Trypanosomatid plastids and uninentional scientific comedy

One need not read past the abstract:

"It is usually assumed that the trypanosomatid plastid shared a common origin with that of euglenids, but Δ4 desaturase phylogenies suggest that it could have originated via an independent, tertiary endosymbiosis involving a haptophyte alga. It is also possible that ancestors of the Trypanosomatidae initially possessed a primary plastid that later was replaced by a secondary or tertiary plastid." – Bodyl et al 2010 J Parasitol (pdf)

I could go on for many, many pages about the implausibility of most entirely unnecessary serial plastid symbiosis theories; I could go on for pages yet on how little a single gene phylogeny means these days; I could go over the typical first few lectures on phylogenetic reconstruction and the fundamental principle of parsimony. But instead, I've quickly thrown together a diagram highlighting the KEY problem with Bodyl et al.'s hypothesis:

Taxa in black – non-photosynthetic and non-plastid-bearing.

Trypanosomes don't have plastids.

Or any reason to suspect they might.

*to be fair, they are (I hope) talking about a plastid in their ancestry, but those things are seldom lost completely due to inevitable strong dependencies.

In fact, unlike apicomplexans, trypanosomes are nested firmly within a completely non-photosynthetic phylum in a predominantly non-photosynthetic subgroup of an almost-exclusively non-photosynthetic supergroup. Furthermore, the many possible phylogenies of euglenid evolution overwhelmingly support a single symbiotic event; character evolution supports this further, in one of the few cases where there's actually little room for dispute. Endosymbiosis may not be excessively rare, but it ain't common either, particularly in a full-fledged form involving vast transfer of plastid genes to the nucleus AND mechanisms of plastid targeting of the synthesised proteins. Too many an ambitious biologist completely forget about targeting, or that there's actual cell biology happening around their beloved gene sequences.

For a properly scientific and civil demolition of an earlier iteration of this ridiculous idea, see Leander 2004 Tr Microbiol (pdf). That smell of something burning? No need to worry – probably just coming from the link.

Lastly, as ridiculous as this hypothesis is and as amusing as it is that this actually survived peer review (no offense to J Parasitol, but phylogenies and evolution do not seem to be their strong point based on some other cases...), I fully support it being published. It is the excessive censorship of atypical theories rather than sketchy papers that "stiffles [scientific] thought"...

(Note: I would've submitted this to the high IF Journal of Are You Fucking Kidding, but I'm out of hard liquor and would thereby fail the author instructions...)

References
Bodył, A., Mackiewicz, P., & Milanowski, R. (2010). Did Trypanosomatid Parasites Contain a Eukaryotic Alga–Derived Plastid in Their Evolutionary Past? Journal of Parasitology, 96 (2), 465-475 DOI: 10.1645/GE-1810.1

LEANDER, B. (2004). Did trypanosomatid parasites have photosynthetic ancestors? Trends in Microbiology, 12 (6), 251-258 DOI: 10.1016/j.tim.2004.04.001

Sunday Protist – Trimastix marina

Before we begin, two things about [current] Trimastix marina – it has four flagella (not three) and is found in freshwater. The taxonomic author, Saville-Kent, is a bit notorious for some rather sketchy descriptions, and Trimastix is one of his 'trophies'. That said, it may be that Kent did actually see a three-flagellated and/or marine thing like this and it just hasn't been found or published yet. But for the time being, feel free to point and laugh at the double misnomer.

This past fall I dumped a bunch of leaves in a dish and kept them wet for a while. Turns out, the abundance and diversity of microbes and meiofauna thriving in that pile of dead leaves in your yard is quite amazing – all sorts of ciliates, myxomycetes (slime moulds), tardigrades, rotifers, springtails, flagellates, amoebae – you name it. Some of this world can be seen with a simple dissecting scope; it helps to put a coverslip or some other piece of glass on the wet leaves to see better. This coverslip is also great for investigating what lives on the surface of the rotting leaves. The other impressive detail was how quickly the leaf tissues rot away, after a couple months leaving little more than the bare skeleton of the vascular system. Dead leaves are the whale falls of the terrestrial microbiome.

