Insights & Perspectives

The rise and fall of Picobiliphytes:

Think again How assumed autotrophs turned out to be heterotrophs

David Moreira and Purificacion Lopez-Garc ıa*

Algae are significant members of Earth’s biodiversity. Having been studied for a been instrumental in attempting to long time, the discovery of new algal phyla is extremely unusual. Recently, the establish a natural, phylogeny-based enigmatic Picobiliphyta, a group of uncultured unveiled using . These studies led to classify eukaryotic into a relatively small molecular tools, were claimed to represent an unrecognized early branching number of lineages [5]. Three algal algal lineage with a nucleomorph (remnant nucleus of a secondary algal groups (Glaucophyta, Rhodophyta, endosymbiont) in their . However, subsequent studies rejected the and ) harbor plastids that presence of a nucleomorph, and single- genomic studies failed to detect any derive from the original endosymbiosis -related genes, ruling out the possibility of plastid occurrence. The of a cyanobacterium within an initial heterotrophic eukaryotic cell (primary isolation of the first picobiliphyte, Picomonas judraskeda, a tiny that endosymbiosis) [6, 7]. The rest of the < feeds on very small ( 150 nm) organic particles, came as final proof of their non- eukaryotic algae have plastids acquired photosynthetic lifestyle. Consequently, the group has been renamed Picozoa. through the endosymbiosis of red or The passage from picobiliphytes to picozoa illustrates the crucial role that within different eukaryotic classical protistology should play to provide sound biological context for the hosts (secondary endosymbioses), al- wealth of data produced by modern molecular techniques. though in some occasions tertiary endosymbioses or secondary plastid replacements may also have oc- Keywords: curred [8]. These secondary and tertiary algae; eukaryotic evolution; phylogeny; Picobiliphyte; Picozoa; . events gave rise to a variety of algal groups that are distributed across the eukaryotic tree. These include the Euglenida, Chlorarachniophyta, Crypto- Introduction microscopic, but the presence of photo- phyta, Haptophyta, various groups synthetic and other accessory pigments within the Heterokonta or Strameno- , , and a variety of in their cells makes them easily visible piles (Bacillariophyta, Phaeophyta, eukaryotic algae are responsible for the under the microscope. This is why, to Xantophyta, etc.) and, within the Alveo- release of oxygen to the Earth’s atmo- the detriment of the many colorless lata, the Dinophyceae and a group that sphere and also for the most significant heterotrophic species that coexist with secondarily lost the photosynthetic part of CO2 fixation into organic matter them, became a favored capacity, the parasitic . [1–4]. Among these , cyano- target of study since the early days of As mentioned above, these groups have bacteria and many unicellular algae are optical microscopy and why they figure been known for a long time, so the among the best known microorganisms. discovery of new algal lineages can DOI 10.1002/bies.201300176 A vast amount of microalgal morpholo- be regarded as highly improbable, gy and cell ultrastructural data has been something only comparable to the collected during the last five decades discovery of new mammalian species Unite´ d’Ecologie, Syste´ matique et Evolution, CNRS UMR8079, Universite´ Paris-Sud, Orsay, and has served for phenotype-based or new groups. In fact, a recent France taxonomy. In more recent years, this analysis using accumulation curves for body of information has been completed high level algal taxa (families, orders, *Corresponding author: Purificacion Lopez-Garcı ´a by molecular phylogeny analyses of and classes) has showed that the curves E-mail: [email protected] conserved gene markers, which has are already close to saturation, which

