Who is hiding behind these square scales? or The mystery of the origin of square shell plates in testate amoebae

Contributed by Anush Kosakyan

 

It would be hard to find a testate amoeba lover who does not know the genus Quadrulella. These vase-shaped species are a very unique group within family Hyalospheniidae (Arcellinida) since they are capable of secreting their own square or rectangular shell scales (or plates). These scales are characterisstic only for this genus, while most other members of the family are predators that build their shell by using mainly recycled plates taken or scavenged from other testate amoebae (i.e. kleptosquamy, Lahr et al. 2015).

***

My story started in the summer of 2013, when Edward Mitchell returned from his holiday in South Africa with a bag of Sphagnum samples. The samples were full of Quadrulella shells, including species that had never been seen since their original description back in 1957. And that was it….”the right time” for single-cell barcoding of this group. From this material, we obtained very interesting results that have been published recently (Kosakyan et al., 2016).

The most surprising result was that organisms presenting square shell plates appeared not to be monophyletic: two different groups of taxa with square scales (genus Quadrulella and newly established genus Mrabella) were indeed placed far from each other on the mtCOI-based phylogenetic tree. We thought of two possible evolutionary scenarios to explain this situation. These two hypotheses depend on whether the square-shaped plates in an organism have an autogenous or exogenous origin. This is a relevant aspect because hyalospheniids are known to engage in the behaviour of kleptosquamy, or recycling of shell plates produced by other organisms (Lahr et al., 2015).

Assuming that all amoebae that present square-shaped plates in their shells have produced them autogenously, the phylogenetic hypothesis presented here indicates homoplasy:

1) either the ability to produce square plates is an ancestral character that has been lost in a number of lineages (reversion); or

2) this character has evolved more than once independently (convergence).

We have assumed that convergence is the most parsimonious alternative (see the detailed discussion in the paper for further detail), with only two steps required – one event in genus Quadrulella and another independent event in genus Mrabella. Although we should not forget another unrelated group of organisms that are capable of producing square plates: the shelled amoeba Paraquadrulla is capable of producing calcareous square plates (the genus has not been sequenced yet, but while it is almost certainly an Arcellinid it is most likely unrelated to hyalosphenids or at best branches in a basal position to the whole group).

Alternatively, considering the fact that majority of hyalosphenids have the ability to scavenge plates from prey and use them to make the shell (kleptosquamy), a possible scenario is that Mrabella is in fact using scales from preyed Quadrulella, or scavenging these scales from the environment to construct the shell. And indeed, this was our first guess when we saw the description of Nebela galeata (current name Gibbocarina galeata) from Africa by Gauthier-Lièvre (1957). It is almost identical to Q. subcarinata in general shape and dimensions of the test (L=180-200, B=98-144, A=31-41 μm) (see Figure). Gauthier-Lièvre, 1957 (in her Fig. 10A) showed that square plates typical for Quadrulella can be integrated in the shell of N. galeata.  This is very easy to explain since Gauthier-Lièvre (1957) documented other Quadrulella species from the same locality where she found N. galeata and Q. subcarinata. Thus, other Quadrulella species could have provided the square plates use by these two species.

 fig10

Legend: A- Line drawing of Gibbocarina (Nebela) galeata from Congo by Gauthier-Lievre, 1957. B and C– Scanning electron microscopy and light microscopy micrographs of Mrabella subcarinata from South Africa. Scale bars =50 µm (in A) and 20 µm (in B). Modified from Kosakyan et al. 2016.

 

However,  things are not that easy in our case for two reasons:

1) Mrabella would specifically select these plates, a trait that is not known for any other hyalosphenid presenting kleptosquamy; these species generally use a mixture of plates from different origins to build their shell (see for instance: Apodera vas in Fig. 69 in Meisterfeld 2002);

2) if not specifically selecting, then Quadrulella would have to be the most abundant prey organism, or Quadrulella plates would have to be the most abundant in the environment, both options are likely not true since Quadrulella tends to be present in low abundance in comparison to Euglypha (although Euglypha was not abundant in our sample where we found Mrabella subcarinata), a genus of filose testate amoebae which is generally abundant and produces oval and ornamented siliceous plates.

Additionally, there are a number of other described species in genus Quadrulella that differ from the tear-shaped morphology of Quadrulella s.str. These also present similar shapes to other genera: Quadrulella vas, Q. constricta Apodera vas, Q. lageniformis Padaungiella lageniformis, Q. tubulata P. tubulata. These “mirror” species could either be a result of convergent evolution or alternatively represent cases of the “classical” hyalosphenids (A. vas, Padaungiella ssp.) that live in environments where euglyphids are rare but Quadrulella are abundant enough to provide material for building their shells. We have suggested that at this point these Quadrulella species must be treated as incertae sedis, and their sequencing will certainly illuminate the conundrum of the evolution of square-shaped plates.