Rotting tissues tend to have relatively low oxygen concentrations, and thus host some unique organisms. Among them was this peculiar flagellate that simply screamed "EXCAVATE" at the top of its lungs, but I couldn't quite figure out what it was:

Trimastix marina. The cell body is about 25-30µm, with a prominent anterior flagellum sticking out in front, and three smaller flagella trailing behind. The nucleus is the little blob at the very anterior tip of the cell, in front of a large circular food vacuole. At the very posterior tip is the contractile vacuole characteristic of freshwater things in general. Along the side of the cell is an exceptionally conspicuous groove, through which one of the recurrent flagella runs – a characteristic feature of excavates. Anoxic, leaf litter moistened with ample water for a couple of weeks. 40x obj, DIC

The part that screamed "EXCAVATE" at me was the distinctive groove (namesake of the supergroup) along the side of the cell. You can often discern it in other excavates like Jakobids, Retortamonads and Carpediemonas-like organisms (CLOs; hey, it beats "Clade B"...), but here you don't even have to look hard. Curiously, the closely related oxymonads (see Streblo, Saccinobaculus) seem to have lost the groove, but that's another story.

Overview of 'basic' excavate cell types. Trimastix marina is the very distinctive one in the bottom middle. There's something distinctive and cute about its thick anterior flagellum and the way it moves. (Simpson et al. 2002 JEM)

Thus far, Trimastix may seem like your garden variety peculiar flagellate. But there's something universally eukaryotic you might have difficulty finding – a proper mitochondrion.

I mentioned earlier the sample was somewhat anoxic. It wasn't irrelevant, because I've never seen anything like this critter in regular pondwater or well-aerated soil. Like many of its excavate relatives, Trimastix has lost the necessity to maintain the elaborate complexity of aerobic pathways and their accompanying structures, like cristae. Furthermore, it lacks a mitochondrial genome. This led to the conclusion that Trimastix lacks anything mitochondrial altogether, and may have diverged prior to mitochondrial endosymbiosis – a perfectly reasonable assumption given the data at the time. This landed Trimastix (along with the better-known sister Oxymonads) a position in then-subkingdom/phylum Archezoa (Cavalier-Smith 1983)
[NB: Archezoa = 'beginning/early animals', not ArchaEzoa, which would be 'ancient animals'. He seems particular about that.]

Trimastix wasn't a major player in the Archezoa Hypothesis (wherein 'amitochondriate' lineages are contemporary representatives of pre-endosymbiotic eukaryotes) since it's rather obscure, but was still a piece of the puzzle. Eventually, better phylogenetic techniques and improved taxon sampling destroyed the Archezoa Hypothesis, particularly as mitochondrial genes and derived organelles (such as mitosomes and hydrogenosomes) were found. Trimastix's mitochondrial genes were found later than those of other anaerobes, perhaps owing to its obscurity – but they're there: mitochondrion-targetting genes in the nuclear genome (Hampl et al. 2008 PLoS ONE). Furthermore, the aftermath of mitochondrial reduction looks like a generic double-membrane bound blob in electron micrographs (Hampl & Simpson 2008 in Hydrogenosomes and Mitosomes: Mitochondria of Anaerobic Eukaryotes) – no wonder it was so hard to find!

All that's left of Trimastix's mitochondrion, as the eons of anaerobic existence devoured its need to maintain one. It is uncertain whether it produces hydrogen gas – which would make it a hydrogenosome rather than a mitosome – though at least some of the necessary genes seem to be present in the nuclear genome. (Hampl & Simpson 2008)

As an aside, there's no known case yet of a reduced mitochondrion that simply disappeared – in addition to aerobic respiration, eukaryotes have also become dependent upon it for some other vital metabolic pathways, such as those involving the Fe-S cluster. In fact, in at least one species of microsporidia, ATP is imported into the mitochondrial relic in order to keep the key metabolic pathways running. (I vaguely recall having written about this before, somewhere...)

Lastly, Trimastix is host to some lateral gene transfer for its glycolytic pathway – it appears to have picked up and replaced at least four of the eukaryotic genes with bacterial versions (Stechmann et al. 2006 BMC Evol Biol). There was a discussion somewhere on the blogosphere lately (Coyne's blog, IIRC) about the relative importance of LGT – it sure as hell does happen in eukaryotes as well, though not crazy enough to wreak havoc on the phylogenies.


And the rain hasn't stopped yet. But I can't skip a second night of sleep... as much as I'd love to keep blogging about stuff.

References
Hampl, V., Silberman, J., Stechmann, A., Diaz-Triviño, S., Johnson, P., & Roger, A. (2008). Genetic Evidence for a Mitochondriate Ancestry in the ‘Amitochondriate’ Flagellate Trimastix pyriformis PLoS ONE, 3 (1) DOI: 10.1371/journal.pone.0001383

Hampl, V, & Simpson, AGB (2008). Possible Mitochondria-Related Organelles in Poorly-Studied “Amitochondriate” Eukaryotes HYDROGENOSOMES AND MITOSOMES: MITOCHONDRIA OF ANAEROBIC EUKARYOTES DOI: 10.1007/7171_2007_107