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suggests that most of those taxa have Accordingly, the authors of these obser- phylogenetic analyses that confirmed already been discovered [9]. vations concluded that these organisms the monophyly of group BP2 and that Unexpectedly, the existence of three were possibly photosynthetic and BP3 and, especially, BP1 were not hn again Think new groups of putative algae has been coined for them the term Picobiliphyta, monophyletic and could be subdivided reported recently. One of them, the in reference to their small size, pigmen- into several smaller clades [21]. Never- , represented by its first tation, and presumed algal nature [19]. theless, the most interesting result of described species Chromera velia,is However, these conclusions were Cuvelier et al. was the absence of clear related to the once-photosynthetic Api- based on indirect observations in natu- evidence for the presence of a nucleo- complexa and contains plastids derived ral environments, since no picobili- morph. This was tested by direct cell from [10–12]. This algal group phyte representative was available in observation after FISH and DNA stain- is now firmly established based on culture. Nonetheless, the discovery of a ing, which did not reveal any DNA in abundant sequence information as well new algal group attracted much atten- the cell area showing phycobilin-like as morphological, ultrastructural, and tion and efforts were made to gain more fluorescence (the putative plastid). In biochemical data, and its diversity has knowledge from this potentially pivotal addition, primers designed to amplify been expanded with the isolation of the algal group. As data began to accumu- nucleomorph and/or rhodophyte 18S new species Vitrella brassicaformis [13– late, doubts were raised as to the rDNA failed to yield any sequence from 15]. The second group, informally called photosynthetic nature of these organ- the “picobiliphyte” cells. Thus, the “rappemonads”, has been characterized isms and, finally, a new conclusion on authors concluded that “understanding by a combination of the analysis of these organisms’ lifestyle was reached. the origin of biliphyte plastids will plastid 16S rRNA gene sequences re- We will discuss here these recent likely require ultrastructural, molecular trieved from environmental surveys of advances as an example that illustrates phylogenetic, and genomic investiga- marine and freshwater environments the necessity of a renaissance of classi- tions” [20]. Such investigations came a and fluorescent in situ hybridization cal protistology to complement modern few years later thanks to the work of (FISH) experiments with specific plas- molecular methods of microbial diver- several teams. tid-targeted probes [16]. Rappemonads sity analysis in order to fully understand appear to contain several plastids in the biology of new microbial eukaryotic their cells and branch as sister-group of lineages. Single-cell genomics and the in plastid rRNA gene phylogenies. Thus, it is unclear whether deep-sea sequences – these organisms actually represent a “Picobiliphytes” are where is the plastid? new algal group or just an uncharac- terized deeply diverging widespread in oceans but Recent technical advances in single-cell lineage. The third group of putative lack a nucleomorph manipulation and in working with small new algae was probably the most amounts of starting material have enigmatic. Initially detected in 18S As mentioned above, “picobiliphytes” facilitated the development of single- rDNA environmental libraries as a (also known as “biliphytes”), were first cell genomics, a discipline that is likely lineage with unclear affinity to any detected as a separate group of sequen- to revolutionize many scientific areas, in other eukaryotic group, their members ces in 18S rDNA libraries constructed particular microbiology [22]. Using this were subsequently visualized using from marine surface DNA approach, Yoon et al. isolated single FISH with specific 18S rDNA-targeted samples [19]. A subsequent study by “picobiliphyte” cells by fluorescence probes. FISH experiments and fluores- Cuvelier et al. [20] retrieved additional activated cell sorting (FACS) and used cent microscopy revealed very small “picobiliphyte” sequences in warm and multiple displacement amplification to cells (<5 mm) showing orange auto- cold surface samples from very distant amplify their genomic DNA. They used fluorescence, which might indicate the marine locations, indicating that this 454 pyrosequencing to obtain 5 Mbp of presence of phycobilin pigments group was globally distributed, since assembled contigs per cell followed by (known to occur in cyanobacteria and they were present in very different Illumina sequencing on one of the in plastids of , red algae, oceanic regions. Phylogenetic analysis amplified genomes, which led to 28 and some other algae containing plas- of the complete set of 18S rDNA Mbp of assembled contigs [23]. They tids derived from red algae [17]). Sur- sequences led these authors to divide then looked for plastid and nuclear- prisingly, DNA staining with fluorescent the “picobiliphytes” into three groups encoded plastid-targeted gene sequen- intercalating agents appeared to show (BP1, BP2, and BP3) containing sequen- ces in all these datasets but were unable the presence of a DNA-containing ces from different locations, without to identify any convincing hit. As is organelle resembling a plastid with a any apparent pattern of distribution often the case for this type of approach, nucleomorph. Nucleomorphs are rem- related to geography or temperature the single-cell genome data obtained nants of red or green algal nuclei in adaptation. In contrast with the strong were partial. However, given that se- plastids derived from secondary endo- statistical support found for group BP2, quenced genomes of eukaryotic plants symbionts [18]. Their occurrence in the groups BP1 and BP3 were weakly and algae contain a significant propor- these tiny organisms would indicate supported [20]. The subsequent accu- tion of genes related to that they are algae containing plastids mulation of new “picobiliphyte” 18S and other plastid-related activities scat- acquired by secondary endosymbiosis. rDNA sequences allowed more accurate tered in the genomes [24, 25], they