References

Gauther-Lievre L. 1957.  Additions aux Nebela d’Afrique. Bull. Soc. Hist. Nat. l’Afrique du Nord 48, 494-523.

Kosakyan, A., Lahr, D. J. G., Mulot, M., Meisterfeld, R., Mitchell, E. A. D. and Lara, E. 2016. Phylogenetic reconstruction based on COI reshuffles the taxonomy of hyalosphenid shelled (testate) amoebae and reveals the convoluted evolution of shell plate shapes. Cladistics, 32: 606-623. doi:10.1111/cla.12167

 

What’s in a name? Something (completely different) to be said about taxonomic nomenclature

By Edward A. D. Mitchell,

Laboratory of Soil Biodiversity, University of Neuchâtel, Switzerland

With the advent of high throughput sequencing, estimates of global diversity are being totally revised as well.  For us protistologists – arguably much more importantly – so is the picture of how diversity is distributed among the different branches of the tree of life. The image that emerges is one that shows a huge unknown diversity among protists, at all levels, from major groups (i.e. “environmental clades”) and within known groups (i.e. from more or less divergent groups to complexes of cryptic and pseudo-cryptic species). This is fascinating and to say the least mind-boggling and a much welcome development for making a case about the need to study protists more intensively. It is indeed impossible today to ignore this diversity and the many functional roles that protists play in all ecosystems.

But this unknown diversity also calls for a massive investment in taxonomy. And as we all know there are only few active protist taxonomists. We therefore need to train a new generation of taxonomists to meet the huge challenge of keeping up with the novel discoveries resulting from molecular studies. And indeed, combining molecular and traditional microscopy approaches is the key to doing this job properly.

But there is also an often overlooked but important aspect of taxonomy, besides the critically important fact that descriptions need to be done correctly in order not to make a huge mess of nomenclature: naming a species. Choosing an appropriate name is indeed not always easy. Should we name a species after an esteemed colleague, the geographical location where the species was found, a morphological feature of the species, or should we try to find a name that also allows non-specialists to relate to the species – thus providing an excellent opportunity to increase the impact of the finding and making a broader audience aware of the sheer existence of our beloved amoebae?

I firmly believe that witty names are useful. They make us happy, allow many lively discussions to take place among colleagues and are much appreciated by journalists always keen to report on “something completely different” (Monty Python, 1971).

The choice of names tells a lot about the personality and cultural references of the author. Here are three recent examples among testate amoebae:

Padaungiella Lara & Todorov 2012.

This genus of hyalosphenid testate amoebae is characterised by an elongated neck (Kosakyan et al., 2012). The idea for this name came to me while riding my bicycle (a great source of inspiration!). I knew about the existence of some African tribes that used metal necklaces to elongate the necks of woman (allegedly, if the woman cheated on her man he would remove the necklaces and she would die due to cervical spine injury). I did a bit of research on this and found out that there were two unrelated tribes using such necklaces, one in Africa, with independent circular necklaces piled on top of each other and one in Asia (Padaung – https://en.wikipedia.org/wiki/Kayan_people_(Myanmar)), with a single spiral necklace. The latter corresponded much better to the shape of the amoeba shell and hence we decided to choose this name.

At the time of writing this post there were 1’110 hits for Padaungiella in Google.

padaungiella

Padaungiella wailesii (left, picture by E. Mitchell) and a Padaung woman (right, source: http://www.chiangdao.com/chiangmai/karenlongneck.htm)

Nebela gimlii Singer & Lara 2015.

This species was named after Gimli (http://lotr.wikia.com/wiki/Gimli), a dwarf in Tolkien’s Lord of the Rings saga (Singer et al., 2015). This dwarf wanders in forests during the saga, something dwarfs are not supposed to do much. This species being the smallest of the species complex and being found in forested bogs the name seemed appropriate. Newspapers as far as Austria wrote about this. http://www.krone.at/wissen/amoebenart-nach-figur-aus-herr-der-ringe-benannt-in-torfmoor-entdeckt-story-497678.

At the time of writing this post there were 188 hits for “Nebela gimlii” in Google.

Arcella gandalfi Féres, Porfírio-Sousa, Ribeiro, Rocha, Sterza, Souza, Soares & Lahr 2016.

