Short Branch Attraction, the Fundamental Bipartition in Cellular Life, and Eukaryogenesis Amanda A

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Doctoral Dissertations University of Connecticut Graduate School

12-16-2016 Short Branch Attraction, the Fundamental Bipartition in Cellular Life, and Eukaryogenesis Amanda A. Dick PhD University of Connecticut, [email protected]

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Recommended Citation Dick, Amanda A. PhD, "Short Branch Attraction, the Fundamental Bipartition in Cellular Life, and Eukaryogenesis" (2016). Doctoral Dissertations. 1479. https://opencommons.uconn.edu/dissertations/1479 Amanda A. Dick - University of Connecticut, 2016 Short Branch Attraction, the Fundamental Bipartition of Cellular Life, and Eukaryogenesis Amanda A. Dick, PhD University of Connecticut, 2016

Short Branch Attraction is a phenomenon that occurs when BLAST searches are used as a surrogate method for phylogenetic analysis. This results from branch length heterogeneity, but it is the short branches, not the long, that are attracting.

The root of the cellular tree of life is on the bacterial branch, meaning the Archaea and eukaryotic nucleocytoplasm form a clade. Because this split is the first in the cellular tree of life, it represents a taxonomic ranking higher than the domain, the realm. I name the clade containing the Archaea and eukaryotic nucleocytoplasm the Ibisii based on shared characteristics having to do with information processing and translation. The Bacteria are the only known members of the other realm, which I call the Bacterii.

Eukaryogenesis is the study of how the Eukarya emerged from a prokaryotic state. The beginning state of the process is represented by the relationship between Eukarya and their closest relative, the Archaea. The ending state is represented by the location of the root within the Eukarya. Neither is known. I attempt to use Eukaryal stem branch length (ESBL) to inform on the relationship between Archaea and Eukarya. The long ESBL found, shows a great deal of evolution, due to a product of time and rate of evolution. This suggests that Eukarya have not evolved from Archaea, but is inconclusive. I propose a new model for evolution, the Watershed of Life. Instead of the data representing a strictly bifurcating tree, it represents two signals: vertical descent and horizontal gene transfer, accounting for opposing signals present in our data.

Both the data that has been claimed to show the Eukarya evolved from within the Archaea and Amanda A. Dick - University of Connecticut, 2016 data that shows that the Eukarya did not, are consistent with only a watershed in which the

Eukarya did not evolve from within the Archaea, but shared genes with the Archaea. Finally, an attempt is made to root the Eukarya using paralogs duplicated along the eukaryal stem branch, because said paralogs provide a closer outgroup. Unfortunately, the paralog datasets did not contain enough data to resolve the placement of the root. i

Short Branch Attraction, the Fundamental Bipartition of Cellular Life, and Eukaryogenesis Amanda A. Dick

B.S., University of California, Irvine 2005

A Dissertation

Submitted in Partial Fulfillment of the

Requirements for the Degree of

Doctor of Philosophy

at the

University of Connecticut

2016 ii Copyright by Amanda A. Dick

2016

iii

University of Connecticut 2016 iv Acknowledgments:

I would like to acknowledge the professors whose mentorship has made this thesis possible. Foremost, is Dr. J. Peter Gogarten, who gave me the freedom to develop my own ideas and hypotheses and the support needed to carry them through. Secondly, is Dr. Paul Lewis, who gave valuable input with regards to experimental design. Additionally, the remaining members of my committee gave valuable input or provided enlightening scientific discussion on the topics of my thesis; namely Dr. Ken Noll, Dr. R. Thane Papke, Dr. David Benson, Dr. Andrew Pask, and

Dr. Jonathan Klassen. Prior to entering the PhD program, I was lucky enough to be mentored by

Dr. C. E. Jones, who gave me the freedom to discover which scientific areas interested me the most. Dr. Luis Villarreal was the instructor of the only writing class that ever made the least bit of sense to me. I credit Dr. Villarreal with teaching me how to write and teaching me how to teach myself. Dr. Douglas Eernisse and Dr. Brandon Gaut were the first to teach me about phylogenetics; a subject that has fascinated me since discovering what the term meant. Dr

Gogarten, Dr. Villarreal, Dr. Nilay Patel, and Dr. Arthur Weis were essential to my employment during this entire learning process. Finally, Dr. Arthur Weis was the first to allow me into his laboratory and teach me how to do science and encourage my intellectual development; without him, my dreams of becoming a biologist may never have come true.

Two of my colleagues provided useful scientific discussion and aided my learning to code; namely Dr. David Williams and Timothy Harlow.

I would also like to thank my family. My father, Carl Dick, has been by my side from the beginning and was for a long time, the only logical adult I knew. My mother, Sondra Dickens, prompted my first scientific hypothesis at the age of four; a hypothesis that was proven right, when my successful experiment forced her to admit she lied. My brother, Andrew Dick, was the v

only sane child I knew growing up and told me not to worry about the rampant insanity amongst the majority of our species. My brother also consistently pushed me to hurry up and finish my

PhD already. My sister, Cyndi Dickens, is probably the reason I survived to adulthood, because she was the only one to seek medical treatment for the pneumonia I could not recover from.

While on the subject of medical treatment, my ex-boyfriend, Tabashi Price, arranged medical treatment for a case of Bronchitis that I was too ill to seek treatment for myself. And most importantly, my daughter, Zaalayka Dick-Price, has provided the reason for me to keep going.

Table of Contents Title Page Page i Copyright Page Page ii Approval Page Page iii Acknowledgments Page iv Table of Contents Page vi Chapter 1 Page 1 Chapter 2 Page 19 Chapter 3 Page 40 Chapter 4 Page 58 Chapter 5 Page 74 Chapter 6 Page 92 References Page 119 Article Permission Use from Elsevier (chapters 2 and 4) Page 129

Chapter 1: Introduction

1.1 Overview of Thesis

The foci of this thesis are the early evolution of life and the origin of eukaryotes. I cover both, the approaches and pitfalls in inferring evolution from the molecular record, and the consequences for taxonomy, i.e., the naming of groups of organisms. This thesis covers work on

Short Branch Attraction, a reconstruction artifact that sometimes is found when BLAST searches are used for phylogenetic inference, recent changes in taxonomy, and Eukaryogenesis, the study of how the Eukarya originated. Research on reconstruction artifacts will be presented first, because an advanced understanding of the issues faced with bioinformatic analyses is vital to doing the complicated type of reconstructions that was undertaken in later parts of this thesis.

Chapter 2 covers Short Branch Attraction (SBA), a feature of BLAST searches, caused by slowly evolving sequences.

Chapter 3 changes course, to discuss the necessary changes that must be made to taxonomy and systematics. Having appropriate terms and definitions is critical in scientific communication. Unfortunately, this aspect of science tends to lag behind the primary discoveries that support them. Names and ranks for critically important clades must be assigned.

In Chapter 4, eukaryal stem branch length is used in an attempt to answer the question of the relationship between Archaea and Eukarya, because if Eukarya evolved from within Archaea and are no more than 1.2 billion years old (Berney and Pawlowski, 2006; Cavalier-Smith, 2002;

Douzery et al., 2004; Eme et al., 2014), as many researchers have suggested, then the amount of time allowed for Eukaryogenesis to occur is short and the eukaryal stem branch length should be 2 correspondingly short, implying a rapid radiation; a somewhat strange, but thoroughly prokaryotic, Archaea practically overnight underwent a series of changes that led to LECA (Last

Eukaryal Common Ancestor), which quickly radiated into Eukarya as we know them today. But this is at odds with the invariable finding, no matter which dataset one studies, of a long eukaryal stem branch (Fournier et al., 2011; Pace et al., 1986). The long eukaryal stem branch shows that there has been a great deal of evolution, reflecting the product of time and rate of evolution, that needs to be accounted for. This suggests a monophyletic Archaea or a polytomy between

Euryarchaea, Crenarchaea, and Eukarya. At the very least, Eukarya must be somewhat older than

1.2 billion years, the age of the oldest clearly Eukaryal fossils (Butterfield et al., 1985).

However, because the rate of evolution cannot be distinguished from the time of evolution when measuring stem branch length (T. H. Jukes and Cantor, 1969), my findings on the long eukaryal stem branch are inconclusive. Overall, even though more and more scientific evidence is amassed each year (Cox et al., 2008; Lasek-Nesselquist and Gogarten, 2013a; Nasir et al., 2015; Williams et al., 2013), the exact relationship between Archaea and Eukarya remains inconclusive. That is why Chapter 5 proposes that the data is not the problem, but the model of evolution is. If instead of looking at the data as representing a strictly bifurcation tree, the data is viewed as representing the two signals of the watershed of life, then the two opposing signals present in the data have been accounted for. The only problem is determining which of the signals represents vertical descent, i.e. universally present with a consistency in divergence times, and which represents horizontal gene transfer, i.e. patchily distributed with inconsistence divergence times. The Eocyte data is consistent only with the pattern seen with horizontally transferred genes in the 3 domain tree, while the 3 domain data is consistent with the vertically 3 inherited genes in the 3 domain trees. At present, there is no data supporting either of the signals expected if the Eukarya evolved from within the Archaea.

After examining Eukaryogenesis from the bottom up, in Chapter 6 I switch to looking at the problem from the top down and the question of where the root within the Eukarya is located.

I attempt to root the Eukarya using paralogs duplicated on the Eukaryal stem branch. The problem with rooting the Eukarya is that the closest living outgroup, the Archaea, is too far away to inform on the ancestral state of the group, because of the long Eukaryal stem branch (Fournier et al., 2011). Genes that were duplicated on that stem branch provide a way around this problem, because they represent closer outgroups than the Archaea. These paralogs can then be used to root each other, and thereby the Eukarya, as was first done when the ATP synthase paralogs were used to root the tree of life (Gogarten et al., 1989).

1.2 Short Branch Attraction

In 1999, twenty-five percent of the Thermotoga maritima genome was thought to be horizontally transferred from Archaea (Nelson et al., 1999a), because unidirectional best BLAST hits were used as a proxy for phylogenetic analysis in Horizontal Gene Transfer detection. But years later when this analysis was repeated, using the exact same genome sequence as query, that number decreased first to 11%, then 10% (Swithers, 2012), showing that BLAST results change over time, even though the sequences being used as query are unchanged. The only change is the size of the non-redundant database, which grows exponentially over time (Kulikova et al., 2007).

Dr. Gogarten hypothesized that ancient horizontal gene transfers were at fault, because transferred genes were originally finding the donor as best match, before a closely related species that also inherited the transferred gene was sequenced. But upon preliminary examination, far 4 more evidence of heterogeneous branch lengths was found as the cause. That led to the hypothesis that short branches cause this artifact in BLAST searches. This hypothesis was tested using simulations that showed short branches return other short branches as top scoring BLAST hits over more closely related sequences.

1.3 Molecular Data and a Natural Taxonomy

Modern taxonomy started with Linnaeus who gave each species two names and assigned them to a series of ranks (Linnaeus, 1758). But back in Linnaeus’ time, Darwin’s principle of evolution through natural selection was not known, so groups were not necessarily based on shared descent. The highest rank was the split between plants, animals, and minerals, because fungi, protists, Bacteria, Archaea, and virus had yet to be discovered or recognized as distinct groups. As these discoveries were made, taxonomy was adjusted to accommodate this new knowledge.

Robert Whittaker proposed the five kingdom system that was widely taught in American high schools (Whittaker, 1969) and championed by Ernst Mayr (Mayr, 1974), but although he won favor with teachers and book writers, it was Willi Hennig’s principles of taxonomy (Hennig,

1965a), written while being held as a prisoner of war by the Allies, that won favor with scientists. The advantage of Hennig’s system was two-fold: the phylogenetic tree was imbedded in the taxonomy, so that the tree could be recreated from the taxonomy, and precise definitions were given, allowing for clearer communication. Precision in vocabulary is one of the prerequisites of good science, because it aids communication between scientists. That is why

Hennig’s system triumphed, despite the disadvantages of being on the wrong side of the war and dying young. 5 In accordance with Hennig’s principles, modern taxonomy needs a name to represent the

Eukarya/Archaea clade. If recent studies finding the Eukarya branch from within the Archaea turn out to be accurate (Nasir et al., 2015; Williams et al., 2013), the Archaea will be a paraphyletic grade (Huxley, 1959a) and therefore cannot be a taxonomic domain. Also, groups linked by horizontal gene transfer are deserving of a name. Finally, there is the issue of a species definition that is applicable for Bacteria and Archaea. Chapter three tackles all of these issues.

1.4. Eukaryogenesis

1.4.1 Eukaryogenesis And What Remains Unanswered.

Eukaryogenesis is the study of how Eukarya originated, likely from a simple anucleate state to the complexity that is seen in all existing members. Although it has been suggested that life arose in a complex Eukarya-like state and evolved through reduction to produce the other two domains (Patterson and Sogin, 1994; Forterre and Philippe, 1999; Hartman and Fedorov,

2002; Kurland et al., 2006), the consensus is that the Last Universal Cellular Ancestor (LUCA) was prokaryotic in nature (Gogarten and Taiz, 1992; Delaye et al., 2005; Martin et al., 2007).

There are two important yet currently missing pieces of evidence with regards to

Eukaryogenesis that prevent progress regarding the evolution of Eukarya. First, there is the bottom up approach, which asks what is the nature of their common ancestor with their closest living relative, the Archaea. Secondly, there is the top down approach, which asks what is the nature of the common ancestor within the Eukarya. If we know the answers to these two 6 questions, then we know where the process of Eukaryogenesis started and where it ended. Only with these two pieces of information in hand, can future researchers hope to tease apart the process that led to the revolution in cellular structure that is Eukaryogenesis.

The main hypotheses regarding Eukarya’s common ancestor with Archaea fall into two categories: Paraphyletic Archaea hypotheses, illustrated in Figure 1, specify that the last common ancestor shared between Eukarya and Archaea was already an Archaeon. For example, an Archaeon ingested a Bacterium and developed an endosymbiosis with it to form the mitochondria, creating Eukarya. An example of this is the Eocyte hypothesis (Cox et al., 2008).

Monophyletic Archaea hypotheses, shown in Figure 2, on the other hand, specify that the last common ancestor was not an Archaeon, but an ancestor shared by the two domains, i.e. the organism that ingested the Bacterium was not an Archaeon. The Archaeozoa hypothesis is an example (Cox et al., 2008).

There is a third possibility and that is that the split in question is a polytomy, with the

Eukarya branching off from the Archaea at the same time that the archaeal domain diverged into its kingdoms, as shown in Figure 3. There is conflicting evidence supporting both monophyletic and paraphyletic hypotheses, leaving, in effect, a polytomy. Whether this polytomy is soft, meaning that there is a meaningful bipartition pattern, but the evidence for it has not been or cannot be identified, or whether this polytomy is hard, meaning that the Eukarya, Crenarchaea, and Euryarchaea all three diverged simultaneously, will require additional evidence to determine.

With regards to the second major question of Eukaryogenesis: where is the root within the Eukarya? The answer is truly unknown. Various research studies have come forth favoring a myriad of possibilities. The first rooting used rRNA and produced a tree where plants, animals, 7 and fungi were at the top of the crown of the tree, with a great many single celled Eukarya branching off first (Pace et al., 1986; Sogin, 1991; Sogin et al., 1989, 1986; Woese et al., 1990).

This reinforced the idea that multicellular organisms are more evolved than single celled organisms and should therefore be higher up on the tree; a modern misconception that shows

Plato’s Great Chain of Being (Lovejoy, n.d.) is still impacting thought processes in science. This rooting of the tree has fallen out of favor, because it appears to be the result of Long Branch

Attraction (Baldauf et al., 2000).

A popular rooting of the eukaryal tree is the unikont/bikont rooting, which splits Eukarya based on the morphology of possessing one flagellum (unikont) or two (bikont) (Derelle and

Lang, 2012; Richards and Cavalier-Smith, 2005; Stechmann and Cavalier-Smith, 2002). The unikonts include Opisthokonta, animals and fungi, and Amoebozoa. The bikonts include everything else. This rooting, unlike other rootings, has two sets of physiological traits that determine which of the two super groups a given Eukaryon belongs. But this distinction does not necessarily coincide with the first split in the Eukarya; a scenario which would mean that the

Last Eukaryal Common Ancestor (LECA) gave rise to one clade that went on to be composed only of members with one flagella and another clade composed only of members with two.

When determining the probability that this rooting is correct, it would help to know the ancestral state of flagella in Eukarya, because many have argued that the root actually belongs within the bikonts (Rogozin et al., 2009) or within the unikonts (Stechmann and Cavalier-Smith, 2002).

Unfortunately, Archaea, the closest living outgroup, do not have flagella of the eukaryal kind

(Peabody et al., 2003) and so are neither unikonts nor bikonts and therefore unable to inform on the ancestral state. 8 Excavates, a weird group of single-celled Eukarya, have also been suggested to be the location of the root within the Eukarya (Arisue et al., 2005; He et al., 2014). Excavates are a highly varied group, united by a ventral feeding groove (Baldauf, 2003). Excavates commonly diverge first from molecular trees, and indeed were among the first to diverge from the rRNA tree, so heavily criticized for Long Branch Attraction (Sogin, 1991). The excavates include many quickly evolving parasites, such as Giardia (Sogin et al., 1989), that lead many to ignore them in analyses completely, in favor of finding a rooting that cannot be an artifact. The problem is exacerbated by lack of sequencing of more slowly evolving members and that monophyly of this group is unproven (Simpson, 2003). Many trees put the root not on the excavates, but within them (Cavalier-Smith and Chao, 2010), making them paraphyletic. Still others suggest multiple, polyphyletic origins for the feeding groove.

The other possibilities are not popular or common, but have all been supported by one dataset or another: rootings within or on the amoeba (Stiller et al., 1998); within or on the

Archaeaplastida (Rogozin et al., 2009); on a supergroup containing the animals, fungi, amoeba, and excavates (Wideman et al., 2013). The lack of consensus is so profound that analyses attempting to determine gene presence/absence in LECA generally repeat the analyses given three different rootings, to ensure results are robust against the location of the root. But even with three rootings, there is still a very real chance that the true rooting is not among the ones used.

Furthermore, the only features that are robust to all possible rootings, are ones that are almost universally present in all Eukarya, because they were present in LECA; conversely features that were not present in LECA cannot be ruled out without knowing the root. Therefore, it is of the upmost importance to know the location of the root going forward. 9 To evaluate a rooting of a molecular tree, it helps to be aware of certain already established supergroups within the Eukarya (Derelle and Lang, 2012). Numerous studies support the Opisthokonta clade (Huang et al., 2005), so any tree that breaks this clade up, is suffering from one of the many known issues with phylogenetic reconstruction, and would therefore not be useful in placing the root within the Eukarya. Additionally, the SAR, Stramenophiles,

Alveolates, and Rhizaria, are another well-supported group (Derelle and Lang, 2012).

Archaeplastida, a clade defined as a group of organisms that all had primary plastid endosymbionts, which includes plants, green algae, red algae, and glaucophytes (Bhattacharya and Medlin, 1995; Cavalier-Smith, 1982; Huang and Gogarten, 2007), should also be recovered in any tree purporting to root the Eukarya, unless this group is in fact polyphyletic (Bhattacharya and Medlin, 1995).

A few years ago, there were gaps in whole genome sequencing for a great many of these groups. Archaeplastida are known for their giant genomes, which are prohibitive to genome sequencing, so that there were only whole genome sequences available for two members of this group when this project started. There were no Rhizaria genomes available at all, because of their complex and repetitive genomes. Amoebas, with their humongous genomes, were represented by only 3 species. And, as mentioned previously, the highly divergent parasitic excavates were the first sequenced, leaving a hole in our knowledge of their non-parasitic more normal relatives. These sequencing gaps limited possible attempts at rooting the Eukarya to the minimal sequence information available, such as rRNA and ribosomal proteins data sets. But recent years have seen advancement in genome sequencing and a concerted effort to sequence

10 the diversity of the eukaryal tree. Now that all of these sequences are available, they will be used in this thesis when attempting to root the eukaryal tree.

Figure 1: Paraphyletic Archaea hypotheses. In this sketch, the Eukarya evolved from within the archaeal domain (the Archaea would be paraphyletic). The tree is rooted on the bacterial branch.

Red arrows indicate acquisition of an alpha proteobacterial endosymbiont (precursor of the mitochondria), and the transfer of tyrosyl RS to the ancestor of the opisthokonts from a relative of Haloarcula. The eukaryal Stem Branch Length (SBL), highlighted in green, is short. 11

Figure 2: Monophyletic Archaea hypotheses. Eukarya and Archaea are sister-taxa. A long independent evolutionary history separates LECA (Last Eukaryal Common Ancestor) from the common ancestor with the Archaea. The eukaryal SBL, highlighted in green, is now much longer.

Figure 3: Archaeal and eukaryal polytomy. Eukarya and Archaea diverged during the same time period that the entire archaeal domain diverged. The Archaea and Eukarya together form a clade to the exclusion of Bacteria, but it is impossible to tell which split occurred first.

13 1.4.2. The Current Scientific Evidence

Now that the knowledge needed to move forward has been established, it is time to review what is already known from all available sources.

The geological record provides some information on the dates of the emergence of the three domains of life. It has been proposed that LUCA, the last common ancestor of cellular life, lived before the late heavy bombardment 3.8 bya, based on trees with the molecular clock calibrated with the fossil record (Boussau and Gouy, 2012). 3.45 billion year old stromatolites were most probably the product of microbial mat formation (Allwood et al., 2009). Cellular microfossils of sulfate-reducers, presumably bacterial in nature, were found in fossils from 3.4 billion years ago (bya) (Wacey et al., 2011). But Bacteria and Archaea are not morphologically distinct (Woese and Fox, 1977), so it is difficult to tell when the Archaea first appeared in the fossil record.

The first unambiguous sign of Archaea are 2.8 bya carbon isotopes signatures from methanogenesis (“The Proterozoic Biosphere,” n.d.). Ancient eukaryal fossils are highly debated, with estimates for the oldest Eukarya as low as 0.850 bya (Cavalier-Smith, 2002) and as high as

3.2 bya (Javaux et al., 2010). The estimate of 3.2 bya comes from morphologically indistinct

Acritarchs; the same authors who suggest that these fossils may be ancient Eukarya also suggest that the fossils may be large Bacteria, although there is a third possibility: these fossils may represent the ancestor of the Archaea and the Eukarya. Other fossil Acritarchs, from 2.7 bya, have been ascribed to the Eukarya (Javaux et al., 2001). Carbon isotope data suggests that

Eukarya were present by 2.0 bya (Des Marais, 1997). And finally, molecular clock analyses give

1.866-1.679 bya as the best estimate for the root of LECA (Parfrey et al., 2011), which agrees 14 with the finding of fossilized red algae at 1.2 bya (Butterfield et al., 1985). Red algae are primary endosymbionts that engulfed cyanobacteria (Stiller and Hall, 1997), which had to occur after

LECA, implying the passage of time on the eukaryal side of the tree prior to 1.2 bya.

