A new approach to testate amoeba paleoecology: reconstructing past environmental conditions from morpholological traits

Contributed by Edward A. D. Mitchell

Laboratory of Soil Biodiversity, University of Neuchâtel, Switzerland

Testate amoebae are well known to be good indicators of micro-environmental gradients and especially soil moisture, water table depth and pH. This has been known since the early 20th century (Harnisch, 1925). The predictable distribution patterns of many species not only systematically result in seeing these variables emerge as being significantly correlated to testate amoeba community data in ecological studies but also allow the development of inference models – so called “transfer functions” – based on testate amoeba community data to reconstruct (infer) these variables either from modern samples or more generally from subfossil communities extracted from peat deposits or lake sediments. As there are few proxies to reconstruct past hydrological changes, testate amoebae have become part of the standard toolbox of palaeoecologists (Charman, 2001; Mitchell et al., 2008). Indeed, developing and using such transfer function has been the main reason to study testate amoebae and indeed most papers dealing with testate amoebae published in the last couple of decades. These organisms were thus being used as tools using correlative approaches rather than considered as study subjects in their own right.
Simon van Bellen and colleagues have just published a paper in which they present a new approach for palaeo-environmental inference based on testate amoeba morphological traits rather than community data (van Bellen et al., 2017). They used nine different traits and related them to the depth to the water table (DWT), the variable most commonly inferred from testate amoebae. Their data set is from Tierra-del-Fuego, but could almost be from anywhere in the World where Sphagnum peatlands occur. The list of traits includes morphological traits such as biovolume, aperture position, test compression, aperture size and test composition, a binary physiological trait (mixotrophy vs. heterotrophy) and two phylogenetic traits (Arcellinida and Euglyphida – there were no Amphitremids in the data). Most traits showed a relationship to DWT. For example, Arcellinida show an almost perfect negative linear correlation to DWT (i.e. the propostion of Arcellinida decreases with increasing DWT, in other words, the wetter the habitat, the more Arcelinida dominate the community). By contrast Euglyphida show a positive correlation to DWT (so the drier it gets the more the testate amoeba community is dominated by Euglyphida).

vabellen

An extract from Figure 3 of van Bellen et al. 2017. Relationship between the community weighed means of Arcellinida (top) and Euglyphida (bottom) vs depth to water table (DWT, horizontal axis – left is wet, right is dry). The two phylogenetic groups clearly show opposite responses to the humidity gradient.

Interestingly this pattern corresponds to a ratio used in mineral soil, the LF index (Bonnet, 1976), where “L” stands for Lobosea (Arcellinida) and “F” stands for Filosa (Euglyphida). By analogy to MacArthur and Wilson’s concept of r (ruderal) and K (competitors) life history classification (MacArthur and Wilson, 1967), Euglyphida are considered as r strategists while Arcellinida are on average more K strategists. Euglyphids are on average smaller and thus more likely to feed on smaller prey such as bacteria. Being smaller also means that they may tolerate dry conditions better than the generally larger arcellinids that require a thicker water film to move.

The study of testate amoebae functional traits is clearly rapidly becoming a dynamic field of research within our community (Arrieira et al., 2015; Fournier et al., 2016; Fournier et al., 2015; Fournier et al., 2012; Jassey et al., 2016; Jassey et al., 2015; Lamentowicz et al., 2015; Marcisz et al., 2016; Marcisz et al., 2014). It will be interesting to follow the next developments in this area!

References

Arrieira, R.L., Schwind, L.T.F., Bonecker, C.C., Lansac-Toha, F.A., 2015. Use of functional diversity to assess determinant assembly processes of testate amoebae community. Aquatic Ecology 49, 561-571.

Bonnet, L., 1976. Le peuplement thécamoebien édaphique de la Côte-d’Ivoire. Sols de la région de Lamto. Protistologica 12, 539-554.

Charman, D.J., 2001. Biostratigraphic and palaeoenvironmental applications of testate amoebae. Quaternary Science Reviews 20, 1753-1764.

