESOURCE AVAILABILITY

### Lead contact

Requests for further information should be directed to and will be fulfilled by the lead contact, Dennis J. Nürnberg (dennis.nuernberg@fu-berlin.de).

### Materials availability

This study did not generate new, unique reagents.

### Data and code availability

* MAGs built during the course of the study, together with newly sequenced FaRLiP clusters, have been deposited in the NCBI database and in Figshare (https://doi.org/10.6084/m9.fishshare.25757274). Accession numbers are listed in Tables S2 and S3 and are available

contains highly conserved 5' and 3' regions. Long motifs (2), ~59 bp, are found upstream of other FaRLiP genes, but their 5' region is less conserved. Yet other genes may only have a short motif (3), 33 bp, equivalent to only the conserved 3' region.

(B) The distribution of motifs (1-3) is shown, both according to their presence upstream of certain genes (top) as well as in particular cyanobacterial orders (left). Full circle: motif present; empty circle: motif absent, gene present; dash: gene absent. Upstream of the gene *psbA*, both long motifs (dark turquoise) and short motifs (light turquoise) may be found. Proposed operons of the FaRLiP cluster in various lineages are listed to the right.

(C) A tree of motifs is shown. All FaRLiP motifs evolved from a motif preceding the gene *chlF* (yellow). From it, motifs preceding other genes were duplicated (colored lines), and their evolution is consistent with the species tree (black lines). Note that this tree does not show the order of gene incorporation (all core genes would be present from the start), just the history of intergenic motifs. Uncertain evolutionary paths are highlighted in dashed lines. Phylogenetic aspects are expanded on in Figure S9, while Table S13 lists motifs by species.
from the date of publication. All other sequence data used in the study (all FaRLIP clusters, genes, and intergenic motifs) are available from the same Figshare repository.

* All original code used in the cluster labeling process is accessible at https://github.com/cyano-lab/GeneGraphics_formatting and is publicly available as of the date of publication.

* Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

## ACKNOWLEDGMENTS

Many thanks to Tal Dagan (University of Kiel), Michael W. Gaunt (Solena Ag), and Paul Johnston (University of St. Andrews) for bioinformatics advice. D.J. N. acknowledges the support of Europlanet 2020 RI for sampling in Morocco. Europlanet 2020 RI has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement no. 654208. The HPC facilities at Imperial College London, Freie Universität Berlin, and the University of Kiel were used during the study. Daniel J. Wilson, Nicholas D. Sanderson, and Leanne Barker are thanked for the sequencing of the CL0 and SAG 4.84 clusters, with sequencing supported by the National Institute for Health Research Health Protection Research Unit (NIHR HPRU) at the University of Oxford in partnership with Public Health England (PHE) (HPRU-2012-10041 to D.J.W.). This research was supported by the Deutsche Forschungsgemeinschaft (DFG; Emmy Noether project award no. NU 421/1 to D.J.N.). L.A.A. was supported by a postdoctoral fellowship from the Freie Universität Berlin and a Schrödinger fellowship from Imperial College London. S.W. was supported by a scholarship from the China Scholarship Council. Access to the CCAP/SAMS collection was possible through the AssemblePlus program. C.R.M. and D.G. were funded in part by the UK Natural Environment Research Centre (project NE/R017050/1). The Pasteur Cultures of Cyanobacteria is funded by the Institut Pasteur. The Mutualized Platform for Microbiology (P2M) is acknowledged for PCC genomes. Open access funding was provided by Project DEAL.

## AUTHOR CONTRIBUTIONS

L.A.A. and D.J.N. designed the study. C.R.-M., D.H.G., and M.G. provided strains and sequence information. S.W. cultured the strains. S.M., S.S., and D.H.G. were responsible for sequencing and data curation. L.A.A. performed the analyses with the help of M.P. and T.O. for the intergenic motif analysis. L.A.A. and D.J.N. wrote the paper, with contributions from all authors.

