Pushing the Limits: Exploring Extreme Phenotypes in

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Pushing the Limits: Exploring Extreme Phenotypes in Soil Protozoa from the McMurdo Dry Valleys. Andrew R. Thompson, Byron J. Adams. Department of ...
Pushing the Limits: Exploring Extreme Phenotypes in Soil Protozoa from the McMurdo Dry Valleys Andrew R. Thompson, Byron J. Adams Department of Biology, Brigham Young University, Provo Utah

The search for life in our solar system involves in part delineating the habitat limits of extremophilic life on earth1,2. One of the many challenges to this approach is distinguishing the difference between the realized habitat and the theoretical limits of the organism in question1,3. This project utilizes cultured soil protozoa from Antarctica and directive selection to explore the phenotypic limitations on these cold desert extremophiles4. Understanding the potential of these organisms to evolve even more extreme phenotypes will better inform our search for potentially suitable habitat on cold desert worlds, like Mars. Finding Extraterrestrial Life

Broad surveys of present-day extremophile diversity provide valuable insight into the habitat suitability and limits of life as we know it1,2,5. However, due to a variety of constraints, there may be phenotypes that are possible yet not present on Earth today3. This space represents those possible phenotypes. Whether this space exists, how extensive it is, what diversity of phenotypes are missing from our catalogs, and how to explore it are all important considerations in the search for life.

Realized Range (includes any Phenotypic Plasticity)

Actual Lower Limit

Physicochemical

Unexploited Phenotypic Space

Theoretical Limit

Ecological Geographical Evolutionary Environmental

Trait Fitness

Phylogenetic

more severe conditions, but are hampered by higher metabolic requirements Genetic Inheritance Adaptation is to a large degree constrained by existing traits Competition Organisms must secure resources more efficiently than others, diverting energy away from exploring more extreme phenotypes Distribution Species with limited distribution tend to have smaller population sizes (which limits variation), and are more susceptible to extinction. Variation Evolution acts on variation, but if an environment is too extreme or static it may limit variability Environmental Organisms only need to do the bare minimum – more extreme adaptations may not be needed in many “extreme” environments



0

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Protozoa are Model Eukaryotes Understanding the limits of eukaryotes generally helps us find more complex forms of life Excellent lab organisms - rapid generation time, usually small genomes and high population density Protozoa include numerous ancient eukaryotic lineages and a diversity of unexplored adaptations Inhabit mostly the same places that prokaryotes do, allowing for comparisons on how different forms of life handle similar environments

Potential Maximum

Unexplored Phenotypes

Phylogenetic Constraint

Dry Valley Soil Conditions5,7

Phenotype Space

High Salinity (10-10,000 mSv)

landscape4,6 consisting

Phenotypes can be displayed visually in a theoretical of peaks and valleys. Peaks represent the greatest local fitness, and it is therefore expected that in general natural selection drives phenotypes towards a peak and away from lower fitness valleys. Various constraints modulate this ideal, however, as can be seen in the graph above – an ecological constraint like competition might prevent an organism from reaching the local maximum fitness, while a phylogenetic constraint like genetic inheritance might prevent its retreat towards a different phenotype3. Not all peaks are equal in fitness height, but often in order to explore novel peaks and phenotypes, there can be a variety of barriers, including low fitness itself6. Evolution’s efficiency at exploring phenotypic space varies with scale (probably less efficient over short time intervals, more so over longer ones) but it will likely never be fully comprehensive because it is constrained by stochastic variables3,6. Thus, there should be phenotypes we can discover that may not occur naturally on Earth but might occur naturally elsewhere. We can simulate natural evolution in the laboratory where we can effectively mediate the effect of at least some constraints4.

Acknowledgments This research was funded by McMurdo LTER NSF OPP grant 1115245, NSF ANT 1115245, and the Utah NASA Space Grant Consortium.

