Evolution of Eukaryotes

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Christopher J. Paradise
Evolution of Eukaryotes
A. Malcolm Campbell, PhD

Christopher J. Paradise, PhD
Copyright © A. Malcolm Campbell and Christopher J. Paradise. 2016.
All rights reserved. No part of this publication may be reproduced, stored

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Abstract
Many people have a vague sense that the hypothesized origin of life, in the
form of bacteria, sounds plausible. However, few people can fathom how the
first eukaryotic cell, complete with nucleus, mitochondria and maybe chlo-
roplast, came into being. This book presents the evidence that reveals the
origins of all three DNA-containing organelles. In addition, this book will
illustrate how DNA, a molecule that is 2 meters (6 feet) long, can fit into all
cells’ nuclei that are only about 2 microns (0.000002 meters) in diameter.
Once eukaryotes evolved, the next obvious question is how multicellular
organism could have evolved from simpler unicellular species. This book
looks at multicellular algae as a case study on the origins of multicellularity.
Keywords
domains, bacteria, archaea, prokaryotes, missing links, evolutionary tree,
bioinformatics, ring of life, chromatin, histones, nucleosomes, epigen-
etic, central dogma, falsification, fabrication, plagiarism, mitochondria,
chloroplasts, orthologs, division of labor
Contents
Preface...................................................................................................ix
Acknowledgments....................................................................................xi
Introduction.........................................................................................xiii
Chapter 1 The Origins of Eukaryotes from Prokaryotes......................1
Chapter 2 Fitting a Genome into a Tiny Nucleus...............................9
Ethical, Legal, Social Implications: Ethical Guidelines
for Scientific Research...................................................14
Chapter 3 The Origins of Mitochondria and Chloroplasts................19
Chapter 4 The Evolution of Multicellular Organisms.......................25
Conclusion............................................................................................33
Glossary................................................................................................35
Index....................................................................................................37
Preface
This book the evolutionary origins of eukaryotic cells is part of a thirty
book series that collectively surveys all of the major themes in biology.
Rather than just present information as a collection of facts, the reader
is treated more like a scientist, which means the data behind the major
themes are presented. Reading any of the thirty books by Campbell and
Paradise provides readers with biological context and comprehensive per-
spective so that readers can learn important information from a single
book with the potential to see how the major themes span all size scales:
molecular, cellular, organismal, population and ecologic systems. The
major themes of biology encapsulate the entire discipline: information,
evolution, cells, homeostasis and emergent properties.
In the twentieth century, biology was taught with a heavy emphasis
on long lists of terms and many specific details. All of these details were
presented in a way that obscured a more comprehensive understanding.
In this book, readers will learn how eukaryotes evolved, how they stuff
all their DNA into the tiny nucleus, the origins of chloroplasts and mi-
tochondria as well as one way that multicellular organisms evolved, as
well as some of the supporting evidence behind our understanding. The
historic and more recent experiments and data will be explored. Instead of
believing or simply accepting information, readers of this book will learn
about the science behind the origins of eukaryotes the same way profes-
sional scientists do—with experimentation and data analysis. In short,
data are put back into the teaching of biological sciences.
Readers of this book who wish to see the textbook version of this
content can go to www.bio.davidson.edu/icb where they will find
pedagogically-designed and interactive Integrating Concepts in Biology for
introductory biology college courses or a high school AP Biology course.
Acknowledgments
Publishing this book would not have been possible without the generous
gift of Dr. David Botstein who shared some of his Breakthrough Prize
with AMC. David’s gift allowed us to hire talented artists (Tom Webster
and his staff at Lineworks, Inc.) and copyeditor Laura Loveall. Thanks go
to Kristen Mandava for project management and guidance on the pub-
lishing process. In particular, we are indebted to Katie Noble and Melissa
Hayban for their many hours of help and attention to detail.
Kristen Eshleman, Paul Brantley, Bill Hatfield and Olivia Booker
helped us with technology at Davidson College. We are grateful to ad-
ministrators Tom Ross, Clark Ross, Carol Quillen, Wendy Raymond,
Verna Case, and Barbara Lom who had confidence in us and encouraged
us to persist despite setbacks along the way.
These books were the product of the shared labor of my two vision-
ary coauthors Laurie Heyer and Chris Paradise. We shared the dream
and the hardships and developed this book from scratch. My family has
been very supportive and I thank Susan, Celeste and Paulina for their
support and patience. I also want to thank Jan Serie, my pedagogical
mentor, who taught me so much about the art and science of helping
students learn. I benefited from the support of the Howard Hughes Med-
ical Institute grant 52006292, the James G. Martin Genomics Program,
and Davidson College. This book would not have survived its first draft
without my students who endured the typos and the early versions of this
book. These undergraduates participated in a bold experiment to see if
beginners could construct their own knowledge, retain what they learned,
and transform the way they see themselves and the discipline of biology.
While many people said that beginning students were not up to the task,
my students proved them wrong.
Introduction
Eukaryotes occupy a unique place in the evolution of life. No other do-
main of life has cells with multiple internal organelles that are membrane
bound. Cells from no other domain of life contain organelles that carry
their own genomes. Eukaryotes are the only organisms that form multi-
cellular species produced after the fusion of two unrelated haploid cells.
And of course, only Eukaryotes can read books about biology. This book
examines four of the unique aspects of being eukaryotes. Perhaps the
most amazing feature of eukaryotes is the nucleus. The first chapter pres-
ents a subset of the data that revealed how the first nucleus evolved from
cells lacking nuclei. Once the nucleus appeared in the first eukaryote, a
new problem had to be solved—how to jam all that DNA into a tiny
compartment. DNA is wrapped up to facilitate mitosis while still allow-
ing transcription to proceed. Plants carry not only nuclear genomes but
also mitochondrial and chloroplast genomes. Chapter three presents data
showing the origins of both of these genome-containing organelles. The
last chapter allows the read to evaluate data explaining one example of the
selective advantage provided by multicellularity. In addition to these four
case studies, this book will present research ethics and how scientists are
entrusted to conduct their research while maintaining high ethical stan-
dards. The four chapters of this book focus on evolution at the organismal
level by looking at the origins of complex life forms.
CHAPTER 1
The Origins of Eukaryotes

from Prokaryotes
When Charles Darwin was formulating his ideas about evolution, he drew
a branching tree to depict how individuals within a species can change
and give rise to several related species, just like we use tree-like pedigrees
to show family relationships among humans (Figure 1). In Darwin’s tree,
the letters A through D represent different species in a large genus. The
dotted branched lines represent the offspring of A, each with some varia-
tion. Species B to D did not diverge and went extinct, as noted by their
lines terminating before reaching the top of the page. Though his drawing
was simple, Darwin’s diagram had a profound effect on the way biologists
think about evolution. Only now, about 150 years later, is this way of
thinking slowly giving way to a new image. Figure 1 was Darwin’s only
picture in the entire 556-page 1859 edition of On the Origin of Species. As
everyone knows, trees sprout from seeds and form a trunk with branches
diverging in many directions. If life on Earth can be drawn as a tree, then
what was the seed and where is the trunk? The tree of life predisposed
biologists to think of evolution as a series of events with gradual, linear
progression toward the existing species. Whenever people go looking for
evidence to support a particular idea, they can bias themselves to find
what they were seeking even if alternatives exist.
In Darwin’s explanation of evolution, he stated, “Only those varia-
tions which are in some way profitable will be preserved or naturally
selected.” A dotted line crossing a horizontal line represents 1,000 genera-
tions. Variation would have accumulated until descendants of A looked
different from the original parent as denoted by a1 and m1. If a branch
reaches the very top line where a10, f10, and m10 are seen, then that spe-
cies avoided extinction. Darwin recognized that his diagram looked too
2 EVOLUTION OF EUKARYOTES
a10 f 10 m10
a9 f9 m9
a8 f8 k8 l8 m8
a7 f7 k7 l7 m7
a6 f6 k6 m6
a5 d5 k5 m5
a4 d4 i4 m4
a3 i3 m3
a2 s2 m2
a1 m1
Figure 1 Tree of life from Darwin’s On the Origin of Species. Species