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interpreted the complete absence of sequences [19]. In these trees, “picobi- due, at least in part, to the large amount photosynthesis-related genes from sin- liphytes” branched close to the crypto- of missing data for this taxon (66% of gle-cell genome sequence data as evi- phytes and a group of essentially the genes employed by Burki et al. were dence against a photosynthetic lifestyle heterotrophic flagellates that had been not found in the single-cell genome data for the “picobiliphytes”. Moreover, the considered for a long available for the “picobiliphytes”), as three “picobiliphyte” cells examined time, the , although missing data can reduce the resolving had been retrieved from the colorless, with moderate statistical support power of phylogenetic analysis [27]. In chlorophyll-lacking planktonic fraction (Fig. 2A). In a subsequent analysis, fact, one problem frequently encoun- of a seawater sample, reinforcing the Cuvelier et al. confirmed this result and tered in single-cell genomic experi- idea that these organisms were not also the proximity of a clade containing ments is the incomplete and biased

Think again photosynthetic. This result was consis- the “picobiliphytes”, cryptophytes and amplification of the genomic DNA by tent with the finding of “picobiliphyte” katablepharids to the glaucophytes multiple displacement amplification or cells recognized by specific FISH probes (Fig. 2B) although, once again, with related amplification techniques, which but lacking the orange phycobilin-like weak statistical support [20]. This rela- leads to incomplete genome cover- fluorescence [16]. tionship was unexpected since glauco- age [28]. Thus, genome sequence data In addition to those studies, the phytes resulted from a primary from other picobiliphyte species, ideally use of “picobiliphyte”-specific primers endosymbiotic event with cyanobacte- from cultured strains to avoid the allowed us to detect a large diversity of ria and are assumed to form a mono- DNA limitation inherent to single-cell “picobiliphyte” 18S rDNA sequences in phyletic group with red algae and green approaches, or from single-cell ampli- different marine samples, including algae and plants rather than with fied genomes using improved amplifi- not only surface but also deep-sea cryptophytes [6, 7]. Indeed, 18S rDNA cation methods, remain necessary to ones. Thus, surprisingly, we found very alone does not contain enough phylo- solve the phylogenetic position of this diverse sequences belonging to the genetic signal as to allow reconstruction group. In this sense, the recovery of groups BP1 and BP3 defined by Cuvelier of all evolutionary relationships among genome sequence data from the newly et al. [20] not only in surface waters, the different eukaryotic groups: access identified deep-branching groups 1 and as expected, but also in very deep to additional genomic information from 2 (Fig. 1) would be of particular interest. samples, down to 3,000 m (Fig. 1). So “picobiliphytes” was necessary to try to far, only one “picobiliphyte” sequence, resolve their phylogenetic position. SSRPB47, a partial sequence retrieved at Thus, in addition to providing Classical protistology 500 m depth in the Sargasso Sea and material to test the presence of a belonging to group BP1, had been found putative plastid, the single-cell genome discloses a lifestyle in deep-sea samples [19]. Thus, whereas study also offered, in principle, abun- mystery group BP2 appears to occur in surface dant sequence data to address this waters, groups BP1 and BP3 do not show question. Among approximately 2,000 Since their discovery, “picobiliphytes” any clear distribution pattern related to picobiliphyte predicted proteins bearing were comprehensively studied using depth, being found all along the water similarity with sequences in other 18S rDNA libraries, FISH and even column. In addition to BP1 and BP3 eukaryotic species, Yoon et al. selected single-cell genomics, but the major sequences, we also retrieved sequences seven conserved markers to carry out question of whether they had a photo- that define two new early-diverging a phylogenetic analysis. It retrieved synthetic or heterotrophic lifestyle groups sister to the already known the “picobiliphytes” as a deep branch remained open since only indirect and picobiliphyte clades (deep-branching related to the telonemids (another partial evidence was available. A formal groups 1 and 2, see Fig. 1). One of them, group of heterotrophic of unre- proof remained necessary. Such a group 2, appears to be dominated by solved position), within a clade also proof has been provided very recently deep ocean sequences, although this containing the cryptophytes, hapto- thanks to the isolation of the first has to be confirmed by more extensive phytes and katablepharids (Fig. 2C), “picobiliphyte” species, Picomonas sampling Altogether, these results sug- although with moderate statistical sup- judraskeda [21]. It was cultured from gest that “picobiliphytes” are at least as port [23]. This study still used a a surface seawater sample previously diverse in the deep dark oceanic regions relatively small amount of data, which filtered through 2 mm pore size filters. as in surface waters, which is difficult to could explain such limited resolution. A Accordingly, Picomonas cells turned out imagine for supposedly photosynthetic more comprehensive phylogenetic anal- to be of tiny size, 2.5–3.8 2–2.5 mmon organisms. ysis was carried out by Burki et al. using average. These cells had a very unusual a much richer dataset containing 258 structure, as they were divided into protein markers. In their tree, “picobi- two very distinct parts separated by a liphytes” emerged close to the primary- deep groove (Fig. 3). One part contained An intriguing phylogenetic plastid-bearing glaucophytes (Fig. 2D), all the major cell organelles (the nucle- position far from the telonemids [26]. Surpris- us, a single mitochondrion, a Golgi ingly, despite the large amount of body and the flagellar apparatus) and The first insights into the phylogenetic sequence data used, the statistical the second part was specialized in position of “picobiliphytes” were based support for the position of the “picobi- feeding, containing the “mouth” (cytos- on trees reconstructed using 18S rDNA liphytes” remained low. This could be tome) and a number of vacuoles and