## DECLARATION OF INTERESTS

The authors declare no competing interests.

## STAR★METHODS

Detailed methods are provided in the online version of this paper and include the following:

* KEY RESOURCES TABLE

* EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

* Cyanobacterial samples

* METHOD DETAILS

* Environmental classification

* DNA extraction

* (Meta)genome sequencing

* (Meta)genome assembly

* Metagenome binning and quality control

* Database sourcing

* Gene-level phylogenetic analysis

* Genome-level phylogenetic analysis

* Synteny analysis

* Taxonomic conventions

* Motif identification

* QUANTIFICATION AND STATISTICAL ANALYSIS

## SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.cub.2025.04.038.

Received: May 22, 2024

Revised: January 20, 2025

Accepted: April 15, 2025

Published: May 13, 2025

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## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3"><strong>Bacterial and virus strains</strong></td></tr><tr><td>Altericista sp.</td><td>Pasteur Culture of Cyanobacteria, Paris, France</td><td>PCC 9706</td></tr><tr><td>Calothrix sp.</td><td>Pasteur Culture of Cyanobacteria, Paris, France</td><td>PCC 7709</td></tr><tr><td>Calothrix sp.</td><td>Pasteur Culture of Cyanobacteria, Paris, France</td><td>PCC 9633</td></tr><tr><td>Chlorogloeopsis fritschii</td><td>Pasteur Culture of Cyanobacteria, Paris, France</td><td>PCC 9301</td></tr><tr><td>Chroococcidiopsis sp.</td><td>Pasteur Culture of Cyanobacteria, Paris, France</td><td>PCC 7432</td></tr><tr><td>Chroococcidiopsis sp.</td><td>Pasteur Culture of Cyanobacteria, Paris, France</td><td>PCC 7434</td></tr><tr><td>Chroococcidiopsis sp.</td><td>Pasteur Culture of Cyanobacteria, Paris, France</td><td>PCC 7439</td></tr><tr><td>Fischerella sp.</td><td>Culture Collection of Algae and Protozoa at the Scottish Association for Marine Science, Oban, Scotland, UK</td><td>CCAP 1427/2</td></tr><tr><td>Gloeocapsa sp.</td><td>Pasteur Culture of Cyanobacteria, Paris, France</td><td>PCC 10905</td></tr><tr><td>Mastigocladus sp.</td><td>Culture Collection of Algae at Göttingen University, Göttingen, Germany</td><td>SAG 4.84</td></tr><tr><td>Phormidesmis sp.</td><td>Culture Collection of Algae and Protozoa at the Scottish Association for Marine Science, Oban, Scotland</td><td>CCAP 1446/5</td></tr><tr><td>Pleurocapsa sp.</td><td>Pasteur Culture of Cyanobacteria, Paris, France</td><td>PCC 7516</td></tr><tr><td>Rivularia sp.</td><td>Pasteur Culture of Cyanobacteria, Paris, France</td><td>PCC 7111</td></tr><tr><td>Stanieria sp.</td><td>Pasteur Culture of Cyanobacteria, Paris, France</td><td>PCC 7304</td></tr><tr><td>Wilmottia sp.</td><td>Pasteur Culture of Cyanobacteria, Paris, France</td><td>PCC 9708</td></tr><tr><td>Xenococcus sp.</td><td>Pasteur Culture of Cyanobacteria, Paris, France</td><td>PCC 7307</td></tr><tr><td>CL0</td><td>This study. See 'Deposited data'.</td><td>CL0</td></tr><tr><td>CL1</td><td>This study. See 'Deposited data'.</td><td>CL1</td></tr><tr><td>WS17</td><td>This study. See 'Deposited data'.</td><td>WS17</td></tr><tr><td colspan="3"><strong>Deposited data</strong></td></tr><tr><td>Initial FaRLiP distribution data</td><td>Antonaru et al.<sup>26</sup></td><td>N/A</td></tr><tr><td>Genbank sequence data</td><td>NCBI Genbank</td><td>Table S1</td></tr><tr><td>IMG/MER sequence data</td><td>IMG/MER</td><td>Tables S4 and S12</td></tr><tr><td>SRA sequence data</td><td>NCBI SRA</td><td>Tables S3 and S14</td></tr><tr><td>MAGs obtained</td><td>This study.</td><td>NCBI WGS: Table S3, FigShare: https://doi.org/10.6084/m9.fishshare.25757274</td></tr><tr><td>FaRLiP clusters</td><td>This study.</td><td>FigShare: https://doi.org/10.6084/m9.fishshare.25757274</td></tr><tr><td>FaRLiP gene alignments</td><td>This study.</td><td>FigShare: https://doi.org/10.6084/m9.fishshare.25757274</td></tr><tr><td>FaRLiP intergenic motifs</td><td>This study.</td><td>FigShare: https://doi.org/10.6084/m9.fishshare.25757274</td></tr><tr><td colspan="3"><strong>Software and algorithms</strong></td></tr><tr><td>BLAST</td><td>Accessed via NCBI, IMG/MER or standalone.<sup>82,83,84</sup></td><td>https://blast.ncbi.nlm.nih.gov/Blast.cgi<br>https://img.jgi.doe.gov/cgi-bin/mer/<br>main.cgi?section=WorkspaceGenomeSet&amp;page=home#genomesettab4<br>https://www.ncbi.nlm.nih.gov/books/NBK279690/</td></tr><tr><td>BBMAP</td><td>Bushnell<sup>85</sup></td><td>https://jgi.doe.gov/data-and-tools/software-tools/bbtools/bb-tools-user-guide/bbmap-guide/</td></tr><tr><td>MEGAHIT</td><td>Li et al.<sup>86</sup></td><td>https://github.com/voutcn/megahit</td></tr></tbody></table>