B, C, and D went extinct, but species A continued to evolve over time
(vertical axis) to give rise to the three species listed at the top.
Source: Reproduced from Darwin, 1859.
regular. He explained, “I do not suppose that the process ever goes on so

regularly as is represented in the diagram.”
Darwin knew it would be difficult to formulate rules in a field where
the object of study changes over time. Biology is an unusual science in that
it is very difficult to make broad statements without citing exceptions. For
example, plants do not move, except algae that can swim; birds fly, except
kiwi, ostrich, and penguins; lizards have four legs, except for the legless
ones, and so on. Similarly, it is difficult to construct a set of rules that
can distinguish all eukaryotes with nuclei from the other two domains
of life: bacteria and archaea. For example, a very common misconcep-
tion is that only eukaryotes have internal membrane-bound organelles,
and prokaryotes lack internal membranes. This misconception is incor-
rect and some prokaryotes do have internal membranes. Cyanobacteria
The Origins of Eukaryotes from Prokaryotes 3
are a very common prokaryote that have stacks of photosynthetic mem-

branes similar to the structures found inside chloroplasts. A quick search
of the Internet for images using the terms “Cyanobacteria EM” will reveal
many internal membranes. Another Internet search for a large bacterium
called Gemmata obscuriglobus will result in electron micrographs showing
internal membranes. This species has an internal double-membrane that
is attached to the inner cell membrane and surrounds a fibrous-looking
genome and a granular matrix. G. obscuriglobus looks like a prokaryote
with an primitive nucleus. Prokaryotes with membrane-bound organelles
reinforce the truism in biology: Rules without exceptions are rare.
In G. obscuriglobus, one can observed a structure that looks transi-
tional, as if the bacterium is trapped between its evolutionary past and a
future species yet to come. Transitional species are sometimes referred to
as “missing links,” as if all of evolution were a chain and each species a
link. If someone were to hold two halves of a chain with an anchor on one
side and a boat on the other, it would be logical to suspect a chain link
was missing that used to connect the two halves. Similarly, lungfish have
lungs and gills and are a link in the evolution of fish from water to land
dwellers and the beginning of amphibians. However, one cannot realisti-
cally expect every transitional species to survive unchanged for millions
of years while other species continue to change and compete for limited
resources. In the absence of live “missing links,” scientists need alternative
data to search for clues about the first eukaryotes and how they came into
being from prokaryotes. This chapter will present DNA evidence about
the origin of the eukaryote nucleus, because gene sequences contain clues
about how our eukaryotic relatives evolved the first nucleus.
When DNA sequencing became readily available, biologists searched
for the best gene to sequence in order to uncover the evolutionary rela-
tionships of all species to complete Darwin’s tree of life. They reasoned
that if each species evolved from a previous species, then it should be
possible to discern the relationship between ancestral and modern species
by comparing their DNA. Let’s look at a simplistic example of five hypo-
thetical species and their contrived DNA (Table 1).
Table 1 shows how the DNAs are similar to each other, indicating they
are closely related, but the middle four bases (bold font) have changed
over time. It should be possible to use these sequences to determine which
Table 1 Simplistic examples of DNA sequences that reveal

evolutionary relatedness.
hypothetical species names contrived DNA sequences
S. singularis GCATGCATGCAT
S. doubletus GCATGCTTGCAT
S. tripletus GCATGTTTGCAT
S. reversus GCATTACGGCAT
S. reversusdoublecus GCATTACCGCAT
GTTT TACC
GCTT TACG
GCAT
Figure 2 Evolutionary tree showing the changes in simulated DNA

(from Table 1) over time, the vertical axis. The original species at the
base is represented by the variable sequence GCAT. Only the four
central bases are shown in the tree for clarity.
Source: Original Art.
species evolved from which ancestor. If we use Darwin’s tree imagery and
focus on the central four bases, it is easier to understand (Figure 2).
To interpret the evolutionary tree in Figure 2, look at its base to find
the oldest species/sequence GCAT. From this ancestral sequence, three
events happened. On the middle branch, the original species remained
unchanged, GCAT. On the right branch, the sequence was first inverted
to TACG and later mutated at a single base to TACC. The left branch
mutated A to T and later mutated C to T. Each branch in this overly
simplified diagram indicates new species evolving due to changes in their
DNA. Remember that evolution is the change in allele frequency in pop-
ulation over time. The five species evolved at different times in the past
as indicated by the different lengths of the lines and the location of the
branch points. Using an evolutionary tree makes it possible to see which
species are more closely related because they share a common ancestor
Eubacteria Archaea Eukarya
Figure 3 Evolutionary tree based only on ribosomal genes from many

species. Each domain is labeled, with archaea and eukarya sharing a
common ancestor.
Source: From Woese et al., 1990. Modified from their Figure 1.
(a branch point), and conversely, which species are more distantly re-
lated. It is possible to imagine that producing an accurate tree would be
much easier if the data for every species was available (Figure 3). When
DNA analysis was conducted on highly conserved ribosomal genes from
many species, a more complex tree emerged. The ribosomal DNA-based
(rDNA, encoding rRNA) tree showed eukaryotes evolved from a com-
mon ancestor shared with archaea, and both domains evolving from a
common ancestor that also gave rise to eubacteria.
The nuclei of eukaryotic cells are surrounded by a pair of phospholipid
bilayers, which are similar to the pair of membranes surrounding pro-
karyote cells. Paired membranes also surround mitochondria and chloro-
plast, each of which contains DNA. For now, remember these membrane
similarities while continuing to learn about the origins of nuclei. When
investigators generated the evolutionary tree using rDNA sequences and
bioinformatics tools, they noticed some odd things. Most notably, the
mammals were barely distinguishable, because their sequences were nearly
identical for this highly conserved gene. The first lesson for generating
evolutionary tree is that highly conserved genes are better at revealing
distant relationships, but rapidly changing genes are better at distinguish-
ing closely related species. Therefore, it is impossible to find one gene that
could be used to determine the relationships of all species in the world.
For example, some evolutionary trees put birds closer to amphibians than
reptiles, even though biologists are certain that birds evolved from ancient
reptiles. It is good to remember that single gene evolutionary trees should
not be over-interpreted to indicate the relationships of all species. Trees
Table 2 Subset of results from BLASTing human protein

sequences against microbial protein sequences.
human protein protein protein best match
number function location domain
NP_001009 translation cytoplasm/rER archaea
NP_003185.1 transcription factor nucleus archaea
NP_001001937 ATP synthase mitochondria bacteria
NP_005521 energy harvesting mitochondria bacteria
NP_000393 energy harvesting cytoplasm bacteria
NP_004138 cell signaling cytoplasm archaea
NP_061816 cytoskeleton cytoplasm bacteria
Source: Original table.
based on gene sequence alignments are best at evaluating the conservation

of sequences for particular genes not entire species.
Rather than comparing just one gene across many species, a group of
genomicists evaluated every human gene by using the BLAST sequence
alignment tool against many prokaryotic genomes sequences (Table 2).
Their goal was to see if they could detect a pattern of evolutionary linkage
between eukaryotes (humans) and prokaryotes. Rather than comparing
nucleotides, they used the genetic code to convert all the gene sequences
to amino acid sequences prior to using the BLAST tool.
From the BLAST results similar to those in Table 2, the scientists
noticed that some human proteins are more closely related to eubacte-
ria, whereas others are more closely related to archaea. Human proteins
involved in energy production are more similar to bacteria proteins, but
human transcription and translation proteins are more similar to archaea
proteins. Signaling and cytoskeleton are both cytoplasmic communica-
tion systems, and they were split between the two microbial domains.
Using information similar to those from the protein BLAST results, sci-
entists found it difficult to draw a single tree that accurately represented
the evolutionary origins of all human proteins. Which gene would be the
best one to help understand the evolutionary origin of eukaryotes? When
investigators finally let go of their bias toward a Darwinesque tree, they
realized what had happened (Figure 4). This “tree” looked very different
from a typical Darwinian tree. Humans and all eukaryotes contain genes
from both eubacteria and archaea that diverged from a common ancestor
long ago, species α. Furthermore, nuclei and prokaryotes are surrounded
eukaryotes
bacteria archaea
species
Figure 4 Non-tree based evolutionary relationships. This diagram