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Figure 1. Phylogenetic analysis of “pycobiliphyte” 18S rDNA sequences. Environmental sequences retrieved from the epipelagic region are shown in light blue; deep-sea sequences are highlighted in dark blue. Sequences from our study are in bold (accession numbers KJ173835–KJ173876). 18S rDNA was amplified using the “picobiliphyte”-specific primer pico1F (50-CGTTTATTTGATGATCTCTTG) and general eukaryotic reverse primers from a collection of marine plankton DNA from the South Atlantic/Southern Ocean and the Marmara Sea used in previous studies following the same technical procedures [33, 34]. The name of the only isolated species, Picomonas judraskeda,is indicated by a black background. Groups BP1, BP2, and BP3 are named according to Cuvelier et al. [20]. The tree was reconstructed by Bayesian analysis using 1,057 positions. The scale bar indicates the estimated number of nucleotide substitutions per site.

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vesicles involved in digestion. These two parts were separated by a large vacuolar cisterna of unknown function, though it has been speculated that it may serve as a physical barrier to protect the cell against infection by viruses ingested by the cell region specialized in feeding. In addition to this unusual structure, Picomonas displayed an atypical movement that

Think again was described as a “jump, drag, and skedaddle” cycle [21]. The combination of all these morphological and behav- ioral traits is unique to Picomonas judraskeda. An important observation in the nicely detailed ultrastructural charac- terization carried out by Seenivasan et al. was to show that plastids were absent from Picomonas. In addition to previous studies that failed to find any typical photosynthetic trait in the “picobiliphytes”, ultrastructure provid- ed the missing proof required to ascer- tain the heterotrophic nature of these organisms. Moreover, the food vacuoles found in Picomonas appeared to contain only particles of less than 150 nm in size, much less than the typical size of prokaryotic cells, which suggested that Picomonas probably feeds on small colloidal food particles (such as bacte- rial membrane debris) and, perhaps, also on small viruses. This is an important observation because it rules out the possibility that “picobiliphytes” may ingest eukaryotic algae to recover and enslave their plastids (a process called kleptoplastidy) or even cyano- bacteria, as both types of cells are larger than 150 nm in diameter. This also excludes the possibility that the pattern of orange autofluorescence observed in a number of uncultured picobiliphyte cells is due to the presence of ingested photosynthetic cells, as had been advanced [19], and rather suggests some kind of fixation or staining artifact [21]. The morphological characteristics of Picomonas are unique among the known eukaryotic species and do not show any specific affinity to those found in groups such as cryptophytes, kata- blepharids, or glaucophytes, which Figure 2. Schematic phylogenetic trees showing the positions proposed for the “picobiliphytes” by A: Not et al. [19], based on 18S rDNA sequence analyses; B: Cuvelier et al. [20], based on appear to be related to “picobiliphytes” 18S rDNA sequence analyses; C: Yoon et al. [23], based on the analysis of seven conserved in phylogenetic analyses, albeit always proteins; and D: Burki et al. [26], based on the analysis of 258 conserved proteins. Circles at with weak support (Fig. 2). The unique- branches indicate the statistical support (bootstrap proportions, BP) found by maximum ness of the cell structure and the lack parsimony [19], maximum likelihood [20, 23], and Bayesian inference [26]. of close phylogenetic relatives justify