SIMPSON, A., RADEK, R., DACKS, J., & O'KELLY, C. (2002). How Oxymonads Lost Their Groove: An Ultrastructural Comparison of Monocercomonoides and Excavate Taxa The Journal of Eukaryotic Microbiology, 49 (3), 239-248 DOI: 10.1111/j.1550-7408.2002.tb00529.x

Stechmann, A., Baumgartner, M., Silberman, J., & Roger, A. (2006). The glycolytic pathway of Trimastix pyriformis is an evolutionary mosaic BMC Evolutionary Biology, 6 (1) DOI: 10.1186/1471-2148-6-101

Anoxic microforay Part II: Everything looks like Bodo

To the untrained eye, all tiny heteroflagellates look the same. To the slightly trained eye, all tiny heteroflagellates look like Bodo.

Anyway, here's a continuation of the smelly marine anoxic sludge microforay I should've finished over a month ago. Taking a break from the much too long Sunday Protist post...

There were lots of bodonids (kinetoplastids, see phylogeny in this post). A lot. Plenty of other excavates around too.

They tend to move about by twisting around their anterior-posterior axis with the anterior flagellum sticking in front, the posterior often wrapping around the body as it trails behind.

Free-living bodonids are quite common and diverse. Also quite understudied, as they don't cause disease. This makes them kind of annoying, as there's so many of them and so little to say. They do have interesting mitochondrial genomes, constituting the kinetoplasts, the group's namesake.

This next thing is another excavate. It reminds me of Carpediemonas (a basal fornicate, near diplomonads et al.), but I'm not entirely sure.

Chilomastix-y thing? Can't tell if there's supposed to be more flagella there...

Unidentifiable mess, but there appears to be a peculiar ridge or line of vesicles or something:

Presumably another bodonid. Wasn't moving quite like the others though...

Trigonomonas or something like it. Hard to catch the bugger in its distinctive pose.

Heteroloboseans! They showed some dramatic eruptive pseudopodial motion; will put up a video once I figure out how to. Though I can't currently rule out their being amoebozoans with eruptive pseudopodia (there are some). The human eye isn't particularly great at distinguishing apart amoeboid things.

Another interesting excavate with a very obvious oral groove. It apparently noticed being noticed, and immediately swam away into a pile of debris, and sneered at me from there. That bastard. The second image shows it from its side. Hard to ID anyway... perhaps some Enteromonad-y thing? Hard to tell how many flagella this one has...

A few more shots of that weird flagellate I mentioned earlier. I think it has four flagella now. Who knows how many tomorrow shall bring...

A heterotrophic euglenid, perhaps Petalomonas sp; it feels its way around with its thick anterior flagellum as it crawls around. The posterior flagellum remains inside the flagellar pocket. The cytostome is also visible inside the cell as the wedge-looking thing.

*shrug* Could be some cercozoany thing. Or maybe not.

Bacterial jungle the above critters seem to thrive in:

Next installment: Misc. non-excavate stuff.

Oh, on the topic of excavates, a jakobid from a freshwater (pond) sample. At least I think it is – looks like Reclinomonas americana. Do they form small stalked colonies like that?

Sunday Protist -- Rostronympha and latest parabasalian taxonomy

Since I just spent hours staring at onychophorans (instead of studying), gonna skimp out on the Sunday Protist this week. So here's a wonderful alien-looking freak with a proboscis, Rostronympha; I totally demand an SEM of this, by the way:

Parabasalid Rostronympha. Image by Guy Brugerolle via Micro*scope.

I can't find the original description at the moment, but vaguely recall having searched for it ambitiously once and failed miserably. It's supposed to be (Duboscq, Grassé & Rose, 1937), with a later mention in PP Grasse, A Hollande (1963) Ann. Sci. Natur. Zool. Ser "Les flagelles des genres Holomastigotoides et. Rostronympha". Might as well order it sometime...

And now onto a really nice taxonomic summary of parabsalians by Cepicka, Hampl and Kulda 2009 in Protist:

Recently revised parabasalian taxonomy.

There are now six classes: Trichomonadea, Hypotrichomonadea, Cristamonadea, Tritrichomonadea, Spirotrichonymphea and Trichonymphea. This will be on the final AND your next weekly spelling test. More importantly, this is how they relate:

Phylogenetic relationships between the six new classes.

Ok, I must run...will definitely get back to parabasalians in much more detail later. Some people in our department happen to be rather obsessed with them, and obsession can be contagious. But for now, feel free to join me in salivating over that really sweet diagram!

Right, finals...

