A Case Study written by Jackson David Reynolds, written in the style of the National Center for Case Study Teaching in Science (NCCSTS): http://sciencecases.lib.buffalo.edu/... University of North Georgia, Gainesville, GA, USA Spring 2016

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A Brief History of Mitochondria
The Elegant Origins of a Magnificent Organelle
A Case Study by Jackson David Reynolds
University of North Georgia, Gainesville, GA, USA
Spring 2016
JACKSON DAVID REYNOLDS
Image modified from: Interesting Facts, 2014.

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Part I – Insights and Hypotheses
In the first decade of the 20th century, the Russian botanist Konstantin Mereschcowsky,1,2
while studying plastids3 in lichen, hypothesized that complex, eukaryotic cells were evolutionarily
derived from early cells which had symbiotically incorporated other, less complex cells into their cytosol
– a phenomenon now known as endosymbiosis. In 1905, and again in 1910, he published work in
defense of this idea (Bock & Knoop, eds., 2012).
In the following decade, an American anatomist, Ivan Wallin, working at the University of
Colorado Medical School, noticed that mitochondria could be stained with staining techniques
identical to those used for some bacterial cells. This led him to conclude that mitochondria were once
independent bacteria which had been taken up by and subsequently co-evolved with “proto-
eukaryotic” cells into their modern incarnation (Wallin, 1922). His scientific contemporaries, however,
rejected his ideas, as he had carried out his staining experiments on cultured mitochondria (isolated
from fetal rabbit hepatocytes) in a shed behind the university’s anatomy lab, and thus contamination
with environmental bacteria yielding false positive staining results was strongly suspected (Cold Spring
Harbor Laboratory, 2011). Whether or not Wallin’s experiments were contaminated, his ideas – as well
as those of Mereschcowsky – were ultimately vindicated by the discovery of overwhelming evidence
amassed in the latter half of the 20th century for the endosymbiotic origin of the modern mitochondrion,
chiefly through the work of American cell biologist and evolutionary theorist Lynn Margulis – a name
now synonymous with the focus of this case study, endosymbiotic theory.
Dr. Margulis, in a now famous 1967 paper4, put forth a detailed theoretical framework of the
evidence she believed provided strong support for the endosymbiotic origin of the Eukarya – an idea
still so radical at the time that her paper was rejected by no less than fifteen academic publications before
finally being accepted by the Journal of Theoretical Biology (Sagan, 1967; Margulis, 1995). Her paper, On
the Origin of Mitosing Cells, brought to bear several main observations and hypotheses – which have
since been rigorously tested and substantiated – which now leave virtually no doubt that mitochondria
did in fact originate from free-living, primitive prokaryotes which were incorporated into – and
1 “Mereschcowsky” also sometimes transliterated into English as “Mereschkowski.”
2 Russian: Константи́н Серге́евич Мережко́вский.
3 “[P]lastid: [A]ny of several types of plant cytoplasmic organelles derived from protoplastids, including chloroplasts, amyloplasts, chromoplasts,
proteinoplasts, and elaioplasts” (Hardin, Bertoni, Kleinsmith, & Becker, 2012, G-18).
4 Dr. Margulis published On the Origin of Mitosing Cells as “Lynn Sagan,” her married name at the time of the paper’s publication.