This large Arcella species bears striking resemblance to Gandalf’s hat and thus logically was named after this even more famous character of the Lord of the Rings (Féres et al., 2016; Tolkien, 1954) (http://lotr.wikia.com/wiki/Gandalf).

arcella-gandalfii

Arcella gandalfi (left, from Féres et al., 2016) and Gandalf, as played by Ian McKellen in The Lord of the Rings triology, (right, source: http://lotr.wikia.com/wiki/Gandalf).

The media picked up on this story even more and this illustrates again how a well-chosen name can significantly contribute to making our field of research more visible.

Arcella gandalfi” has 31’600 hits on Google at the time of writing this post. To this date, news agencies in more than 15 countries reported on this, including: Brazil, USA, Germany, France, Russia, India, Mexico, Spain, Turkey, Argentina, Hungary, Indonesia, Croatia, South Korea, Ukraine.

Gandalf clearly wins! Well done fellows!

Who’s next? There’s no end to the fun!

References

Féres, J.C., Porfírio-Sousa, A.L., Ribeiro, G.M., Rocha, G.M., Sterza, J.M., Souza, M.B.G., Soares, C.E.A., Lahr, D.J.G., 2016. Morphological and Morphometric Description of a Novel Shelled Amoeba Arcella gandalfi sp. nov. (Amoebozoa: Arcellinida) from Brazilian Continental Waters Acta Protozool. 55(4).

Kosakyan, A., Heger, T.J., Leander, B.S., Todorov, M., Mitchell, E.A.D., Lara, E., 2012. COI Barcoding of Nebelid Testate Amoebae (Amoebozoa: Arcellinida): Extensive Cryptic Diversity and Redefinition of the Hyalospheniidae Schultze. Protist 163, 415-434.

Monty Python, 1971. And Now for Something Completely Different, in: MacNaughton, I. (Ed.), Monty Python’s Flying Circus. Columbia Pictures, United Kingdom, p. 95 minutes.

Singer, D., Kosakyan, A., Pillonel, A., Mitchell, E.A.D., Lara, E., 2015. Eight species in the Nebela collaris complex: Nebela gimlii (Arcellinida, Hyalospheniidae), a new species described from a Swiss raised bog. European Journal of Protistology 51, 79-85.

Tolkien, J.R.R., 1954. The Fellowship of the Ring, The Lord of the Rings, Boston. Ballantine Books, New York.

Flying amoebae

Contributed by Edward A. D. Mitchell

Laboratory of Soil Biology, University of Neuchâtel

I’m learning to fly but I ain’t got wings

Coming down is the hardest thing

Jeff Lynne/ Tom Petty

Since the early days of protistology it has been known that testate amoebae can be transported passively by wind. Darwin had collected dust that had fallen on the Beagle while sailing off the coast of Africa. He sent the sample to Ehrenberg who observed it under his microscope and found many protists (Darwin, 1846). Observations such as this have led to the idea that microscopic organisms could travel far and colonise all potentially favourable habitats (i.e. everything is everywhere, but, the environment selects; (Baas Becking, 1934, de Wit and Bouvier, 2006)). It is therefore perhaps surprising that not much experimental research has been done to quantify the amoebae that are transported by wind. A recent modelling study showed that the medium to large sized testate amoebae (i.e. 40-60µm diameter) were unlikely to travel over large distances and certainly incapable of crossing oceans while the smaller ones (e.g. 9 µm diameter) could potentially do so (Wilkinson et al., 2012). But, to my knowledge, observational and experimental studies on wind dispersal of testate amoebae are very rare. An elegant recent study by Wanner and colleagues contributed to filling this important gap (Wanner et al., 2015).

Wanner and colleagues used sticky traps: 15cm diameter plastic petri dishes in which filter paper was attached using a paraffin-based balm, which also covered the filters. The petri dishes were used as passive, sticky traps for airborne organisms and contained no growth media. The system was initially designed to study seed dispersal but proved to also be useful to study microorganisms. They exposed traps for periods ranging from 16 to 42 days and recorded 12 testate amoeba species (excluding unidentifiable specimens): Centropyxis aerophila, C. elongata, C. sphagnicola, C. ambigua, C. eurystoma, Difflugia lucida, Phryganella acropodia, Tracheleuglypha dentata, Trigonopyxis arcula, Trinema complanatum, Trinema lineare, and Trinema penardi. The two most commonly found species were respectively ca. 40µm and 60µm in diameter (Phryganella acropodia and Centropyxis sphagnicola). The overall abundance was low with little over 80 specimens recorded in total. Therefore although this study shows that amoebae can “fly”, they don’t do so in massive numbers even close to the ground and near source populations. The probability for long-distance (e.g. across 100-1000km of ocean) passive dispersal must therefore be extremely low.