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<figure><img src="image_33.png" /></figure>

## Continued

<table><thead><tr><td>REAGENT or RESOURCE</td><td>SOURCE</td><td>IDENTIFIER</td></tr></thead><tbody><tr><td>vamb</td><td>Nissen et al.<sup>87</sup></td><td>https://github.com/RasmussenLab/vamb</td></tr><tr><td>CheckM</td><td>Parks et al.<sup>88</sup></td><td>https://ecogenomics.github.io/CheckM/</td></tr><tr><td>GTDB-Tk</td><td>Chaumeil et al.<sup>89</sup></td><td>https://github.com/Ecogenomics/GTDBTk</td></tr><tr><td>Anvi'o</td><td>Eren et al.<sup>90</sup></td><td>https://anvio.org/</td></tr><tr><td>IQ-TREE</td><td>Minh et al.<sup>91</sup></td><td>http://www.iqtree.org/</td></tr><tr><td>Dereplication strategy</td><td>Evans and Denef<sup>92</sup></td><td>https://doi.org/10.1128/msphere.00971-19</td></tr><tr><td>Orthofinder</td><td>Emms and Kelly<sup>93</sup></td><td>https://github.com/davidemms/OrthoFinder</td></tr><tr><td>MAFFT</td><td>Katoh et al.<sup>94</sup></td><td>https://anaconda.org/bioconda/mafft</td></tr><tr><td>iTOL</td><td>Letunic and Bork<sup>95</sup></td><td>https://itol.embl.de/</td></tr><tr><td>FaRLiP cluster labeling code</td><td>This paper.</td><td>https://github.com/cyano-lab/ GeneGraphics_formatting</td></tr></tbody></table>

## EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

### Cyanobacterial samples

Cyanobacterial strains were used from the CCAP/SAMS (UK) and PCC (France) culture collections. Strains and culture conditions are listed in Table S2. The presence of FaRLiP genes was shown through BLAST searches of ApcE2 (or other FaRLiP protein sequences such as ChlF) against a local genome database. Three additional strains were recovered from environmental samples. The CL0 and CL1 isolates were originally sampled from Lake Clifton (Australia) as previously described.²⁶ For WS17, salt crust fragments were sampled from the Sebkha Oum Dba, West Sahara, Morocco, in May 2019. All cultures were enriched in chlorophyll *f*-containing cyanobacteria by growing them under 730 or 750 nm light (Shenzhen Gouly LED Limited; Epitex, L750-01AU) at room temperature in liquid or on solid media (1% (w/v) agar). Strains were repeatedly re-inoculated in culture media for isolation, as previously described for CL0; for CL1 and WS17, modified DSMZ brackish medium BA+50+B+N/2 (#1678) containing a range of NaCl concentrations (0, 0.6 and 1.8% (w/v)) was used instead for the isolation, with standard BA+50+B+N/2 for later culturing. Cycloheximide (50 µg/mL) was initially added to reduce eukaryotic contamination.

## METHOD DETAILS

### Environmental classification

A subset of 86 FaRLiP clusters was entered into the environmental analysis. The clusters had to be either complete, near-complete (missing up to four genes in total, and up to three genes >600 bp), or (in a minority of cases) be of particular interest due to their gene rearrangements (Nostoc sp. C052, *Chroococcidiopsis* sp. CCMEE10). To limit the effect of sampling bias while preserving sequence diversity, virtually identical clusters from the same environment were filtered out. This includes various sequences from South African stromatolites, as well as many *Fischerella* strains from hot springs. The phylogeny of *Fischerella* strains is briefly mentioned in a previous publication,⁹⁶ with a few additional ones listed in Table S1. Identical clusters were not removed if sampled from a different environment. The working dataset was classified into four categories, as follows: microbialites, microbial mats, other, and unknown. The 'other' category included many hot spring samples which are likely to have been microbial mats, but it was not specified.

### DNA extraction

For isolates: Genomic DNA from enriched samples was extracted using Quick-DNA Fungal/Bacterial Miniprep Kit (Zymo Research). A longer bead-beating step (25 min) was used in order to homogenize the microbial mats, together with prior fragmenting with a pipette. For CCAP strains: Cells were lysed by mechanical grinding using mortar and pestle, and genomic DNA was extracted using the DNeasy Plant Mini Kit (Qiagen), following the manufacturer instructions. For PCC strains: After pelleting by centrifugation, cells were washed twice with sterile distilled water, or sterile saline solution (1% (w/v) NaCl) for marine strains, and the pellets were immediately frozen in liquid N₂ prior to being lyophilized. DNA of the lyophilized pellets was extracted using NucleoBond AXG 20 columns (Macherey-Nagel) according to the manufacturer's instructions for bacterial DNA.

### (Meta)genome sequencing

For isolates: Sample CL0 was sequenced with both Illumina and Oxford Nanopore as previously described for other strains.⁴⁸ It was sequenced according to vendor protocols on a MiSeq and MinION (MinKNOW version 1.10.23), the former using Nextera XT v2 and the latter a R9.4.1 flowcell with base-calling via Albacore (version 2.1.7). Samples CL1 and WS17 were sequenced using Illumina. For these two samples, library preparation was done using NEBNext Ultra II DNA Library Prep Kit (New England Biolabs) in combination with NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors) according to the manufacturer's manual. The libraries were sequenced on Illumina MiSeq using the sequencing kit v3 600 cycles (PE 2x300). For CCAP strains: Paired-end libraries of
150 bp (PE150) were built from genomic DNA, and sequenced using Illumina NovaSeq 6000 to a depth of ca. 40 million raw reads (ca. 6 Gb raw data) per library (Novogene Ltd). For PCC strains: The genomes were sequenced with Illumina NextSeq500 system from the Mutualized Platform for Microbiology (P2M) of the Institut Pasteur or from GATC.