Endosymbioses and HGTs restrict the Eukaryogenesis timeline. Eukarya have mitochondria (Roger, 1999), which group within and have evolved from alpha-proteobacteria

(Roberts, 1996). Since all present-day Eukarya have evolved from a common ancestor that had mitochondria, alpha-proteobacteria diversified before Eukarya (Roberts, 1996; Roger, 1999).

Some Eukarya have primary chloroplasts (Bhattacharya and Medlin, 1995; Cavalier-Smith,

1992), which group within Cyanobacteria in 16s rRNA phylogenies (Roberts, 1996), so

Cyanobacteria diversified prior to the diversification of Archaeplastida. Horizontal gene transfer from Haloarcula to Opisthokonta (Huang et al., 2005) and from Chlamydia to Archaeplastida

(Huang and Gogarten, 2007), indicate that Haloarcula and Chlamydia diversified before

Opisthokonta and Archaeplastida, respectively.

Protein trees, such as one made from concatenating 45 conserved proteins

(Cox et al., 2008), weakly support archaeal paraphyly. But trees rooted using ancient gene duplications tend to favor archaeal monophyly. The elongation factors Tu and G (Baldauf et al.,

1996a), the signal recognition particle (SRP) and SRP receptor protein (SR) (Gribaldo and

Cammarano, 1998), and multiple tRNA synthetases (Brown et al., 1997; Brown and Doolittle,

1995), all show support for archaeal monophyly, although support is often dependent on reconstruction algorithm used. Simple models tend to favor archaeal monophyly, while those allowing for rate heterogeneity tend to favor archaeal paraphyly (Cox et al., 2008).

1.4.3. Addressing Major Questions in the Field of Eukaryogenesis 15 With conflicting evidence, questions remain. Do the Eukarya have a unique history or are they simply Archaea with endosymbionts? Are the Archaea monophyletic or paraphyletic? And where is the root within the eukaryal domain?

One of the problems previous analyses have had in answering these questions is due to the extremely long branch separating the Eukarya from their closest living relatives, the Archaea

(Derelle and Lang, 2012). An analogy used to describe this problem is throwing a dart at a phylogenetic tree across a football field and expecting it to find the root. Long branches create artifacts in phylogenetic analyses and long branch outgroups fail to do the very thing outgroups are used for: inform on the ancestral state. Therefore, an outgroup with a long branch is liable to attach at random to the ingroup of the tree, accounting for the seemingly random collection of rootings that are available in the literature.

A dataset with an eukaryal outgroup that is closer than the Archaea would be invaluable.

If instead of throwing a dart across a football field, we could throw it across the room, the dart would have a much better chance at aiming true. The closer our dart to our dartboard, i.e. the closer the outgroup to the ingroup, the better the chances of correctly rooting the ingroup. Our ability to root the Eukarya would greatly increase. Unfortunately, there are no surviving lineages that meet this criteria and a time machine cannot be used to go back in time to obtain these sequences. But what can be used are ancient paralogs that were duplicated on the eukaryal stem branch.

Paralogs that were present in LECA and diverged from each other along the eukaryal stem branch, can be used as outgroups for each other, to break up the incredibly long eukaryal

16 branch. In this way, a closer outgroup for living Eukarya can be found within those same

Eukarya than it is possible to find elsewhere. There is the additional benefit that many of these paralogs work in concert with each other in one macromolecular complex, so that they are more likely to share the same evolutionary history than random proteins. This minimizes the problem with concatenation that using sequences with differing histories creates trees that average the signal, representing the history of none of the individual genes. Being able to concatenate sequences has a huge advantage when it comes to recovering signal, because it adds the signal present in each individual gene and on many occasions, has led to the recovery of strong support for a split that the individual genes were not able to support (Bapteste et al., 2005).

Genes that have undergone duplication along the eukaryal stem branch and that function together in one complex will be used for these analyses. Three gene families that meet these criteria are the Tubulin, Histone, and Proteasome families. In the Tubulin family, gamma, alpha, and beta tubulin are present in all modern Eukarya (Fournier et al., 2011). In the Histone family, histone 2A, histone 2B, histone 3, and histone 4 are universally present paralogs in Eukarya that duplicated along the eukaryal stem branch (Bell and White, 2010; Thatcher and Gorovsky,

1994). Analyses done in Chapter 6 indicate that in the Proteasome family, there are 14 subunits present in LECA. It is possible that the first duplication within this family, that between the 7 alpha and the 7 beta subunits, occurred before the Eukarya split from Archaea, because there are a handful of unrelated Archaea that possess a proteasome composed of 7 identical alpha and 7 identical beta subunits (possible, because the patchy distribution of the proteasome in Archaea indicates it is also possible that these Archaea acquired this primitive proteasome through horizontal gene transfer with early stem branch Eukarya). But after the alpha subunit diverged

17 from the beta subunit, each went on to duplicate into 7 more subunits; duplications that undoubtedly occurred along the eukaryal stem branch. Because of the uncertainty regarding where in the tree the split between the alpha and beta subunits occurred, the desire to keep the outgroups as close to the eukaryal root as possible, and the fact that both alpha and beta types have 7 paralogous subunits to work with, they will be separated into two independent datasets.

All 4 of these datasets possess closer outgroups than the Archaea and multiple sequences with the same evolutionary history, perfect for concatenation (modified from (Hilario and

Gogarten, 1993). With 4 paralogs, the histones contain 4 sequences that can be concatenated, increasing resolution power. Because the 4 subunits are homologous, 4 different concatenations can be made, placing the root of the Eukarya on the tree 4 times, giving 4 chances of finding the root. The same is true of the 3 Tubulin subunits, with 3 sequences available for concatenation and 3 chances to find the root. For both the alpha set and beta set of proteasome subunits, each has 7 sequences to concatenate and 7 chances to find the root.

But before going after the root within the Eukarya, an attempt will be made to use these datasets to evaluate the likelihood of the various relationships between Archaea and Eukarya.

This will be done using stem branch length. Long branches leading to the eukaryal line and divergent sequences within Eukarya in protein phylogenies would indicate copious eukaryal evolution in isolation from the Archaea, as first proposed in Chapter 4.2. This would favor the monophyletic Archaea scenarios.

Datasets with their many paralogs contain a portion of the eukaryal stem branch multiple times, allowing for multiple measures of partial eukaryal stem branch length to constrain the time of Eukaryogenesis. These measurements will necessarily be underestimates of eukaryal 18 stem branch length, because the subunits were duplicated somewhere along this branch, not necessarily at the beginning of this branch. But even so, if these measurements are still found to be long, then that is all the more evidence for copious evolution on the eukaryal stem branch.

For measuring eukaryal stem branch length, only the Histone and Tubulin datasets will be used, because of the uncertainty of the evolutionary history within the Proteasome family when it comes to the Archaea. If the alpha and beta proteasome subunits present in Archaea are there as the result of horizontal gene transfer, then there in not a true archaeal stem branch for comparison.

19 Chapter 2: Short branches lead to systematic artifacts when BLAST searches are used as surrogate for phylogenetic reconstruction.

[This chapter was published in modified form in Molecular Phylogenetics and Evolution, 2016 doi: 10.1016/j.ympev.2016.11.016. [Epub ahead of print]]

2.1 Abstract

Long Branch Attraction (LBA) is a well-known artifact in phylogenetic reconstruction when dealing with branch length heterogeneity. Here we show another phenomenon, Short

Branch Attraction (SBA), which occurs when BLAST searches, a 19honetic analysis, are used as a surrogate method for phylogenetic analysis. This error also results from branch length heterogeneity, but this time it is the short branches that are attracting. The SBA artifact is reciprocal and can be returned 100% of the time when multiple branches differ in length by a factor of more than two. SBA is an intended feature of BLAST searches, but becomes an issue, when top scoring BLAST hit analyses are used to infer Horizontal Gene Transfers (HGT)s, assign taxonomic category with environmental sequence data in phylotyping, or gather homologous sequences for building gene families. SBA can lead researchers to believe that there has been a HGT event when only vertical descent has occurred, cause slowly evolving taxa to be over-represented and quickly evolving taxa to be under-represented in phylotyping, or systematically exclude quickly evolving taxa from analyses. SBA also contributes to the changing results of top scoring BLAST hit analyses as the database grows, because more slowly evolving taxa, or short branches, are added over time, introducing more potential for SBA. SBA can be detected by examining reciprocal best BLAST hits among a larger group of taxa, including the known closest phylogenetic neighbors. Therefore, one should look for this 20 phenomenon when conducting best BLAST hit analyses as a surrogate method to identify HGTs, in phylotyping, or when using BLAST to gather homologous sequences.

2.2 Introduction

BLAST stands for Basic Local Alignment Search Tool (Altschul et al., 1997) and is freely available from the NCBI. It is currently the most frequently used bioinformatics tool, because of its speed and ease of use. The user needs to supply a query sequence and choose a database and with a few simple clicks, BLAST will search the database for matches in sequence similarity to the query. This is incredibly useful for a number of purposes. For example, if an unknown gene is sequenced and a close match is present in the database, BLAST results will indicate what type of gene it is and what species it came from. If only distant relatives are in the database, BLAST can still provide information on the function of the sequence by identifying functional domains present. This works, to a point, even if the domains are used in a novel way.

BLAST is also used in identifying genes in whole genome sequencing and is used in the most effective gene calling programs.

BLAST can do all of these things because it is a tool for detecting sequence similarity. If what a researcher wants to do is detect sequence similarity, BLAST is a great program to use. A problem only arises, when researchers overreach and try to use BLAST for more than identifying sequence similarity. In recent years, BLAST has been used with increasing frequency to inform on phylogeny. This first started by using BLAST to generate datasets with appropriate orthologs from various sequenced genomes, for further analysis with phylogenetic reconstruction programs. The unspoken assumption in this case was that the later appropriate phylogenetic reconstruction would erase any bias from using BLAST to choose the sequences included in the 21 dataset. As sequencing ability increased, ability to analyze data lagged. In the beginning, due to the infeasibility of whole genome phylogenetic reconstruction, the easiest analysis to preform was to BLAST the entire genome. This led to BLAST being used to detect Horizontal Gene

Transfer, by assuming the top hit was phylogenetically informative. As analyses needed to be done on larger and larger scales, BLAST was put to new and unintended uses.

But has the usefulness of BLAST overreached when it comes to phylogenetic inference?

What can a program built to determine sequence similarity tell the user about the underlying phylogeny of the sequences? What is the link between phylogeny and sequence similarity? In this paper, we propose that that link is total branch length. Furthermore, it is the total amount of substitution between any two homologous sequences that determines the strength of a match when it comes to sequence similarity. This has implications for branch length heterogeneity interfering with phylogenetic inference by sequence similarity detection algorithms, such as

BLAST. The following experiments were done with the aim of illustrating total branch length as the link between sequence similarity and phylogeny and to clarify the situations in which sequence similarity can be used as a proxy for phylogeny. To explore how BLAST results vary with respect to branch length, we use simulated sequence evolution along a family of trees depicted in Figure 1 Panel H.

Many studies have screened for horizontally transferred genes based on top scoring

BLAST hit analyses (McNulty et al., 2012; Nelson et al., 1999b; Ruepp et al., 2000; Worning et al., 2000; Zhaxybayeva et al., 2009b). When unidirectional analyses were shown to be phylogenetically uninformative (Koski and Golding, 2001), a switch was made to the more restrictive reciprocal top scoring BLAST hit approach (e.g., (Dagan et al., 2010)). Reciprocal 22 Top Scoring BLAST hits are also used in gene family reconstruction to determine homologous gene families (Zhaxybayeva et al., 2006). In addition, BLAST is used in phylotyping or ribotyping analyses where environmental sequences are assigned a taxonomic affiliation based on top scoring BLAST hit (Basaran et al., 2001; Maloy et al., 2009). In doing these types of analyses one assumes that the top scoring BLAST hit will be the sister taxon, especially if the hit is reciprocal. BLAST was developed to find regions of homology and pick out the most similar sequence from a database, not to inform on phylogeny (Altschul et al., 1990). It is well established that the top scoring BLAST hit does not necessarily represent the nearest phylogenetic neighbor (Eisen, 2000; Koski and Golding, 2001) and this is indeed expected, because the nearest neighbor is not always the most similar. However, the question of how useful

BLAST searches might be in matters of phylogeny is still left unanswered. Specifically branch- length space has never been mapped with respect to the ability of BLAST to recapitulate phylogeny.

BLAST searches work by comparing two sequences at a time and looking for local alignments using the Maximal Segment Pair (MSP) score (Altschul et al., 1990). In a gapped

BLAST search (Altschul et al., 1997), the current standard for BLAST, it first matches identical residues between two sequences and stores this information. Continuous regions with high scoring segments are identified, using a threshold score as a cutoff to speed up the process, and are then extended, using a similarity criterion. The highest scoring regions are joined using dynamic programming. The best match made in the pairwise alignments is reported as the top scoring BLAST hit and is a factor of the length and identity of the match. Longer matches are considered good measures of homology and these matches can include indels and small regions of mismatch. Fewer mismatched positions result in a higher percent identity and a stronger 23 match, as measured by E-value, which gives the expected number of matches of a given quality due to chance alone. It has been shown that the phylogenetic nearest neighbor does not always produce the top scoring BLAST hit (Koski and Golding, 2001), but given the pairwise nature of the BLAST algorithm, might it be that the taxon with the shortest total evolutionary distance will be the top scoring hit?

With a normal BLAST search (blastn or blastp), an entire database is searched, but the other sequences in the database do not influence the results of an individual search, except that

E-value is proportional to the size of the databank (Altschul et al., 1990). This differs from phylogenetic reconstruction programs, in which other sequences are used to give weight to conserved residues and calculate the evolutionary model when calculating the tree (Guindon and

Gascuel, 2003; Ronquist and Huelsenbeck, 2003). Inclusion of an outgroup can allow for rooting the tree and identification of synapomorphies and symplesiomorphies. Without knowledge of the outgroup, it is impossible to distinguish synapomorphies from symplesiomorphies and the two categories of matches are therefore given equal weight in the BLAST search. The importance in using synapomorphies to the exclusion of symplesiomorphies in phylogenetic reconstruction traces back to Willi Hennig, who first expounded on their importance in forming monophyletic groups (Hennig, 1965b, 1975a). If monophyletic groups are not used, then there is increased ambiguity in detection of HGT. With phenetic programs, such as BLAST, where unrooted trees are used, there is no way of differentiating a synapomorphy from a symplesiomorphy. The remaining question is at which sets of branch lengths will this cause a problem?

If time that passed is proportional to branch lengths, then we expect that the length of interior branches should ensure that the shortest total branch length is between sister taxa, as 24 shown in Figure 1 panel A. If some branch lengths are shorter, the shortest total branch length may be between the two short branches. For the example tree given in Fig 1 B, the condition for non-sister taxa to be connected by the shortest overall branch is given by Equation 1.

Equation 1: Y + X > Z + 2X

Where Y is the length of the invariable branches (0.1 substitutions per site in Figure 1 panels A,

B, and H), Z is the length of the central branch, and X is the length of the variable branches.

Equation 1 reduces to Equation 2. Because the relationship between sequence similarity and phylogenetic reconstruction using BLAST is dependent on total branch lengths, these equations mean that Short Branch Attraction is likely to occur, if the combined lengths of one of the short branches and the central branch are shorter than the length of the invariable branch, as seen in

Figure 1 panel B.

Equation 2: Y > Z + X

Short Branch Attraction differs from the well-known Long Branch Attraction (LBA), because Long Branch Attraction is a phenomenon of phylogenetic reconstruction. Short Branch

Attraction is a phenomenon of phenetic reconstruction. In cases of Short Branch Attraction, it is the lack of evolution between slowly evolving taxa that causes the artifact. Conversely, LBA has been demonstrated in phylogenetic reconstruction and is caused by numerous convergent apomorphies resulting in homoplasies and the attraction of long branches, while the short branches behave normally (Felsenstein, 1978; Huelsenbeck and Hillis, 1993; Swofford et al.,

2001).

As the number of taxa included in the database grows, and the phylogenetic relationship between them increases in complexity, the behavior of the BLAST search becomes more difficult to predict. If only one taxon is on a short branch, as shown in Figure 1 panel C, then the 25 shortest pairwise distances are still between sister taxa. In these cases, top scoring BLAST hit analyses will be phylogenetically informative and should minimally return a top scoring BLAST hit to a taxon within the same domain as the query. Then top scoring BLAST hits to another domain really would represent Horizontal Gene Transfers (HGTs), or even ancestral HGTs, as shown in figure 1 panel D. In the case of ancestral HGTs, a closely related group of organisms share, through vertical inheritance, a HGT. BLAST would then return the other members of this clade before returning members of the domain from where the transferred gene originated. It causes a problem when a second clade is evolving as slowly as the one of interest, as shown in

Figure 1 panel E with the Archaea on short branches. Here the top scoring BLAST hit will return the Archaea when given a slowly evolving bacterial query, even in cases where there is no HGT.

In this tree, the red clade on the left is still the sister clade, but the shortest total branch lengths are to the Archaea, thus the Archaea will be returned as the top scoring BLAST hit.

Short Branch Attraction explains some instances of why BLAST searches return different results, in terms of HGTs, as the non-redundant database grows. The apparent HGTs observed from the short bacterial branch attracting to the short archaeal domain, is no longer present when other slowly evolving Bacteria are sequenced, as shown in Figure 1 panel F. This is because the shortest total branch lengths are now between the two slowly evolving Bacteria, creating a new stronger Short Branch Attraction. This phenomenon can also work the other way around, if before there were no slowly evolving taxa from other domains sequenced and then one is sequenced and added to the database, as shown in Figure 1 panel G. Here Short Branch

Attraction is created when before there was none.

26 Figure 1: Relating branch lengths in phylogenetic trees to expected BLAST results. Panel A shows a homogeneous tree with the shortest total pairwise distances between sister taxa. The top scoring BLAST hit is expected to be the sister taxon. Panel B shows a heterogeneous tree where the shortest total pairwise distance is between the two slowly evolving taxa. BLAST is expected to return Short Branch Attraction in this case. Panel C shows a hypothetical three domain tree with only one slowly evolving clade on short branches, where the shortest pairwise distances are still between sister taxa. The top scoring hit is not expected to be out of domain. In cases where the out of domain top hit has branches of normal length, as shown in Panel D, we can conclude that there really was a horizontal gene transfer event. But if the Archaea are all on short branches, as in Panel E, then the top scoring BLAST hit will return them even in cases where there is no HGT. Panel F shows how Short Branch Attraction might be the cause of discrepancies in top scoring BLAST hit analyses when the database grows, as more slowly evolving sequences are added, creating new, closer, Short Branch Attractions. Panel G shows how adding new slowly evolving species of Archaea to the database could introduce Short

Branch Attraction when before there was none. Panel H shows the true tree used for the simulations. The root is indicated by the ^ and is at the midpoint along the central branch. Taxa

B and D are on branches of length X. The length of the central branch is represented by Z. clade before returning members of the domain from where the transferred gene originated.

Problems arise when a second clade is evolving as slowly as the one of interest, as shown in

Figure 1 panel E with the Archaea on short branches. Here the top scoring BLAST hit will return the Archaea when given a slowly evolving bacterial query, even in cases where there is no HGT.

In this tree, the red clade on the left is still the sister clade, but the shortest total branch lengths are to the Archaea, thus the Archaea will be returned as the top scoring BLAST hit. 27

28 With gene family reconstruction, Reciprocal Top Scoring BLAST Hit analysis is normally a stepping stone to further phylogenetic analysis and not a proxy for phylogenetic analysis itself. Since homology is an all or nothing trait, technically there are no degrees of homology and any homolog included into the family is appropriate for gene family reconstruction. Inappropriate results are only possible when homologs are excluded. A bias in exclusion introduced at this step will likely be perpetuated further down the line, with certain homologs being left out of their appropriate larger gene family. There is currently no way to measure the rate of false negatives with a BLAST search and if these false negatives were subject to a systematic bias, then it would be very difficult to determine that it was happening at all. Similarities shared with an ancestral sequence, due to Short Branch Attraction, could reasonably account for a systematic bias in gene family reconstruction.

To study the effect of Short Branch Attraction on BLAST searches, simulations of nucleotide sequence evolution were performed using various branch lengths to determine where

in branch length space BLAST searches are subject to Short Branch Attraction and where the top scoring BLAST hit represents the sister taxon. Three sequence lengths, 10000, 1000, and 200 nucleotides, were simulated, to demonstrate the effect of sequence length on branch length space.

With increasing sequence length, there is more data available for the program to use when deciding between two matches and the transition zone between returning the phylogenetically informative results and the Short Branch Attraction results is expected to be sharper. 1,000 nucleotides is a common sequence length and should demonstrate what the BLAST program can be expected to do in the case of typical real gene sequence data. 200 nucleotides is the length of typical environmental sequences and will be applicable to phylotyping or ribotyping analyses. A 29 short sequence length should provide less data and therefore less information when choosing between the various possible matches. Thus the shorter sequence length is expected to show a more gradual transition between the Short Branch Attraction and the phylogenetically informative states.

2.3 Methods

Nucleotide datasets were simulated using Seq-gen (Rambaut and Grassly, 1997) using four taxa trees, as shown in Figure 1 panel H. Central branch lengths vary from 0.004 to 0.1 substitutions per site and variable branch lengths varying from 0.0001 to 0.1 substitutions per site. The lengths of the 2 invariable branches were held at 0.1 substitutions per site for all simulations. These trees were rooted midway along the central branch, as indicated by the ^, to ensure symmetry. Three replicates of 100 simulated datasets were performed for each tree. Each dataset contained four sequences and was generated using the Jukes Cantor 69 substitution model, because of its simplicity (T. Jukes and Cantor, 1969). Three analyses were performed that vary with respect to sequence length: 10000, 1000, and 200 nucleotides. Each of the four taxa of each dataset were used as query against a database comprised of the other three taxa in the dataset to determine the top scoring BLAST hit in all four possible directions. Classic nucleotide

BLAST, as implemented in blastall 2.2.19 (Altschul et al., 1997), was used. The results for the

100 datasets in a replicate were totaled to produce the percent of time the top scoring BLAST hit returned the sister taxa, which was graphed using rgl, a 3-dimensional graphing package, in R

(Adler and Murdoch, 2012; R Developmental Core Team, 2008). Perl scripts were used to perform repetitive tasks en masse.

2.4 Results and Discussion 30 Figure 2 Panel A shows the results of the top scoring BLAST hit analysis using the simulated datasets of 10000 nucleotides in length and the mapping of branch-length space with respect to BLAST result. This graph is a 2-dimensional projection of a 3-dimensional plot. There are three taxa in each database for the query to possibly match, but BLAST almost always returns either the sister taxa or the Short Branch Attraction taxa. Whenever the sequence belonging to the sister taxon is not returned, the sequence belonging to the slowly evolving taxa is returned. When the sister taxon is returned in 0% of the simulations, then the Short Branch

Attraction artifact occurs in 100% of the simulations.