illustrates how a primitive species a could diverge into two branches,
which later merged genomes to produce a third branch that became
eukaryotes. Note the atypical branching structure with a hole in the
middle.
Source: Data from Campbell and Heyer textbook Discovering Genomics, Proteomics, and
Bioinformatics. Figure 3.11.
by two membranes. The simplest solution to make sense of all these data
was to propose that an archaeal cell and an eubacterial cell combined
genomes with one cell engulfing the other. The original, abiotically pro-
duced cell was species alpha, which evolved and became what we would
recognize as a prokaryote. Later, a subset of prokaryotes diverged and
evolved into archaea and eubacteria. Millions of years later, one archaea
and one eubacteria fused their genomes and gave rise to eukarya.
With redundant genes in this new hybrid genome of the early eukary-
otes, portions of DNA were ejected to be more efficient by minimizing
the burden of replicating duplicate genes. Genes encoding complex pro-
cesses were retained in blocks from one genome rather than a mosaic of
individual genes from both combined genomes. For example, eukaryote
pathways related to DNA replication, transcription, and translation all
appear to be archaea in origin. Energy harvesting genes were retained
predominantly from the ancestral eubacteria genome rather than the ar-
chaea genome. Some eukaryotic processes, however, were retained as a
mosaic of genes from both archaea and eubacteria. It is time to stop using
the metaphor “tree of life” and time to consider the “ring of life,” which
represents more accurately how the three domains evolved. Humans tend
to prefer tidy rules and clear distinctions. However, evolution does not
follow a set plan, nor is it linear in the adaptations that are selected over
time. The “ring of life” is probably more accurate than the “tree of life,”
and so Darwin was off a bit on his model. However, a scientific model
does not have to be correct in order to be useful. Darwin helped society
see how species were related to each other through heredity. He com-
municated how species alive today provide a partial clue to the evolution
of species over millions of years. The genome sequences available today
are similar to a puzzle with several pieces missing, because we have not
sequenced all possible genomes. It may be impossible to determine all the
details, but each experiment makes it easier to understand the origin of
life and the evolutionary relationships between species.
Bibliography
Fuerst JA, Webb RI. Membrane-bounded nucleoid in the eubacterium
Gemmata obscuriglobus. Proc Natl Acad Sci USA 88(18):8184–8188,
1991.
Horiike T, Hamada K, Kanaya S, et al. Origin of eukaryotic cell nuclei by
symbiosis of Archaea in bacteria is revealed by homology-hit analysis.
Nat Cell Biol 3(2):210–214, 2001.
Martin W, Embley TM. Evolutionary biology: early evolution comes full
circle. Nature 431(7005):134–137, 2004.
Rivera MC, Lake JA. The ring of life provides evidence for a genome
fusion origin of eukaryotes. Nature 431(7005):152–155, 2004.
CHAPTER 2
Fitting a Genome into

a Tiny Nucleus
If it were possible to take all the chromosomes from one cell and stacked
them end-to-end, they would stand about 2 meters (6 feet) high. But the
diameter of a typical human nucleus is only about 15 microns, 15 × 10-6
meters. Stuffing DNA inside a tiny little sphere is a critical challenge be-
cause the DNA needs to be accessible for replication and transcription.
The stretched-out length of the DNA in a typical chromosome is more
than 100 times longer than the length of the chromosome packed into a
nucleus. How do cells compact their genetic information at least one hun-
dredfold so that it can fit into a tiny space without tangling all the strands?
In 1974, the wife and husband team of Ada and David Olins isolated
chromosomes from chicken and rat cells and took electron micrographs
of the DNA. The methods section of their publication listed a reagent
that one might expect to find in a kitchen rather than a lab—Joy dish-
washing detergent. Biologists have known for many years that detergents
dissolve membranes and release the chromosomes from the cells. They
knew DNA was very thin and difficult to see, but scattered along the
DNA the two biologists saw beads that looked like “pearls on a string.”
Like any good scientists, they were curious what these beads could be, so
they began to measure the beads’ dimensions.
In the 1970s, biologists had already discovered and characterized many
of the proteins that bind to the DNA of chromosomes. Chromosomal
DNA and its associated proteins are called chromatin, which is the native
state of a person’s genome, rather than DNA devoid of proteins. The most
abundant class of chromatin proteins is histones, so biologists suspected the
“beads” might be histones. In their electron micrographs, the could see thin
pieces of DNA “string” connecting the beads. The beads were about 70 Å
(Å = angstrom or 10-10 meters) in diameter and the string was about 15 Å
wide, which is about the width of DNA devoid of any protein. The team
of biologists used geometry to calculate the circumference of the beads and
used biochemical estimates others had made for chromatin pieces to predict
that about 400 Å of DNA was wrapped around each bead. Given these data,
400 Å of DNA would be packed into a space 70 Å in diameter, approxi-
mately a 6 to 1 packing ratio. The investigators had calculated the first level
of DNA compaction, but there was much more still to be discovered. Ada
and David Olins opened the door for a new area of research. Never before
had scientists measured how one meter of DNA could fit into a tiny nucleus.
In 1975, another biologist decided to use a simpler system to visualize
the packing of DNA—simian virus 40, or SV40. The improved quality of
the micrographs permitted slightly more accurate measurements, in part
because he was working with a small viral genome of known length. He
measured chromatin beads to be 110 Å in diameter, the DNA string 20 Å
wide, and each bead was separated by protein-free DNA of about 130 Å in
length. The virus DNA could be isolated with or without the histone pro-
teins, and he measured the lengths of the two forms of SV40 DNA. The
genome was compacted sevenfold and measured only 2100 Å long when it
looked like beads on a string. Later that same year, a different biochemist
used a centrifugation method to isolate chromatin fragments of DNA and
histone in units of 1, 2, 3, or 4 beads. This new method revealed the DNA
wrapped was around beads in multiples of 200 base pairs. The biochemist
published a series of papers in close succession that described the beads as
200 base pairs of DNA wrapped around a histone complex with quater-
nary structure. He determined that a histone complex in chromatin was
composed of two copies of four different histone protein subunits, for a
total of eight histone subunits. The biochemist combined his findings with
those of many others and succinctly summarized what was known about
chromatin. For the first time, biologists were beginning to understand
how DNA could be stuffed inside nuclei and remain functional.
The biologist working with the SV40 genome reported 21 beads of
DNA-wrapped histone complexes. The genomic database at NCBI in-
dicates the SV40 genome contains 5,243 bp. If each bead is wrapped by
Fitting a Genome into a Tiny Nucleus 11
200 bp of DNA and there are 21 beads per SV40 genome and 38 bp of
DNA between beads, the measurements lead to a calculated size of 4,960 bp
of DNA in the SV40 genome, which is only slightly short of the real
number. Upon closer examination, the biochemists realized the collection
of quadruplet beads was more compacted than expected if the beads had
to be stacked end-to-end. By rotating slightly, the viral genome could be
stacked into a smaller space, which is one reason the calculated length of
SV40 DNA was too short. The next set of experiments revealed how the
beads are arranged to maximize their packing.
In 1976, biologists visualized higher levels of chromatin compaction
thanks to improved electron microscopy techniques. As people refine meth-
ods, the quality of data generally improves. Similarly, the quality of the elec-
tron microscopes continued to improve, which allowed greater resolution.
When chromatin was isolated very gently, biologists could see many beads,
but they were struck by the thicker “coils” of chromatin. They could see the
familiar beads, now called nucleosomes, but the chromatin seemed to have
a second level of wrapping associated with it much like a rubber band can
adopted a highly condensed shape if it is wound very tight. After study-
ing many different views of chromatin, they produced a drawing to convey
what they thought was happening to DNA to achieve higher compaction.
A few years later, an international team from the United States and
Canada isolated chromatin from chicken cells in two different degrees of
compaction. In their electron micrographs, this team could see the nucleo-
somes in a zigzag pattern. Under certain conditions, the investigators saw
larger and more complex stacking of DNA into double coils of ribbons com-
posed of nucleosomes. It is difficult to see some of the shapes when first
studying electron micrographs, but these authors spent many hours studying
the images, and were able to draw their interpretation. Their images helped
identify a key step in the multiple levels of compacting DNA. They estimated
about a fivefold increase in DNA density caused by the coiled ribbons in ad-
dition to the compaction provided by DNA wrapped around nucleosomes.
Many biology students share a common misconception that typical
DNA exists in the mitotic stages found in textbooks as a pronounced “X”
shape of condensed threads. In a micrographic montage of hamster nuclei,
teams of cell biologists from Illinois and New York labeled the DNA so
that it would fluoresce white and took photographs as the chromosomes
progressed from G2 stage of the cell cycle up to the earliest part of metaphase.
Their photographs revealed that the DNA seems to move toward the nuclear
membrane as it condenses. No one knows why, which opens another area for
more research. What is the functional benefit of localization to the periphery
of the nucleus? Between the coiling and movement of chromosomes to the
periphery of the nucleus, chromosomes do not tangle during mitosis or mei-
osis. When thinking about chromatin compaction, do not make the mistake
of thinking of DNA moving in discrete steps during mitosis. To be more bio-
logically accurate, imagine chromosomes as always moving around, adapting
to the cell’s local environment. Chromosomes are large dynamic molecules,
composed of DNA and protein, that transition from one configuration to
the next. As a cell progresses through stages of cell division, it needs to con-
dense its DNA and then unwind it again without getting tangled into knots.
During most of the cell’s life, DNA needs to be transcribed; and during S
phase of the cell cycle, DNA polymerases have to travel the entire length of
every chromosome. On YouTube you can find DNA compaction anima-
tions that illustrate what happens during prophase of mitosis.
Nearly everyone has tried to organize string, yarn, or rope and knows
these long strings are likely to get tangled. No matter how careful a per-
son is, knots form despite his or her best effort. A collaboration of cell
biologists hypothesized that the long polymers of DNA were held in place
during mitosis by a glue-like protein. They wanted to know whether a
recently discovered chromatin protein was functioning like a temporary
glue to hold the chromosomes into their condensed states during mitosis
but could disassemble quickly after telophase. The cell biologists used
an antibody against the glue protein to determine where it was located
once a chromosome was fully condensed. The scientists had formulated a
hypothesis, and the data supported their hypothesis; however, they could
not “prove” the glue protein was holding the chromosome together be-
cause it is impossible to prove anything. Over time, more investigators
will challenge their hypothesis and if the glue protein continues to be sup-
ported, then the cell biologists can feel more comfortable that they were
right about what holds chromosomes together during mitosis.
Basic physics and the second law of thermodynamics tell us all things
tend toward randomness unless energy is used to produce order. From these
laws of nature, it could be predicted that condensing chromatin would
require energy and probably motor proteins to force the chromosomes