Source:
Cepicka, I., Hampl, V., & Kulda, J. (2010). Critical Taxonomic Revision of Parabasalids with Description of one new Genus and three new Species Protist DOI: 10.1016/j.protis.2009.11.005

MM17 Answer - Spironucleus: double cells with twisted nuclei

Really need to take care of the long lineup of overdue Mystery Micrographs. And clean up a bit of this huge drafts pile that has accumulated lately. Because I'm lazy, let's do Spironucleus first, from MM17. It goes well with laziness as not very much is known about it, which means there isn't too much to write about it. Shit, now you know why I blog about obscure organisms like those various protists...my secret is out!

SEMs of diplomonad fish parasite Spironucleus vortens. cr - compound lateral ridge.lpr + rpr - left and right peripheral ridge, respectively. Note their rope-like form. pp - posterior papillum. Note flagellar pockets in (4). r - recurrent flagellum. 5 - an atypical specimen with transposed posterior structures. 6 - laterla view of posterior end. A fairly complicated flagellate! (Sterud & Poynton 2002 JEM)

To clarify the complicated morphology:

Drawings of the 'double-celled' Spironucleus vortens. The two recurrent flagella pass inside the cell and emerge at the posterior end. (Poynton et al. 1995 JEM)

Spironucleus may strike you as being particularly symmetrical. In fact, it very well is a 'double cell', containing two nuclei (slightly wrapping around each other helically, hence Spironucleus), and two sets of flagella. The path of the recurrent flagella makes sense when considering that the single cells of the group have three flagella pointing one way, and the fourth pointing another. These double cells are case of the two cells being 'stuck together' at the side of the recurrent flagellum. Here's a TEM to show the elongate nuclei and the flagella passing between them:

Top: longitudinal section at the anterior end of the flagellate. n - nuclei; k - kinetosomes. Bottom: cross-section of the anterior end. Note the recurrent flagella (r; circled in red) passing between the nuclei. (Poynton et al. 1995 JEM)

Here's another species of Spironucleus, S.berkhanaus, in arctic char blood:

Parasitic Spironucleus barkhanus in the blood of arctic char, as well as in isolation. Note that it's a different species from the one above, which may explain the lack of lateral ridges.(Sterud et al. 2003 Dis Aquatic Organisms)

Note how the two species seem a bit different from each other. This shows the morphological diversity in the group. In fact, Spironucleus seems to be a bit polyphyletic or paraphyletic at best (Jørgensen & Sterud 2007 Protist; Kolisko et al. 2008 BMC Evol Biol).

Phylogeny of Spironucleus. This poor genus seem to be ruthlessly strewn all over Diplomonadida. Note position of S.vortens and S.berkhanus. (Jørgensen & Sterud 2007 Protist)

Diplomonads have an interesting tale involving cell cycle defects and duplications of the nucleus and flagella, but I'll leave you in suspense for a while. That's a bigger topic, and I should probably introduce our cute friend Giardia first (cute friend in SEM, and horrible foe in the intestine...). Giardia is a independent case of cell 'doubling', and is organised quite differently. Further discussion of diplomonads should happen...eventually. Feel free to nag me about it if you're really interested!

References
JORGENSEN, A., & STERUD, E. (2007). Phylogeny of Spironucleus (Eopharyngia: Diplomonadida: Hexamitinae) Protist, 158 (2), 247-254 DOI: 10.1016/j.protis.2006.12.003

Kolisko, M., Cepicka, I., Hampl, V., Leigh, J., Roger, A., Kulda, J., Simpson, A., & Flegr, J. (2008). Molecular phylogeny of diplomonads and enteromonads based on SSU rRNA, alpha-tubulin and HSP90 genes: Implications for the evolutionary history of the double karyomastigont of diplomonads BMC Evolutionary Biology, 8 (1) DOI: 10.1186/1471-2148-8-205

POYNTON, S., FRASER, W., FRANCIS-FLOYD, R., RUTLEDGE, P., REED, P., & NERAD, T. (1995). Spironucleus vortens N. Sp. from the Freshwater Angelfish Pterophyllum scalare: Morphology and Culture The Journal of Eukaryotic Microbiology, 42 (6), 731-742 DOI: 10.1111/j.1550-7408.1995.tb01625.x

Sterud E, Poppe T, & Bornø G (2003). Intracellular infection with Spironucleus barkhanus (Diplomonadida: Hexamitidae) in farmed Arctic char Salvelinus alpinus. Diseases of aquatic organisms, 56 (2), 155-61 PMID: 14598991

STERUD, E., & POYNTON, S. (2002). Spironucleus vortens (Diplomonadida) in the Ide, Leuciscus idus (L.) (Cyprinidae): a Warm Water Hexamitid Flagellate Found in Northern Europe The Journal of Eukaryotic Microbiology, 49 (2), 137-145 DOI: 10.1111/j.1550-7408.2002.tb00357.x