Branch lengths are equal in the rear right corner of Figure 2 Panel A and as predicted, the top scoring BLAST hit did return the sister taxon with 100% success in these simulations. As branch lengths shorten, either along the central branch or along the peripheral branches, Short

Branch Attraction plays an increasing role until the Short Branch Attraction taxon is returned in

100% of cases. There is a very sharp transition between the two states, as expected with such long sequences. Short Branch Attraction is a major problem when either the central or peripheral branches are one-tenth the length of the invariable branches. It is interesting to note that the effect of short branches is additive so that the effect of decreasing the length of the central and variable branches together is combined (cf. equation 1). When the central and variable branches are both half the length of the invariable branches, indicated in the graph with an asterisk, the

Short Branch Attraction taxon is already returned in approximately 50% of simulations; branches that are the smallest amount shorter return the Short Branch Attraction artifact 100% of the time.

This is cause for concern, because branches that differ by a factor of 2 are not normally considered so heterogeneous as to be a problem. Slightly heterogeneous branch lengths like these are commonplace in real data where both evolutionary rate and time between nodes, can vary. 31 Figure 2: Branch length versus the percent of time the top scoring BLAST hit recapitulates phylogeny. Panel A shows the results of the simulations of 10000 nucleotides in length. The percent of time the top scoring BLAST hit returns the sister taxon is graphed on the Y axis, with

100% meaning that the BLAST search always returns the sister taxon. At 0% the BLAST search always returns Short Branch Attraction; the BLAST search almost never returned the third possibility. Each dot represents the result of 100 simulations. The Z axis gives the length of the central branch, and the X axis the log base 10 of the length of the variable branches. Both the results of the analyses performed using Taxon B as query and those with Taxon D as query are shown, with Taxon B in the bright rainbow colors and D in the darker rainbow colors. The shape of the surface formed by the results is identical for the two analyses, showing that the BLAST hits are reciprocal. For the majority of branch length space, the BLAST search is subject to Short

Branch Attraction. It is only free from this phenomenon when the branches are relatively homogeneous in length. Panel B shows the result of using 1000 nucleotide long sequences in the simulations and demonstrates a more gradual transition between the two states and a smaller region in which the sister taxon is returned at a high frequency. Panel C shows the result of using sequences of 200 nucleotide in length. A much greater proportion of branch length space is now occupied by the transition zone, while the area where the sister taxon is returned the majority of the time is now virtually non-existent.

33 The top scoring BLAST hit analyses were reciprocal, down to the individual simulations, meaning that either Taxa B and D both returned the phylogenetic sister or they both return the

Short Branch Attraction taxon. The observed reciprocity results in the curve of the planes for the two analyses having identical shapes and colocalization in branch length space, as shown in figure 2 Panel A.

When the nucleotide sequences are average in length, 1000 nucleotides per sequence, as shown in Figure 2 Panel B, the transition is less distinct between returning the closely related sequence and the slowly evolving sequence. There is still a region in the rear right of the graph, where branch lengths are equal and BLAST returns the sister taxon 100% of the time, however, this region occupies a slightly smaller portion of branch length space. There is an extended region where BLAST transitions between the two possible answers, where any simulation stands a chance of returning the Short Branch Attraction taxon or the sister taxon, but not the third possible choice. And there is an extensive region of branch length space where Short Branch

Attraction reigns as the most probable top scoring BLAST hit result.

As sequence length decreases even further to 200 nucleotides, as shown in Figure 2 Panel

C, the transition between sister taxon and the slowly evolving taxon becomes even less distinct.

There is no region within the coordinates of this graph where the sister taxon is returned in 100% of the simulations and the region where BLAST returns the sister taxa in the majority of simulations is quite small. There is still a region of branch length space where Short Branch

Attraction is returned in 100% of simulations, but this region is less than that seen for the other sequence lengths. There is a much greater proportion of branch length space where it is possible to return either the slowly evolving taxa or the sister taxa as the top hit. This means that shorter sequence lengths are less likely to suffer from Short Branch Attraction, but are also less likely to 34 return the sister taxa. Because of the minuscule amount of information contained in such short sequences, the top BLAST hit of short sequences are increasingly likely to be a result of random chance. The top BLAST hit cannot return any sequence, however, because Taxon C, the third possibility, does not occur even with this short sequence length, when BLAST results seem so intensely ruled by chance, as seen in Figure 3.

It is possible to provide a mathematic approximation to indicate where in branch length space Short Branch Attraction is likely to be a problem, as shown with the solid curved line in

Figure 4, panel A and Equation 2, where Y is set at 0.1 substitutions per site. This mathematical description closely coincides with the observed simulation data in figure 2, as shown by the Xs.

Light colored Xs indicate that the sister taxa is returned in greater than 80% of simulations, while dark Xs indicate that the Short Branch Attraction artifact is returned in greater than 80% of cases. This relationship between branch lengths can be used to alert one to the dangers of Short

Branch Attraction. When the length of the variable branch plus the length of the central branch is less than the length of the invariable branch, then the shortest evolutionary distance in the tree is between the two short branches and Short Branch Attraction is likely to be a problem.

Figure 3: Percent of time Short Branch Attraction is return versus branch length for sequences of

200 nucleotides in length. At 100%, Short Branch Attraction is always returned and occurs when branches are short. The sister is never returned in 100% of simulations, but when branch lengths are equal, the sister taxon is returned the majority of the time, illustrated by the minimum point on this graph. This graph is the inverse of the graph in Figure 2 Panel C, where the percent of time the sister taxa is returned, is graphed.

Figure 4 panel B graphs Equation 2 using values of the length of the invariable branch, Y, ranging from 0.01 to 0.15. The invariable branch is the sister to the variable branch, so when this value is small, the shortest evolutionary distance is to the sister taxon in much of branch length space. As the length of the invariable branch increases, the proportion of branch length space where the shortest total branch length is between the two variable branches, increases. Therefore,

Short Branch Attraction dominates branch length space when the invariable branch is long, but not when the invariable branch is short. 36 LBA is a well-known phenomenon in which heterogeneous branch lengths introduce artifacts into phylogenetic reconstructions, but this is not what is happening here in this phenetic reconstruction. The longest branches are not saturated with substitutions and they are behaving as expected in analyses when used as query. In 100% of cases where Short Branch Attraction is shown grouping the two short branches together, the two longer invariable branches are not subject to this phenomenon and instead return the sister taxon as the top scoring BLAST hit (data not shown). This confirms that LBA is not the cause of the phenomenon. This observation also suggests a means to detect Short Branch Attraction. In the four taxa tree shown in Figure 1,

Panel H, the two taxa picking each other as top scoring BLAST hit as a result of Short Branch

Attraction are each the top scoring BLAST hit of their true sister taxon, as shown in Figure 4

Panel C. Therefore, using all taxa included in the analysis as queries will result in non-reciprocal best BLAST hit relationships with the taxa giving rise to Short Branch Attraction. If the sister taxon also returns the taxon of interest as the top scoring BLAST hit, then this reveals possible

Short Branch Attraction and a phylogenetic tree should be constructed to elucidate the evolutionary history of the gene of interest. It is interesting to note that the program DarkHorse is already attempting to promote matches to more closely related taxa above those to more distantly related ones (Podell and Gaasterland, 2007). Doing so will likely reduce the effect of Short

Branch Attraction in BLAST-based analyses.

37 Figure 4: Ways to determine if Short Branch Attraction is a problem in a particular BLAST search. The solid line in Panel A plots Equation 2: Y = Z + X, where Y is set to 0.1 substitutions per site. The region filled with dark colored Xs shows where in branch length space Short

Branch Attraction occurs when the invariable branches are of length 0.1 substitutions per site.

The region filled with the light colored Xs shows where in branch length space the sister taxon is returned. The Largest Xs represent areas where all three simulations return the result greater than

80% of the time. The medium size Xs represent areas where only the simulations of 10000 and

1000 nucleotides in length return the result greater than 80% of the time. The smallest Xs represent areas where only the simulations of 10000 nucleotides in length return the result in greater than 80% of cases. If data falls into the region of branch length space with the dark colored Xs, top BLAST hit should not be used to inform on phylogeny. Panel B plots Equation 2 for values of Y between 0.01 and 0.15 substitutions per site. The area under the curve represents where Short Branch Attraction is expected with BLAST searches. The area above the curve represents where BLAST is expected to return the sister taxon as the top scoring hit. As the length of the invariable branch, Y, is increased, an increasing proportion of branch length space is dominated by Short Branch Attraction. In Panel C, the sister taxon indicates when Short

Branch Attraction might be occurring. In instances of Short Branch Attraction, both the sister taxon and the other slowly evolving taxon return the taxon of interest as the top scoring BLAST hit.

2.5 Conclusion

If BLAST is used only for its original purposes of detecting homology and finding the most similar sequence from a database, then no issues result from returning a sequence that is not the most closely related one. When using BLAST, or any other measure of similarity, as a proxy for phylogeny, slowly evolving taxa attract each other as top scoring hits, creating Short Branch

Attraction. Erroneous detection of HGT events or incorrect phylotyping can result from this phenomenon. Slowly evolving paralogs due to an increase in rate heterogeneity, but a similar overall rate of substitution, are more likely to be assembled into gene families. Paralogs with a more homogeneous substitution rate or an overall increase in substitution rate might be systematically left out of gene family assemblies.

Short Branch Attraction, a phenetic artifact, is different from LBA, a phylogenetic artifact and is the result of slowly evolving sequences retaining the same character state. Short

Branch Attraction can occur with as little as a factor of two in terms of branch length heterogeneity and contributes to changing results in the detection of putative gene transfers as the non-redundant database grows. To test for Short Branch Attraction one should use all target sequences as queries, or at least the homolog from the expected sister taxon. If the sequence of interest is the best (non-reciprocal) BLAST hit from another homolog, further analyses need to be performed to elucidate the phylogenetic neighbor.

Chapter 3: The Fundamental Bipartition of Cellular Life and its Implications for Systematics and Taxonomy

3.1. Abstract

In 1989, the root of the cellular tree of life was placed on the bacterial branch using ancient duplicated genes, meaning that the Archaea and eukaryotic nucleocytoplasm form a clade. A quarter century of further investigation has upheld this finding. Because this split is the first one in the cellular tree of life, it represents a taxonomic ranking higher than that of the domain, which we here term the realm. There are two realms of life, just as there are two sides of the cellular tree of life. We name the clade containing the Archaea and eukaryotic nucleocytoplasm the Ibisii based on a number of shared characteristics having to do with information processing and translation. The Bacteria are the only known members of the other realm, which we call the Bacterii.

The taxonomy at the highest levels of the cellular tree of life has been muddled in recent years by the discovery of new major lineages and by rampant horizontal gene transfer between lineages. Here an attempt is made to extend Hennig’s principles of taxonomy to apply to the entire cellular tree of life. We call attention to the importance of paraphyletic grades and federations (groups joined by frequent horizontal gene transfer) to evolution. Finally, we revisit the domain level and suggest adjustments to the taxonomy that may be necessary, if the Archaea prove to be paraphyletic and in fact represent a paraphyletic grade of life.

3.2. Principles of Taxonomic Classification

In 1965 Hennig established the gold standard of modern systematic classification as one that reflects phylogeny, names only monophyletic groups, provides a name for the clades 41

resulting from every bifurcation in the tree, and names taxonomic rankings that contain clades approximately equal in age (Hennig, 1965c). These principles were developed in insects, but work well with most multicellular Eukarya. However, Horizontal Gene Transfer (HGT), fusion of lineages, and the absence of polarized characters complicate matters when trying to classify all of cellular life, leading some to question the usefulness of a bifurcating tree of life (Doolittle and Brunet, 2016). To work around the first two complications, it has been suggested that primacy should be given to one gene or a small subset of genes (Ciccarelli et al., 2006). But using only one gene ignores the contribution of every other gene in the genome and often makes resolution difficult. Using a concatenation still ignores the majority of the genome and has the added disadvantage that if genes with different histories are used, the resulting tree may reflect neither the history of the organisms nor the history of the genes (Gogarten and Townsend, 2005).

In an ideal approach, any taxonomic unit should be comprised of a group of organisms that have been traveling together through shared ancestry and all forms of gene exchange (sex and HGT, which is often mediated by the mobilome and at the whim of evolutionary forces playing out in the virosphere (Villarreal, 2016)) for a long enough period of time so that they separate themselves from other organisms in gene content, way of life, and exhibit loose reciprocal monophyly, i.e. for any given gene, accepted taxa are monophyletic and do not violate the monophyly of other accepted taxa, excepting where it is reasonable that these violations occurred due to HGT. Which parts and how much of the genome reflects the organismal history will necessarily vary over the tree of life and will be up to the discretion of each field to determine.

This is similar to the bacterial species definition given by Dykhuizen and Green in 1991

(Dykhuizen and Green, 1991), in that HGT replaces sex as the mechanism for gene exchange in 42 some organisms. But while Dykhuizen and Green restricted HGT to primarily being of importance within a species, we acknowledge that it is a major force driving evolution that may affect a large fraction of the genome and having affected all levels of the tree of life, is not restricted to species. As such, a true representation of evolutionary history of life must take HGT into account. However, because HGT is more frequent between close relatives, it tends to reinforce relationships, building them up, not tearing them down (Andam et al., 2010a; Pace et al., 2012)

HGT functions at two levels. At the species level, mentioned above, it allows close relatives to share genes in a manner analogous to sex. The second, broader level, is across distantly related groups in close physical contact, often due to shared niche or close ecological association, creating highways of gene sharing (Beiko et al., 2005). With our modified taxonomic principles, highways of gene sharing are allowed without any addition to the taxonomy, violating strict reciprocal gene monophyly, up until the point where the distinction between the two contributing taxa is eroded. This is necessarily a subjective criteria and will change as science progresses and the evolutionary history of life on Earth is better understood.

Only when gene exchange is so prevalent that the true organismal history cannot be detangled do

Hennig’s taxonomic principles break down and our extensions are needed.

Often over time, this intense gene exchange will wane or the taxa will become so thoroughly mixed that they become one organism (Timmis et al., 2004), so that loose reciprocal monophyly is restored and our taxonomic principles hold again, as with Eukarya after incorporation of the mitochondria. For an depth discussion on how to treat such groupings in phylogenetics, see (Mindell, 1992). 43 For example, the number of genes horizontally transferred between Thermotogae and

Archaea through a highway of gene sharing is on the order of 10% of the genome (Zhaxybayeva et al., 2009c) and gene phylogenies place these genes inside the Archaea. Nonetheless, these

HGT events are not sufficient to erode the distinguishing bacterial characteristics of the taxon

(Zhaxybayeva et al., 2009c). Therefore, they have not violated loose reciprocal monophyly and are still distinct taxa.

On the other hand, with our same Thermotoga example, HGT with Clostridiae was so rampant that the majority of Thermotogae genes are placed among clostridial homologs. One could use special genes, such as genes encoding the translation machinery that are only infrequently transferred between divergent organisms to provide a reference tree relative to which gene transfer events can be mapped (Williams et al., 2011). However, it seems premature to give one cellular process primacy in defining taxonomy. Rather than arbitrarily assigning primacy to one gene or a small subset of genes, we prefer to remain agnostic, leaving the split between Thermotogae, Aquificae, and Clostridiae unresolved.

Two similar cases were described as highways of gene sharing overwhelming the phylogenetic signal retained in the genome: Aquificae (Boussau et al., 2008) and Sphaerochaeta

(Caro-Quintero et al., 2012). These highways of gene sharing between different bacterial phyla make it difficult to determine some between phyla relationships in Bacteria, and we consider it prudent to leave these relationships unresolved at present.

When a group has spread out into multiple niches and begun to differentiate into multiple groups, but does not yet meet the criteria of loose reciprocal monophyly exhibiting sufficient phylogenetic distinction to tease the two apart, then they are to be considered sub-taxa of a taxon 44 in the process of diverging into two or more; conversely, this divergence may be the prelude to future condensation back into one single taxon through frequent gene exchange (Sheppard et al.,

2008; Zhaxybayeva et al., 2009a) or through extinction of groups with derived characters. If the sub-taxa continue evolving independently, then they will eventually resolve into loosely reciprocally monophyletic groups. This applies to all taxonomic rankings, so that any level of ranking in which taxon amalgamation results in unbreakable polytomies is not a valid ranking worth delineating. But, the conglomeration is in some ways a unit and as such, equally deserving of a name as paraphyletic grades, because they represent a lineage fusion event in life’s evolutionary history. To ignore them is to brush aside a significant part of evolution on Earth.

Clades are groups that share synapomorphies. Paraphyletic grades are groups that share

Sympleisomorphies. But what are groups that share horizontally transferred genes through highways of gene sharing (including endosymbiosis followed by endosymbiotic gene transfer)?

We propose to call them federations, because they bring together and link formerly separate groups, just like formerly independent states are joined politically in a federation of states. A monophyletic federations is thus composed of a composite ancestor, itself resulting from a reticulation in the tree of life, and all of its descendants (Mindell, 1992). This is an extension of the term used for Prochlorococcus, a group of bacteria united by horizontal gene transfer (Biller et al., 2015).

The Thermotogae and Clostridia form a federation linked by a massive highway of HGT so great that it has all but erased the underlying vertical branching pattern of the organisms that engaged in these HGT events. Therefore, it is useful to name this federation and others that we

45 can observe in nature. Lichen is a federation of algae and fungi (de Bary, 1879). The Eukarya are a federation composed of the Bacteria endosymbiont that gave rise to the mitochondria and a pre-mitochondriate stem-group Eukaryeon (Sagan, 1967). The list goes on and on. However, if the reticulation in the tree is easily teased apart through current phylogenetics and the partners are not interdependent, then it is not necessary to create a federation.

3.3. The Clade Comprising Archaea and the Eukaryotic Nucleocytoplasm

When discussing Eukarya, we recognize that Bacteria (in the form of mitochondria and plastids, and through individually transferred genes(Pittis and Gabaldón, 2016)) made important contribution to the modern eukaryal cell, and that the uptake of the mitochondrial symbionts may have triggered key events in Eukaryogenesis. Summarizing both the symbiont and host contributions to the eukaryotic cell, a rooted net undoubtedly is an appropriate representation of gene flow (Margulis, 1995; Rivera and Lake, 2004; Williams et al., 2011). Nevertheless,

Bacteria derived cell organelles remain clearly distinct from the nucleocytoplasmic component of eukaryal cells and genes of bacterial origin are usually distinguishable from those of host origin. Hence, hereafter only the nucleocytoplasmic component will be referred to as Eukarya.

In 1989, the tree of life was first rooted on the bacterial branch using the ancient gene duplication of the catalytic and non-catalytic subunits of the ATPsynthase (Gogarten et al.,

1989). This split was suggested a decade prior when it appeared in a phylogenetic tree of the 5S subunit of the ribosome (Hori et al., 1982; Hori and Osawa, 1987, 1979) and exhibited shared primary sequence in a well known loop in region A and the base-paired region B of the 5S rRNA

(Hori et al., 1982). Similarities between Archaea and Eukarya were also observed in the 46 sequences of L7/L12-type acidic ribosomal protein, ferredoxins, RNA polymerase, and the sequence of initiator tRNA ((Hori et al., 1982) and references therein). Similarities are also present in the Signal Recognition Particle (SRP), the exosome, a number of ribosomal proteins, and DNA replication, including helicase ((Gribaldo and Brochier-Armanet, 2006) and references therein). Additionlly, Archaea and Eukarya share DNA polymerase delta (Olsen and Woese,

1997). Unfortunately at the time these molecular trees remained unrooted, so it was not until the ancient gene duplication in the ATPsynthase was used that the divide of cellular life into

Bacteria on one side and Eukarya and Archaea on the other was recognized as a proper clade

(Gogarten et al., 1989). The Eukarya and the Archaea were shown to share two synapomorphies in the ATPsynthase: the decay of the walker motif in the Rossmann fold of the non-catalytic subunit (Zhaxybayeva and Gogarten, 2007), and a large insertion in the catalytic subunit

(Gogarten et al., 1989). The 1989 paper had far-reaching implications in evolutionary thought throughout biology. A problem is that the authors, including one of the authors of the current manuscript (JPG), did not name their discovery and we have forever since been relegated to describing this aboriginal bifurcation every time we wish to mention it. Given that this is the first split of all cellular life that resulted in forms surviving to present day, we often find ourselves with need to mention this bifurcation.

The rooting of the tree of life was confirmed by analysis of additional ancient gene duplication events, starting with the elongation factors Tu and G (Baldauf et al., 1996a; Iwabe et al., 1989), the signal recognition particle (SRP) and the SRP receptor protein (SR) (Gribaldo and

Cammarano, 1998), and different aminoacyl tRNA synthetases. When the isoleucyl-tRNA synthetase was rooted using two of its ancient paralogs, the valyl- and leucyl-tRNA synthetases,

47 the Bacteria were again shown to diverge first, leaving Archaea and Eukarya as sister taxa

(Brown and Doolittle, 1995). The tryptophanyl- and tyrosyl-tRNA synthetases were reciprocally rooted and both exhibited a rooting along the bacterial branch (Brown et al., 1997).

Fournier and Gogarten (Fournier and Gogarten, 2010) used amino acid composition bias in ribosomal proteins to root the ribosomal tree of life and again the root was squarely placed along the bacterial branch, and again the taxon formed by Eukarya and Archaea went without a name. To be able to discuss a phylogenetic tree, all monophyletic groups, clades, must have a name (Hennig, 1966). Even if the monophyly of each individual domain is still in question, the monophyly of the clade comprising the Archaea and Eukarya is almost universally agreed upon, as are a number of shared characters, including the use of TATA-binding proteins (Marsh et al.,

1994) and the RNA polymerase (Huet et al., 1983).

In 1987, T. Cavalier-Smith first gave name to a clade containing the Eukarya and

Archaea, two years before Gogarten et al. and Iwabe et al. rooted the tree of life. Cavalier-Smith called them the Neomura, or new-wall, based on an almost unanimously rejected hypothesis that the root of life is within the Gram Negative Bacteria and that Eukarya and Archaea therefore share the derived trait of having lost muramic acid from their cell walls. There is no evidence to suggest that having the ability to synthesize muramic acid is the ancestral state of life, which was a significant factor in Neomura’s inability to stick as a name; the other factor being Cavalier-

Smith’s controversial Gram Negative bacterial root of the tree of life (Cavalier-Smith, 1987).

29 years later google can produce hits to only 160 articles that contain the word

Neomura, 20 by Cavalier-Smith himself. Even the man who coined the term acknowledges that it has not caught on (Cavalier-Smith, 2014). The support for placing the root of the tree of life on 48 the bacterial branch falsifies Neomura as a hypothesis. We thus consider Neomura inappropriate as a name and submit that it is time for a new name. A name with a hypothesis behind it that is scientifically supported by data; a name that fits and has a chance of taking off into the memetosphere.