into such a small space. Once the DNA is coiled very tightly, the cells
must have a way to keep it condensed until after mitosis and meiosis. A
glue protein makes sense, but biologists need more information to under-
stand this protein’s function. Does this “glue protein” also act as a motor to
pull the DNA inward? Is this protein necessary and sufficient, or do other
proteins play critical roles? Cell biology is a popular area of research where
basic research and biomedical applications intersect frequently.
A very active area of research is histone modification, which is part of
epigenetic regulation. Histones can be methylated, acetylated, or phos-
phorylated. The portions of the protein that protrude from the nucleo-
somes are prime targets for covalent modulation. Covalent modulation
of histones in a nucleosome can alter gene transcription and DNA rep-
lication. Histone modification can be passed on to a parent’s offspring
and is another form of epigenetic information. The high degree of amino
acid conservation among histones of different species indicates that his-
tone structure is critical to its function, but that is true of all proteins
(Figure 5). However, remnants of nuclear evolution can be seen in this
evolutionary tree because archaea use histones but eubacteria do not. Fur-
thermore, histones from unicellular eukaryotes are more closely related
to archaea than the multicellular plants and animals. As was shown in
Chapter 1 of this book, genes involved in central dogma are more closely
Sponge symbiont (Archaea)

Thermococcus (Archaea)
Pyrococcus (Archaea)
Dictyostelium (slime mold)
Entamoeba (ameoba)
H. sapiens (human)
sea lice (crustacian)
D. melanogaster (fruit fly)
Zea mays (corn - monocot)
A. thaliana (weed - dicot)
Shepard’s purse (weed - dicot)
african palm oil (tree - monocot)
Figure 5 Evolution of histone proteins. Amino acid sequence

alignment of histone proteins from archaea and eukaryotes, along with
the corresponding evolutionary tree.
Source: Original art.
related to archaea than eubacteria. Bacteria such as E. coli do not need to

compact their DNA as much as eukaryotes and archaea.
All eukaryotes must compact their DNA in order to keep the chro-
matin from getting tangled during mitosis and meiosis. Furthermore, not
all genes are active at any one time in a given cell, and chromatin moves
around in the nuclear space, which may be functionally important too.
Based on the multiple forms of histone modification, scientists know that
epigenetic regulation is an important component of gene regulation. His-
tone sequence conservation indicates DNA compaction must follow an
orderly process that has been retained since before archaea and eukaryota
diverged more than one billion years ago.
All organisms have evolved mechanisms to control their genomes
so they are not tangled but are still accessible by RNA and DNA poly-
merases. By looking at DNA compaction in eukaryotes, this chapter de-
scribed how organisms can be linked by lines of descent from common
ancestry. The conservation of histones and DNA compaction is benefi-
cial for organisms as shown by the conservation of essential components
and processes across diverse species. The origin of eukaryotes occurred
by natural processes, and life continues to evolve within a changing en-
vironment. Chapter 3 addresses the evolutionary origins of the other two
DNA-containing organelles, mitochondria and chloroplasts.
Ethical, Legal, Social Implications: Ethical Guidelines

for Scientific Research
Being a scientist carries with it the responsibility to perform work hon-
estly and ethically. A majority of science is funded by federal money,
which means that scientists have a social contract with every citizen to use
their research funds responsibly. Until recently, graduate students received
no formal training about what behavior was acceptable and unacceptable.
The unspoken assumption was that professional ethics would be taught
to each graduate student by his or her research mentor. The educational
philosophy of most graduate programs is that students learn best when
mentored as an apprentice by more experienced scientists.
The three major forms of research misconduct are falsification,
fabrication, and plagiarism, which are often referred to collectively as
FFP. The US Office of Science and Technology Policy defines misconduct

as the activities related to “proposing, performing or reviewing research or
in the reporting of research results.” Falsification includes omitting or alter-
ing data or equipment so that the results do not accurately present what is
being studied. Fabrication means an investigator claims data were generated
through experimentation when they were not. Plagiarism happens when a
person claims ideas or text as his or her own when they were actually the
product of someone else. Participating in any of the FFP unethical behavior
is a breach of the social contract and undermines the integrity of science.
One of the most famous cases of misconduct in recent times were the
2004 and 2005 published reports claiming to produce human embryonic
stem cells from adult human cells. These papers were published in Science,
one of the world’s most prestigious journals, and they were peer reviewed
by nine experts. Donald Kennedy, Science’s editor-in-chief at that time
said, “Peer review cannot detect [fraud] if it is artfully done.” The scien-
tists fabricated data, falsified data, and unethically concealed the source
of donated human eggs. It is difficult to understand why a successful sci-
entist would commit so many ethical violations on a high-profile paper.
Did the authors think that no one would try to reproduce their results
related to human stem cells? Were they so convinced they were right that
they did not feel compelled to perform all of the experiments and present
the results honestly? Scientists are human, and anyone can be tempted
to take shortcuts and embellish the truth. However, shortcuts and em-
bellishments are not permitted in scientific communication, nor should
they be tolerated by others. The misconduct involving human stem cell
research was deliberate and initially denied. Interestingly, the truth about
the misconduct was the result of “whistleblowers,” young scientists associ-
ated with the laboratories posting anonymous tips on blogs and websites
that initiated the investigation and ultimate retraction of both papers.
Although FFP are the three most serious ethical infractions, two oth-
ers are worth mentioning here. An increasing problem is full disclosure.
Did the authors of a scientific publication have any financial stake in
their research? Imagine one of the authors is paid by a drug company to
conduct a clinical trial and the results are not favorable. Would the author
be tempted to interpret the results more favorably either consciously or
subconsciously? Does the reader deserve to know when an author has
financial interests in the outcome of an experiment? The scientific com-