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subsequently co-evolved along with – primitive “proto-eukaryotes” remarkably rapidly after abiogenesis
on Earth.
While, in the clear light of retrospection, it may be tempting to scoff at the initial resistance to
early endosymbiotic hypotheses, it is important to always keep in mind the nature of the scientific
process. The engine of scientific progress is fueled by an unwavering commitment to skepticism and
critical thinking on the part of scientists, and no idea – regardless of how “obvious” or well-reasoned it
may seem to be – should ever be accepted without first being subjected to a rigorous gauntlet of
meticulous testing. Scientific knowledge – which is always tentative – therefore exists at different degrees
of certainty, each of which has an associated term to indicate the level and context of its surety based
on the best available evidence. The terms “hypothesis,” “theory,” and “law,” thus, denote these
contextual distinctions of confidence.
In the common parlance, the words “hypothesis” and “theory” are casually employed as virtual
synonyms, doing a damaging disservice to the large distinction between the actual definitions of these
words in a scientific context. Similarly, people often confuse laws with theories, such as in the frequent
misstatement of gravitational theory by non-scientists as “the law of gravity.5”
To expound upon this problem, as well as to provide clarification as to the actual scientific
meanings of these words, watch the short video Theory vs. Hypothesis vs. Law… Explained! from the PBS®6
series It's Okay To Be Smart at https://youtu.be/lqk3TKuGNBA (Nicolosi, dir., 2015).
ASSESSMENT ITEMS
A working familiarity with the technical connotations of these three terms is of the utmost
importance in one’s understanding of any scientific writing. To further cement your grasp of the
similarities and differences between hypotheses, theories, and laws, construct a tripartite Venn diagram
of the three terms in which you provide levels of evidence which apply to each.
After constructing the diagram, briefly write a short paragraph as to why you believe that theories
are far more common in the biological sciences than are laws.
5 There are individual gravitational laws, to be sure (e.g., Newton’s famous Universal Law of Gravitation: {Fg ∝ (m1m2/r2)}), but the sum of these laws and
their associated experimentally verified observations together comprise gravitational theory.
6 Public Broadcasting Service.

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Part II – Discoveries and Evidence
Endosymbiotic theory now provides the most plausible and well-supported explanation for
mitochondrial origins, but how did this once heretical idea become widely embraced by the scientific
community? While On the Origin of Mitosing Cells was a largely speculative work, Dr. Margulis followed
up her ideas with a more experimentally substantiated paper nine years later in the journal Experimental
Parasitology entitled Genetic and evolutionary consequences of symbiosis (Margulis, 1976). This publication, which
was less speculative in nature, served to dismantle the doubts held by many in the scientific community
at the time with regard to the validity of endosymbiotic theory as a legitimate avenue of study.
Several observations similar to those made by Mereschcowsky and Wallin led Lynn Margulis to
strongly suspect prokaryotic origins for mitochondria and other modern organelles, such as plastids.
When, as an undergraduate, she learned that one of her professors had observed DNA in chloroplasts
(a type of plastid), this only served to further pique her curiosity and embolden her to more deeply
investigate eukaryotic evolution (Zimmer & University of California Museum of Paleontology, n.d.).
Margulis observed that mitochondria contained their own DNA which was contained in a circular
chromosome like that of bacteria, that some mitochondria were evidently similar in size to certain
modern prokaryotes, and that, like bacteria, mitochondria have two separate plasma membranes
(Sagan, 1967).
Throughout the following decades, it was further uncovered that mitochondria possess their own
ribosomes – ribosomes which, interestingly, are comprised of subunits equivalent in size to those of
extant prokaryotes – and that they replicate independently (by binary fission, no less!) in a manner
chronologically independent from the mitotic activity of their host cell. Perhaps even more intriguingly,
like bacterial DNA, mitochondrial DNA (mtDNA) is devoid of introns (Cummings, Spencer, &
Paladino, 2012, pp. 225-228; Gillen, 2009, A-37-A-39)! Most astounding to some, however, was the
discovery that while nuclear DNA does encode some mitochondrial proteins, the specific sequences
responsible for these gene products have been shown to be of ancestral bacterial origin (Gillen, 2009,
A-37-A-39)!
To acquire a deeper grasp of the multiple lines of evidence which seamlessly converge in strong
support of the endosymbiotic theory’s explanatory model for mitochondrial origin, read the abstract
and the sections entitled “1. Introduction” and “4. The origin of mitochondria (and chloroplasts)” from
the review article Endosymbiotic theories for eukaryote origin by Martin, et al., paying careful attention to the
authors’ focus on the natural history of mitochondria and the experiments which shed light on this
organelle’s remarkable beginnings.