27 years after the fact, we are putting forth this manuscript in an effort to finally undo the injustice that was done to the clade comprising the Archaea and the Eukarya by offering them a proper, fitting name. Recently there has been a movement to do away with naming the rankings in taxonomy (Group, 1998), because rankings are supposed to indicate clades of a similar age

(Hennig, 1966, 1965c), but often do not. But because this is the first split in cellular life, involving only two clades of similar age, we indeed have a rank deserving of a name. Therefore, we propose that this taxonomic level be called the realm, because it is composed of many kingdoms and falls between empire and domain in taxonomic classification schemes. There are two realms of life, one of which is composed of the Bacteria and the other the Eukarya and

Archaea. Finally, we have chosen the suffix -ii for all proper taxonomic names belonging to the realm ranking.

When choosing a name for the realm containing the Archaea and the Eukarya, we focus on traits present in all members of this clade and in the clade’s common ancestor, and that are exclusive to this clade. Not many features fit these criteria, but the majority that do have to do with the machinery of replication, transcription, and translation. This machinery allows the cells to process the information stored in DNA into functional RNA and protein molecules. Synonyms for information processing include cognition, knowledge, comprehension, and reasoning. The

Archaea and Eukarya share the baroque machinery used in what is in essence their cognition at 49 the cellular level. It is not that their machinery is necessarily better than that of the Bacteria, and in many ways the complexity seems gratuitous, but that it is shared by all members of this clade, was present in the clade’s common ancestor, and is not found outside the clade. Undoubtedly some of the individual genes that make up this shared machinery were present in LUCA and lost in Bacteria. Currently we cannot determine which of the many shared traits fall into this category, but others will certainly prove to be synapomorphies. By choosing a name that encompasses the entire cognition of these cells, we are resting our name on a collection of genes and not just a single gene. But finding a name based on cellular cognition is a challenge.

We looked to ancient Egypt for a name, because it is the first recorded realm of human civilization, just like the realms of life represent the first bipartition in cellular life. The Egyptian god of cognition, knowledge, comprehension, reasoning, and wisdom is Dhwty, a god named after the ibis. Owing to the difficulty of pronouncing the name of this ancient Egyptian god, the realm suffix -ii was added to Ibis, giving us Ibisii. Thus we propose to name the realm containing the Archaea and the Eukarya the Ibisii. It is interesting to note that the bill of the ibis, the symbol of Dhwty, is the same shape as the TATA Binding Protein (TBP), which binds to the promoter of

DNA and recruits transcription factors and the RNA polymerase in all Ibisii. TBP curves around the DNA, like the bill of an ibis, to initiate transcription. Also, the word transcription comes from the word scribe, or writing. The Ancient Egyptians are famous for their writing, the god of which is Dhwty. This leads us back to Ibisii and the wise ibis as the perfect symbol for a clade based on information processing and writing.

Finally, we propose that the other half of life, comprising only the domain Bacteria, be called the Bacterii. It is possible that some as yet unknown domain that groups on the bacterial 50 side of the tree may be hiding in the remote regions of the biosphere, as suggested by (Hug, et al.

2016) or that future studies of existing Bacteria may result in splitting Bacteria into multiple domains, possibly confirming previously reported deep splits (Battistuzzi and Hedges, 2009;

Boussau et al., 2008). If either occurs, then these additional domains would too belong to the

Bacterii, just like if a hidden domain is discovered that is not an Eukaryeon and not an Archeon, but branches on the ibisial side of the tree, would too belong to the Ibisii, because it is the split between Bacteria and Archaea/Eukarya that we are delineating. It is conceivable that there will come a time when additional levels of classification are needed between realm and domain to describe additional splits in the tree of life.

3.4. The Domain Level

While the question of how many realms of life there are was solved by Gogarten et al.

(Gogarten et al., 1989) and Iwabe et al. (Iwabe et al., 1989) back in 1989, the question of how many domains of life (sensu (Woese et al., 1990)) there are is still under debate. Woese et al. distinguished and named three domains, shown in Figure 1, Panel A, based on unrooted 16S rRNA grouped by SAB similarity criteria. In doing so they found a group of non-nucleated cells that was as different from typical Bacteria as Eukarya. The authors hypothesized that this taxon was one of three aboriginal descendants of a primitive ancestor called the progenote, explicitly rejecting the idea that Archaea could be considered “Proto-Eukaryotes” (Woese et al., 1978); this was superseded by Gogarten et al. (Gogarten et al., 1989) and Iwabe et al. (Iwabe et al., 1989) when Archaea were shown to be the sister group to Eukarya. The validity of Woese’s findings were first called into question on the basis of computer simulations that suggested that SAB 51 values might be virtually meaningless (Hori et al., 1982) and change in substitution rates in the

16S exaggerating the difference between Archaea and Eukarya (Evolution of Life - Fossils,

Molecules and Culture | Syozo Osawa | Springer, n.d.), but the 5S ribosomal phylogeny also found this non-nucleated taxon to be distinct from Bacteria (Hori et al., 1982; Hori and Osawa,

1987, 1979) and showed it to be monophyletic (Hori and Osawa, 1987). Hori named this group the metabacteria (Hori and Osawa, 1979), but that name did not catch on. Woese had historical precedence on his side when he first named them the Archaebacteria. He later changed it to

Archaea, which is most popular today. Both of Woese’s names were based on the later rejected hypothesis that Archaea directly emerged from an ancient paraphyletic grade of life, the progenote, that also gave rise to both the Bacteria and the Eukarya (Kandler, 1995; Woese et al.,

1978). Hori turned out to be correct when it came to Archaea not being old, but the inappropriate name remains to this day.

In 1990, Woese wrote, “The archaebacteria are called Archaea to denote their apparent primitive nature (vis a vis the eukaryotes in particular).” While there is no way of knowing precisely what Woese meant by this, one interpretation is that the term Archaea describes a paraphyletic group defined by shared primitive characters. Given this, if Archaea are found to be paraphyletic as has been argued in recent years (Cox et al., 2008; Foster et al., 2009; Spang et al.,

2015), then the fact that Woese had an inkling that the term describes a paraphyletic group lends strong support for the term to revert to the name of a paraphyletic grade. On a side note, paraphyletic grades are not proper taxonomic units and should not be used as such; systematics sensu Hennig utilizes only proper clades in assigning taxonomic names (Hennig, 1975b, 1965d).

Paraphyletic grades are given non-taxonomic names. 52 Recently several studies (Cox et al., 2008; Foster et al., 2009; Lasek-Nesselquist and

Gogarten, 2013b; Spang et al., 2015; Williams and Embley, 2014) have called into question whether or not the Archaea are monophyletic, or whether the Eukarya actually group within the

Archaea, as shown in Figure 1, Panels B, C, and D. If the Archaea are paraphyletic, then this is not a proper taxonomic group, and a four (Figure 1 Panel B), five (Figure 1 Panel C), six (Figure

1 Panel D), or more domain system would be needed. Depending on which particular tree wins out, Bacteria, Crenarchaea or Proteoarchaea, Euryarchaea, Eukarya, Lokiarchaea, DPANN, and possibly any number of the newly sequenced Archaea may all be at the domain level, resulting in three or more ibisial domains. The name Archaea as a level of taxonomy would have to go, but would persist as designation for a paraphyletic grade within the Ibisii.

Trees showing paraphyletic Archaea have been described as 2 Domain trees (Embley and

Williams, 2015; Gribaldo et al., 2010; Koonin, 2015; Williams et al., 2013). This in essence suffers from the same problems that were faced back when the domain level was created (Woese et al., 1990). With only 2 Domains of life, Eukarya are demoted to the level of kingdom, or possibly a subdomain. Animals and plants are then demoted from their status as kingdoms, which they have held for centuries. Long recognized phyla would also be demoted and the shifting of taxonomic categories would continue throughout the entire ranking system. Assigning

Eukarya to the subdomain category is also problematic, because of the bipartitions between

Eukarya and the classical kingdoms of plants, animals, and fungi that are currently being delineated. There just are not enough intermediate levels to satisfy the bipartition pattern.

Therefore, we prefer to retain Eukarya as a domain and have 4 or more domains, depending on the precise topology of the Ibisii. The question is not two or three domains, but three or more. 53 Certain 2-domain papers have gone so far as to suggest the Eukarya are unfit to hold the ranking of domain, because they result from a fusion between two lineages and are a federation

(Williams et al., 2013). We reject this idea for two reasons. Firstly, lineage fusions may occur throughout the tree of life (Lake et al., 2015) and such level of pervasiveness combined with throwing out all resulting taxonomic units would necessitate throwing out the entire tree.

Secondly, at some point after the fusion, gene exchange with both parental populations was severed, resulting in a monophyletic eukaryal clade that has been evolving independently for over a billion years. Once loose reciprocal monophyly has been restored, as is the case with the

Eukarya, then taxonomic efforts should proceed as normal, as described in the Principles of

Taxonomic Classification, delineated earlier in this work.

There is also data and scientific evidence suggesting that the Archaea are monophyletic and group as sister taxa to the Eukarya (Baldauf et al., 1996b; Brown et al., 1997; Brown and

Doolittle, 1995; Gribaldo and Cammarano, 1998; Hori and Osawa, 1987). If this proves to be correct, then the name Archaea would remain, since it would be a proper monophyletic group.

Woese would still have first claim to their discovery and the three domain system would hold.

This, however, is a separate issue from that of the realm.

3.5. The Paraphyletic Grade Archaea

Mayr’s concept of ecofunctional adaptiogenetics of paraphyletic groups is no longer acceptable for naming taxonomic groups (Hennig, 1975b), and rightly so, but has been repurposed as the term evolutionary grade (Huxley, 1959b). There are two types of grades, polyphyletic and paraphyletic. Polyphyletic grades are virtually useless, because they are joined by convergent traits and therefore have no predictive power. Paraphyletic grades are useful when 54 they define an ancestral state of a group, because knowing the ancestral state has its value in determining evolutionary history. If a group is proven to be paraphyletic, then the exclusive traits they share, symplesiomorphies, are ancestral to the group.

The question of what it means to be an Archaeon is a complex question that depends upon the topology of the tree of life. In the 3-domain tree, shown in Figure 1 Panel A, Archaea are a domain. With the 4-domain tree shown in Figure 1 Panel B, Archaea do not form a clade and are therefore not eligible as a name for any taxonomic unit. Just like the term amniote replaced the term reptile as a taxonomic term when reptiles were shown to be a paraphyletic grade of life rather than a clade, the term Archaea must be replaced in taxonomic classification.

We do, however, still mean something when we use the term Archaea. Archaea is still the name that describes the Crenarchaea, the Euryarchaea, and ancestor of the two, but not the Eukarya.

Archaea possess a number of similarities to each other that they do not share with Eukarya, such as ether-linked lipid membranes and derived tRNA and rRNAs (Woese et al., 1978). Under this topology, these similarities are either sympleisomorphies, meaning that the shared traits represent the ancestral state, or products of HGT, which means the traits can be shared between domains; both scenarios are intriguing, because the traits in question are so rarely changed throughout the tree of life. In conclusion, under the condition that the Archaea prove to be paraphyletic, the name Archaea stays only with the paraphyletic grade of organisms it was originally assigned to.

3.6. Summary:

The first bipartition in the cellular tree of life defines two realms of life. These are the

Bacterii, presently composed of only the Bacteria, and the Ibisii, composed of the eukaryotic 55 nucleocytoplasmic component and all of the archaeal domains. If Archaea prove paraphyletic, then the term names a paraphyletic grade composed of prokaryotic Ibisii with ether-linked lipid membranes.

Clades represent the underlying organismal phylogeny. Federations represent HGT, lineage fusion events, and the flow of genes outside of the underlying organismal backbone of the tree of life. Paraphyletic Grades represent the ecofunctional levels and stages of adaptation that life has progressed through in its journey through time. Here we expand the bipartite system that names clades and paraphyletic grades(Huxley, 1959b) to a tripartite system that also names federations, because the combination of all three fully reflects the complexity of the evolutionary history of life on Earth.

56 Figure 1: LUCA (Last Universal Cellular Ancestor, LACA (Last Archaeal Common Ancestor,

LAECA (Last Archaeal Eukaryal Common Ancestor, LECA (Last Eukaryal Common Ancestor).

Panel A: 3-Domain Tree showing the cellular empire and corresponding terminology. In this tree, Archaea are monophyletic. Archaea and Eukarya are the domains of the ibisial realm.

Bacteria is the only domain in the realm Bacterii. Panel B: 4-Domain Tree showing the cellular empire and corresponding terminology. In this tree, Archaea are not a proper taxonomic name, but a paraphyletic grade. Crenarchaea, Euryarchaea, and Eukarya are the domains on the Ibisial side of the tree. Bacteria are the only domain on the Bacterii side of the tree. This topology requires one additional taxonomic level between domain and realm and a name for the clade composed of the Crenarchaea and the Eukarya which we will leave to the discovers of this split to assign. Panel C: 5-Domain tree, if proposed deep branching archaeal lineages are verified as proper clades. Here Lokiarchaea are promoted to a domain (Spang et al., 2015), but there are other possible 5-Domain trees, depending on which Archaea is found to be sister-taxon to the

Eukarya. These trees require 2 additional taxonomic levels and contain two new clades that require names. Panel D: 6-Domain tree, if the so-called DPANN group (Williams and Embley,

2014) is verified as a proper clade and not just a Long Branch Attraction artifact. In this case,

DPANN is also promoted to a domain and thus would require a proper name. This topology requires 3 additional rankings and contains 3 new clades that require naming by the discoverers.

Chapter 4: The Relationship Between Archaea and Eukarya

4.1. Eukaryal Stem Branch Length

This chapter contains one article (Fournier et al., 2011). The article uses three tubulin paralogs that diverged along the eukaryal stem branch to investigate the length of this stem branch as a measure of eukaryal evolution. The chapter also contains an addendum that repeats the analysis using the four histone paralogs, which were also duplicated on the Eukaryal stem branch.

The research presented here was conceptualized with the assistance of Dr. J. Peter

Gogarten and Dr. Greg Fournier. The phyml tree (figure 1) in the article was produced by myself, while the rest of the analyses were carried out by Dr. Greg Fournier and Dr. David

Willams. The research in the addendums was carried out by myself. Dr. J. Peter Gogarten supervised the research and assisted with writing the article. Permission for reprinting the article was granted by Elsevier, see appendix. 59

4.2. Evolution of the Archaea: Emerging Views on Origins and Phylogeny

64 65

4.3. Addendum: Eukaryal Stem Branch Length in Histones

4.3.1. Introduction

Histones are a good candidate protein family for examination when looking for long eukaryal lines of descent. There are 4 histone paralogs, H2A, H2B, H3, H4, common to all

Eukarya, with an archaeal histone homolog outgroup (Bell and White, 2010; Thatcher and

Gorovsky, 1994). Each of these 4 paralogs can therefore provide 4 independent measures of the eukaryal SBL, in addition to the 3 provided by the tubulins.

If phylogenetic reconstructions show much more divergence in eukaryal histones compared to archaeal histones, and long branches along the eukaryal lines compared to branch lengths within the crown group, then Eukarya are uniquely eukaryal and are at least as ancient as

Archaea or have undergone an increased rate of evolution. This will provide a time constraint on the origin of Eukarya and circumstantial evidence in favor of archaeal monophyly, although a polytomy cannot be ruled out. If long branches are not observed, then the circumstantial evidence will be in favor of archaeal paraphyly and the origin of Eukarya from within the

Archaea, because short Eukaryal stem branches are expected under this topology.

4.3.2. Methods

Histone amino acid sequences were downloaded from NCBI and T-COFFEE was used to modify taxa names (Notredame et al., 2000). Deepview was used to create a structural alignment of the four histone subunits (Kaplan and Littlejohn, 2001). Profile alignments in Clustal X 2.0 were used to add histone sequences to the structural alignment (Larkin et al., 2007). Jalview was used to manually adjust the histone alignment (Clamp et al., 2004). Clustal X 2.0 was used to 67 convert alignments to phylip format. Treeview X was used to convert trees to nexus format

(Saldanha, 2004). FigTree1.2.2 was used to visualize trees (Rambaut and Drummond, 2008).

Archaeal histone sequences were used to root trees.

PhyML was used to produce Maximum Likelihood trees (Guindon et al., 2005), using the

WAG substitution model, 100 bootstrap replicates, estimated proportion of invariable sites, 4 categories of substitution rates, and an estimated gamma distribution parameter. MrBayes was run for at least 1000000 generations to estimate Bayesian trees (Huelsenbeck et al., 2001), with the following priors: unconstrained branch lengths, with a mean of 0.1 substitutions per site, 4 categories for the gamma shape with an exponential distribution and a mean of 1.0, and 2 categories of rate substitution. If MrBayes failed to converge by 1000000 generations, additional

250000 generations were run at a time until convergence.

Extreme divergence and short sequences make histone alignments difficult. To judge the quality of the alignment, Maximum Likelihood trees were generated and examined for eukaryal monophyly. Since no one believes that the Eukarya evolved independently more than once or that a Eukaryon has reverted to a prokaryote (Blackstone, 2013), it is universally agreed upon that the Eukarya, and therefore LECA, are monophyletic. LECA is present four times on the histone tree, one for each of the four subunits, and only when all four instances are resolved as monophyletic with high bootstrap support value, will the alignment be accepted.

4.3.3. Results

The histones were easy to align within subunits, but extremely difficult to align between subunits, which was why the structures were used. In order to obtain a high quality alignment, the sequences were first aligned within subunit and then to the structural alignment. The N- 68 terminus of the H2A sequences did not correctly align with the homologous region in the H2A sequence from the structural alignment, so Jalview was used to manually correct this error.

Figure 1 shows that branches within a histone subunit are relatively long compared with those of archaeal histones, indicating copious evolution in isolation from Archaea along the eukaryal line, which indirectly supports monophyletic Archaea, although the very long branches joining subunits could be due to increased evolution associated with gene duplications

(Goodman et al., 1987). The same Maximum Likelihood tree is shown in Figure 1 and 2, unrooted and rooted on the archaeal histones, respectively. Figure 3 shows the same dataset analysed in Mr. Bayes. The Bayesian tree is in complete agreement with the Maximum

Likelihood tree when it comes to long eukaryal stem branches.

Figure 1: Maximum Likelihood tree after manual adjustment of the structural alignment and histone profile alignment. All four instances of LECA are resolved as monophyletic with high bootstrap support value and all four eukaryal SBLs are long. 69

Figure 2: Another view of the Maximum Likelihood tree, with bootstrap values, of the manually adjusted profile structural alignment, this time with the archaeal histones (AH) added. Archaeal histones appear paraphyletic, possibly due to unresolved paralogy. Long branches are exhibited along the eukaryal stem and within the Eukarya.

Figure 3: Bayesian tree of the manually adjusted profile structural alignment with the archaeal histones (AH) added. The posterior probabilities are shown in blue and branch lengths shown in red. The archaeal histone mono or para-phyly is not resolved. As with the Maximum Likelihood tree, long branches are seen along the eukaryal stems and within the eukaryal crowns.

71 4.3.4. Discussion

All 4 eukaryal histones show longer stem branch lengths than the branch lengths observed within the archaeal histones. All 4 eukaryal histone stem branch lengths are longer than their respective eukaryal crown branch lengths. If the rate of evolution were constant, then this would imply that the duration of the eukaryal stem’s existence, from the time the Eukarya split with the Archaea to the time of LECA, when the Eukarya radiated out into its living descendants, is equal to the age of LECA. Since red algae fossils tell us that the time of LECA was prior to 1.2 billion years ago, then the eukaryal stem would be approximately 1.2 billion years old. Summing the two, would indicate that the Eukarya diverged from the Archaea at least 2.4 billion years ago, around the time Archaea first appeared in the fossil record. If this were the case, archaeal paraphyly would be extremely unlikely, because the Archaea would have to be much older than evidence currently supports. This evidence supports the hypothesis of Archaeal monophyly, or possibly a polytomy between Archaea and Eukarya.

Unfortunately branch lengths are the product of both time and evolutionary rate and there is no way to tease the two apart. The branch lengths in the tree indicate that evolutionary rate is not constant, because the branch lengths within the Archaea are so short. Under any tree topology, except one in which the Archaea evolved from within the Eukarya which there is no evidence to support and no one believes, the sum of the length of the branches in the archaeal crown and the archaeal stem would be expected to have branch lengths at least as long as the corresponding measurement for the Eukarya. But the Archaeal stem is very short, making the total archaeal branch length quite a bit shorter than total eukaryal branch length. Given the three topologies under consideration, I must conclude that the Archaea are evolving more slowly than the Eukarya. 72 If the Eukarya are evolving faster than the Archaea, then part of the long stem branch lengths seen in Eukarya is likely due to this increase in rate of evolution. Unfortunately, there is no way of knowing how much of it is due to the increase rate of evolution and how much is due to time. The shorter the time, the higher the rate of evolution. But as estimates of divergence time get to the shortest extremes that have been suggested in the literature, the rate of evolution must increase so much as to seem unbelievable. Therefore, at the very least, I argue that scenarios where the Archaea diverged from the Eukarya shortly before the lineages descending from

LECA radiated from each other, can be ruled out.

4.3.5. Conclusion

The 4 measurements of long eukaryal stem branch length in the 4 histone subunits are in agreement with the 3 measurements of long eukaryal stem branch length in the 3 tubulin subunits. A long eukaryal stem branch is a common feature in phylogenetic trees (Derelle and

Lang, 2012; Fournier et al., 2011). And although variation in rate of evolution prevents placing a time estimate on these long stem branches, they cannot be ignored. This is a piece of the puzzle that needs to be considered when discussing possible scenarios for the origin of the Eukarya.

The extreme divergence within Eukarya indirectly supports the monophyletic Archaea hypothesis or archaeal polytomy. It is possible that the two archaeal kingdoms diverged from each other before the split with Eukarya, but remained a cohesive unit through continual HGT. If that were the case, some genes could show support for monophyletic Archaea, others for paraphyletic Archaea, and they could both be right. It is also possible that these two events occurred so closely in time, and so long ago, that we will never be able to recover with certainty 73 which split occurred first. But whatever the case, Eukarya have evolved in isolation from

Archaea for a long period of time.

Chapter 5: The Watershed of life

5.1. Abstract

Additional data do not seem to be solving the problem of whether the Eukarya evolved from within the Archaea or as sister to the Archaea. In light of this, the problem may not be the data, but how we interpret the data. Here, life is described as a watershed with two types of signals, one from vertical descent and the other from Horizontal Gene Transfer, giving rise to the complex patterns we see in molecular gene trees. This new perspective sheds light on the different signatures of the two signal types, which could lead researchers to tease the two apart sufficiently to answer the question of whether the Archaea are indeed a monophyletic group.

