Crossing the Algal Divide

This post is the direct result of a brief exchange in the comments to an earlier post which has nothing in itself to do with this one. Isn't it funny how tangents work?


Glaucocystis, a member of the primary chloroplast-carrying glaucophytes. Photo by Jason Oyadomari.

It has become pretty much universally acknowledged that at least two of the organelles found in eukaryotic cells, mitochondria and chloroplasts, are derived from endosymbiotic bacteria that progressively gave up more and more of their vital functions to their host cells until they became inextricably linked to them. Mitochondria are probably derived from Alphaproteobacteria (Gray et al., 2004), while chloroplasts are certainly derived from Cyanobacteria. Endosymbiotic origins have been suggested for other organelles, most notably the eukaryotic flagellum, but have not reached the same level of acceptance. While a number of eukaryotes lacking mitochondria are found in the world today, the weight of current evidence suggests that most if not all are descended from mitochondria-carrying ancestors, and the origin of the mitochondrion pre-dates the known eukaryote crown group. The origin of the chloroplast, however, is not quite so simple.


Cryptomonas, another unicellular alga from a different group, the cryptomonads. Photo from here.

The chloroplast was undoubtedly a later innovation than the mitochondrion. As I've alluded to before, the basalmost division in eukaryotes currently seems to be between unikonts (including animals, fungi and amoebozoans) on one side and bikonts (plants and most other protists) on the other. All eukaryotes with chloroplasts are bikonts (with the exception of sequestered chloroplasts in some marine molluscs and flatworms), so chloroplasts at least post-date this division. Unfortunately, bikonts are a much more disparate bunch than unikonts, and our understanding of how the various major groups of bikonts are related to each other is correspondingly less. Among the bikonts, chloroplasts or clear chloroplast derivatives are found in twelve well-supported monophyletic groups (as a cautious maximum). However, different groups have chloroplasts with different physiologies and ultrastructures, indicating different modes of origin. Some groups have what are called primary chloroplasts, derived directly from endosymbiotic cyanobacteria. Primary chloroplasts have two membranes separating the host cell and chloroplast cytoplasm, corresponding to the two cell membranes of a free-living cyanobacterium. Most cyanobacteria contain a single type of chlorophyll, chlorophyll a, and so do the primary chloroplasts of glaucophytes* (a small group of unicellular algae), rhodophytes (red algae) and the shelled amoeboid Paulinella. The fourth group of eukaryotes with primary chloroplasts, Viridiplantae (green algae and land plants), differ in having two types of chlorophyll, both the original a and an additional form called chlorophyll b**. Glaucophyte, rhodophyte and viridiplantaean chloroplasts share a number of genetic signatures absent from cyanobacteria, suggesting that their chloroplasts are derived from a single endosymbiotic event (Kim & Graham, 2008). The chloroplast of Paulinella, on the other hand, is more similar to a cyanobacterium, and Paulinella has clear and close non-photosynthetic relatives among the group of unicellular protists known as Cercozoa. Paulinella is therefore believed to have acquired its chloroplast recently and completely independently of the other groups.

*As an intriguing aside, it was long debated whether it was more appropriate to regard the photosynthetic enclusions in glaucophytes as "chloroplasts" or "endosymbiotic cyanobacteria", and a number of glaucophyte chloroplasts were given names as taxa in their own right.

*Just to confuse matters, there are also three species of cyanobacteria that possess chlorophyll b. Current indications are that these species are not closely related to Viridiplantae chloroplasts - nor, indeed, are they closely related to each other. The odd scattered distribution of chlorophyll b remains as yet completely unexplained.


Diagram of the origin of secondary chloroplasts in chlorarachniophytes through the engulfment of one eukaryote by another. From ToLWeb.

The remaining groups of photosynthetic eukaryotes, in contrast, have what are called secondary chloroplasts (or, in a few cases, tertiary or even quaternary chloroplasts). Secondary chloroplasts have three or four membranes surrounding them, and are not derived directly from a cyanobacterium, but from a eukaryotic alga containing a primary chloroplast. In those secondary chloroplasts with four membranes, then, the membranes represent the two membranes of the primary chloroplast, the outer cell membrane of the endosymbiotic eukaryotic alga, and the membrane surrounding the vacuole in which the secondary host contained its endosymbiont. Clear support for this complicated origin can be seen in the two secondary-chloroplast groups, the amoeboid chlorarachniophytes and the flagellate crytomonads, where a small dark mass sits wedged between the second and third membranes. This mass contains DNA, and is nothing less than the degraded remnants of the endosymbiotic alga's original nucleus.