munity is developing a consensus that authors must disclose any finan-
cial or personal stake in their research, and failure to do so is considered
unethical.
Perhaps the most common ethical lapse might seem the least harm-
ful. Who should be an author on a publication, and who should be given
credit in the acknowledgements? Unfortunately, there are no clear rules
defining the amount of work that warrants authorship. If someone per-
formed a procedure for one experiment, is that sufficient? What if the
head of the lab did not set foot in the lab but helped analyze the data?
What if the senior member of the lab, wrote a grant to fund the research
but had no role in the research or its publication? Research has become
more collaborative, and the number of authors on papers has increased
over the last fifty years. Some papers have so many authors that their
names don’t appear near the title, and they are relegated to a footnote.
Failing to give someone credit is plagiarism, but what term should be
applied to the ethical lapse of giving people more credit than they de-
serve? In many labs, the litmus test for authorship is whether the indi-
vidual had any “intellectual contribution” or not. If all a person did was
load a gel and take the picture, this should qualify for an acknowledge-
ment but not authorship. If the person helped design the experiments,
interpret the results, or contributed to the writing of the paper, then
authorship would be warranted.
No one expects perfection from individual scientists, but society does
expect perfection from the scientific endeavor. The scientific process of
publishing results and methods allows others to reproduce published re-
sults which can lead to the correction of honest mistakes. Honest mis-
takes based on misinterpreting the data or limitations of the technology
are not a failure of the system. As long as scientists continue to disclose
their methods, the original data, and their financial connection to the
research, science will continue to make life-saving discoveries and provide
new technology to improve the quality of life. It is important to maintain
the public’s trust so that politicians have the opportunity to rely on sci-
ence as the cornerstone of policy decisions instead of ideology or precon-
ceived ideas. Data should be the ultimate arbitrator of good government
just as it is the cornerstone of good science.
Bibliography
Bednar J, Horowitz RA, Grigoryev SA, et al. Nucleosomes, linker DNA,
and linker histone form a unique structural motif that directs the
higher-order folding and compaction of chromatin. Proc Natl Acad
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Belmont AS, Bruce K. Visualization of G1 chromosomes: a folded,
twisted, supercoiled chromonema model of interphase chromatid
structure. J Cell Biol 127(2):287–302, 1994.
Finch JT, Noll M, Kornberg RD. Electron microscopy of defined lengths
of chromatin. Proc Natl Acad Sci USA 72(9):3320–3322, 1975.
Finch JT, Klug A. Solenoidal model for superstructure in chromatin. Proc
Natl Acad Sci USA 73 (6):1897–1901, 1976.
Griffith JD. Chromatin structure: deduced from a mini chromosome.
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Kireeva N, Lakonishok M, Kireev I, et al. Visualization of early chro-
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tones. Science 184(4139):865–868, 1974.
Kornberg RD. Chromatin structure: a repeating unit of histones and
DNA. Science 184(4139):868–871, 1974.
Olins AL, Olins DE. Spheroid chromatin units (v bodies). Science
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Ethical, Legal, Social Implications: Ethical Guidelines for Scientific

Research
Chong S, Normile D. Stem cells: how young Korean researchers helped

unearth a scandal . . . Science 311(5757):22–25, 2006.
Martinson BC, Anderson MS, de Vries R. Scientists behaving badly. Na-
ture 435(7043):737–738, 2005.
CHAPTER 3
The Origins of Mitochondria

and Chloroplasts
This chapter focuses on the evolutionary origins of two important or-

ganelles that have greatly expanded their internal membranes. A quick
Internet image search for chloroplasts and mitochondria will produce
electron micrographs that reveal many membranes inside these organ-
elles, which indicates that they evolved to increase surface area while
constraining growth of their overall volume. All eukaryotic organisms
have mitochondria, but plants contain chloroplasts in addition to mito-
chondria. Mitochondria convert metabolites into adenosine triphosphate
(ATP), whereas chloroplasts convert sunlight into molecules that store
chemical energy. Close examination of the mitochondrion outer mem-
brane will reveal two distinct membranes, each of which is composed
of two layers of phospholipids. Like mitochondria, chloroplasts also
have two outer membranes as well as internal layers of membranes that
provide extra surface area while not increasing the overall surface area or
volume of the organelle.
Like the nucleus, chloroplasts and mitochondria also contain their
own genomes. This fact should seem odd, because other organelles (such
as endoplasmic reticulum, Golgi and ribosome) don’t contain their own
DNA. Why are the mitochondria and chloroplasts so different? If one
were to isolate a single chloroplast and purify its DNA, one would have
purified many small, circular chromosomes of exactly the same sequence.
In short, mitochondria and chloroplasts are the only organelles to have
double outer membranes and their own genomes. Could these traits be
evidence of their evolutionary origins?
Before determining the origins of these organelles, it is important to

be familiar with a visualization tool called evolutionary trees which were
introduced in the two previous chapters. Evolutionary trees use shared
traits to display the evolutionary relationships of different individuals or
species. Evolutionary trees are similar to common family trees that con-
nect people with their parents and grandparents and show relationships
across time.
When conducting biology research, it is impossible to have access
to every ancestral species over the course of their evolutionary history.
Which data are the most reliable for generating evolutionary trees? Anat-
omy can be deceiving. When looking at divergent breeds of dogs, it is
understandable for a novice to think a German Shepard and a Chiguagua
are different species when in fact they are only different strains of the same
species that diverged rapidly through selective breeding. To determine the
evolutionary origins of species, many investigators use DNA as their data
for generating evolutionary trees. In this simplistic example, each genome
sequenced is represented by a small segment of DNA. To analyze real ge-
nome sequences for evolutionary relationships, investigators use millions
of bases from a very wide range of diverse organisms (Figure 6). Look at
this DNA-based evolutionary tree to see the original sequence on the far
left, and the mutations that occurred to the right of each branch point.
For clarity, the mutations are depicted as a lighter gray color. This DNA
evolutionary tree has an additional numerical feature called bootstrap
values. Bootstrap values indicate the percent frequency that a particular
branch point was produced on multiple iterations of producing the tree.
For example, if a computer independently produced 100 evolutionary
trees using one set of data and a particular branch point was identical
each time, the bootstrap value for that branch point would be 100%. In
Figure 6, the tree was generated by a computer algorithm that was run
1,000 times in a row on a subset of each sequence, and the percentage of
identical branch points among the 1,000 iterations are displayed as boot-
strap values, which range from 100% down to 88%. A bootstrap value of
88 means that in the 1,000 trees generated by the computer, 120 of them
did not agree for the branch nearest the base of the tree.
The branching pattern of an evolutionary tree reveals the relatedness
of species, whether the species are represented by anatomy or DNA.
The Origins of Mitochondria and Chloroplasts 21
GGGTGCAA
GGATGCAA
100 GGTTGCAA
GCATGCAA
98
GCATGCAT
88 CCATGCAA
GCTTTAGC
GCATTAGC
99 GCAAAAGC
100
GCATTACG GCAATAGC
100
GCATCTACG TCAATAGC
100
GCATTTACG
GCATTTACGG
Figure 6 Deducing the origin of evolved DNA sequences. Original

DNA is on the far left side. Bases that changed with each branch
are lighter gray to facilitate detection. Bootstrap values given at each
branch point.
Source: Original art.
It is tempting to consider the terminal adjacent sequences that come

from different branch points but are located near each other vertically as
closely related, but comparison of two sequences will reveal many differ-
ences despite them being spacially near each other. The evolutionary dis-
tance between two adjacent sequences is indicated by there being several
branch points in the tree to the left of each sequence. In fact, the most
recent common ancestor of two sequences may be the original sequence
at the base of the tree. Now it is time to evaluate some real genomic data
to see if it is possible to deduce the evolutionary origin of mitochondria
and chloroplasts.
Most prokaryotes have circular chromosomes just like mitochondria
and chloroplasts. Prokarotes (bacteria and archaea) have two outer mem-
branes between their cytoplasm and the outside world. Mitochondria
and chloroplasts are contained within paired membranes too, which is
100 Prototheca wickerhamii

Marchantia polymorpha
100 mitochondria
100 Reclinomonas americana
98 Acanthamoeba castellanii
100 Rickettsia prowazekii
100 Rhodobacter capsulatus a-proteobacteria
Paracoccus denitrificans
Escherichia coli
95 Thermus thermophilus
Mycobacterium tuberculosis
Synechocystis sp. cyanobacteria
100
100 Marchantia polymorpha
chloroplast
Zea maize
Figure 7 Deducing the origin of chloroplasts and mitochondria.