5.2. The Watershed of Life

The problem is whether the Archaea are paraphyletic or monophyletic and the intense debate with equal evidence on both sides. My attempt in Chapter 4 at using stem branch length to resolve the issue is inconclusive, even with positive results, because the nature of branch length is a factor of two variables, time and rate of evolution, that cannot be teased apart (T. H. Jukes and Cantor, 1969). As I have done this work, others have also gathered additional evidence regarding this question, in the form of genes that were formally thought to be specific to Eukarya being found patchily distributed in Archaea. But even though the evidence has increased, there has been no progress towards an answer. We remain just as uncertain as ever and researchers have even asked if we are at a phylogenetic impasse; if we have a question here that cannot ever be answered. This conundrum led me and my advisor, Dr. Gogarten, to consider the nature of cellular life itself. Might the problem not be the data, but how the data is interpreted? Is a simple 75

bifurcating tree the best representation of cellular life, or is a rooted net of life (Olendzenski and

Gogarten, 2009; Williams et al., 2011) the better metaphor?

One of the issues with determining whether the Eukarya group within the Archaea or as sister taxon to the Archaea, is that the splits between the Euryarchaea, Crenarchaea, and Eukarya happened with relatively few molecular changes to support them (Rochette et al., 2014). In addition, these splits likely occurred over two billion years ago, allowing ample time to erode the phylogenetic signal. Add to that the web of biased HGT (Andam et al., 2010b; Lester et al.,

2006) and the picture is very muddled. Perhaps reexamining the existing evidence in light of a new model, that of the Watershed of Life, will shed new light on the problem. The two alternative hypotheses are depicted in Figure 1 Panels A and B: a monophyletic and a paraphyletic Archaea. Both result in data that produce similarly muddled messes as scientists are currently observing with regards to the Archaea.

Figure 1: The underlying webs of HGT in Figure 1 Panels A and B are depicted as a watershed that runs underneath the tree of vertical descent. Each set of colored lines represents a gene family. The shades of grey represent the underlying watershed of HGT.

77 In both Panels A and B of Figure 1, there is an underlying mostly, but not strictly, bifurcating tree that depicts the unbroken line of vertical descent of cellular life. The lightest shade of grey in Panel A and B of Figure 1 represents the taxon boundaries and can be thought of as similar to the species boundaries with multiple genes (represented as colored lines, coexisting within each species) except that the focus of evolution is zoomed out to the various kingdoms or domains. The kingdoms of Eukarya are not individualized, because current research suggests that

LECA was a complex modern Eukaryeon (Koumandou et al., 2013), so the core features of the

Eukarya evolved on the stem eukaryal branch. The kingdoms of Bacteria are simplified into a polytomy comprising five kingdoms to depict the current consensus that Bacteria underwent a rapid radiation at the base of the domain (Hori and Osawa, 1987); the actual number of bacterial kingdoms and their phylogenetic relationship to each other matters little in this case, because we are interested in the evolution of the Ibisii.

The watersheds of HGT are shown as multi-dimensional surfaces, depicted as varying shades of grey. The lightest shade can be thought of as the deepest region of this surface, where genes flow the most freely, like water flows most swiftly through deeper canals. This represents genes passing from ancestor to descendant in a vertical manner. The second lightest shade of grey represents the second deepest region of the surface, where genes flow quickly due to highways of HGT. These canals link kingdoms that more readily share genes. The next shade of grey represents a shallower region of the canal, where genes can flow, but only rarely. HGT, represented by dashed lines, occasionally reaches across vast expanses, represented in these figures by the fact that only once does a gene cross the darker region of grey. The black represents dry land, where genes cannot flow and are thus not exchanged. The challenge with

78 this multi-dimensional watershed of HGT is that it can change over time. For example, the acquisition of the mitochondria by the Eukarya, represented by the channel connecting Eukarya and Bacteria, made HGT from Bacteria to Eukarya more conducive, resulting in additional gene transfer events traveling through the channel. The sum of the watershed of HGT through time is what is important in fully characterizing HGT. Unfortunately, this integral is difficult or even impossible to calculate, obscuring our ability to subtract it out from the big evolutionary picture.

That is why parsing out the vertical signal in trees is so difficult.

Everything within the lightest shade, or the quickest flowing canal, represents vertical descent and is left out of Panels A and H of Figure 2, which depict only the underlying watershed of HGT. HGT tends to reinforce cellular ancestral relationships, because homologous gene replacement occurs more frequently between close relatives (Andam et al., 2010b).

Therefore, the waterways are deepest surrounding the lines of the species tree. In addition to biased HGT, there are also highways of gene sharing, creating links of rapidly flowing genes between parts of the tree that are connected for reasons other than recent shared ancestry (Beiko et al., 2005), where the underlying web of HGT does not closely mimic the cellular tree.

The ribosomal phylogeny is often assumed to represent only the vertical signal in the data. Therefore, a hypothetical ribosomal phylogeny, shown by the black line, is used in Figure 1

Panels A and B to represent the purely vertical signal. This signal is isolated in Figure 2 Panels B and I. Together with the underlying watershed of HGT shown in Figure 2 Panel B, they make up total evolutionary history of all the genes in the tree of life.

Panels A and B of Figure 1 show the two possibilities for how the tree of vertical descent and the underlying web of HGT interact to produce the molecular patterns that can be observed

79 in cellular life today. In both possibilities, vertical descent and HGT play significant roles in the history of cellular evolution. Figure 1 Panel A, represents the monophyletic, or 3-domain, hypothesis, in which all three domains are monophyletic and the paraphyletic signal is the result of HGT. Figure 1 Panel B, represents the paraphyletic Archaea, the so called “2 Domain” (but really 4 or more), or Eocyte (Cox et al., 2008; Poole and Neumann, 2011) hypothesis, in which the Eukarya evolve from within the Archaea and the signal linking Crenarchaea and Euryarchaea is a result of HGT and of sympleisomorphies inherited from their shared ancestor, but lost in

Eukarya.

Each of Panels C through N of Figure 2 depict the molecular history of one gene found at the base of the cellular tree, pre-LUCA. These trees represent the types of phenomenon seen in molecular data, such as the ancient duplication of Gene 1 in Panels C and J of Figure 2 that results in all living species having both version a and version b. Recent duplication by itself results in multiple copies of the gene with a similar history being found in living species, such as

G1b version a and b found in Eukarya in Figure 2 Panel C, and G2 versions a and b found in

Eukarya in Figure 2 Panels D and K. Gene duplication and differential loss or incomplete lineage sorting can be seen in the Bacteria with Gene 3 in Figure 2 Panels E and L, resulting in a gene history that does not directly correspond to the species tree.

Figure 2: The individual gene trees that make up the watershed of life. Panels A through G correspond to the 3-Domain watershed. Panels H through N correspond to the 4-Domain watershed. Panels A and H show the underlying watersheds of life. Panels B and I show the ribosomal trees. Panels C and J show Gene 1. Panels D and K show Gene 2. Panels E and L show Gene 3. Panels F and M show Gene 4. Panels G and N show Gene 5.

HGT is pervasive in the watersheds shown in Panels A and B of Figure 1, represented by the dashed lines crossing grey regions. This results in multiple copies with different histories being found in living species, such as G1a version a and b found in B3, G2 versions a and b and version c in Eukarya in Figure 2 Panel D, G2 version a and b in Euryarchaea in Figure 2 Panel K,

G3 versions a and b found in B1 in Figure 2 Panels E and L, and G4a versions a and b found in

B2 and B5 and G4b and G4a version b found in B4 in Figure 2 Panels F and M.

Occasionally genes can be horizontally transferred across the dark grey region of the watershed, i.e. G4a being found in the Ibisial branch in Figure 2 Panels F and M. In addition, highways of HGT can form bridges between distantly related kingdoms, allowing for easy flow 84 of genes. This can be seen with G3 version b and c in Eukarya in Figure 2 Panel E, with G4a in

Eukarya in Figure 2 Panel F, and with G4a version b in Eukarya in Figure 2 Panel M. These genes link Eukarya with Bacteria and may have come in with the mitochondria or have been picked up during phagocytosis (Doolittle, 1998).

HGT alone leads to difficulty in interpreting phylogenetic trees, but it is even worse when transfer is followed by loss of the original copy, as shown in Figure 2 Panel C with G1a versions a and b in B5, in Panels E and F with G3 versions a and b found in B2 and B3 and versions a, b, and c in Eukarya, and in Panels F and G with versions a and b of G4a found in B4. Even more difficulty is caused when these transferred genes are first transferred to the Mobilome or now extinct lineages, diverging from the standard vertical copy, and then transferred back to surviving cellular lineages, shown by dashed lines that drift out into the grey regions of the web of gene sharing, before drifting back. An example where a gene from the Mobilome or extinct lineage is transferred to only one surviving cellular lineage is G1b version c and d found in

Eukarya in Figure 2 Panel J. Examples where two surviving cellular lineage both pick up the same gene from the Mobilome or from extinct lineages are G1b versions c and d found in

Eukarya and G1a version b in Eukarya and G1a version b in Crenarchaea of Panel C of Figure 2, version c in Eukarya and version b in Crenarchaea of G5 in Panel G of Figure 2, and version b in

Eukarya, Crenarchaea, and Euryarchaea in G5 in Panel N of Figure 2.

Examination of the gene trees shown in Figure 2 Panels C through G and J through N reveals that there are differences between vertically and horizontally acquired genes in terms of the appearance of their respective phylogenies. HGT leads to patchy, inconsistent data, where one gene unites a clade with one specific lineage, while another gene unites the clade with a different, although equally specific, lineage (Lane, 2011). Then when a supermatrix of all of 85 these genes is constructed, strong support is found for either the average of these signals or the tree with the least conflict with both topologies, because this is a feature of the matrix concatenation approach (Lapierre et al., 2014). On the other hand, genes mostly undergoing vertical descent show broad presence in the clade that is the proposed sister group, especially when the gene is postulated to have been present in the ancestor. These genes then consistently show the same picture, grouping in the same position, with the same sister taxa, and similar divergence times (although not necessarily identical).

5.3. Applying the Watershed of Life

Given that the true evolutionary history must either be that shown in figure 1 Panel A, or that shown in Panel B, we can examine existing evidence in a new light. The one place where the sort of patchy gene distribution characteristic of the HGT network can readily be found is in the work of the proponents of the Eocyte hypothesis (Cox et al., 2008; Ettema et al., 2011; Makarova et al., 2010; Nunoura et al., 2011; Spang et al., 2015; Williams et al., 2013; Wolf et al., 2012;

Yutin et al., 2009; Yutin and Koonin, 2012). Oddly enough these researchers have compiled rather strong evidence in favor of the 3-Domain tree and a monophyletic Archaea, by showing that shared Crenarchaea-Eukarya Signature Proteins (ESPs sensu (Hartman and Fedorov, 2002)) all show the pattern associated with HGT. The evidence collected by the 3-Domain supporters is consistent with the vertical signal expected with the 3-domain tree. (Baldauf et al., 1996a; Brown et al., 1997; Brown and Doolittle, 1995; Gribaldo and Cammarano, 1998). No evidence has been found that is consistent with either the vertical signal expected with the Eocyte tree nor the underlying web of HGT expected with the Eocyte tree. Although researchers have searched for

86 the vertical signal, there has been no concerted effort to look for the horizontal signal. Thus, the question of how many patchy genes result from frequent HGT between Euryarchaea and

Crenarchaea, has been left largely unanswered.

Owing to the dearth of research on HGT between the Euryarchaea and the Crenarchaea, this quantity cannot be compared to the quantity of HGT between Crenarchaea and Eukarya.

Only when this comparison can be made, can the question of which scenario has led to the unique relationship between Eukarya and Archaea be answered. Support for the Eocyte tree should be looked for, by examining Crenarchaea and Euryarchaea for shared, but patchily represented genes. Although this endeavor is likely obscured by the fact that HGT networks are not stationary, nor are they set in stone for all time, there should still be evidence in the form of patchily distributed genes that do not closely recapitulate the known phylogeny (i.e. the type the

Eocyte group has already gathered supporting the 3-domain tree). Likewise, if the Eocyte tree correctly represents the true linear history of cellular life, then there should be some genes left in the genome to show this. Such genes would be broadly represented in all Eukarya and

Crenarchaea, having come from their hypothetical common ancestor. These genes would produce phylogenies that support known clades. No known genes fit these criteria.

It has been suggested that although Eukarya have exchanged HGT for Meiosis as their primary method for gene sharing, they went through a phase in their early evolution when they readily acquired horizontally transferred genes. The fact that there are more bacterial-related genes in Eukarya (Rochette et al., 2014) implies that Eukarya at one point had a very high rate of

HGT. Phagocytosis of a wide range of prey may have provided the mechanism by which this happened (Doolittle, 1998). These genes were then incorporated into the eukaryal cell and put to 87 new, innovative usage. This might explain why Eukarya have acquired so many crenarchaeal genes from various sources, even though they no longer appear to be continuing acquisition of horizontally transferred genes on the same scale.

There are either synapomorphies or horizontally acquired genes common to the Archaea that give this group its key signature way of life. These features were acquired after the split with

Eukarya, or shared via HGT. These traits include the unique ether linked lipid membrane, derived tRNA and rRNAs, the ability to colonize various extreme habitats, (Woese et al., 1978), and a slow evolutionary rate.

The reason why the branch separating Eukarya from Archaea is so short, could be that

Archaea evolved a slower rate of evolution immediately after branching from Eukarya. Tighter control of mutation rate might have been important in adapting to extreme environments or simply the result of a more accurate polymerase. Or a slower rate of evolution could have evolved in the stem Ibisii and then changed back in Eukarya, speeding evolution back up. Once evolution was stuck at a slower rate, Archaea were less able to compete with the more quickly evolving Bacteria. HGT from Bacteria and Eukarya would then have become an important work- around to acquire new functional genes in order to remain competitive.

Note that the direction of transfer is not known. Stem Eukarya may have picked up these shared genes from a variety of Crenarchaea, or a variety of Crenarchaea may have gained genes from stem Eukarya, or both. If the genes are crenarchaeal gains, then this could be a valuable way for slowly evolving lineages to acquire more genes. Per (Brown, 2003), “If gene occur in most species of one domain but only a few species of another, then it is probable that a species from the more populous group was the donor.” If this is the case, genes shared between Eukarya and Archaea most likely originated in Eukarya. 88 On the other hand, if patchy genes are eukaryal gains, then they could be the result of phagocytotic life-style (Doolittle, 1998). In the latter case, this is also a valid explanation for how and why the genes of non-mitochondrial bacterial origin entered the eukaryal line (assuming they are not in fact vertically inherited genes present in LUCA, but lost in Archaea). The eukaryal stem would thus have had a tendency to pick up and retain genes. Such tendency may be a pre- requisite to the type of morphological complexity exhibited by various eukaryal cells (Koonin,

2010).

5.4. Implications for Symbiosis in the Origin of Eukarya

Symbiotic hypotheses for the origin of Eukarya in which the Eukarya arose from within the Archaea (Martin and Müller, 1998; Moreira and López-García, 1998) are generally thought to be more parsimonious, because they do not require the existence of a proto-eukaryal lineage, but they are not simpler. The latest symbiotic fusion papers claim that the Last Archaea Eukarya

Common Ancestor (LAECA) was the proto-eukaryal lineage, and as such was a fully-fledged

Archaeon that was more complex than any modern Archaeon (Wolf et al., 2012; Yutin et al.,

2009). An important point is that the Last Eukarya Common Ancestor (LECA) was a complex modern Eukaryeon (Archibald et al., 2000; Collins and Penny, 2005; DeGrasse et al., 2009; Field and Dacks, 2009; Koumandou et al., 2013; Neumann et al., 2010), so the list of eukaryal features that need to either be present in LAECA or acquired shortly after is close to the list of eukaryal signature proteins sensu (Hartman and Fedorov, 2002). If all the genes found in desperate archaeal species were present in the LAECA, then gene loss was rampant. This is in fact a very convoluted scenario, because the proto-eukaryal lineage is still proposed to have existed. It has only been moved further back in time and given less time to evolve, in addition to requiring a 89 large number of independent gene losses and convergent genome simplification events to explain the genomes of the present-day Archaea. The most parsimonious choice is that Archaea are monophyletic and shared genes are the product of HGT. But, evolution is not necessarily parsimonious and the Eukarya could very well have evolved from within the Archaea. However,

Archaea/Bacteria fusion scenarios for the origin of Eukarya require various implausible intermediates with unlikely membrane loses, that are not supported on a cell-biology level

(Cavalier-Smith, 2014) (Jekely, 2006; Poole and Penny, 2006). Fusion hypotheses, relying on extensive HGT from various branches of a tree to create a complex, undocumented hypothetical host ancestor that was then involved in a symbiosis with implausible intermediates, should not be considered a simpler explanation to autogenesis of eukaryal traits (Cavalier-Smith, 2014; Jékely,

2007; Keeling, 2014; Poole and Penny, 2007; Rochette et al., 2014) with a highway of gene sharing. And, there is significant scientific evidence supporting a link between Eukarya and

Euryarchaea, the archaeal Kingdom proposed to be most distantly related to Eukarya per the currently favored fusion hypotheses, including Supertree whole genome analyses (Pisani et al.,

2007). If the Eukarya are more closely related to the Crenarchaea, then Supertrees, which have the advantage of not suffering from the inherent concatenation artifact of combing the signal of

HGT with that of vertical descent to produce a phylogeny that represents neither, should not group Eukarya with Euryarchaea to the exclusion of Crenarchaea. The fact that Eukarya go with

Euryarchaea in some analyses and Crenarchaea in others mildly supports the hypothesis that

Eukarya go with both, equally, but the branch length separating them is too short to produce a reliable signal. 90 It has been said that the Eukarya did not exist before the formation of the federation with the Alphaproteobacteria that became the mitochondria (Williams et al., 2013). In this way of thinking, the Eukarya are nothing more than a fusion between an Archaeon and a Bacterium, but this is not the case. Even if the Eukarya evolved within the Archaea and not as sister to them, they still evolved from a very derived ancestor that possessed many or possibly all the eukaryal innovations. These innovations are not found in Bacteria and did not come with the endosymbiont; complex molecular machineries and membrane bound compartments do not simply spring up when an endosymbiont is gained. Even if these traits were gained after the mitochondria was acquired, even with simpler homologs performing similar, but fundamentally simpler functions were acquired and built upon, the full interwoven cellular and molecular complexity of it all was still invented by cells that gave rise to LECA. Eukaryal features are fundamentally eukaryal innovations and the eukaryal clade is much more than just the fusion of the parts of the federation. Even if the Eukarya evolved from within the Archaea, they are not

Archaea. They lack the level of cellular simplicity and specific features shared by the Archaea.

They are the only known living members of a more cellularly complex grade, called the

Eukaryotes.

5.5. Conclusion:

There is hope that one day a definitive answer can be obtained with regards to the topology of the split between Eukarya and Archaea. But the type of data that is missing is not the type of data that researchers are currently looking for. An effort needs to be made to examine and

91 quantify the network of Horizontal Gene Transfer that joins Crenarchaea and Euryarchaea. When this last piece of the puzzle is obtained, then a monophyletic archaeal scenario can be teased apart from the paraphyletic archaeal scenarios.

The present data, with an ever-increasing number of Eukaryal Signature Proteins found patchily distributed in distantly related Archaea, supports only the 3-Domain topology, because patchy genes are a sign of Horizontal Gene Transfer. Furthermore, symbiotic fusion scenarios for the origin of the Eukarya are less parsimonious than autogenesis scenarios for the origin of

Eukarya, explaining less while requiring unnecessary and convoluted steps. Unless new evidence is found that supports symbiosis or archaeal paraphyly, then the 3 Domain tree and autogenesis as the mechanism for the origin of Eukarya must remain the null hypotheses.

92 Chapter 6: The Root of the Eukarya

6.1. Introduction

Currently no consensus exists as to where the root within the Eukarya lies (Cavalier-

Smith and Chao, 2010; Derelle and Lang, 2012; He et al., 2014; Pace et al., 1986; Rogozin et al.,

2009; Stechmann and Cavalier-Smith, 2002; Stiller et al., 1998; Wideman et al., 2013; Woese et al., 1990). The fossil record is no help in this regard, because the first definitive eukaryal fossil belongs to the red algae (Butterfield et al., 1985), which belong to a larger group, the

Archaeaplastida, and are not even the deepest branch within this group (Huang and Gogarten,

2007), making them an unlikely candidate for the location of the root (unless one believes figure

7). With no fossil evidence to inform on the problem, one is left only with molecular sequence information and phylogenetic reconstruction.

The main problem with rooting the Eukarya using phylogenetic reconstruction has to do with the long eukaryal stem branch discussed in Chapter 4. Because of this long branch, the closest outgroup, the Archaea, are incredibly far away. Using Archaea to root the Eukarya is analogous to throwing a dart across a football field and trying to hit a target. But some phylogenetic reconstruction programs, such as phyml (Guindon et al., 2010), must return a bifurcating tree, even if there is no evidence in the dataset to support any bifurcation pattern. So the result of such an exercise is that the long branch to the archaeal outgroup attracts to some random branch of the eukaryal tree, creating a Long Branch Attraction artifact.

In order to root the Eukarya, a closer outgroup is needed. It is impossible to find a lineage that is a closer outgroup than the Archaea, because all such lineages went extinct. But although whole cell lineages did not speciate during this time period or have not yet been sequenced, many individual genes did proliferate through the processes of gene duplication. Paralogs that 93 diverge from each other on the Eukaryal stem branch are closer outgroups to the Last Eukaryal

Common Ancestor (LECA) than Archaea and should therefore be more likely to accurately locate the root within Eukarya.

An additional problem with rooting the Eukarya is that individual protein trees lack resolution when it comes to branching order. A multi-protein concatenation may multiply phylogenetic signal and allow for deeper resolution, so a concatenation of paralogs could provide a closer root and increase the phylogenetic signal. On the other hand, concatenation will also multiply the signal from artifacts and could produce a consensus tree that does not correspond to the actual evolutionary history of any of the component proteins (Bapteste et al., 2005). In this chapter, I attempt to counter these problems by producing protein concatenations that are limited to actual evolutionary units, such as the histone complex, the tubulins, and the proteasome.

The four histone subunits come together to form the histone complex and have likely evolved together throughout the history of the Eukarya, as have the three tubulins, which interact to produce the microtubule system, and the 14 subunits of the proteasome, which form a complex macromolecule responsible for protein degradation. Using histone, tubulin, and proteasome protein concatenations will increase resolution and allow for independent determinations of branching order at the deepest level in the eukaryotic tree.

There are 4 histone paralogs, H2A, H2B, H3, H4, common to all Eukarya (Bell and

White, 2010; Thatcher and Gorovsky, 1994). Alpha, Beta, and Gamma Tubulin are present in most Eukarya and believed to have been present in LECA (Fournier et al., 2011). It was unknown whether the fourteen subunits of the proteasome were all present in LECA, or if gene duplication continued after LECA, but my preliminary analysis indicated that all fourteen 94 subunits each are monophyletic. If gene duplication continued after LECA, one or more subunits would group inside another, creating a paraphyletic subunit. Thus, because all fourteen subunits are monophyletic, all fourteen must have been present in LECA and are suitable for placing the root within Eukarya.

6.2. Methods

6.2.1. Histones

Although the histone data had previously been used for analyzing the eukaryal stem branch length in chapter 4, the data were reassembled from scratch in 2013 for this project.