Also it is rather surprising that the dominant taxa recorded on the traps were not small euglyphids but mid-size arcellinids. Indeed small euglyphids are likely to be more numerous in the upper soil horizons and the better drained and exposed microsites from which they can be expected to have more chances to be lifted up by the wind. Their small size and lighter shells should potentially allow them to be transported more easily than larger taxa, but this is not what Wanner and colleagues observed.

Extrapolating form their results, Wanner and colleagues estimated that on average 61 individual amoebae (living + dead) were deposited per square meter each day. Therefore a viable population can become established rather rapidly on a newly exposed surface, provided that a source population is present nearby. These results also suggest that initial colonisation will be rather stochastic but that as more and more amoebae are deposited on a given place the full potential community will soon be present and community composition will therefore soon be controlled by local processes such as environmental filtering. The authors estimated that the shift from stochastic to deterministic community pattern takes place after ca. seven years of soil development and therefore concluded that testate amoebae are valuable indicators of initial ecosystem development and utilisation.

Such research is important at the local as well as global scales. Locally it informs on the mechanisms that determine primary and secondary colonisation of soil and other habitats and at which temporal scale testate amoebae can be used as bioindicators. Globally it provides useful data on actual wind dispersal of amoebae. The first step is indeed for an amoeba to become airborne and this may not be trivial depending where the species live.

The study of Wanner and colleagues also shows that it is not straightforward to design traps for studying aerial dispersal of testate amoebae. The traps were indeed initially not designed for such a study but were nevertheless useful. However it may be possible to develop an optimal type of trap for studying aerial dispersal of testate amoebae. This study clearly gives food for thoughts and hopefully will stimulate the community of testate amoeba researchers to further explore how amoebae colonise new habitats.

References

Baas Becking, L.G.M. (1934) Geobiologie of inleiding tot de milieukunde. W.P. Van Stockum & Zoon The Hague, the Netherlands.

Darwin, C. (1846) An account of the fine dust which often falls on vessels in the Atlantic Ocean. Quartely Journal of the Geological Society, 2, 26-30.

de Wit, R. & Bouvier, T. (2006) “Everything is everywhere, but, the environment selects”; what did Baas Becking and Beijerinck really say? Environmental Microbiology, 8, 755–758.

Wanner, M., Elmer, M., Sommer, M., Funk, R. & Puppe, D. (2015) Testate amoebae colonizing a newly exposed land surface are of airborne origin. Ecological Indicators, 48, 55-62.

Wilkinson, D.M., Koumoutsaris, S., Mitchell, E.A.D. & Bey, I. (2012) Modelling the effect of size on the aerial dispersal of microorganisms. Journal of Biogeography, 39, 89-97.

Opening the Pandora box of community ecology – The value of long-term data sets and collaborative research

Community ecologists study how communities of plants, animals and other organisms vary in space and time, how they interact and what controls these patterns. To do this they usually either observe (more or less) natural communities or conduct experimental manipulation in the field (in situ experiments) or in controlled conditions (mesocosms, microcosms). Observational studies of natural communities have the longest history and have contributed to major (and often controversial) theories in ecology such as the intermediate disturbance hypothesis (IDH). Starting with the more easily studies taxonomic groups such as vascular plants observational studies of natural communities have expanded to covering numerous taxonomic groups, including microbes and of course testate amoebae.

In order to describe the ecological preferences of species numerous plots need to be studied, typically in the range of 50-100 or more if possible. And even so, most studies end up with a fair number of rare species, which are found in few samples and are usually excluded from the data analyses. A further complication is that patterns observed at a single site (where many plots may be sampled) may be misleading and it is clearly preferable to study fewer plots in multiple sites to circumvent the problem of spatial autocorrelation (pseudo-replication within individual sites).

Studying numerous sites and ideally distant ones means that it is all but impossible to visit these sites on many occasions with the budget limitations most ecologists have to live with. Community ecologists therefore often collect their data and samples during a single visit. The timing of such field campaigns inevitably ends up being a compromise between ideal season and weather and the agenda of the various participants. The problem with this approach is that as many environmental factors vary over time -and this is clearly the case for soil pH, temperature, moisture content, water table depth, which are key ecological factors in terrestrial ecosystems – the available data will only represent a snapshot of what happens over the growing season or the year. This raises the questions: how representative is this snapshot of longer-term patterns? Are the relative values of the measured variables comparable among samples at different times?