Phylogenetic tree for mitochondria from four species, some bacterial
genomes, photosynthetic cyanobacteria genome, and two different
chloroplast genomes. Bootstrap values at branch points.
Source: Modified from the original tree with permission of Macmillan Publishers Ltd: Andersson,
Siv G.E., Alireza Zomorodipou, Jan O. Andersson, Thomas Sicheritz-Pontén, U. Cecilia M.
Alsmark, Raf M. Podowski, A. Kristina Näslund, Ann-Sofie Eriksson, Herbert H. Winkler,
and Charles G. Kurland. 1998. The genome sequence of Rickettsia prowazekii and the origin of
mitochondria Nature. Vol. 396: 133–143. Figure 5b p. 138.
twice as many cell membranes as eukaryotic cells have. These anatomical

features are compelling, but it would be better if biologists used DNA to
generate an evolutionary tree. Evolutionary trees are easier to understand
when using simple examples of short DNA sequences, but considerably
more difficult when using real chloroplast and mitochondrial genomes.
How can their evolutionary origins be deduced from chromosomal se-
quences? A group of biologists used a gene from chloroplast and mito-
chondrial genomes and compared these to the same gene (orthologs) in
other organisms. They built evolutionary trees to represent which ortho-
logs were most closely related to each other (Figure 7). In this example,
they used four mitochondrial genomes, along with two chloroplast ge-
nomes and six bacterial genomes. The software was programmed to draw
1,000 trees to generate bootstrap values. By following the branching
patterns, it is easy to determine the evolutionary origins of these two
eukaryotic organelles that have their own genomes.
The Origins of Mitochondria and Chloroplasts 23
Based on the data in Figure 7 and many other studies, it is clear that
mitochondria evolved from a subtype of bacteria called α-proteobacteria,
with the species Rickettsia prowazekii as the most closely related living
species. Their genome sequences are most similar, which reveals their re-
latedness. Chloroplasts evolved from photosynthetic prokaryotes called
cyanobacteria, which are the same organisms that produce the black
streaks on roofs of homes after several years. The nearest living relative
in Figure 7 comes from the genus Synechocystis. Because every eukaryotic
cell has mitochondria and only plants have chloroplasts, it seems logical
to speculate that mitochondria evolved before chloroplasts. However, ani-
mals may have evolved from simple plants after losing their chloroplasts.
Science is a discipline that begins with observations and is followed by
hypotheses and research, which means more data will need to be collected
before we can be sure which organelle is older.
The current model explaining the origin of both organelles is that a
primitive eukaryote (see Chapter 1) swallowed a bacterium and rather
than digesting it, the swallowed bacterium proved to be beneficial. Natu-
ral selection led to the evolution of mitochondria and chloroplasts from
prokaryotic cells, because they provided their eukaryotic hosts with ben-
efits that led to more offspring. Over time, the organelles have lost many
genes from their ancestral genomes because the nuclear genome carried
the duplicate genes. In short, the organelles evolved first as symbionts,
although the distinction between a symbiont and an organelle is a very
fuzzy line.
Chapter 3 demonstrated that by comparing DNA from many differ-
ent species, it is possible to piece together evolutionary trees showing the
relationship of all species and organelles. The large surface area within
mitochondria has proven very adaptive because every eukaryotic cell still
retains this organelle that evolved from an engulfed bacterium. Plants also
benefited from the even larger increase in surface area within chloroplasts.
Perhaps the original cell that was engulfed and evolved into chloroplasts
looked similar to the cyanobacteria found in the world today. Having
learned about the evolutionary origins of all three DNA-containing or-
ganelles, it is time to examine evidence from one case study that explains
how eukaryotes evolved to form multicellular organisms.
Bibliography
Andersson SG, Karlberg O, Canbäck B, et al. On the origin of mito-
chondria: a genomics perspective. Philos Trans R Soc Lond B Biol Sci
358(1429):165–177, 2003.
Andersson SG, Zomorodipour A, Andersson JO, et al. The genome
sequence of Rickettsia prowazekii and the origin of mitochondria.
Nature 396(6707):133–140, 1998.
Bisalputra T, Bisalputra AA. Chloroplast and mitochondrial DNA in a
brown alga Egregia menziesii. J Cell Biol 33(3):511–520, 1967.
Chu KH, Qi J, Yu ZG, et al. Origin and phylogeny of chloroplasts
revealed by a simple correlation analysis of complete genomes. Mol
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Coleman AW, Heywood P. Structure of the chloroplast and its DNA in
chloromonadophycean algae. J Cell Sci 49:401–409, 1981.
Esser C, Martin W, Dagan T. The origin of mitochondria in light of a
ﬂuid prokaryotic chromosome model. Biol Lett 3(2):180–184, 2007.
Gray MW, Buger G, Lang BF. The origin and early evolution of mito-
chondria. Genome Biol 2(6):1–5, 2001.
McFadden GI. Chloroplast origin and integration. Plant Physiol 125(1):
50–53, 2001.
Ris H, Plaut W. Ultrastructure of DNA-containing areas in the chloro-
plast of Chlamydomonas. J Cell Biol 13:383–391, 1962.
CHAPTER 4
The Evolution of
Multicellular Organisms
The evidence of life’s origins points to the first organisms being unicel-
lular prokaryotes that existed as long as 3.8 billion years ago. All species
were unicellular prokaryotes for over a billion years; the fossil record indi-
cates that eukaryotes evolved around 1.6 billion years ago. Multicellular
organisms appeared in the fossil record close to 1 billion years ago. The
evolution of multicellularity is important if we want to understand how
plants and animals evolved. Scientists from many different disciplines
have collaborated to deduce the evolutionary history of life that relies on
fossils, comparative anatomy, and DNA sequences.
Biologists do not know exactly how multicellularity evolved, and
there is debate about the steps involved. Despite the lack of agreement on
the mechanism, there is agreement that multicellularity evolved indepen-
dently several times. Animals and plants are both multicellular eukaryotes
and are thought to have evolved independently from different lineages of
unicellular eukaryotes. Because some of these evolutionary events took
place over 1.6 billion years ago, the steps have been obscured by time.
However, there is at least one group of green algae in which multicellular-
ity evolved more recently. This chapter explores the evolution of the green
algae called Volvox to gain insight into how multicellularity may have
evolved in other plants as well as animals.
There are about 40 species in the Volvox family distributed by taxono-
mists into about ten genera (plural of genus). The Volvox family of green
algae includes colonial plants that use flagella to swim. Colonial living by
a population of cells is considered a possible evolutionary link between
unicellularity and multicellularity. Colonial cells of the same species live
16 species of > 32 cells/colony
8 species of 8 or 16 cells/colony
5 species of 8,16, or 32 cells/colony
2 species of 4 cells/colony
4 species of unicellular Volvox genera
Figure 8 Summary of the evolutionary tree for 35 members of the

Volvox family generated using five chloroplast genes.
Source: Modified from Nozaki et al., 2000. Figure 2.
together, often for mutual benefit. What evidence is there that all the
Volvox species evolved from a common unicellular ancestor and belong
to a single taxonomic family? One Japanese team led by Hisayoshi Nozaki
used DNA sequences from several different genes to construct an evolu-
tionary tree of the Volvox species (Figure 8). This simplified evolutionary
tree shows how similar each collection of species on a branch is similar
to groups on the other branches. The evolutionary distance between the
groups is represented by the horizontal distance of a branch point on the
right side of the tree.
All species in the Volvox family are hypothesized to have evolved from
a common ancestor. Nozaki and his colleagues used 6,021 nucleotide
bases from five Volvox chloroplast genes to produce their evolutionary
tree. For clarity, individual species have been lumped together based on
similarity of DNA sequences. Each group of species on a branch is de-
scribed by the number of cells that live in a single colony.
Nozaki and his colleagues produced DNA evidence that all members
of the Volvox family evolved from a common ancestor. Branch points
further to the left were species that existed farther back in time. As seen
in Figure 8, descendants on the lower branches are unicellular species
or species that live in very small colonies. Species with larger colonies
evolved from ancestors that are more recent with branch points further
The Evolution of Multicellular Organisms 27
to the right. The Japanese team of biologists concluded that the ancestors
of colonial green algae were unicellular because their branch points are
closer to the base of the tree on the left side. An example of a unicellular
alga related to the Volvox family is Chlamydomonas. In fact, it is thought
that the first colonial species evolved from unicellular green algae only
about 75 million years ago.
The number of cells per colony does not correlate with the evolu-
tionary relationships—as demonstrated by three separate branches that
contain species with 16 cells per colony. This distribution of colonies
with similar numbers of cells indicates that colony size differences evolved
multiple times. Presumably, each species evolved in ways that were ad-
vantageous given its habitat, which means that colony size was selected
for repeatedly over time. In colonial species with a small number of cells,
colonies never exceed a maximum size of 4, 8, 16, or 32. Within the colo-
nies, Volvox cells exhibit division of labor among the individual cells; a
subset of the cells is involved in reproduction and the rest of the cells work
on other tasks. The greater the number of cells, the more likely colonies
will exhibit division of labor.
In the larger colonies of Volvox, most cells are small and cannot re-
produce, whereas a few large cells are specialized for reproduction. Large
Volvox colonies form the shape of a hollow ball with the somatic cells held
together by proteins and carbohydrates forming the surface of the ball and
their flagella pointed outward. Internet searches for videos of Volvox will
help illustrate the complexity of these small colonial species. The spheri-
cal Volvox colony structure allows coordinated movement, as the flagella
propel these colonial organisms through the water. The next generation
of colonies are retained inside the hollow sphere, which can be seen in
the videos, until the parental colony breaks apart. Some species of Volvox
are either male colonies that produce sperm or female colonies that pro-
duce eggs. These species reproduce sexually when the egg and sperm fuse.
Regardless of the reproductive details, these large colonies have evolved
through natural selection to produce multicellular organisms that contain
different cell types with specific functions. Now consider the evidence that
points to the selective advantage for multicellularity and division of labor.
Graham Bell combined information from the scientific literature with
his own data in a comparative study to measure the selective advantage of
1.0
colonial
relative daily growth rate