Histone amino acid sequences were downloaded from NCBI’s list of fully sequenced genomes.

If more than one genome of a closely related group was available, one was chosen at random.

Histone sequences were first identified from the list of annotated proteins and then verified using reciprocal best BLAST hits. An in house Perl script was used to modify taxa names. Clustal X

2.0 was used to create initial alignments within a subunit and also profile alignments of the subunits to the structural alignment (Larkin et al., 2007). Deepview was used to create a structural alignment of the four histone subunits (Kaplan and Littlejohn, 2001). Jalview was used to manually adjust the histone alignment (Clamp et al., 2004). Clustal X 2.0 was used to convert alignments to phylip format (Larkin et al., 2007). FigTree1.2.2 was used to visualize trees

(Rambaut and Drummond, 2008). An in house python script was used to concatenate the subunits. Permutations of concatenation order were used to root the concatenations, as depicted in figure 1 (modified from (Hilario and Gogarten, 1993)). With 4 histone subunits, only 4 permutations are needed so that each individual protein in the concatenation is rooted with all of its paralogs, to break up the long branch to the outgroup as much as possible. 95 PhyML 3.0 was used to produce Maximum Likelihood trees (Guindon et al., 2005), using the WAG substitution model, 100 bootstrap replicates, estimated proportion of invariable sites, 4 categories of substitution rates, and an estimated gamma distribution parameter.

Extreme divergence and short sequences make histone alignments difficult. To judge the quality of the alignment, Maximum Likelihood trees were generated and examined for eukaryal monophyly. Since no one believes that the Eukarya evolved independently more than once or that a Eukaryeon has reverted to a prokaryote, it is universally agreed upon that the Eukarya, and therefore LECA, are monophyletic. LECA is present four times on the histone tree, one for each of the four subunits, and only when all four instances are resolved as monophyletic with high bootstrap support value, will the alignment be accepted.

6.2.2. Tubulins

The tubulin dataset was curated in the same manner as that of the histone dataset. The data were reassembled from scratch in 2013. Tubulin amino acid sequences were downloaded from the same genomes used in the histone analysis. Tubulin sequences were first identified from the list of annotated proteins and then verified using reciprocal best BLAST hits. An in house Perl script was was used to modify taxa names.

When taxon sampling of NCBI proved insufficient to root the Eukarya, transcriptome datasets and whole genome sequences freely available were searched for additional sequences using TBLASTN (Gertz et al., 2006). Because the initial sequence used as query affects the results of TBLASTN searches, multiple searches were performed on the virdiplantae using

Angomonas deanei sequences (an euglenozoa-type excavate) as seed and Arabidopsis thaliana sequences as seed. A. deanei sequences were used under the hypothesis that the excavates are the 96 root of the Eukarya and therefore would be an outgroup to all the newly sequenced genomes under consideration, making them an equally good seed for all. A. thaliana sequences were used under the hypothesis that as a far closer relative to the virdiplantae sequences, being a member of the virdiplantae themselves, they would make for a better seed and be more likely to return the full sequence of the desired ortholog. Upon comparison of the two sets of TBLASTN searches, the excavate seed performed equally well as the viridiplantae seed, when analyzing multiple virdiplantae genomes. The excavate, A. deanei was used as the only seed for all further

TBLASTN searches. Alpha and Beta subunits were identified, but gamma TBLASTN searches returned only partial matches. Preliminary trees showed all partial gamma sequences grouping together, to the exclusion of the full gamma sequences, even though both sets were interspersed throughout the eukaryal tree. This was ruled to be an artifact caused by the truncated sequences and all partial gamma sequences removed from further analysis.

An in house PERL script was written to modify taxa names. Protein alignments were performed using Muscle (Edgar, 2004). Clustal X 2.0 was used to create profile alignments

(Larkin et al., 2007). Clustal X 2.0 was used to convert alignments to phylip format.

Protest 2.4 was used to determine that the LG + Gamma +I model maximized the likelihood of the tree (Abascal et al., 2005). PhyML was used to produce Maximum Likelihood trees (Guindon et al., 2005), using the LG substitution model, 100 bootstrap replicates, estimated 97 proportion of invariable sites, 4 categories of substitution rates, and an estimated gamma distribution parameter. Although aligning tubulins was much simpler than histone and proteasome alignment, Maximum Likelihood trees were still generated and examined to judge alignment quality; mostly sequences causing Long Branch Attraction artifacts were removed.

6.2.3. Proteasome subunits

Proteasome amino acid sequences were downloaded from NCBI, as described for the histone and tubulin data sets. When taxon sampling of NCBI proved insufficient to root the

Eukarya, transcriptome datasets and whole genome sequences freely available were searched for additional sequences using TBLASTN (Gertz et al., 2006). As described for the tubulin data set,

Angomonas deanei sequences (an euglenozoa-type excavate) and Arabidopsis thaliana sequences were both used as seed for multiple virdiplantae genomes. When they performed equally well, A. deanei was chosen for all further TBLASTN searches. An in house perl script was used to modify taxa names. Each subunit was first aligned individually using Muscle (Edgar,

2004). Then Promals3D was used to create a structural alignment for the 14 subunits (Pei et al.,

2008). The number of subunits stymied the profile alignment capabilities of popular alignment programs, so Seaview was used to manually preform a profile alignment.

Clustal X 2.0 was used to convert alignments to phylip format (Larkin et al., 2007).

FigTree1.2.2 was used to visualize trees (Rambaut and Drummond, 2008). An in house python script was used to concatenate the subunits. Permutations of concatenation order were used to root the concatenations. With 14 proteasome subunits, 14 permutations would be needed so that each individual protein in the concatenation is rooted with all of its paralogs. But because it is unknown whether the alpha subunits diverged from the beta subunits on the eukaryal stem or 98 before the Last Archaeal/Eukaryal Common ancestor, and because the branch separating the alphas from the betas is long, the dataset was split in two: The alpha proteasomes and the beta proteasomes, so that alpha subunits are concatenated and rooted with only alpha subunits and the beta subunits are concatenated and rooted with only beta subunits.

Protest 2.4 was used to determine that the LG + Gamma +I model maximized the likelihood of the tree (Abascal et al., 2005). PhyML 3.0 was used to produce Maximum

Likelihood trees (Guindon et al., 2005), using the LG substitution model, 100 bootstrap replicates, estimated proportion of invariable sites, 4 categories of substitution rates, and an estimated gamma distribution parameter. When PhyML failed to produced trees in time,

FastTree (Price et al., 2010) was used with default settings (JTT, the "CAT" approximation, and

"SH-like local supports") to create quick and dirty place holder trees.

Proteasome alignment was complicated by the misnaming of the subunits for which crystal structure was available. Additional subunits were found to be misannotated, with top

BLAST hits also misannotated, so placement in Maximum Likelihood trees was used to confirm subunit identity. Additionally, taxa and or subunits causing long branch attraction artifacts in

Maximum Likelihood trees were removed. 99

Figure 1: Using Histones concatenations to root LECA. Each of the 4 histones subunits is rooted using concatenations composed of the other 3 subunits in every other possible order, as well as a concatenation made from repeating the archaeal histone sequence 4 times.

100

Figure 2: Using tubulins to root LECA. Each of the 3 eukaryal tubulin subunits, alpha, beta, and gamma, is rooted using concatenations composed of the 2 subunits in every other possible order, as well as a concatenation made from repeating the FtsZ sequence 3 times.

6.3. Results

6.3.1. Histones

The histones were so conserved within a subunit that they could be aligned by hand.

However, they were so divergent between subunits that a structural alignment had to be performed. Unfortunately, histone sequences are extremely short ~200 bp per sequence, so not even concatenating all four together provided a reliable branching order within the Eukarya, shown in Figure 3. I therefore conclude that the histones do not contain enough phylogenetic signal at the level of LECA to root the Eukarya.

101

Figure 3: One of the 4 histone concatenations, rooted with the other 3 histone concatenations.

The other 3 are not shown, because they are suffering from the same problems as this one.

102 6.3.2. Tubulins

Obtaining alpha and beta tubulin sequences from new genomes was straightforward, owing to the high levels of sequence conservation in these subunits. The same cannot be said for the gamma subunit, whose sequences were highly divergence across the Eukarya. When

TBLASTN yielded a sequence, it was almost always a partial sequence. A preliminary tree of these sequences showed the new partial sequences grouping together to the exclusion of the full gamma sequences. Therefore, the partial gamma sequences were thrown out of the dataset and only alpha and beta tubulin were used to represent the newly sequenced genomes and transcriptomes.

Figures 4, 5 and 6 show the three tubulin concatenations. They were calculated as one

Maximum Likelihood tree, but broken into three parts, in order to display them. All 3 exhibit a paraphyletic excavate root, but all three show signs of artifacts. The deepest branch is always

Euglena, but this looks like Long Branch Attraction. The Archeaplastida and SAR supergroups are not present as monophyletic groups. One or two taxa grouping in unexpected places could be due to errors in the phylogenetic assignment to supergroup or horizontal gene transfer, especially in taxa known for horizontal gene transfer or known to harbor eukaryal endosymbionts, such as the chromalveolates. But there are just so many taxa out of place that there is little hope for this dataset in resolving the split at the deepest levels in the eukaryal phylogeny.

To verify that this tree is suffering from some irreconcilable artifact, all of the deepest branches were removed and the tree rerun, checking to see if the interior of the tree was stable. It wasn’t, as seen in Figure 7. The topology of the tree completely changed, likely because there is no support for any of the deeper nodes, and the root shifted to within the red algae. The data is

103 not shown, but this process of removing deeply branching taxa was continued until there were so few taxa left that it was clear that the instability of the tree was not a factor of a few long branches.

It is possible that a combination of identifying long branches to remove, adding the full sequences of the gamma subunit from newly sequences genomes, and adding data from taxa yet to be sequenced, might provide new information that would allow the tubulin family to resolve the branching order at the deepest levels within the Eukarya. But as it is now, with only the alpha and beta subunits available for half of the dataset, there just is not enough phylogenetic signal to produce a reliable branching order.

104

Figure 4: The first concatenation of the three tubulins, rooted with the other two. 105

Figure 5: The second tubulin concatenation, rooted with the other 2. Colors are the same as in

Figure 4. 106

Figure 6: The third tubulin concatenation, rooted with the other 2. Colors are the same as in

Figure 4. 107

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2 2 C n f l

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0.6 Figure 7: The deepest branches from figures 4, 5, and 6, the Euglena, were removed and the tree re-estimated. Now the root is within the red algae and known supergroups are still not recovered.

Colors are the same as in Figure 4.

108 6.3.3. Proteasome

Figure 8 shows a phylogenetic reconstruction of all 14 of the eukaryal proteasome subunits and the 2 archaeal proteasome subunits. From this tree, it appears that either the ancestor of the Archaea and Eukarya had a 2 subunit proteasome, or, the Archaea acquired a 2 subunit proteasome from a stem group Eukaryon, prior to the divergence of the 7 alphas and the

7 betas. Although the topology within the eukaryal subunits is not resolved, all 14 subunits are clearly resolved as monophyletic and therefore had to have been present in the Last Eukaryal

Common Ancestor (LECA).

Figure 9 shows only the 14 eukaryal proteasome subunits, but with the addition of a number of new sequences obtained from newly sequenced genomes and from transcriptome projects. All 14 subunits are still monophyletic. The topology within each subunit is still unresolved.

Figure 10 shows the 7 individual beta subunits that are concatenated in Figure 11. Neither shows resolution at the deepest nodes in the eukaryal tree, but occasionally known super groups are recovered. One of the subunits in Figure 11 is scaled up in size in Figure 12, to increase visibility. Support values, representing "SH-like local supports,” are shown in black. All support values are so low as to be unusable, represent known clades whose topology is not in question, or represent obvious cases of Long Branch Attraction.

Figure 13 shows the 7 individual alpha subunits that are concatenated in Figure 14. One of the subunits in Figure 14 is scaled up in size in Figure 15, to increase visibility and support values, representing "SH-like local supports,” are shown in black. All support values are so low as to be unusable, represent known clades whose topology is not in question, or represent obvious cases of Long Branch Attraction. 109

1 2

e 1 0 6 2 5 t 0 0 1 a 1 1 0 1 1 5 0 0 B 1 6 6 1 1 1 6 3

e a a 8 0 a 6 t t 6 2 B 7 t t 2 2 a e e a 0 a 1 e t t e 0 2 5 3 1 B B B 0 r 0 e e t 1 t 1 0 3 a 0 1 4 6 B s 2 4 B B 50 B 0 a 1 0 6 t 1 1 2 B a 6 B 0 e 11 1 1 e t 1 a e 0

6 0 0 at e 2 8 t 05 e 6 0 a t e 6 t B 1 1 B a 0 1 5 e 1 r t 0 1 3 0 t 8 a a e 3 t B 5 a a B 0 7 5 6 t t 0 0 3 a e 3 B 1 B s 0 a t B 1 5 5 B 9 1 e 1 e t B t 1 a e 04 1 a 3 B B B 6 t t e 0 a1 e 0 0 2 t e 1 B 2 e 6 1 3 a 0 B 0 a a 0 e B e B e B e 1 1 t1 e1 50 B et B t 0 t 0 a a 6 a B 5 t a B 0 e 0 t 4 0 t 7 2B e3 t t t 6 B B a 7 10 e B 1 e0 e e 7 a a a a 1 1 2 5 t 1 6 B 5 t 2 e0 0a e e B B 00 a a e 5 t 5 t 3 20 t 3 eB 5 t 51 5 t B 0 1 a a 6 2 0 t a e 0 B1 e 1 0 5 a 3 e t B B t 1a e 0 a ea 0 10 1 e t 1 t a 0 5 5 a 0 1 3 t 5 t 0 1 B 0 1 7 B 1 B a 9 05 t 3 0e e 5t5 B 2 02 0 1 e a 4 1 1 a 3 a 1 1 B e 13 t 2 5 0 0 t 1 0s B e a 2 B 1 1 28 0 6 B a e 0 0 6 a 5t B 5 9 0 3a e 0 1 1 t 4t 0 B 0 0 0 t t t 2 4 6 e a e 03 5 r 0 0 1 1 0 a 1 5 e 0 a 1 B t e a B 1 1 9 0 B 33 1 t e 00 1 t 0 3 1 0 0 7 3 B 0 0 t 6 0 3 5 8 e 3 a 8 a 10 e 6 B e 000 0 e B 1 0 6 B 1 a 0 0

2 2 1 1 2at 5 5 3 11 t B 1 2t 3 a B 3 t 1 18 a 0 a t 1 0 e 1 1 e 0 0 333 1 2 e 30 9 3 0 a 3 5 6 3 1 a t e t B aa 0 0 2 1 0 at a a 0 B tt t 1 1a 1 e 3 t 2 5 1 e 1 9 e B B 5 B 1 t e e 0 0 0 3 a e 3 3 0 B B e e 1 6 e t B B 8 B a t B 2 1 a e t a t 0 2 a t 7 eB a 6 t 3B e B 2 5 e B 1Bt 5 B B ae 7 e 1 a 0 e7t 1e t B 3 t a a B e B 3 2a17 l B 0ta B e 0 0 p t 0 B 7 e e 3 1 1 a B 3 e 12 h t e 5 1 2 B 0 a 2 t 0 06 a B t 1 6 0 t a ea 22 2 7 aB 3 1 1 7 3 e 0 1 ta 9 B 1 7 6 a 1 11 1 1 t Be 1 t B 7 s 0 a 0 B0 e a0 t 1 t ta3 7 e 1 t 7 eta 1 e 0 4 3 1B B 1 3 e 1 e a r 0 e 1 ta 021 1t Be 19 t 7 B 1 0 0 e 7 B a 2 B B 1 7 a B 2 12 1 7 7ta 1tea0e 71 a3 11 tar1 0 17e0t7a2 10 7 0t a st 1 01ta27ta5 01 7 1e t 1e 8 1 7117 B7 2 B1 2 e taB B 02 1001 0e 0 a 0B 10 e e 1 01 0 4 1 t 0 00 B ta 1 0 ta 70 1 e 01 01 7 870 7 01 a B 21 151 5 1 9 1 0 et 0ta 0a3t1a 0 00 0 1 1e0e10te10 B 6 0 1 B 1aB1 a1B1B1 2 e 5 ta 02 t 1t ta 2 Bta e 1 e tae e 109 e7 B 1 B eB B 100 ta10 B B ta B ta1 72 B e e1 Be10 e B Bta ta00 eta Be18 Be 712 ta7 10 ta 0 71 6a1 7 28 10 0e0t 10 01 1B 1 0 05 ta1 1 0 3 e 1120 B t1a01 Be tBae1 ta7 Be 031 10 a21 01 2Bet 103 ta2 3 Be 6 01 102 a2310 ta29 011 e1t0 B0e120372 0t1a21 taB2 a2ta11202a2B1e Be BeBettea2 Bet B 010 ta0271 09 aB2e10ta21201 Bet Btae2120100619 0141002 BeBtaet1a008 BetaB21eta2 Beta2 010003 BBeBetaetaB2ta2e121t0a102015 5 Beta21004 B Ba Beta2100Beta2a120s1tr7 ar1 Br1 Bet 100 a00 Beta210B2e0ta21024 083 r10 Beta21028 06

B ar Ba10 r105B 00a4r Ba 100Ba r10 1 r10 07 02

Beta41032 Beta41013 Beta41022 BetBa4e1ta04016 Beta4B10e00t1a994 Beta4 1025 B a4101007 eta410 B08 BeBt eta410 ae4t1a0411012 B 05B B eta eta410 eta 41B01 B0e4ta 41 BetBete5aettaa etta44c101 B0e29 eta41414401100 str 7 ta4 4B1e0t2000013 BBeeta 102 2a441 ta441101 6 028 0024

Alph Aal5pA1lAplp Al h0a3h52ah5a phaAllp 101350130 51h0a5 1A013 25910 Alph Alphlpha 121 Alpha51A0lph Aalp5A1lp0h aA51lp05102 A1l6pah5a1501A1llp4lphhhaaa5525a1185010h0a9517 AlphAalp5hstar50102 5100100530124 006 AlphaA501lp10h70a451 AllphaAlpha510085A1l0p1h00a705015020 AlpAhlap5h1a0519022

A ar1 A 00 ar1 A2 00 Aara 0 1r010 010 A A A 4 a Aaraar r10 1r01010 05 030706 25 lpha2101a9lphaalp2h1a0321070091 a lpha21a0lp2h6a2lp1lph0ha2a2h21a102011032 1806 8 a aa ahl1pa023100210a2a712010 00 alp0ah3al2p2alphalaplhph 0 21 2 ph0a321 02ha 01 hal21 405 53010 21alp 21 alp 17120100 10012108 ha 22 ha aa22s1thrp0ah2a haah22a0120402alp 10 lp alplphhalpal alplalphalph210221 a2 a A 016 apahp2h1a lph a21 aplhaal a al1p4h al 210 ha 8 alp 0 Al 0 0 ph 2 61 a 053 8407 7004 61 A 000101 24211 1001 a 9 0 lp 11000110a00 004 h 0 0 h 441 1 51 8 6 a aa4042aa4p4h4 041halp 0 6 pphha04hh l a40a4p2A3 1 0 A 10 llph11 pAh 1hal 4 7 0 l AAlp40 lpl lptr 04 hA 2 17a 0 A ph 26 Aa1 AA s 0alplp 0 02 2 10 12 1 A A a h A1h 1 h 1 0 0 67 lpAlpAlp p 4 44 AA 100p 1 2 3 0 9 A Ah l h6 l a alp 4 43 l 8 01 1 00 0a A lAh p 1 Ah h a a 1 0 0 0 1 0 lp Aplaa Aha 0 p hA a04A 1 1 7 01 0 8 A l phl6p6 l6 0 l p h 1 7 5 h 0 p ap l p h1 1 a 0 1 h lphah1 1 8 A l p a 0 4 7 07 40 p 0 l a hA 1 6h0 l 1 a 1 a6 AA 2lp4h 1 a4 h 4 1 7 l p ah 0a0 1a2 a 8 a 00 0 6 l a61060 06 A a a 2 7 h 75 h p 3Alp 4 h hlp 227h a A 1 6 10315 011 0 h h p01 1 a 1h6 a p 0 a a2 0 pA l pA 00ap l1 h A 0 Aa100 70 1 l p l 1 l 7 1 6 0 001 1 h l 5h 0 0 41 A 10h A7 p h l 10 lp26 1 5 0 A p A 7 1p a l a pA 10 l A A 1 9 a3 71pl p 0 h4 a4 l a l 02 A h 0 hA h h l A A a 7 7 0 A 9 p 2 a lp h 1 A hp 7 a A 2 lp1 h a1 l A p h 5 lp6 h a l 8 p 0 p 2 A 6 4 l 7 l 2 2 lp 1 a A p h 1 0 a Ah 6 l A h a 1 2 0 A 1 0 a 0 A h a A p A 2 l A 6 l 1

A p l r 1 3 p 0 p h A7 3 1 p a 60 0 h t 3 l 1 0 1 A A h 0 l A l 1 h 4 1 p 0 h 2 A aA p 1 l 6 l p s 1 7 p 0 a 1 0 p a l 1 7 A A 3 l h 1 1 0 l 1 0 1 3 a l 6 A 7 0 h p p h 2 1 a l p AA 6 h A A 7 a p 0 c1 p 7 2 7 2 l A 2 A l 0 1 a A a 1 a h ph s 7 h 1 h l 7 a A 1 a h h l l a A p l A t 1 0 a 0 p l p 1 p 3 h 0 a l 7 a h A 1 p r a p 1 5 a p A 9 h p 1 3 a h h l h l l

h 1 7 1 l A l h ph 1 p 0 a 0 a3 p 1 1 h h l p h 0 1 1 p l a 1 0 1 p ap 7 l p 1 A 2 aa l 1 ha 0 A h 6 0 31 l h A 0 a a 6 0 1 l A p 1 0 1 h h

0 p 0 1 a 1 h a 3l 2 33 2 A a a310 3 l 3 6 p 1 A 2 1 0 p Al 1 1 0 a a6 0 h p 0 1 0 0 1 1 h 0 1 1 0 h 3 0 1 l A A 0 1 1 6 0 h 0 0 a 1 2 1 1 1 a p A 1 7 1 0 0 4 1 0 101 0 l p 0 aA 1 1 a A l 0 9 h l A 1 a 7 A 2 p 1 7 a 3 1 1 0 1 0 00 3 03 7 2 A h A A l 7 p 0 h p

A l h 1 l a l h 0 05 p6 3 a a p 7 1 2 0 p 2 l h p l l 1 a 0 p A h 8 p p l h A h h A 3 A0 h l A 0 a 6 A A A 1 3 h h a l p 2 0 p A a l p l A l 3 p l l 1 p l A p l a 3 lp 1 0 a l 5 p 0 1 p p h 1 h A 1 h A h 3 1 h A r 3 1 0 a 1 h h A0 t a a a a 1 1 0 3 a 1 1 0 a a 3 l1 s 3 A h A p 3 3 0 0 0 1 9 7 a 2 3 3 1 1 1 h 1 p l 1 l 1 9 0 4 1 l p p 1 1 0 h 3 0 0 a a 0 9 1 2 4 1 h h 0 0 0 p A A 3 1 h l 2 3 2 0 2 4 a a 0 s 1 p l 2 A l A p 1 2 5 t 1 3 r h l 1 A 1 1p

a A a h 0 0

1 h 2a l 3 p

1 p 3 2 8 h l A 0 1 a

A l 3 0 p 3 h 1 1 2 4 a 0 3 3 1 0 0 2 3 0.9 Figure 8: Phyml Maximum Likelihood tree of all 14 of the proteasome subunits and the 2

Archaeal proteasome subunits. Colors are the same as in Figure 4, with the addition of pastel blue for the archaeal alpha subunits and seafoam green for the archaeal beta subunits.