## (Meta)genome assembly

For isolates and datasets from sequence databases: The metagenomes were quality-trimmed with BBDuk (software package BBMap).⁸⁵ Settings for Illumina: `qtrim=r trimq=10 minlen=30 ref=adapters.fa` (default) `ktrim=r k=23 mink=11 hdist=1 tpe tbo`. Settings for Nanopore: `trimq=9 ktrim=l k=11 hdist=0 edist=2 mm=f rcomp=f mkf=0.51 restrictleft=100 copyundefined=t`. The results were assembled with MEGAHIT,⁸⁶ with the exception of less-diverse CL1 and WS17 samples which used SPADES,⁹⁷ and the sample CL0 which used Unicycler⁹⁸ to incorporate the long reads. Paired-end information was used if available. For the cluster shape analysis, sequences in the same environment were co-assembled where computationally possible (Table S14), while for building MAGs, they were assembled individually and then co-binned (Table S3). The latter is expected to be more accurate, while the former led to more complete FaRLiP clusters. The two methods agreed on the sequence level. For CCAP strains: A metagenomic pipeline based on metaWRAP⁹⁹ was used to pre-process sequence data for quality and assemble using metaSPADES (v3.13.0).¹⁰⁰ For PCC strains: Assemblies were built using `fq2dna v21.06`,¹⁰¹ with coverage ranging from 58x to 438x. PCC cluster sequences are available under accession numbers listed in Table S2.

## Metagenome binning and quality control

For CCAP strains, ensemble metagenome binning was based on Concoct,¹⁰² MaxBin²¹⁰³ and MetaBat².¹⁰⁴ For isolates and datasets from sequence databases, a different method was used. Independently assembled samples from the same environment were co-binned with `vamb`.⁸⁷ Bin quality was checked with `CheckM`.⁸⁸ Bins with >70% completion were further manually refined with `ani'o`,⁹⁰ and the final refined dataset aimed for bins with >70% completion and <5% redundancy, also based on `CheckM` (Table S3).

Duplicate bins from the same environments were removed with MASH and `pyANI`, using a de-replication strategy and code previously described.⁹² Remaining bins which were cyanobacterial in nature and contained FaRLiP genes were used in the study. A sequence similarity analysis was performed for groups V and VI with `EzAAI` at both genome and cluster level, in order to better understand a divergent group V lineage.¹⁰⁵ In this case, the cluster was approximated as the concatenation of 19 core genes.

For all assemblies, the binned metagenome assembled genomes were verified using `CheckM`,⁸⁸ taxonomically identified with `GTDB-tk`⁸⁹ and annotated using `Prokka`.¹⁰⁶ All bins used in this study are available at via NCBI accession codes listed in Table S3, and in FigShare (https://doi.org/10.6084/m9.fishshare.25757274).

## Database sourcing

The main databases used in this study: NCBI (nr, WGS and SRA) and IMG/MER.⁸²,¹⁰⁷,¹⁰⁸ For the NCBI nr (non-redundant) and WGS (Whole Genome Shotgun) databases, `BLASTp` and `tBLASTn` were used to recover new genes and clusters.⁸³,⁸⁴ The far-red specific `ApcE2` motif `VIPEDV` was used as a query, as previously described.²⁶ IMG/MER and SRA data is based on accessions originally recovered through `BLAST` or `SearchSRA`¹⁰⁹ in previous work with the same query.²⁶ The IMG dataset also contained metatranscriptomics (Figure S7; Table S12). SRA data included all runs from the projects previously identified to contain a `VIPEDV`-like motif,²⁶ which were then downloaded with `fasterq-dump`.¹¹⁰

Other FaRLiP genes besides *apcE2* were recovered largely via co-localization with *apcE2*. However, `BLASTp` searches of other FaRLiP genes and motifs (Figure S1; Table S5) were also conducted, in order to test that the previously recovered genes were indeed far-red paralogues, and to recover split clusters, partial clusters (Figure S8) and pseudogenes. This was applied to fragmented data, either from public databases or locally assembled. Gene alignments were constructed with `MAFFT` in Jalview (default settings).⁹⁴,¹¹¹ `SplitsTree 5` software¹¹² was used to build networks of the aligned genes and to rapidly identify sequences clustering with known FaRLiP paralogues. It was also used to analyze *psaA* and *psaB* alignments from a previous publication³⁸ (Figure S3).