Volvox
0.5
0
unicellular
Volvox family
members
–0.5
–1.0
–2 0 2 4 6 8
log body volume (mm3)
Figure 9 Relative population growth rates in relation to organism

size. All the unicellular algae species fall within the lighter gray area,
and all the colonial species fall within the darker area. Each species
is graphed with growth rates on the y-axis and average individual
organism volume on the x-axis.
Source: Modified from G. Bell, 1985.
division of labor and multicellularity. Bell focused on Volvox cell volume

and the population growth rate. He first examined the distribution of
cell volumes of unicellular species and colony volumes for colonial spe-
cies of Volvox. He collected growth rates for the same species and plotted
the growth rates as a function of unicellular volume or colony volume
(Figure 9).
The relative frequency distributions of cell sizes in unicellular species
and colony size for colonial Volvox family members overlap over several
orders of magnitude (Figure 9). The largest unicellular species are bigger
than the collective size of some of the smallest colonial species. On average,
however, individuals of unicellular species are smaller than whole colonies.
From Figure 9, it can be seen that small unicellular species (for example
102 µm3) have fast population growth rates, but there are no colonies that
small. When one compare sizes that include both single cells and colonies
(for example 106 µm3), the colonial Volvox species grow faster than the
unicellular species. This indicates that there is a selective advantage for
colonial species with larger body size (at least in some habitats) and thus an
advantage to multicellularity. Bell hypothesized that colonial species might

have a selective advantage if the majority of cells specialized in cellular
metabolism. The larger reproductive cells could use the energy supplied to
them by the majority of the smaller colonial cells so that the reproductive
cells could focus their cellular processes only on reproduction.
In collaboration with other biologists, Bell tested his hypothesis by
conducting growth experiments in an attempt to measure the selective
advantage of multicellularity. The investigators compared growth rates
of intact Volvox carteri colonies to growth rates of isolated reproductive
V. carteri cells that would have to conduct their own cellular metabolism
as well as perform reproductive processes. The scientists predicted that
reproductive cells would not grow as quickly on their own compared to
reproductive cells within functional colonies.
The investigators produced three types of V. carteri cell populations
from colonies: Isolated reproductive cells; Partially disrupted colonies;
And intact colonies. The algae were grown in constant light and tem-
perature to promote maximum growth rates. The scientists estimated re-
productive cell volume by measuring individual cell diameter under a
compound microscope. The volume was calculated from the measured
diameter (d) using the formula: π/6 * d3. The experiment ended at
21 hours because some reproductive cells were beginning to divide, which
would have confounded cell volume data collection.
For intact colonies, the biologists estimated the total volume of so-
matic cells per reproductive cell by multiplying the number of somatic
cells by their average volume and dividing by the number of reproductive
cells. Keep in mind Volvox cells live in a hollow sphere, which makes
measuring colonial cell volume much more difficult than measuring iso-
lated reproductive cell volume. The investigators calculated an average
of 2,385 somatic cells per intact colony at this stage of the colony’s life.
What they found was that the total cellular volume increased at a much
faster rate for intact colonies than for isolated reproductive cells or dis-
rupted colonies, ones that had their normal structure physically disrupted
but the cells remained connected to each other. In rich media, repro-
ductive cells grew faster in intact colonies than in disrupted colonies or
in isolation. Growth rates of isolated reproductive cells and reproductive
cells growing in disrupted colonies were not different from each other.
1.6
1.2
growth rate ± SE
0.6
0.4
Figure 10 Relative change in growth rates in response to nutrient