110

a t e 01 3 3 4 B 3 37 3 30 7 B 31 a0 1 B 2 378 3 a63 3 1 3 3t 2 r B 3a ta 7 3112 t10

1 2 2 e 1 4 3 0 1 1 a 1 a t at aae 0 4 1 a e 2 3 t t a03 s0 B t t e 0t 1 e 1 t 3 6 a 0 t3 3 0 0 B 0 1 t 0 0 a 0 0 0 0 te3 e 1 1B t 0 B 1 a 1 a t8 e e0e 1a 33 6 e 2 B 1 e a 0 t e 0 a a t B ea 3 e 1 t 3 0 54 1 1 0 B 3 11 t 5 3 1 3 ae 0 t ea t 0 1 B 2 3 3 6 7 0 3 e B 3 aa10 e 0 1 1 1 1 a 0 t B B 0 6 B 53 Be t t 2 B B eB 1 1 a t B 8 e 3 0 9 4 0 5 7 e Ba 0 6 t 3 6t e a a e e 7 7 5 0 t 5 6 3 a a 1 a 1 1 1 B t t 5 0 e 13 3t30 t 0 a 1 B 58 3t ee0020 1 t B 3 6 4 a 03 6 t a 0 B a 8B 7 4 B B a e e e e B 0 1 B t 1 a 3 5tB0 3 a 7 6 a t Be a 0 3 5 a a e 0 r B t a e1 5 0 4 1 t t 5 5 100 1 6 1 1 1 1 6 3 0 4e a a 6 1 0 t e5 5 B4 t t1BtB1 1 6 4 B 6 t 1 t 6 e 9 t t 1 aB 0 e 0 e5 e 0 0 B B 1 e B 9 e 3 6 e 7 1 3 3B1 0 1 e 2 3 a e 3 B t1 a 7 0 0 0 3 1 a e 4 0 0 B 60 1 0 a a 1 e3e3 0 e a 0 60 s e0B 3 B 0 a t 0 0 2 1 1 B e a 1 1 B t 4 B t t 4 e t 1 t 14 a t 1 1 t a303 B 4 1 8 t B e 0 0 1 B 1 0 6 a t 7 a 4 a B1 1 0 B B 0 6 1 0 1 1 B e B 1 e a 6 0 ata

4 0 5 t 1 a B a B B a 1 41 1 Bt B B 2 B 1 4 B 1 a 5 6 e t B l 0 0 5 t 0 4 B 0 7 e B e 0 0 tBt a 3 e 5 5 a p t e 9 0 0 e e 5 0 3 0 e 0 1 B e B0 0 a B 0 4Bt 6 0 e t 0 6 e 1 1 t e a 6 t 1 1 0 1 2 0 e

e 1 Be e 0 6 0 e 5 1 6 e 0 B 3 t e 5 4 7 a e e e 4 a h Be B a tB a0 B 51 1 e0 4 11 3 t t 2 t t 3 t 1 B e 5 3 0 0B B 2 e e 1 5 0 a 1 B t 1 1 t 1 t 1e 0 a 6 t 0 B BB B 6 t t a 6 0 1B 1 et 1a5 0 5 e 1 a 0 B 1 B aB1Ba a t 9 71t B e B1 0 a B a a ae 6 t 0 B BB e B t 00 6e 1aB e e 0 a 18 1 e 1 B B 8 B e 0t0 0 B 6 1 B B 0 4 2 B 6 7 0et 17 0 2 a 0 7e02a 707B3 4B 7 t 0 4 9 6 1 7 e a 1 e 6 7 7 e 1 a 4 Bt 7t B e et 5 a B B e t B7 t t e 1 1 1e 3 134 B 7 64 a 1e t 0 a e a 1 4 1 e 7 2 4 1 1 e4 7 a e 2 4 4 t t a11 B a1 a 9 B 2 1 a1 3 t 7 e 71 0 t a 48 e t 0 0tt a e 1 7 e t t e 0 4 1 t 0t B 3 t 3 055 1 B a 1 t a0 0 4 7e 7 a aa t1 B 0 a 0 1 a 70 e 7 0 0 0 0t 6 5 0 2 3 a77s0 t 1 B a 1 a 2 e 0 0 7 aB 0et 0 3 t r1 0 B at 0 a1 e 25t 10 3BB t a 7 6 0 B 0 77 B 1ta 1 6 0 1 1 17 1 6 1 11t120a t 1 e 2 6 1 10 1 e a 1 1 B 01 17 01a7 6 t 1 1 s r ee t a1 70 e e 07 01 1 0111e10B7800a 0 B 3 100a0ca 1 00701e67ee7 0 3 B 0 e 0 t0 8 t 6 60 1a 0t0051t a0 07 e 0 1 a 5000tt4tt Ba7 79 2 a t a 1t1211 tt 10a 07 B 1 e1 t 0a1tea tt 2 1 a t 110 1e1010aa e 3 Ba 21022 a a79a 1 2 2e0a101tta e0 2715t7e8 7 6 B a t1B110Bt 7 B52 a 5 6 e at0a1a1aae BB1 e 380 7 77 0 3 t tBteat1at1e8 16 12 B t 1 t 1 B e1e111tetB11 a7313 e 7a 2 2 e eea1B 5t5a a 0 aBate a1 t30 e B0 t 7 B1Bt5t teBa8t 0160e0 10 7 a 2 2 B BBet 1a01e17 1 t 2 3 5ae0eB 4etta1B10111 1 Ba1 e1 97 10 7t15 Be0Be1a061Ba01a2a11t9a ta B50 t0 2 e34BBB a1t tattatt e0 e eBt eB14 a2 ta1 003 1t01Be4ae11ee1eet1aB0 3B 1 B BaBBet07 B 7 e 1 10B 4t1ea5B4 BtaBBBtea21027 B011Be 11e15eteaa1 Be 16 ta 11 0eB854 e5t3a9410eaB41104 BBBee50t07tea100t01taat5t5a17B eBt 0 B 150 B00e54teB13at 11 ee1ttaetBat1a55B551t850a13415055121500e511tae 2 0 e 01B1B1B1050eB0et 054Bae505te41B05059034B11B10s0ta60045ta 3 4B 1 201B B BB1e11e01500eB1at10at540a4ee1060B35B02e020ter45B4315 0 051 e1t0aBtat5a310e104151B05tt0a6aeB281eeB2t082t e1B01 1 1 451e21B0t515412Bt1Ba85103B0505tea8tat0ae5545tB130 B BB0ae5e05e0tae0e51t0te00t5a55taaeet3013 25 04 eBtaeB5Bta91taB5055a0e1a755t1a5555 taa 2 10 1107 2 tae15te0a1t2a10t90a25 55 a2 9 ta1s0250t0r0104 50915201385 et ta020Bt81taae022a202100153 22 Be 20627 B B2e1B0aeB2e1212et01a02t100284ta1220 ta21210 ta51 4e2ttaB2eteatteaBt2tat21a2102B1e1et0ta0a02702B0B11ee02t6a1 104022 10BeBBeeBeBtate02e5t45ate1a5B2tBaBe1t1a2e0022t4a211t39a02 1 0Be B0151B10B603eBetB0ta24e21e0110te1aB02t9ae27221 Be10B4e0ta1011500850555B121B05 Be ta5 1BB1eB2Be0t0Baet105a32te40a41t8B2eaBBt12ea20et2tata22ta2 Bteat4a14B04e241t231a001400B2b47eeB4Btet0aeBaet4at4eta14taB04Be ta41 1B10101e50501ta3515402B7B451Be1B8e10t0tae020a42t46a672BBeeta2 BBeBetB1eata0te4Ba41e1B40ta05e461taB4etea1at4a04140e60ta8t4a4 032 110ta042591B20e1t03a32 1031 BetBae4Bt1aBe04te1astt4a7atr1440110200138220401554 BetaB21eB0e3t0a Beta2 B1e0tta414B501Be50tea11t4aB421141e040ta01470 10513B0B1e43e0t95at534aB4984eBtae4ta4 19 10Beta14B014e02tta2B45e1t20a940 1013 BBeetata4411002B6eta4

AlpAhAAalp5Alp1h0a2571047Alpha5 1AA1A1l0plApl0p4hhl5phalap2ha55hAa511a51l0p015101h10103a30205132 AA10AAl1lp04llpl0lph50Ah3h1Aa5a1591aA54l5Ap01Al5lp01lp31h0hA0a11a4l52p59 Allp1AA0hlpllA1pplAphAhalhpllapa5aAh5h15ll1ph1a0ha50h1a50a8045589hAAalpl5phhaa55 h1a50h50h4plaA1pa25hAha1a10a500250120a104507s01t4r 410505lAp101lph50lp50h101a01350287 2 2lp26a1hh059a950511240 7h1a770711311h00100a13h32730a5079 1 a5 16 AlApAhlpllpalphh04lhapaA2ahlA7p0a2l701p28450002037ah7160a77a717 7 104AAl A 1A01h5A0aa77h1s1a70t074lr1p01A0l5hp0hphla0hpaha00aa7a7827a7277779ha A01101Al0pl1Aplhp0lp10 AAlllplpAhh7alap1h780ha1Aa85A7A2A7llplpl71pllphp10hhh0h02a12213lp AA1l1p50A101A4515hlAp6ah5aA1l4h00a47 pAhllpAahAl0lpll4p0145h5095h4h1aAAa7AAlp1lpl71lp10h01A A Al l1A0Ah32l1Ap040Al5p0AhApa4lp1h4A404l1Al AlA 1A111A010l0p5l3p36354a7A1A6aaA777l12p163 llppAplp10l5aA0lphl51p54l14phA4lapa8lp1h0a102p10Ah01ph 110A0lp0303h4p5h0haaA70704 ph1hAllphAhp34A3lhap5A8h7A80Ahllpap4h4Aha4a24319lap48 a4 1A111l0pl4phh11 3 ha0alhpaalh716la4hAlaAlp45lph4h1alp44710ha AA0lpllp7 171 1 a 44pah44aAp0A4p1al4lplplph9p3haA10a4h 0324 A1Aha 60002a91 811 2 4 01ah4a114lhp01h041hhha0Aha4l0117sa431 p 02130h9a1 01a1aa a 1 A041a4001laph80aaaa40alp400tr l 3011p1h11a 00a1a1h1h h8 0 2142204h1474433h50 A 21a21110llph2 7101h1a 1 2 l 0142a 06 a 4 01h1aa11A15pa2a321ahalha6lpp p02 1p00108a24 4 aa33aAhlh1 500ah1laph0h a 2l 2a 26a 1 100 4 1p0hh101a 00p p 2 280 2h 10 1hh0lph3lpApa010hp1lhhlAAh 00 a 5031 5 l 81 p0p a 3 a p1l pa0h1r 201l All 1Aah 10h A 2 laplA1lpl 52ht 1palpp 12727a912 A 4 1Ah01lpAA 20011l Apl1A46p 0hh13h A 4 AhA1 A05ps1 1 Al4 l 4h2p 20p 2 aAp16l 0411h5 AA 2l0a2h50l l l 11 0a 9 74 a 0 6 lpa 2l1Aa 1AaA 14 1pp010p0 0 p pA h 01 65A a 3 2 13 A a A p 5 0 5 121 p 58 ll l01 5 A l A A h a4A 11hl 3 la0hp 8 6 4A6 h 1h8 500 Ah 021 1 p A Ap a a A1 2 A h 1a 309 2 130l 0 0 A 1 l l A h 0 0 h 4 0 0 0 2 4 A p h0 1a A 0 A l p 1 5 040 6 A 1 l 0 3 ap 0 5 22 1 A A 5 p 2 A 0 A1p 1 1 p l l5pA3 01 Ah A 2 a l 1 l lh h p 1 0 A 1 h A4l 2 0 h Aap A p 1 l 014 0 82 A p a p a 1 0 0 l l 1 3 l A 1 h 1 p 0 l 4 a 4 1 12 a h0 0 0 A 0 A A 0 hl 0 l lp 1 A p 1 200 a p 00 2 3 aa 0 2 l h l p 1 9p2 2 l p1 p l A 0 0 l8 3 1 A p 55 1 99 h l hp l A hl 1 0 2 1 p h p 7 l a 0 0 l a 4 p pl 1 h A 1 A l 0 h 1 2 4a p p a4 lp 1 2aA2 1 1 l 4 5 hp A p l A11 1 A 5 0 l 6 1hAhl 0 l a 5 011 p a 3 a0 hh 1 h h101 p a 1 a 1 a 1 p A p h 3 l A 1 3 h Ap 0 4 161 p 0 A a l A 1 a 21 p Ah 30 p a 1 h A 6 1 l 1 2 h r 8 h 0 p A A h3a a h 0 h h h a l l a l 4 2 2 6 8 hA 1 2 ah 1 0a 3 1 l 50 1 A 3 00 t l a0h p a h 12 h 0 1 hpp 2 2 p6 3 h 0 p 1 0 0 0 h a 1A A a A 2 1 A p l h a0 12 h 0 0 l A l l l a 1 l l a a 0 1 p a p h p5 1 A p3 h h p h a 3 a a p 1 5 0 a a l A 33pp 10 a 1 l 0 6 a 3 11 l l l s A 0 1 1 34a 2 A A p 2 p A1 2 h 0 1 2a a p a a a h 1 6 3 l p l 1 2 4 3 a l 3 p 1 a a 6 h 3 h 0 1 0 4 a 0 l 1 p p 0h l a 1 l 6pl 0 hh 0 5 p Ap 6 1 A0 0 l a 6 hh s 9 6 p A 1 A 6 91 153 h h h l a l a 1 16 0 1 l l a 2 pA 0 0 l 0 2 1 l a a a a h al1 h h a 2 h h 6 1 p a l p a 2 1 p h 2 a 5 6 a 5 0 p h 1 5 1 p 1 h 5 2 t 6 a 6 a 0 1p 1 1 p 2 a a 1 1 a 0 2 a h A ah A l 1 0 AA0 0 l 6 7 a l 4 A 1 1 4 0 r a p p p 9 0 A 6 1 6 4 p h a h a h 0 3 0 0 5 A a a p hp h a 1 h l 6 p l l p 1 l h h 0 A 0 h 0 4 1 h a A h A 6 0 3 p2 p 0 6 2 0 l 6 l a 3 1 3a 5 l 0a l 0ahp 6 4 03 11l a l 6 0 A 2 a l 5 1 0 0 1l ll 1 0 h1 1 3 0 0 p a p 0 5 a a 1 h p h l l p0 p a p 0 6 p p a0 a 0 1 a 4 1 l a 0 h p l 3 l p p 0 p1 A l 11135 a l aa l 0 A 1 A a h a1 0 2 h 6 0 l 1 h l l 6 a p p A 3 a0 l 0 0 a a 0 0 3 a A 0 0 h 0

p 0 A l 4 p 5 31 0 p p h 1 6 1 h h 3 a 1 3 0 7 0 3 2 p 4 01 l p 6 2 00 ah a l h h0 0 3 a 0 9 h p 1 l1 2 0 4 a h lAA h 1 a 1 p l 1 6 4 h 1 5 l 3 0 2 1 l 0 A p A a h 5 1 1 A h 1 a l p 5 1 1 p 0 l h A aAA 0p2100 1 a 1 l 8 1 a p a a p 4 3 0 4 p a a A 9 0 s6 a A 9Aa 9 l p l A 0 5 7 l 3 l h 0 6 0 3 h 4 6 a 7 a a 0 3 0 h p l 22 l 2 A a p 95 2 h 0 3 l p ll 6 6 h 7 0 1 A 3 a h A 0 t 6 p pp 2 0 aA 5 l la 5 l 1 8 4 a 6 6 1r 1 h 2 a h p A 2 3p h p p l A 1 a A 1 0 hh 4 p 4 1 3 h 3l 3 1 0 l 0 0 h 7 al ap 6 6 h h 1 1h p a 4 0 A 1 6 l a aa 0 p A2 a 1 3 h 1 p 3 a a 4 a 0a h 3 l 3 7 3 3 33 4 3 h 1la73 3 h 1 4 0 1 3 a p 1 0 A 2 a 3 0 3 1 0 1 p 1 3 h 0

1 l 6 0 1 0 2 a 1

A 1 3 3 3 1 2 0 0.5

Figure 9: FastTree with default settings of the 14 eukaryal proteasome subunits with a broader sampling. Colors are the same as in Figure 4.

111

e B

t B a e B

e 3 r t 7 a e t t 1 1 3 t a s a B 0 0 1 3 3 3 1 8 e 1 0 1 a t 1 1 4 t 3 1 0 B

a 4 0 0 e 3 6 6 1 a e t 1 2 1 3 0 a 3 3 B t 2 3 0 t e 3 3 a 1 1 0 1 a a 3 6 t 3 e3 a t 1 B a a 3 3 0 2 1 B t 3 0 t e 3 a t e 3 1 B a a 7 t B a a 0 B t t 0 3 e e a 0e e t t B a 1 1 5 3 0 B t 5 0 B t e B e e 1 2 e e a e e t 1 B 2 0 0 0 3 a 0 e 3 B B 3 t 4 a e 5 3 t e t 3 e 9 B B 5 t B 3 B 4 a a B1 4 2 0 7 B a 7 e 3 0 a 3 t 0 a a 3 t e B 0 t t 0 6 a8 t 3 8 t 1 a 2 3 7 3 5 0 1 a 0 B a 3 1t a 3 1 e 1t 9 B 4 t e 5 e 5 0 5 B e 5a 1 2 4 B 1 3 0 3 3 2 3 0 e t 1 e e 0 4 0e B 0 B 8 5 0 B0 B 1 0 0 1 1 B 0 0 a a 1 e e 1 2 0 t 2 t 0 B 1B B 7 t 0 1 1 e 3 11 3 5 3 1 a 0 1 1 t 0 0 8 3 0 B 2 1 4 e1 9 t a e 4 00 4 a 3 3 5 a 3 0 a 3 0 t 4 0 0 t 4 5 a 3 0 11 B 0 1 t B 0 1 0 2 a 0 e0

1 e t 1 e 1 B 1 3 6 1 3 3 1 B1

e a a 0 B a 4 t Bt Be t 4 0 0 e e t B 5 ea e B 4 1 B B B t 2 0 e a B 1 e t 21 ta a 0 B 2 B2 s 1 e1 t1 e B r 7 1 ta 0 t 0 1 3 a e 0 2 20 0 1 1 2 t 5 0 1 B a 4 8 2 B 0 eB 2 7 2 B B 1 1B e 1 Bta B e t 4 e 0 0 1 t ae B2 e 0 B a t eta 0 5B e 4 2a et1 1t e 2 B a2 5 7a t 0 1 12 t 0 0 e a B ta e0 a21 1 2 B0 1 0 Bt 2 e 2 t 20 210 0 15 a t 1 e3 a 13 1 5e 1 a B0 2 242 01 3 0 2 a 2 e ta 0 0 Bt t 1 1 8 05 5 4 a1 B t1a 2 B 0 4 e e 1 0 0 B 4e20 t 2 1 e 2 1 6 B a 1 0 Bt 0 1 7 2 0 0 1 B ta 0 e B a 1 4 3 t e 6 0 4 0 1 1 e 2 1 a e 2 0 4 2 2 1 t 5 1 B ta 0 2 t a 1 1 7 6 B 0 3 a 1 0 B B e 2 5 a 2 t 1 1 e e et 0 B1 e 1 0 t t ta 1 8 6 2 0 1 1 a a a2 0 0 B9 2 ta 1 2 2 2 B e 1 3 e trta 1 1 3 4 20 1 0 1 1 t 0 2 0 B se 0 0 0 e a 40 a 0 005 1B 0 2 2 0 9 01 t1 011 a 14 9 3 2 t 2 e1 0 t B B 08 7 a B 11 a 11110 e et e 3B 2 ta Bt 10t1a1 B a2 10 ta24eBt e e e ta0atea1 1 5 a t B 1et 1e2 a e B 5 00 0B 0ta B B 1Be200 1 19 B e 2 2 2 1 taB 0 a BeB1e 04 etata101 ta e 101 et ta0t2a52 2B 22 52 e B 1a1 B 101101B0 e 10 B B 1 taet 48 451e0t7 ta2 19 e 3 0 ta eB 0 Bea2 ta2 2 5 e B 1 1 ta2 0 0 B ta eta 1 1 6 Be1 57B 1 5 4102 01 10 ta 10 101111 0e10ta7 e taetata a111B10 ta1 B B5eBBe ta11ee0tt5a 6Be 018 00337 Be B 104 ta11 11 45 etata01106Be 10 45e45tBaB11e1 1810B0Beta 105 1 539BBeeteatata11 110100454B 1030 ta1 013 Beta1 1052Be Beta1B1etae1t1a01311032 Beta1102B7 B10e4ta01B1e0t2a21

Be Btae4t1 Beta eta040110 1 BB4e1ta02 0030Beta 052Bet eta44181024 410 a4104 020 15 Be1tBeta4 1BB0ee5tat1a4B41e1t0a047 a41008 104081B0eta4 BBeetat1045Beta4a4411001121 3Beta41049Beta544Beta4 105 110e0ta349Beta4 1035B 056Beta4 1 9 eta4101 ta41025a4B 02 Be 050Bet 006 10 1 teat4ae4tata441 1009 4 005 14 ta4 05055taB74eB4BBe eteat4a4 eta 0441 10 Be 05181be 104 4B2B 43B 10eta ta4 1 10 10 eta4B Be 17str B 41e0ta4 BetaB