Fournier, B., Coffey, E.E.D., van der Knaap, W.O., Fernandez, L.D., Bobrov, A., Mitchell, E.A.D., 2016. A legacy of human-induced ecosystem changes: spatial processes drive the taxonomic and functional diversities of testate amoebae in Sphagnum peatlands of the Galapagos. Journal of Biogeography 43, 533-543.

Fournier, B., Lara, E., Jassey, V.E.J., Mitchell, E.A.D., 2015. Functional traits as a new approach for interpreting testate amoeba palaeo-records in peatlands and assessing the causes and consequences of past changes in species composition. Holocene 25, 1375-1383.

Fournier, B., Malysheva, E., Mazei, Y., Moretti, M., Mitchell, E.A.D., 2012. Toward the use of testate amoeba functional traits as indicator of floodplain restoration success. Eur. J. Soil Biol. 49, 85-91.

Harnisch, O., 1925. Studien zur Ökologie und Tiergeographie der Moore. Zoologisch Jahrbuch (Abteilung Systematik) 51, 1-166.

Jassey, V.E.J., Lamentowicz, M., Bragazza, L., Hofsommer, M.L., Mills, R.T.E., Buttler, A., Signarbieux, C., Robroek, B.J.M., 2016. Loss of testate amoeba functional diversity with increasing frost intensity across a continental gradient reduces microbial activity in peatlands. European Journal of Protistology 55, Part B, 190-202.

Jassey, V.E.J., Signarbieux, C., Haettenschwiler, S., Bragazza, L., Buttler, A., Delarue, F., Fournier, B., Gilbert, D., Laggoun-Defarge, F., Lara, E., Mills, R.T.E., Mitchell, E.A.D., Payne, R.J., Robroek, B.J.M., 2015. An unexpected role for mixotrophs in the response of peatland carbon cycling to climate warming. Scientific Reports 5, 16931.

Lamentowicz, M., Gałka, M., Lamentowicz, Ł., Obremska, M., Kühl, N., Lücke, A., Jassey, V.E.J., 2015. Reconstructing climate change and ombrotrophic bog development during the last 4000 years in northern Poland using biotic proxies, stable isotopes and trait-based approach. Palaeogeography, Palaeoclimatology, Palaeoecology 418, 261-277.

MacArthur, R.H., Wilson, E.O., 1967. The Theory of Island Biogeography. Princeton University Press.

Marcisz, K., Colombaroli, D., Jassey, V.E.J., Tinner, W., Kołaczek, P., Gałka, M., Karpińska-Kołaczek, M., Słowiński, M., Lamentowicz, M., 2016. A novel testate amoebae trait-based approach to infer environmental disturbance in Sphagnum peatlands. Scientific Reports 6, 33907.

Marcisz, K., Lamentowicz, L., Slowinska, S., Slowinski, M., Muszak, W., Lamentowicz, M., 2014. Seasonal changes in Sphagnum peatland testate amoeba communities along a hydrological gradient. European Journal of Protistology 50, 445-455.

Mitchell, E.A.D., Charman, D.J., Warner, B.G., 2008. Testate amoebae analysis in ecological and paleoecological studies of wetlands: past, present and future. Biodivers. Conserv. 17, 2115-2137.

van Bellen, S., Mauquoy, D., Payne, R.J., Roland, T.P., Hughes, P.D.M., Daley, T.J., Loader, N.J., Street-Perrott, F.A., Rice, E.M., Pancotto, V.A., 2017. An alternative approach to transfer functions? Testing the performance of a functional trait-based model for testate amoebae. Palaeogeography, Palaeoclimatology, Palaeoecology 468, 173-183.

Testate amoeba CSI

Now here is something you don’t see everyday. In fact, I am pretty sure this has never been done before.  Ildiko Szelecz and colleagues set out to answer a question that very few of you must have already pondered: could testate amoebae be used in forensic studies?