enrichment of four related green algae species in the Volvox family.
From left to right, they grew unicellular (Chlamydomonas), planar
colonies (Gonium), spherical colonies but no specialized reproductive
cells (Eudorina), and Volvox with specialized reproductive cells.
Source: Modified from Koufopanou and Bell, 1993. Figure 2.
Therefore, the Bell and his colleagues deduced that the non-reproductive
cells transferred nutrients to reproductive cells within the structure of a
colony, which facilitated a higher rate of growth of reproductive cells in
intact colonies.
Once the biologists had devised a method to measure growth rates
of Volvox cells, they wanted to compare growth rates of different species
of related green algae as a function of nutrient enrichment (Figure 10).
Biologists had noticed that Volvox were found more often in ponds
containing a lot of organic matter, such as farm ponds. They quanti-
fied growth rates in ponds with different levels of nutrient enrichment.
They studied unicellular Chlamydomonas which is small and each cell
is autonomous, Gonium which produces colonies that are flat, and
Eudorina which produces small spherical colonies. Volvox was the only
species studied that produces specialized reproductive cells (division of
labor). The investigators determined the growth rates of all four species
at different nutrient levels, and Figure 10 shows the results for the richest
nutrient level tested.
V. carteri grew the fastest of the four species tested in Figure 10. The
data show that multicellularity with division of labor is better adapted to
growth in a rich environment. The biologists found that Volvox did not
grow faster in nutrient-poor water, which indicates that Volvox probably
did not evolve until some bodies of water were rich with organic mol-
ecules produced by other species. The investigators speculated that the
energetic cost of swimming was too great in low nutrient water for the
large colonies compared to the smaller colonies and unicellular species.
Although scientists still do not know exactly how multicellularity
evolved, this chapter presented compelling evidence of advantages to
multicellularity in one family of plant species. The complex colonial or-
ganization of Volvox likely evolved several times independently as indi-
cated by the complex distribution of colony sizes in Figure 8. This chapter
demonstrated that there are growth advantages to multicellularity and
division of labor among cells within the colony, at least under conditions
of high nutrient availability. A nutrient-rich ecological condition may not
be the only driving factor in the evolution of multicellularity in Volvox,
nor would it be expected to be the driving factor in the evolution of
other multicellular species. Using a family of related species that evolved
recently helped us to understand the mechanisms and factors involved in
evolution of multicellularity.
Bibliography
Bell G. (1985). The origin and early evolution of germ cells as illustrated
by the Volvocales. In H. Halvorson and A. Monroy (eds.) The origin
and evolution of sex. pp. 221–256. Alan R. Liss, New York.
Coleman AW. Phylogenetic analysis of “Volvocacae” for comparative ge-
netic studies. Proc Natl Acad Sci USA 96(24):13892–13897, 1999.
Koufopanou V, Bell G. Soma and germ: an experimental approach using
Volvox. Proc R Soc Lond Biol Sci 254:107–113, 1993.
Nozaki H, Misawa K, Kajita T, et al. Origin and evolution of the colo-
nial Volvocales (Chlorophyceae) as inferred from multiple chloroplast
gene sequences. Mol Phylogenet Evol 17(2):256–268, 2000.
Conclusion
Over time, ancient cells reproduced and diversified to produce differ-
ent domains of life. Later, a bacterium and an archaea fused and merged
their genomes to produce the third domain of life—eukaryotes. The first
nucleus evolved from the fusion of two cells from different domains.
Although it may be hard to image this happening, simply noting that
some data are missing is insufficient to refute our understanding of evolu-
tion. All data were missing until a scientist discovered them. To overturn
a hypothesis, one needs to analyze available data that directly contradict
current understanding. The data need to be reproducible and founded on
scientific standards of credibility. Hearsay and secondhand accounts are
unacceptable to be considered scientific data.
Nature solved the problem of stuffing six feet of DNA inside every
nucleus of a human’s 50 trillion cells. Human cells use the same mecha-
nism that archaea use for DNA compaction, because both domains of
life have histones around which their DNA is wrapped. Like the nucleus,
mitochondria and chloroplasts are the consequence of one cell engulfing
another. Evidence of their origins is visible in the double membranes sur-
rounding the organelles as well as the DNA they carry. Finally, Chapter 4
presented one example of how multicellularity evolved multiple times in
Volvox. Multicellularity and division of labor were adaptive when these
species were grown in nutrient-rich waters. The four case studies pre-
sented in this book demonstrated how eukaryotes show evidence of evo-
lution at the organismal level. Each case illustrated three of themes of
evolution: the origin of living systems occurred by natural processes, and
life continues to evolve within a changing environment; organisms can be
linked by lines of descent from common ancestry; and natural selection is
a mechanism of evolution that accounts for adaptation.
Glossary
archaea. one of the three domains of life (Eubacteria and Eukaryotes are the other
two); similar to bacteria except they often live harsh conditions, and evolved sepa-
rately from Eubacteria such as E. coli.
bacteria. unicellular cells without nuclei that live in common places.
bioinformatics. the field that uses mathematical, statistical, and computational
tools to extract meaningful information from biological data.
bootstrap values. it quantifies the confidence for branch points in evolutionary
trees.
central dogma. it refers to the critical role of converting DNA information into
RNA and then into protein.
chloroplasts. DNA-containing organelle in photosynthetic plants, contains
green pigment chlorophyll and is composed of many layers of membranes.
chromatin. the mixture of DNA and protein that comprise chromosomes in
living cells.
division of labor. the specialization of cooperative labor in specific tasks.
domains. the three broadest classifications of organisms.
epigenetic. epigenetic changes to DNA are chemical modification that do not
change the sequence of DNA nucleotides.
evolution. the scientific explanation for the origin of life and its continual change
over time.
evolutionary tree. an evolutionary tree is a diagram that shows the evolutionary
relationships among various species.
fabrication. the act of inventing data from experiments never performed.
falsification. the act of changing data so that the results no longer reflect actual
experimental evidence.
histones. protein complex of eight subunits around which DNA is wrapped in
chromatin.
missing links. as yet unknown species that is evolutionarily located between two
known species; its existence is hypothesized based on known species.
mitochondria. DNA-containing organelle that eukaryotes have (including plants)
that are instrumental in cellular respiration; contains internal undulating mem-
brane and central area called matrix.
multicellular. organism consists of many interdependent cells, which cannot
exist on their own.
nucleosomes. the bead of chromatin composed of DNA and histone proteins.
orders of magnitude. the power of 10, which is signified by plus-or-minus 1 in
the logarithm of the quantity.
36 GLOSSARY
orthologs. two genes with very similar sequences found in two different genomes.
plagiarism. the act of taking credit for someone else’s ideas or words.
prokaryotes. unicellular microbes that include bacteria and archaea.
replication. making copies of DNA using existing strands as template for new
double-stranded DNA molecules.
ring of life. it describes how eubacteria and archaea cells fused and integrated
their genomes to form eukaryotes; better visual image than tree of life.
transcription. the enzymatic production of RNA using DNA as the template.
Index
Adult human cells, human embryonic nuclei of eukaryotic cells, 5–6
stem cells from, 15 overview of, 1–2
α-proteobacteria, 23 packing of, 10
proteins importance of, 2–3
Bell, Graham, 27–28 sequencing of, 3–4
Bioinformatics tools, 5 stretched-out length of, 9
BLAST sequence template for RNA production, 1–8
alignment tool, 6 visualize packing of, 10
human gene evaluation by, 6–7
Bootstrap values, 20 Energy harvesting genes, 7
Brenner, Sydney, 3 Epigenetic regulation, 13, 14
Eukaryotes from prokaryotes,
Cell biology, 13 origins of, 1–8
Chlamydomonas, 27 Evolutionary trees, 20
Chloroplasts, origins of, 19–23 branching pattern of, 20–21
Chromatin, 9 interpretation of, 4–5
from chicken cells, 11 of Volvox, 26
higher levels of, 11
histone complex in, 10 Falsification, fabrication, and
protein as temporary glue, 12 plagiarism (FFP), 14–15
Chromosomes, 12
Colonial cells, 25–26 Gel electrophoresis, 2, 3
Colonial green algae, 27 Gemmata obscuriglobus, 3
Covalent modulation, of histones, 13 Genes encoding complex
Cyanobacteria, 2–3, 23 processes, 7
Cytoplasmic communication Genome
systems, 6 ethical, legal, social implications,
14–16
Darwin, Charles, 1 into nucleus, 9–14
Darwinesque tree, 6–7 Gonium, 30
Division of labor
among individual cells, 27 Histones, 9–10
selective advantage of, 27–28 amino acid conservation among,
DNA 13–14
accessible for replication and complex in chromatin, 10
transcription, 9 covalent modulation of, 13
based evolutionary tree, 20 Human embryonic stem cells from
BLAST sequence alignment tool, 6 adult human cells, cases of
of chromosomes, 9–10 misconduct in, 15
compaction in eukaryotes, 14 Human eye cells, 2
genes encoding complex, 7–8 Human gene, by BLAST sequence, 6
genetic information, source of, 2 Human nucleus, diameter of, 9
38 INDEX
Intact colonies, total volume of Ribosomal RNA (rRNA), 4

somatic cells, 29 Ribosomes, 3–4
Isolated reproductive cells, growth Rickettsia prowazekii, 23
rates of, 29–30 Ring of life, 7
RNA polymerase, 2
Jacob, François, 3
Second law of thermodynamics,
Kennedy, Donald, 15 12–13
Simian virus 40 (SV40), 10–11
Meselson, Matthew, 3 SV40 DNA, 10
Missing links. See Transitional species genomic database at, 10–11
Mitochondria, origins Synechocystis, 23
of, 19–23
Multicellular organisms, evolution Transcription, 2
of, 25–31 Transitional species, 3
Multicellularity species, selective
advantage of, 29 Unicellular species
relative frequency distributions of
Nozaki, Hisayoshi, 26 cell sizes in, 28–29
Nucleosomes, 11 US Office of Science and Technology
Nucleus, localization to periphery Policy, 15
of, 12
Volvox
Orthologs, 22 division of labor among individual
cells, 27
Partially disrupted colonies, 29 evolutionary tree of, 26
Phospholipid bilayers, 5 family of green algae, 25–26
Plagiarism, 14–15 growth rates of, 30–31
Prokarotes, 21–22 in hollow sphere, 29
Proteins spherical structure of, 27
-coding RNA, 4–5 Volvox carteri
cytoplasm of eukaryotic, 3 growth rates of, 29, 31
importance of, 2 three types of, 29
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EBOOKS Evolution of Eukaryotes

FOR THE A. Malcolm Campbell • Christopher J. Paradise

Engineering ticellular algae as a case study on the origins of multicellularity.

A. Malcolm Campbell, PhD

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Printed in the United States of America

The Origins of Eukaryotes

Figure 1 Tree of life from Darwin’s On the Origin of Species. Species

regular. He explained, “I do not suppose that the process ever goes on so

are a very common prokaryote that have stacks of photosynthetic mem-

Table 1 Simplistic examples of DNA sequences that reveal

Figure 2 Evolutionary tree showing the changes in simulated DNA

Eubacteria Archaea Eukarya

Figure 3 Evolutionary tree based only on ribosomal genes from many

Table 2 Subset of results from BLASTing human protein

based on gene sequence alignments are best at evaluating the conservation

Figure 4 Non-tree based evolutionary relationships. This diagram

Fitting a Genome into

require energy and probably motor proteins to force the chromosomes

Sponge symbiont (Archaea)

Figure 5 Evolution of histone proteins. Amino acid sequence

related to archaea than eubacteria. Bacteria such as E. coli do not need to

Ethical, Legal, Social Implications: Ethical Guidelines

FFP. The US Office of Science and Technology Policy defines misconduct

financial interests in the outcome of an experiment? The scientific com-

Ethical, Legal, Social Implications: Ethical Guidelines for Scientific

Chong S, Normile D. Stem cells: how young Korean researchers helped

The Origins of Mitochondria

This chapter focuses on the evolutionary origins of two important or-

Before determining the origins of these organelles, it is important to

Figure 6 Deducing the origin of evolved DNA sequences. Original

It is tempting to consider the terminal adjacent sequences that come

100 Prototheca wickerhamii

Figure 7 Deducing the origin of chloroplasts and mitochondria.

twice as many cell membranes as eukaryotic cells have. These anatomical

16 species of > 32 cells/colony

5 species of 8,16, or 32 cells/colony

Figure 8 Summary of the evolutionary tree for 35 members of the

relative daily growth rate

Figure 9 Relative population growth rates in relation to organism

division of labor and multicellularity. Bell focused on Volvox cell volume

advantage to multicellularity. Bell hypothesized that colonial species might

Figure 10 Relative change in growth rates in response to nutrient

Intact colonies, total volume of Ribosomal RNA (rRNA), 4

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