Coccolithophores, shelled algae of the Haptophyta. Image from here.

Two groups of secondary-chloroplast algae, the chlorarachniophytes and the euglenoids (Euglena is probably about the most commonly-illustrated flagellate in any textbook), possess chlorophylls a and b, indicating an ancestor among the Viridiplantae for their chloroplasts. For the remaining groups, phylogenetic analyses indicate a rhodophyte origin for their chloroplasts. The recently discovered Chromera only has chlorophyll a, like a rhodophyte, while Chromera's relatives in the parasitic Coccidiomorpha (a subgroup of the Sporozoa) possess chlorophyll-less chloroplast derivatives. The remaining four groups - cryptomonads, haptophytes (coccolithophores), ochrophytes (which include brown and golden algae and diatoms) and dinoflagellates - possess two types of chlorophyll, a and a form called chlorophyll c that is unique to these taxa.

The big question hovering over eukaryote phylogenetics is how many times these secondary endosymbioses occurred. One of the most prolific authors in this field has been the British researcher Tom Cavalier-Smith. Cavalier-Smith's writings can induce feelings of great admiration or extreme loathing (sometimes both over the course of a single page)*, but one certainly can't go very far without coming up against them. A lot of Cavalier-Smith's views (some of them since adjusted) were summarised in what was published in 2002 as two papers (Cavalier-Smith, 2002a, 2002b) but should really be read as one single gigantic über-paper on the origins of life, the universe and everything (well, not the universe, but you get the idea). Using a combination of molecular and morphological interpretations, Cavalier-Smith divided the bikonts into five major clades, all but one including both photosynthetic and non-photosynthetic major subgroups - Excavata (including euglenoids, among others), Rhizaria (to which belong chlorarachniophytes and Paulinella, as well as foraminifera and radiolarians), Plantae (the remaining primary-chloroplast organisms), Alveolata (dinoflagellates, sporozoans and ciliates) and Chromista (cryptomonads, haptophytes and heterokonts - the last includes the ochrophytes). He further proposed that the Alveolata and Chromista together formed a clade called chromalveolates, uniting all the chlorophyll c-containing organisms. Supposedly, the rhodophyte endosymbiosis giving rise to the chromalveolate chloroplast happened just once, and the non-photosynthetic chromalveolates are derived from ancestors that lost their chloroplasts.

*At least in the late 1990s and the early 2000s, a rough indication of the amount of ire that Cavalier-Smith's publications generated in some circles could be gained by scanning the works of other protistologists and noting the lengths some of them went to not to cite Cavalier-Smith.


Paulinella. This genus is the only eukaryote lineage to have acquired its chloroplasts separately from the archaeplastid lineage. Photo from here.

A major factor in Cavalier-Smith's proposals has been the idea that chloroplast acquisition is far more difficult than chloroplast loss, because gaining a working chloroplast requires not only the endosymbiont but the evolution of appropriate molecular channels for transporting metabolites between the endosymbiont and the host cell, so the phylogeny that minimises the number of chloroplast acquisitions is most likely to be true (as an extreme example, in 1999 he also suggested that Excavata and Rhizaria formed a clade derived from a single green algal endosymbiosis, which the resulting chloroplast lost in all members of both clades except chlorarachniophytes and euglenoids. Because chlorarachniophytes and euglenoids are both nested reasonably deeply within their respective clades, necessitating a fairly large number of chloroplast losses in this scenario, nobody except for Cavalier-Smith himself seems to have given it a huge amount of credence). Other researchers, on the other hand, hold the opposite view - that chloroplasts perform such a significant role in their host cells that losing them would be a Very Bad Thing - and point to the fact that many photosynthetic groups have clear closest relatives among non-photosynthetic groups. Unfortunately, most phylogenetic analyses in this field have lacked strong resolution or support, probably simply due to the incredibly long time since the lineages diverged.