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ASSESSMENT ITEM
Imagine that you are a graduate student tasked with assisting in writing the cell biology textbook
on which your doctoral advisor is currently working. This hypothetical text, The Eukaryotic Cell: A
Molecular Perspective by Reynolds, et al., is aimed at undergraduate students and will contain a chapter
entitled “Endosymbionts: A Natural History of Mitochondria and Plastids” for which you and your
fellow doctoral candidates must write a subsection from the chapter section “Evidences for
Endosymbiotic Mitochondrial Origins”. Choosing one from the following list of hypothetical
subsections, write a mock text of no more than 450 words for this subsection, using what you have
learned thus far to inform your writing. Focus on conciseness, readability, and clarity of thought.
• “Morphological parallels between mitochondria and modern prokaryotes”
• “Mereschcowsky and Wallin: early perspectives”
• “Margulis and Goksøyr: an idea develops”
• “Primitive phagocytes engulfed H2-producing α-proteobacteria”
• “‘Proto-eukaryotic’ metabolism as a selective advantage”

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Part III – Implausibility of the Autogenous Model
While we now know that the wealth of evidence for the endosymbiotic origins of mitochondria
makes this theory virtually undeniable, such has obviously not always been the case. Recall that
Margulis’ publication of On the Origin of Mitosing Cells – which, we should again take note, was largely
hypothetical – was met with marked intransigence – and this in 1967 (Lake, 2011)! At the time, the
prevailing hypothesis was that all of the Eukarya arose from a single, ancestral prokaryote and that all
membrane-bound organelles arose from the infolding of existing cytosolic elements and plasmid-
mediated compartmentalization events which ultimately led to the intricately connected organellular
systems we today observe in all modern eukaryotes. This idea is referred to as the autogenous
hypothesis (The George Washington University, n.d.; Keeling, 2014). Initially, observational evidence
did seem to suggest that mitochondria may have evolved autogenously. For instance, not all
mitochondrial DNA is in fact circular; the mitochondrial chromosome of members of the protozoan
genus Paramecium is linear (Pritchard & Cummings, 1981). Furthermore, most mitochondria exhibit a
reticular morphology far removed from that of the prokaryote-like, bacillus-shaped drawings of
mitochondria commonly found in textbooks (Davison & Garland, 1977, p. 233s; Hardin, et al., 2012,
pp. 254-257). This school of thought, of course, has fallen greatly out of favor in view of the many
evidentiary indications of the validity of the endosymbiotic model, as detailed in Part II of this case
study. To quote evolutionary biochemist Nick Lane’s superb paper Energetics and genetics across the
prokaryote-eukaryote divide on the issue, “There is little doubt that all known eukaryotic cells share a
common ancestor that arose only once in four billion years of evolution. Common traits range from the
conserved position of many introns, to the structure of nuclear pore complexes, to complex traits such
as syngamy7 and two-step meiosis. It is implausible that all of these shared properties arose [largely] by
lateral gene transfer (which is inherently asymmetric in mechanism) or convergent evolution (which
implies that traits like intron position are dictated by selective constraints, rather than historical
contingency). Common ancestry is [...] the most parsimonious explanation”8 (2011). Common ancestry
(and therefore endosymbiosis) is the most parsimonious explanation for the evolution of the modern
mitochondrion, indeed.
Interestingly however, over the past several years, evidence is beginning to be brought to light
that some eukaryotic cellular elements, namely the Golgi complex and endoplasmic reticulum (among
other members of the endomembrane trafficking system), may have indeed originated in an autogenous
7 “Syngamy[:] The fusion of two cells, or of their nuclei, in reproduction” (Oxford University Press, 2016).
8 (Underlining added.)