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and sequencing their genomes will be crucial to understand the evolution of these eukaryotic lineages and resolve hn again Think their phylogenetic relationships as well as to understand their ecological role.

Conclusions and outlook

Picozoa represent a novel diverse group of small marine heterotrophic protists branching deeply in the eukaryotic tree. Unfortunately, the Picomonas strain isolated by Seenivasan et al. [21] was lost after a relatively short time of successful cultivation in the laboratory. Nevertheless, their work demonstrated two important things. First, that new eukaryotic lineages can be put into culture not only by chance but also targeting them using FISH, flow cytom- etry sorting or other techniques to enrich the desired group from natural samples. Second, that, as could be expected, having the organisms in culture allows their accurate character- ization, including morphological, ultra- structural, behavioral, genomic, and other aspects. This illustrates that, despite the amazing power of new molecular techniques, traditional pro- tistology remains fundamental to un- derstand the biology of eukaryotic microorganisms. Regrettably, at the Figure 3. Scanning electron microscopy image of a cell of Picomonas judraskeda.An same time as molecular approaches optical microscopy image is shown in the inset. The difference in cell size between the two became more and more prevalent, images is due to the chemical fixation prior to optical microscopy, which slightly increases classical specialists about many micro- the actual cell size. Photographs courtesy of R. Seenivasan and M. Melkonian. bial eukaryotic groups retired, so that the vast body of classical knowledge on these organisms became highly the inauguration of a new , up to 50% of the dissolved organic endangered and can get lost forever. which Seenivasan et al. proposed to carbon [29]. Although quantitative data Of course, this problem is not limited call “Picozoa” in order to highlight are scarce for this group, it has already to protistology but to all branches of the heterotrophic nutrition mode of these been shown that picozoan cells can biology, as traditional whole-organism organisms, in clear contrast with the be abundant in certain locations. approaches are being increasingly semantics of the previously proposed For example, FISH experiments have deserted [30–32]. The story of the raise name “Picobiliphyta”. The dismissal of revealed that they can reach densities and fall of the Picobiliphyta, a poten- the photosynthetic nature of these between 80 and 300 cells/mL and tially novel unique algal group, that organisms may be perceived as a account for >1% of all planktonic suddenly became the Picozoa, a phylum disappointing result. However, Picozoa picoeukaryotic cells in several surface of heterotrophic protists feeding on tiny constitute a bona fide new eukaryotic marine locations [19]. Thus, they are particles, is a paradigmatic example of lineage, widespread across very different most likely significant actors of the importance of combining molecular oceanic locations and depths. This organic matter recycling in oceans. In and classical tools to fully characterize makes them good candidates to play a addition, they seem to encompass a new lineages. relevant ecological role in marine eco- large diversity, which remains to be systems, may be as specialists in the fully explored, and branch in a very degradation of very small particles, difficult-to-resolve region of the eukary- Acknowledgements including marine colloids which, inci- otic tree, where photosynthetic and We thank Ramkumar Seenivasan and dentally, are the most abundant par- heterotrophic lineages appear inter- (University of Co- ticles in the ocean and represent mixed [26]. Thus, isolating new species logne) for the generous gift of Picomonas

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