So where do things stand now? In the last couple of years, analyses of sometimes quite huge amounts of data have been released. Of Cavalier-Smith's (2002b) five groups, the Rhizaria and Alveolata have continued to receive support from almost all angles. The Excavata continue to cause a bit of hemming and hawing (though Hampl et al., 2009, recently presented the first molecular analysis to support excavate monophyly), but with only one photosynthetic subgroup they're not really relevant to the current discussion anyway. The monophyly of the Plantae (renamed Archaeplastida in the eukaryote classification of Adl et al., 2005, to avoid the confusion of the many different uses of the name "Plantae") is at a bit of a draw - Patron et al. (2007) and Burki et al. (in press, 2008), for instance, found it as monophyletic, but Kim & Graham (2008) and Hampl et al. (2009) did not. None of the recent analyses, however, have found a monophyletic Chromista. The cryptomonads and haptophytes look to form a clade that may be close to (Patron et al., 2007; Burki et al., in press, 2008) or even within (Kim & Graham, 2008; Hampl et al., 2009) the archaeplastids. The heterokonts seem to form a clade (with a certain degree of irony) with the alveolates - which brings up the possibility that, depending on how you choose to use the names, "chromalveolates" may be monophyletic even if "chromists" are not. A surprising result of a number of recent analyses (including most of the ones cited above) is that this reduced chromalveolate clade may be sister to the Rhizaria.



As shown in the figure above from Bodył et al (2009) summarising all this, this implies a number more chloroplast origins than Cavalier-Smith's model. Does this vindicate those who hold that chloroplast acquisition is easier than chloroplast loss? Well, as often happens in biology, there is a third possibility. As well as chloroplast gain and chloroplast loss, there is also chloroplast replacement. Dinoflagellates, the one group of eukaryotes that never manage to do anything sensibly, include some members with secondary rhodophyte-derived chloroplasts, and others with tertiary chloroplasts that seem to be derived from haptophytes. It seems that these serial hosts have shucked out their original secondary chloroplasts in favour of a new endosymbiont. Chloroplast replacement sidesteps some of the theoretical difficulties of acquiring a chloroplast entirely de novo, because the host already possesses the biochemical pathways to communicate with its new chloroplast. If the cryptomonad-haptophyte clade is nested within archaeplastids, as indicated by some phylogenies, this may represent a case of chloroplast replacement rather than chloroplast gain.

REFERENCES

Adl, S. M., A. G. B. Simpson, M. A. Farmer, R. A. Andersen, O. R. Andersen, J. R. Barta, S. S. Bowser, G. Brugerolle, R. A. Fensome, S. Fredericq, T. Y. James, S. Karpov, P. Krugens, J. Krug, C. E. Lane, L. A. Lewis, J. Lodge, D. H. Lynn, D. G. Mann, R. M. McCourt, L. Mendoza, Ø. Moestrup, S. E. Mozley-Standridge, T. A. Narad, C. A. Shearer, A. V. Smirnov, F. W. Spiegel & M. F. J. R. Taylor. 2005. The new higher level classification of eukaryotes with emphasis on the taxonomy of protists. Journal of Eukaryotic Microbiology 52: 399-451.

Bodył, A., J. W. Stiller & P. Mackiewicz. 2009. Chromalveolate plastids: direct descent or multiple endosymbioses? Trends in Ecology and Evolution 24 (3): 119-121.

Burki, F., K. Shalchian-Tabrizi & J. Pawlowski. in press, 2008. Phylogenomics reveals a new ‘megagroup’ including most photosynthetic eukaryotes. Biology Letters.

Cavalier-Smith, T. 1999. Principles of protein and lipid targeting in secondary symbiogenesis: euglenoid, dinoflagellate, and sporozoan plastid origins and the eukaryote family tree. Journal of Eukaryotic Microbiology 46 (4): 347-366.

Cavalier-Smith, T. 2002a. The neomuran origin of archaebacteria, the negibacterial root of the universal tree and bacterial megaclassification. International Journal of Systematic and Evolutionary Microbiology 52: 7-76.

Cavalier-Smith, T. 2002b. The phagotrophic origin of eukaryotes and phylogenetic classification of Protozoa. International Journal of Systematic and Evolutionary Microbiology 52: 297-354.

Gray, M. W., B. F. Lang & G. Burger. 2004. Mitochondria of protists. Annual Review of Genetics 38: 477-524.

Hampl, V., L. Hug, J. W. Leigh, J. B. Dacks, B. F. Lang, A. G. B. Simpson & A. J. Roger. 2009. Phylogenomic analyses support the monophyly of Excavata and resolve relationships among eukaryotic "supergroups". Proceedings of the National Academy of Sciences of the USA 106 (10): 3859-3864.

Kim, E., & L. E. Graham. 2008. EEF2 analysis challenges the monophyly of Archaeplastida and Chromalveolata. PLoS ONE 3(7): e2621.

Patron, N. J., Y. Inagaki & P. J. Keeling. 2007. Multiple gene phylogenies support the monophyly of cryptomonad and haptophyte host lineages. Current Biology 17: 887-891.