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manner! Read (in its entirety) the 2008 paper Phylogeny of endocytic components yields insights into the process of
nonendosymbiotic organelle evolution by Dacks, et al. for a solid overview of some of this recent work.
ASSESSMENT ITEM
Now that you have had the opportunity to survey the emerging evidence for the likely
autogenous evolutionary origin of some organelles such as the Golgi complex, compare the strength of
the evidences detailed in the paper by Dacks, et al. for this likely scenario to those historically proposed
in defense of an autogenous mitochondrial genesis by writing a brief, informal paper of no more than
700 words, laying out what specific evidence you think would need to be uncovered in order for the
autogenous hypothesis to regain support as a valid explanation for the natural history of mitochondria.

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Conclusion
Thanks to the tireless work of Mereschcowsky, Wallin, and their many scientific successors such
as Lynn Margulis and the previously quoted Dr. Lane (amongst many others), we now know that
mitochondria almost certainly evolved from the symbiotic metabolic relationship of an early prokaryote
and a larger, “proto-eukaryote” which had engulfed it.
To now bring the fascinating issue of mitochondrial evolution into a broader context, re-read
the review article Endosymbiotic theories for eukaryote origin, this time in its entirety, focusing principally on
thinking about what connections you can make between the authors’ discussions of mitochondrial
evolution and those pertaining to the evolution of plastids (especially the chloroplast).
ASSESSMENT ITEM
In order to better interpret the review article you just read in the broader context of scientific
review literature in general, construct a bulleted outline of a hypothetical review article on the biological
topic of your choice. This outline should be generally representative of extant review literature in both
format and contents. You may wish to view other published review papers in order to gain a better feel
of their usual layout. An excellent resource for this is the National Center for Biotechnology
Information’s 9 (NCBI) PubMed 10 literature database which can be accessed by anyone at:
http://www.ncbi.nlm.nih.gov/pubmed. (To access only review articles, enter your search term(s) into
the search bar, click ‘Search,’ then click “Review” under “Article Types” in the left-hand pane, then
click “Free full text” under “Text Availability” in the same options pane.)
9 Part of the United States National Library of Medicine (NLM) (a branch of the National Institutes of Health {NIH}) (https://www.nlm.nih.gov).
10 Part of the United States NLM and NIH (http://www.nih.gov).

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References
Cold Spring Harbor Laboratory. (2011). Higher cells incorporate an ancient chromosome.
Retrieved March 14, 2016, from http://www.dnaftb.org/30/bio.html
Cummings, M. R., MS, PhD, Spencer, C. A., PhD, & Paladino, M. A., PhD. (2012). Knowledge of
mitochondrial and chloroplast DNA helps explain organelle heredity. In W. S. Klug
PhD (Author) & B. Wilbur, M. Early, L. Allen, B. Pessagno, J. Tringali, D. Friedman, et al.
(Eds.), Concepts of Genetics (10th ed., pp. 225-228). San Francisco, CA: Pearson Education,
publishing as Pearson Benjamin Cummings.
Dacks, J. B., Poon, P. P., & Field, M. C. (2008). Phylogeny of endocytic components yields
insight into the process of nonendosymbiotic organelle evolution. Proceedings of the
National Academy of Sciences, 105(2), 588-593. doi:10.1073/pnas.0707318105
Davison, M. T., & Garland, P. B. (1977). Structure of Mitochondria and Vacuoles of Candida
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Johnson, et al. (Eds.), Microbiology: An evolving science (2nd ed., pp. A-37-A-39). New York, NY:
W.W. Norton & Co.

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Hanson, J., PhD (Writer), Nicolosi, J. (Director), & Eads, K. (Producer). (2015, September 21). Theory
vs. hypothesis vs. law… explained! [Video file]. Retrieved March 13, 2016, from
https://www.youtube.com/watch?v=lqk3TKuGNBA
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Metabolism: Aerobic Respiration: Are Mitochondria Interconnected Networks
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