## Gene-level phylogenetic analysis

DNA and protein alignments for the 19 core genes (present in all complete FaRLiP clusters) were constructed with `MAFFT` (–maximum 1000 –localpair). Alignments included 81-96 sequences each, depending on the gene. General sequence management such as renaming was performed with `SeqKit`.¹¹³ Trees were inferred with `IQ-TREE` (bootstrap 1000), with the best substitution model being chosen automatically using `ModelFinder`.⁹¹ As per the `HoT` method, both ‘head’ (default) and ‘tails’ (reverse-complement, align, then reverse-complement again) alignments and trees were built, for DNA sequences, in order to assess alignment reliability.⁵⁴ To further test possible effects of alignment artifacts, head and tails trees were compared with `IQ-TREE’s` –sup argument. Graphical comparisons together with `Newick trees` are available in Figshare (https://doi.org/10.6084/m9.fishshare.25757274). Trees were rooted with `Minimal Ancestral Deviation (MAD)`,¹¹⁴ with the best-scoring root point (lowest Ambiguity Index in multiple trees) supporting vertical descent (Table S15). Trees were illustrated with `iTOL`⁹⁵ and `Inkscape 1.2.1`. `SplitsTree 5` was used to group multiple trees into networks.¹¹²

## Genome-level phylogenetic analysis

The genome tree was built with `Orthofinder`,⁹³ using single- and multi-copy protein orthologues, and is consistent with recent work.¹³ It focused on FaRLiP genomes and MAGs, but also included entries lacking the cluster (*Gloeobacter violaceous* PCC 7421,
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'Chroococcidiopsis' sp. CCMEE 29) or containing only some FaRLiP genes (Hyella patelloides LEGE 07179) for increased phylogenetic accuracy.

**Synteny analysis**

Annotated, manually corrected clusters were submitted to the Gene Graphics webserver for visualization,¹¹⁵ and then compared for similarities. This required a change in data formatting (from the.gbk Prokka output, to a.tsv tabular format for Gene Graphics). The code used can be found at https://github.com/cyano-lab/GeneGraphics_formatting. Final figures created with Inkscape 1.2.1. Clusters were submitted to NCBI with the help of GB2sequin.¹¹⁶

**Taxonomic conventions**

This paper uses a genome-based taxonomy.¹³ For ease of cross-referencing with the GTDB classification, or NCBI taxonomy, see Table S16.

**Motif identification**

The chlF motif and several long motifs were identified by aligning intergenic areas upstream of their respective genes with MAFFT, in Jalview. Less-conserved motifs were recovered by aligning the remaining intergenic areas with multiple copies of the conserved chlF motifs, thus forcing alignment to this region. Results were checked with the MEME webserver, which was also used to obtain the consensus logos. Since MEME cannot use gapped data, occasional single-nucleotide deletions were replaced with “X” in the dataset. Sequence similarity networks were built with SplitsTree 5 (Figure S9). The presence or absence of these intergenic motifs per each strain and FaRLiP operon under study is listed in Table S13.

**QUANTIFICATION AND STATISTICAL ANALYSIS**

A calculation of synonymous and non-synonymous substitution rates (dS, dN) was executed with a combination of MAFFT (ClustalW format), pal2nal (aligning protein to nucleotide sequences) and codeml (rate calculation)¹¹⁷,¹¹⁸ (Figure S2; Table S7). GC percentage for clusters and genomes was calculated with SnapGene 8.0.1 (Table S11).