Growth Rate Comparison

Mild Salinity Temperature (low) Freeze-thaw Cycling Radiation Resistance Desiccation High pH

A B C

A

B Fast growth, dense population

C

Slow growth, low density

Slow growth, high density

Set Time

Meta-population Incubation

Absolute Maximum

Potential Maximum Current Unexplored Phenotype Phenotypes Ecological Constraint

Sample Parameter Strength

Employing Directed Evolution

Constraints and Trait Landscape Model

Metabolism Consumers may be able to directly handle

Determining Phenotype Range

Once culturing parameters are established for cultured organisms, a series of tests will identify the upper and lower limits of their ranges, as well as their optimum growth conditions. For simplicity’s sake, only one parameter will be explored at a time, though some (temperature) will require the simultaneous modification of another (salinity) to explore the full range.

Exploring Unexploited Phenotypic Space Physiological

Samples from ~25 different locations were stored at 4°C until culturing began7. 50g per sample were wetted periodically for one week8. Runoff from wetted soil was sieved, centrifuged and incubated at 10°C for one week, and then extracts were examined. To obtain pure cultures wells of a 96-well plate were seeded with extracted protozoa and a variety of media. DNA was extracted and 18S rDNA amplified for sequencing in order to approximate the genus.

Extreme

Even across all presently extant lineages on earth, have there been sufficient evolution in the appropriate environments for enough time to fully explore this unexploited space?

Examples of Categorical Constraints3

Over time, mobile protozoa will move into the supernatant from the pellet

Short Growing Season (Dec-Feb) High pH (8-10) Low Summer Temperatures (-5°C – 15°C)

Minimal Nutrient Availability Frequent Freeze-Thaw Cycles High Solar Irradiation Low Moisture Extreme Winter Temperatures (-50°C – 10 generations3,4.

Tracking Changes in the Genome Control

Scenario A Same genes, different activity

Expression

Identify Matching Environments

Approach

Questions

Unidentified Ciliate

Context

Gene

Up and down-regulation of the same genes or change in gene copy number

Possible Explanation:

Scenario B Novel genes appear

Scenario C No detectable change

Possible co-option of existing Increase in efficiency of phenotype‡ (via upregulation) proteins, which would or a novel phenotype detectable in the sequence

To identify the effect of selection on the genome, RNA will be extracted from subsamples at various time points throughout the directed evolution step. Changes in the relative expression rate of genes potentially involved with the extreme parameter will be tracked using transcriptomic analyses and gene function predictive software9.

References 1. Board, Space Studies, and National Research Council. The limits of organic life in planetary systems. National Academies Press, 2. 3. 4. 5.

2007. Rothschild, Lynn J., and Rocco L. Mancinelli. "Life in extreme environments." Nature 409.6823 (2001): 1092-1101. Hoffmann, Ary. "Evolutionary limits and constraints." The Princeton guide to evolution. Princeton University Press, Princeton (2014): 247-252. Romero, Philip A., and Frances H. Arnold. "Exploring protein fitness landscapes by directed evolution." Nature Reviews Molecular Cell Biology 10.12 (2009): 866-876. Courtright, E. M., D. H. Wall, and R. A. Virginia. 2001. Determining habitat suitability for soil invertebrates in an extreme environment: the McMurdo Dry Valleys, Antarctica. Antarctic Science 13:9-17.

6. 7. 8. 9.

Orr, H. Allen. "The genetic theory of adaptation: a brief history." Nature Reviews Genetics 6.2 (2005): 119-127. Bamforth S.S., Wall D.H., Virginia R.A. (2005) Distribution and diversity of soil protozoa in the McMurdo Dry Valleys of Antarctica. Polar Microbiology 28:756-762. Foissner, W. 1992. Estimating the species richness of soil protozoa using the “non-flooded petri dish method. Pages B-10.11B-10.12 in L. RJ and S. AT, editors. Protocols in Protozoology. Allen Press, Lawrence, Kansas. Adhikari, Bishwo N., Diana H. Wall, and Byron J. Adams. "Effect of slow desiccation and freezing on gene transcription and stress survival of an Antarctic nematode." Journal of Experimental Biology 213.11 (2010): 1803-1812.