This should in fact be a very ordinary question if you know something about soil biology and protists. A dead body is a huge influx of carbon and nutrients  into the soil that it is sitting atop.  The bacterial communities will basically go wild, and all of that must influence very heavily the community of eukaryotic microorganisms that used to inhabit there.  Interestingly enough, it appears that the idea of checking out what happens to testate amoebae communities has never popped into anyone’s head before.  Testate amoebae are abundant, somewhat easily recognizable micro-eukaryotes, which despite a few taxonomic issues are quite useful as indicators of environmental conditions.  Well, I think we have to thank Miss Szelecz for kindly looking into this gory issue!

Basically, the authors hung pig cadavers of approximately 20 kilos each in a nicely setup experiment.  For each pig replicate, there were two controls – a similar sized are with nothing in it, and an area where they added a plastic bag filled with 20 kg of soil to mimic possible micro-climatic effects that do not come form decomposition itself.  They then sampled soil from beneath the cadavers at regular intervals and counted testate amoebae – what could be more fun?

pig

What happens then is truly incredible.  At first, the testate amoebae die.  The changes introduced in soil chemistry are simply not tolerable to the amoebae and by the 22nd, all amoebae under the dead body are also dead.  The two controls behaved different from each other, presumably because microclimatic conditions (evaporation, etc) are quite different under a plastic bag filled with litter.

Screen Shot 2014-03-12 at 4.54.55 PMIn the image above straight from the paper, the plain line indicates live amoebae, the short-dashed line indicates encysted amoebae, and the long-dashed line indicates dead amoebae.

After the massive amoeba genocide caused by the dead pig body, it is apparently very difficult to recolonize and rebuild the original community.  Even after almost one year, the effects of the cadaver were still noticeable in the composition of the testate amoeba community.

The authors conclude that testate amoebae may actually be good forensic tools to estimate post-mortem intervals.  I’ve heard a bigger experiment exploring distinct types of habitats is in the works.  Perhaps the authors shouldn’t be surprised if they receive a phone call from the producers of CSI or another forensic show where they are looking for some kind of new forensic evidence…

Article:

Biogeography, cryptic species and amoebae

If you are lucky enough to know a protistologist, you have certainly heard her (or him) utter the statement: “not much is known about protist _________”, where the blank can be filled with your choice of a biological subject, such as “meiosis”, “ecology”, “paleontology”, and many others. The statement gets staggeringly truthful when the blank is filled with the word “biogeography”.

To me this has always been very interesting because biogeography is one of the most intuitive biological concepts: anyone knows that there are some biological species that simply won’t show up in their backyards. If you live in South America, you can expect to see a Capibara, even (or specially) in a large metropole like Sao Paulo. This is not true for London, Berlin, or Moscow, unless you are at the local Zoo. Similarly, people living in Sao Paulo know that they won’t see a Grizzly Bear, or any bears at all – leading to the very intuitive notion that animals have restricted geographical ranges. This is also true for plants, one of my personal dreams is to see a Baobab, but I would have to travel to one of the other pieces of Gondwana to see it.

Which brings us to the first thing that may influence how species are distributed on the planet: the geological history of the place where a given biological species had first appeared. If you check the following map, depicting what the supercontinent Gondwana must have looked like in the past, you can understand how different iconic fossils were distributed. The logical assumption by looking at these ancestral distributions is that the descendants of these biological species will inhabit the same places. The difference is that by now, those continents have moved apart.

ImageSource: Wikipedia

The same kid who knows that they won’t find a bear in Sao Paulo also knows that they will not find a Great White shark roaming the streets (although that would certainly be a cool sight). But that is because Great Whites are exclusively marine animals that can not survive on land. Although this may seem like a simplistic analogy, it illustrates a distinct issue altogher: ecological requirements. Besides the geological history of the biological species distributions, ecological requirements will also restrict the places on earth where a given species can be found.

Therefore, biological entities should be distributed across the globe according to geological history and ecological requirements, right? No. Protists are more difficult. Firstly, you generally can’t see them without a microscope. So, people need to get trained on microscopy to start seeing protists. Even then, it may be difficult to identify species among them. Take a look at the following images:

ImageImage

Source: micro*scope

Although very similar, these are two distinct genera of amoebae. If you know who they are, you certainly have a few of years of protistology under your belt. This is the first difficulty in protist biogeography – we must live with the possibility that different species live in different places, but we can’t tell them apart because trained morphologists are hard to come by [1]. To overcome this difficulty, people came up with the concept of “flagship species”: these would be easy to identify, large and abundant species [2]. That way, if the organisms are somewhere in the world that has ever been sampled by a human being, those organisms have a high probability of having been recorded. This is where the testate amoebae come into play. I use the following example in classes:

Image

Quadrulella symmetrica. Source: Ferry Siemensma.