As species may respond to environmental factors over more or less long-time periods patterns of community structure may not necessarily be best explained by one-time measurements of environmental variables but rather by more or less long-term averages or some measure of variability as done recently by Sullivan and Booth in a study of the relationship between the relative humidity of mosses and testate amoeba communities (Sullivan and Booth, 2011). The fact that some organisms are able to enter dormancy (e.g. encystment) during part of the year further complicated the story. However, only a small proportion of community ecology studies have addressed these longer-term patterns. This is of course understandable as the collection of long-term data at dozens of sites bears a cost that cannot be covered by classical funded research projects (i.e. typically 3 years) or regular budget of research groups (if they have any at all).

Community ecologists are usually taxonomically competent in one or two groups of organisms and therefore community ecology studies are typically limited to one of very few taxonomic groups. This makes it very difficult to assess how different communities respond to ecological gradients or perturbations as studies always differ in one aspect or another (e.g. slightly different methodologies used either to record the species data or the environmental variables). Yet sound comparative studies of multiple taxonomic groups would allow addressing important ecological questions such as how life history traits (e.g. dispersal potential, generation time) determine the responses of communities to ecological factors. This of course requires collaborative research efforts. And these are surprisingly rare!

A recent study published in the journal “Freshwater Biology” by Martin Jirousek and colleagues (Jiroušek et al., 2013) from the Czech Republic stands out by its focus on both long-term (15 years) environmental data and the comparative analysis of community patterns for four taxonomic groups, vascular plants, bryophytes, diatoms and (last but not least for this blog!) testate amoebae in 51 plots located in 12 Czech peat bogs in two mountain ranges.

Taxonomic groups included 1) short-lived microscopic organisms (diatoms and testate amoebae) and long-lived macroscopic organisms (vascular plants and bryophytes), 2) organisms dispersing easily (diatoms, testate amoebae and bryophytes) and less easily (vascular plants), and 3) photosynthetic (vascular plants, bryophytes, diatoms) and heterotrophic (testate amoebae – with a few mixotrophic exceptions) organisms.

The long-term ecological data also offered a unique opportunity to conduct a study on the effects of aerial liming (Ca, Mg), which has been used in the study area since the 1980’ as a forest amelioration practice (following damage caused by acid rain) and influenced the studied bogs. Such unintentional experiments can be highly valuable for ecologists, for example, as in this case to untangle ecological gradients that are usually strongly correlated (e.g. pH and calcium gradients in peatlands).

Martin Jirousek and colleagues hypothesised that long-term data would overall explain the community data better than short-term or one-time measurements and that this would be especially true for the longer-lived vascular plants and bryophytes. Following this they further hypothesised that the newly established pH and Calcium gradients would be better reflected in the shorter-lived communities of diatoms and testate amoebae. They compared the significance of environmental variables for different time spans: single time point, three, five, 10 and 15-year averages.

The results only partly supported the hypotheses. Micro and macro-organisms were correlated to both short- and long-term water chemistry variables (but not the same ones). Water table was correlated to all four communities but in agreement with the hypothesis, only the short-lived organisms reflected the recently established pH and Ca gradients. The results therefore generally support the idea that life-history traits condition the response of species communities to environmental gradients.

Although long-term ecological data sets such as the one used by Martin Jirousek and colleagues are not very common, they are perhaps not that rare. Ecologists would be well inspired to search for such data sets and if the monitoring program that generated them is still in place they should try to conduct similar studies (and encourage the monitoring program to continue!). Despite all technological advances we still can’t go back in time and therefore such data sets should be considered as a highly valuable asset. Although they involve some costs these can be very well justified if ecologists make good use of the data to address currently debated ecological questions such as the factors controlling community assembly and other questions related to metacommunity theory. This study also shows how valuable multi-taxa studies can be and hence how much added value there is to collaborative research!

Reference

Jiroušek, M., Poulíčková, A., Kintrová, K., Opravilová, V., Hájková, P., Rybníček, K., Kočí, M., Bergová, K., Hnilica, R., Mikulášková, E., Králová, Š. & Hájek, M. (2013) Long-term and contemporary environmental conditions as determinants of the species composition of bog organisms. Freshwater Biology, 58, 2196-2207.

Sullivan, M.E. & Booth, R.K. (2011) The Potential Influence of Short-term Environmental Variability on the Composition of Testate Amoeba Communities in Sphagnum Peatlands. Microbial Ecology, 62, 80-93.