This is a Quadrulella symmetrica. If you can’t remember the look of an amoeba that lives inside a shell she made herself out of internally mineralized square pieces of glass, then maybe biology isn’t for you. Testate amoebae are really good models to ask biogeographical and ecological questions, because they are so conspicuous. The problem is that we do not know much about their taxonomy, but that is quickly changing.

These easily identifiable, large and abundant species, are found everywhere that present their ecological requirements. So in the protist world, the sentence in bold above is generally translated to everything is everywhere, the environment selects. A simplistic way to explain this paradigm is that because protists are small, they should be able to disperse to all places, which seems to be a reasonable assumption [3]. This basically destroys the assumption that geological history must have an effect on the distributions of organisms, simply because they can potentially override this constraint. The second part of the paradigm, is quite similar to the shark analogy, and is to me the least understood part, not only in protist biology (well, not the extreme example of sharks on land, but more sutile things like pH gradients).

Recently, Thierry Heger and colleagues published a very intriguing study dealing with these questions [4]. They chose a single “morphospecies” to test some of these predictions — the testate amoeba Hyalosphenia papilio, a quite “flagshippy” species.

Image

Hyalosphenia papilio. Source: Dan Lahr.

They sampled 42 Sphagnum dominated locations in 11 northern countries, obtaining over 300 individuals! They then went on to sequence a marker gene from each of these individuals and tried to correlate the genetic divergences with either geographical or ecological factors.

It may be surprising to some that even though Heger and colleagues sampled individuals that look almost exactly identical, they actually found 12 distinct genetic lineages. Now, the people who performed this work are experienced testate amoebae researchers, so we can rule out the possibility that they lumped together a bunch of morphologically distinct taxa. This may be a typical case of a “cryptic species”, lineages have diverged but the morphology has not yet changed. There is a small possibility that each of these 12 lineages actually have a distinguishing characteristic, but these would be so minimal that would not be of much help.

Even more surprising may be the fact that geographical distribution, or rather vicariance, cannot be an explanation to the divergence of these 12 distinct lineages. The following map is a figure from their paper, and shows how the different lineages were spread in the globe.

ImageAlthough you can already see that a particular place in the world doesn’t contain a single type of Hyalosphenia papilio, they went further ahead and tested this statistically, also testing if environmental variables they collected in specific sites could explain the distribution pattern.  They found out that climatic factors alone were responsible for 21% of the variation, while spatial factors (geography) were responsible for 3% of the variation.  The two combined factors explain an additional 13%.

Amazingly, this means that 63% of the variation is due to unkown factors.  In my view, this is solid proof that when a protistologist tells you that “not much is known about protist __________“, you should take them very seriously, because we can still learn a whole lot from the wee-beasties.

Literature

[1] Mitchell EAD, Meisterfeld R. 2005. Taxonomic confusion blurs the debate on cosmopolitanism versus local endemism of free-living protists. Protist 156(3): 263-267.

[2] Foissner, W. 2006. Biogeography and dispersal of micro-organisms: a review emphasizing protists. Acta Protozoologica 45(2): 111-136.

[3] Wilkinson, D. M., Koumoutsaris, S., Mitchell, E. A., & Bey, I. (2012). Modelling the effect of size on the aerial dispersal of microorganisms. Journal of Biogeography, 39(1), 89-97.

[4] Heger, T. J., Mitchell, E. A., & Leander, B. S. (2013). Holarctic phylogeography of the testate amoeba Hyalosphenia papilio (Amoebozoa: Arcellinida) reveals extensive genetic diversity explained more by environment than dispersal limitation. Molecular ecology, 22(20), 5172-5184.