1043Beta5 B 1 e 105023B8Be1tea0t53a5Beta5Beta51002 ta B 1 Be BBeeta 5 e 0 taB5e1t0B2etat5a150s10421 1 B ta 4 1 a5410 tr B 0 5 B 1 0 1 28 eta5 3 Be e B 5 041 1017 eta 1 e 1 1 6B044 1 t 0 ta 6 0 0 e 1Be a5 2 5 ta B 5 4B8 tBa51005t5aB5 104 51 7 B1 5 e B 4 eB eta85BeB1ta 7Be 10 e t e B Btae 1B 5 eet0at5a51 ta5 1 0 a t1 11B e5ta 03et 10 5570B06 0 ta2 5 a0 e00 eB ta105 4aB5 18 eta 33 56 55 ta459tea1 5B10 1e10Be 5 0 1 10B 53BBt5B0B e01 0t4a508ta B0 0Be eae155e1e Bt0a0 5B 51 e2 2et tt012taBta e5t BBe 03 3 5at aa 005e5 a15 eeta 2 ta a5 1 55 0 1t1a 001 ata55 3 1 00 5 5 00 5 051 301 1100 2 1 4 70 5 11 0 1 2 21 0 2 9 BB 1 9 ee 6 ttaa a 55 t 1 e 00 B 9 B e ta 6 1 7 0 a7 7a 2 et 7 tat 6 B ta e e 54 3 e 7 B 7 1 0ta2 058 2 5 7 a B 6 1 1 1 t 8 7 0 2 aaB 7 3 e 00 0 1 0 tt 2 a 7 6 0 10 1 e 7 t B 1 2 0 6 7 3a 0 2 3 177107 10ta 0 ee a 5 t 0 1 7 4 a e 5 7a 70B 0 3 a 1 1B t 0 0 ta tata t1 7e B 0 0 e 1 t B 0 1 et e7 a7 74 0 0a5 e 1 9 e 8 7 0 1 e B 4 t 1 B t e 1 7 B BBeBtaeta4 0ta456 1 14 B 1e 5 e B a 0 1 7 1 0 4 B3 e 7 0 7 t a B 0 3 1 e 3 0 0 5 B t 0 4 B 10e 70 0 t 0 B1 1 7 a 0 a 0 B B0 1 a e 5 4 B 0 1 1a0 t 1 3 e 0 0 B t 1 0 t 7 e 6B 1 1 1 14 1 a B 1 7 4 1 2 1r 1 0 1 t e e B0 t t e 7 a 5a 20 5 e 5 B 1 7 7t 7 t 7 7 B aB e B e s 9 0 a 1 t ta B B0 71 e a 01 2 0 a t B 0 7e a 8 B e t a e a 2 e B 6 t t 4 B 4 t Be a 5 1 1 3t 0 t 9 e a e a 6 et B 1 3 B 1 2 3 B 5e B e t 1 6 0 e 2 e t 6 B 77 e 2 a e B t 6 e 0 1 0 2 a B1 a 5 0 B 9 t 0 B 1 aa 1 0 0 t B B 7 B a 6 t 1 1 tt 1 0 ea 6 0 6 7 1 a 0 1 8 e 0 6 1 1 e 1 e a 6 1 0 e 7 16 7 t 6 1 5 t0a 7 1 6 4 1 2 a t t 7 6 t 7 3 0 a 2 0 BB e a 72 a 1 s 0 7 a 9 1 a 0 t 0 6 a 0 e a t B a 3 6 t 1 0 6 1 t t a 0 t B6 1 7 0 t 0 B t 4 e 1 1 e B 1 r 4 e 5 B e 1 e 0 8 e 2 5 0 a 1 e e e 1 0 t 6 0 B B 0 B 6 B e 4 0 t 4 B 7 B 0 t a 7 B B 1 e B a 1 a 5 a e 0 t 8 0 3 a 6 t B t 5 a 6 B t 6 B0 e 7 6 t 4 B 6 B 0 a e a e e B 6 e t 1 1 a t e 1 0 B t a e e 6 t B 1 B e B 0 a B 1 e t

e t a e 6 t 0 0 a 6 5 e t a B 6 t a

B 0 6 1 a 0 t 3 6 7 1 a 6

7 0 3 0 6 4 0 6 1 1 3 1 1 1 0 1 0 1 0 0 0 1 2

1 1 2 0

5 7

2 0.6 Figure 10: FastTree with default settings of only the 7 eukaryal beta proteasome subunits, used for the beta concatenations. Colors are the same as in Figure 4.

112

6 1 0 1 C 3 4 B B 1 3 1 2 0 C

1 1

1 0 0 C 7 0 1

1 2 1

3 4 B 0 0 C

3 B C 3 7 0 tr1

B B

C B 1 sC

3 C 1 3 3 3 3 1 C 4CB B 3 1 B B B 1 0 6 0B3 3 B B 00 C 3

C 5 5 3 3 0 3 4 8 03 C 4 C B 9 3 500 B C 0 0 8 0 1

7 1 B 3 5 0 3 22 5 1 9 1 3 0 3 1 3 1 C 2 C 1 1 04 0 5 3 0 0 2 1 1 00 5C 1 1 4 1 C 0 7 0 5 0 B 0 0 B 0 C 1 0 0 1 2 B 1 1 1 0 1 1 11 0 0 B 1 0 1 1 B 1 3 C 3 1 1C 0 1 3 B 3 1 6 C 3 C 1 3 1 B 0 0 C 0 2 C 1 3 0 8 5 0 C C C 0 CC 1 00 1 BC CB3 5 B C 1 5 6 0 C C C 1 2 1 0 1 C B 1 1 B B 0 C 1 1 B 3 B 3 C B B 8 1 3 C 8 B B 0 3 C 0 6 1 11 0 B 3B 3 1 0 3 3 1 1 3 C C 3 B B 3 B 0 3 9 3 2 4 C2 B 2 1 B0 2 1 3 3 3 B B C C 3 B 4 B 0 049 9 4 5 3 3 C 4 C1 3 0 B B0 50 C 1 C B 3 0 3 0 1 4 7 4 3 1 31 010 2 Bs 3 0 1 1 1 C 4 6 11 1 t 0 C C1 2 0 Cr 0 5 6 8 C 0 B 2 B CCC 1 0 4 0B B 51 1 1 3 3 1 7 B 13 B3BB0 0 BC 2 1 7 4 C 1 0 0 1B 3 33310 4 B 1 C1 B0 1 C B 1 B 1 1 0 1 B C 1 B C 3 BC BB C10 C4 B 1 C BC C1 C 10 B C 3 C 1 C 10 1 3B 0 1 C B 1 1 B 1 0 1 B 1 1 1 1 1 00 0 1 3 3 1 0 1B 0 0 0 3 B B 9 B C 13 1 0 B B 21BC 02 C C 5B C 11 2 5 0 5 2 1 C 1 48 C 10 11 C11B1 0B1 0 3 2 0 0 10 8 1 1 1 1BB5 140CB 5CB 1 B 0 4 3 C 0 2 CBC7C 00231C 11 1 11C11 59 01 B01 4 B0 0 4 0 1 B 0 100 50 C0 1BB C11C05 64 90 1 11 C 18B5165 1 8 B 11BB1C1 10BC80 1 0 C BB1CBCBB1100 2C14 5 11BB10 CC1C01CC14 1 04 4 2 9 1CC3 1100130311005 104 6 0 3 1 B112 340960 5 56 9 2 310 05 1B C0033 4835371 2 230 03 1 102 1C1B101 021 10C3 C1 B1C0C113 10C C02B1 BC 6 12060BC CB1 B1C 2B2 7 01 2231 BC27 2CB 2049 01 1 9 02 2B2 B2 50043 str1 1B4C 7 20 2 10100 3 4CC 102 1 CC161 5 00B2B 2C C B2BBCC51 910 5 12 020B B 22 B04 4C 0BC C1 2 C20110B 102 220482B BC15C4028C 1100 050301 B2120B102BBBCC 110C10101 2CC 22 2B2BCB2CBC 2B2B 2 036 B305C319 CB10C2031704384 001121 2BBC221BCC111010 22BBC11 8 2 C2B1C0511045 8 C103 2B2B2BC101 7 2B BC2B12C022B41BB6C0CC4114102005B5085C6105 2 2BC1021 2BC1052 2BC1047 2BC120B4C01022

5B 5B C105B C51 16C1 B0C17 0514 5BstCr 5 BC1 1500BC 50B50C2 BC41103 5B5BCB1C010 0056 55BBC10101000013 C1100225480 55BBCC51B0C1 5BC150B5C31033957008 55BBC11001112 150B4C91055B4C1038 55BBC5C1B10C011400551 55BBCC11500B3244C181052 5B7C1000565584446 5BC150B55BC5C1B1C508B1C010 1 5BC10 5BCC1015064 C511B004530 55BBC1004029 CC10110925B5BCC1 5B5B 5 027 047 03113BC1 77BBC7CB11C00212309 5BC1 B0C312C01305602293 7BC10277B7B 26 BC5B15CB1 10C2110 040 7CB1C70B103310 5 5 5B5C5BBC C21 C1032 51B02 7 BC 7B 7 B7CB17C0476BC1 7BC 5 C10 BC170B77B1BBC0CC414105018 1047 38 7B7B5C21012700B5085C61 7BC7B1C0C104 1 057 7B B77CB7105110 5 7B C1 7BB1BC0C31710 77BB C 00CC131501303 CC11 7 1 78 040396 001121 B7 70 7B 84 B B5 7B C1 CC7 C4 77BBCC 700 7 1777B 17B 7BBCC110BC5 B 010BBC 04C 7CBC10220 1 C 45CC1 9 1C010284 700 1 161110 10 1005 B74C 70 00057 5 03 BCs B4 404907B 3 7B0 t1r C0 32 BC C1 01 1 C1 1 7 0 102 07 2 05 0B2 2 19 C 107 1B4C 040 10 C1 16 40B22 C1 41 B 290 056 4 8 4 004005 10 5 3 C111 BCC31 108 0 4BBCBC 4B04 4C90 1 4945 C41 0B10 C 0102 B 5C314C B 11 4 10B4B 4 CC 4 2 BB C 6 7 6 44 B 5 0 57 1 5 B 4 3 48 8 3 5 4 2 6 0122 5 3 40 0 C 100 02 0 44 0 05 0 6 B 10 0 90 0 41 1 01011 0 03311114 1 B C 1 66 01 1 1 4 CC 1 10 0 80C C 0 BB 1CC 06C0C 5 C 3 6 6 6 2C C 1 C 1 2 0 5 C 1 2 BBB 4C C 1 1 1 65 B 9 B 6 6CC 0CBB B 3B B 5 B 2 B 66 B 0 6 4 4 0 B 0 8 0 3 B 4 0 1 7 0B44 4 B BCC04 4 C C 4 2 6 6 B B B1 0 4 C 0 4 1 0 BB C 6C 5 1 1 14 4 4B 01 1 1 4 0 1 C 6 0 6 6 1 B1 B 1 B 0 6 6 B6 B 6B C2 C C00 B 1 1 9 C 6 C 4 1 00 1 4 6 C B 146 B B 4 r 0 C 2 6 1 22 tC 4C 4 4 C4 1 C 1 B B C BB 1 1 1B C7 3 4 C C06B1 1 13 2 0 B C C 39 s C C 5 1 C0 0 6 1 0 04 7B B B B C 0 C C 60 C B C 0 B5 0 0 0 1 C4 2 1 1 5 1 C 2 1 1C 0 0 1 4 4 B4 C4 2 C B1 B 0 C 0 1 2 B11 0 1 B 0 6 6 4 0 4 1 B 1 0 1 6 C 0 6 1 0 1 1 10 0 0 B 1 3 6 4 9 3 1 3 4 C 6 8 1 C 2 3 3 3 6 B B 6 0 1 1 C 6 5 C B 1 1B 4 1 4 6 0 0 4 005 3 3 C 1 2 B 37 0 C 0 C 5 1 0 B B 4 4 5 6 04 4 6 B 1 1 C 1 0 B C 0 5 5 C B B C 3 08 1 0 1 4 1 1 4 6 9 0 B1 C 0 C B 0 B B 0 0 0 0 B C 5C B 1 6 3 4 B C 8 6 6 8 B 0 C C 1 B 4 1 4 4 10 1 C 3 C B 1 1 6 1 4 B C 4 6 1 1 7 C 3 2 1 1 6 0 C C 4 1

C 1 6 C 0 0 1 B C C 0 4 8 B 6 B 1 0 0 6 B 0 0 C B 0 2

1 5 0 4 5 B 2 B B B 4 6 0 2 C5 B 4 4 1 0 0 7 1 2 6 B 0 9 6 4 6 6 C 0 8 7 5 C 1 1 0 6 0 1 B C 3 1 6 0 1 0 1 0 C B C 0 B 0 0 1 6 4 C B 1 1 4 BC

6 2 C B 1 0 6 Cs B 6 0

B t 6 1r 2

C 0

1 1

7 0 6

1 B

0.5 Figure 11: FastaTree with default settings of the beta concatenation. Colors are the same as in

Figure 4. 113

Figure 12: Same as Figure 11, but zoomed in to show only one of the 7 concatenations. Support values represent "SH-like local supports.”

114

p 2

A h 0 3

A l

1 4 p 1 a A l 1 2 7 p 1 l 0

h 1 p 7 7 a l 0 r h t p 1 0 A 1 1 a 0 2 A h 1 a h 1 2 s A a 1 5 h 0 4 7 0 8 7 0 l 1 a 0 p h 7 7 l A p 2 1 a 7 7 l 7 2 p l 5 2 4 1 a 0 1 p 1 7 4 7 3 a h p a 0 6 7 0 h4 l 1 l a a 00 2 0 A 1 0 h 50 0 0 h p a 30 h 1 1 h 1 1 A a 1 0 a h 1 0 1 1 Aa1 h 0 1 7 0 A A 3 7 0 A 4 p 7 00 1 p 4 a h h 7 0 l 7 7 l 0 p h 7 0 7 7 7 0 p0 7 3 1 l 1 40 l p a 7 2a 0 A l 4 0 l 00 l 2 p l A 3 0 7 a l p a a 4 5 p p 3 a p a p l 1 a A A a 1 1 7 0 1 13 A a 0 0 1 0 l h 0 h a0 11 0 l A 1 7 Ah A h Ah 7 0 h lh 1 A h 4 p 2 h h a h 2 7 7 l 0 A p 01 0 h h 1 1 7 1 A A h p A p 1 p 1 A l 77 8a 9 1 Aa a 0 01 a l p pp h 7 5 h l l 6 p a al l l a 0 A a 0 7 l h p 7 l 1 l l 3 p p 2 pl p 7 aa 0h A h l l 7 8 a 5p 1 0p p4a 4 1 4 20 7 l A hp 1 0 6l 5 A hh a l a 1l a 7 1 A p a p 2 h h 9 A a hhA 0 3h l p0 15h A a 4 lh 1 h 0 1 p 0 5 A 4 1 1 72 A A p h 0 1 0 3 A pp h lA 1 4 7 h 6 Aa h 0 4 h a a l l8 pp p A 0 h155 1a a 3 9 h 0 ll pAl 1 58 10 A 5 0 4 1 p 7l 2 14 a 0l 1 1 5 p l 3 1 a a 5 l A l 0 8 4A p 7 7 1l 6 p A 0 p a75A 4 4 4 4 pAA A A 0 9 3 0 A a 60 4 4 0 5 5l A 3 2 h 4 0 0 l 0 3 1 AA l 3 h 4AA51 h4 h 0 A 7 2 1 a p 1A5 1 A lp 1 20 A p 0 2 A h 2 a0 1 l l 7 41l A1 1 9 0 0 50 2 2 p h 0 a p 1 1 A 1 3 3 p l l 0 a p 0 2 A 4 A 4 0 l 0 h p h1 h Alp App0p h A 7 l 1 5 l31 0 a 0 h a 3 4 h 0 l 1 1 0 11 9p52 3 lp1 l A5 h l 0 4 3 p h l 1 A aa0 4p5lhp lh0pa 0 a 0 0 2 1 2202a 0 A Ah a l A A1 pA 2 A Al 44 1hAalp2paah7 p h 2 220l00h2 1 llpAlp la0 h a6 4 9 7 1 1 1 2 a0 2a0 p hlpl hps14 0a4hAh3 h a aa1ha11lpa7h33 h aphah4tr0 24lpa 4a a 0 a 225 h2pa10 Aa 4ha a10 h4alpa 4 a 1 h hh2lp0ap0h0l1 A 1 Al4 a4 0 21 4 4 7 hp 8ppa aal1lp1a2 lp 0 plh11041415 0a h 4 2 pl 0 ll ahh1A2a2a 10 ha48pha0101001107 04 a a 2 Al 0aAhlplp3aah68 47 A 41Ala44142003502 8 4 h a A51 2lpaa 5hhlp00a122 072 Alp Allp 01ph10 1 0 p h 392 5a 1l0plpa211hh2a001h0a 05 7 ha ha 8 a04228 l lp 03a 0 aaaA2Allphp2aA211lp 102a01240s1tr161014 4 41 4 aA 10h 1 lphh5A9hlp5ha1aha02h0ap2hlpaha2p1h0a2 012 4 1lp aal0p0a44a1lplA0plp21allpahalAlph00a2l 1 3 a 10154p8ha h2a1120010010135 0 110al aaalplaplphlhphaaa22110020824 1 alpahlpaha21 alpha21011 1047Alpha2 A A 1lp0h5a61A04Al Allpha6 0A1l0p1h2p9aAhlpa 1047Alpha6 104061A0l312 a6A1h60a12600 Alphp1ah6a6101 lpha569 0516Alp3ha6 1006 1041Alp1h01a40695A3l0Ap5lhp4ahA6alp6ha6 AAAlpha61007lplphhaa6611001201 Alplphha6a1601st10r074 000103 AlphAaA6lplplhphaaha66a611610010108252045 a6 AllpAphhha6a616061210Allp6h A11l0p103h1035a039A136A1l8Apl0phlp0hah8a11 61014AlpAhlplapha05428ah60a8 6 103A4lpAAhallpa1h1 Alpha A A A119l0pAhl1p0 02 A1lph phaa111011 013053 a6 2 61 100552Aa01Al 10l 04029 116lph 02ha 1 101510A4pl1phphah1aA1lpha 0h1aA 61 lp 1 10 A 60A5l6Aalp110191 61lp ha A 0 014 A1Alp AAlA 1pAhalpha1 ha4A lp 5 41016 lp0lph5 AlpphAhlph0A401ha5 lp03 A 14 0450A Ahha11 lp alpa11al1phalp A1 0A A5A75lp Al aaA1 ha h1a1010a1ha 41 l5Apl8ph Aplh11l1p011A01 0112726101 A l9p0 AhlAha lppaAA00ha0 4lp1h0 A3l03 23 l Ah5 pala1 hh1lplp2071 3Aa2Aplh0 1 p a3 lphp11 aa1hAh0 l1p2 pha1 0 h lp1A hah1 110alap1 shtr a1 A 4 A a hl a1a 101h a 103 A p 01 00311a0 1 02 lp 1 l 5 a h 10 10 0 3 1 pl 1 1 6 50 120 1 A h A phA a 182 A 0 0 0 01 0 1 5 0 9 A h 5 9 A a l l A l a 2 1300 A 1A p Ap 2 2 1 1p 4

0 5 l2 1 000 1 a 4 0 a 0 p A 5 5h l 7 011 5 5 l h l 1 l 0 05 2 l pp 0 h l 5p p 0 0 1 1 0 333 0 p p 1 A 1 a h 0 h a a3aa 3 0 a 5l A h 0 h 3a 7 6 2 h 5 3 p 1 a h 0 2 0 h 3 3 1 h A 0 hh 1 h 5 5 4 a A 1 p 1 a A h 0 l p 0 h a l pp 03 7 l 5 1 0 l 1 a a p 4 5 hl 1 l h 0 0 a p a 4 9 0 1 A a a 1 0 5 l 8 0a0 3 5 a p l 1 A 3 7 A 0 p h 1 5 5 5 p AA1 l 1 3 1 l A1Aa pl 33 2 0 l 5 h A 0 1h h p 4 A 1 5 p 6 1 a 0 A 5 4 a 5 5 a h A 3 p a 5 1 0 l 1 30A3p3l a 3 a 1 1 h 0 a 2 l h 1 0 p 0 l5 3 p 1 a h 0 0 5 h 1 l 1 1 0 l a l A 1 p 5 a a h aa h 0 h 0 A h 1 l p l 0 p 0 0 p 1 ar h 5 a 3 A lp 1 r p p 1 h hh l 3 h 0 1 a p 1 t 0 5 0 5 0 5 9 a t a 0 l p 1 h 5 2 3 2 0 h a 1 p p 5 p l A 0 h 0 p h 3 1 2 h 5 l p a 3 l p2 A 3 a 9 3 l l h h 4 a 8 0 A l A 5 h s l 5 1 1 1 1 0 s 0 A 2 h 5 0 a a 5 4 A 2 A 8 5 4 0 9 p 3 a p 6 1 a 1 A 0 p a 5 a a 0 h 4 a lh 0A h a l 0 1 l A 5 0 0 3 A 0 5 3 0 7 3 1 5 1 3 4 A p l 1 0 5 l 1 3 2 a 1 0 3 1 A 5 A 5 0 p p h h l 5 5p p 0 a l 1 03 3 3 h p 5 7 0 A l 1 0 1 a l A a 0 aA 2 0 3 a 3 0 A 3 a p 1 h 0 1 1h 10 7 1 p h 1 1 h l 1 a l 3 p l A l 0 p a ha 1A1 a h hl 8 p h h A 0 1 2 3 p 4 1 0a h3 a 0 h p l 2 3 p A a h p l h 3 h p p A l 1 h 0 p 0 p l a a l l A 2 8 25 p 4 l al 2 h l 5 a p a A a A 1 A p 0 4 l A 9 A h lh A 4 A 1 h A 9 A 5 p 1 5 24 3 p1 hl 4 A 5 1 A p 3 0 p3 l 0 A A l 3 al 4 p l 0 p 01 43 l A a 0 0 h a5A 1 2 1 10 A 0 A 1 l h h 4 h 1 p 1313lp9 06 A 4 3 1 6 a 3 1 p 0 p h a A4l 15 0 2l a 0 1 5 1 h 0A 0 1 a hp 1 1 1 0A 0 pl 3 3 1 5 4 Al a 5 0 7 A h 0 35 A p0 1 a 0 l3 h lp A0 1 h 1 lp a 3 A 1 a h lp A

0.5 Figure 13: Phyml Maximum Likelihood tree with only the 7 eukaryal alpha proteasome subunits.

Colors are the same as in Figure 4.

115

1 6

1 1

0 0

1 1

C C

A A

A 4

1 1 C 1 6

1 0 r 1 t A 1 0 A s C 4 C C C

1 0 2 A A 0 1 1 1 1 1 1 0 A 2 6 0 6 3 7 1 C 0 A 1 4 A 1 8 1 1 0 C 2 2 4 1 1 A 1 0 C C 1 0 0 0 A 0 1 1 C A 1 A 5 1 0 0 1 1 1 C 7 6 C 3 C 8 C A 1 1 16 0 1 A 1 5 A 2 1 A CC 0 0 1 3 A 0 6 4 6 C 1 0 A C C C A 1 1 A 1 1 1 2 0 A 0 1 0 A 0 C 0 3 1 A A 1 s 1 C 1 4 3 3 1 0 C 5 2 C C 1 C 0 0 1 1 8 t 0 5 C 3 r 0 A 1 A 0 1 2 9 0 2 0A0 0 1 A 6 1 4 0 6 1 1 C 1 4 0 8 1 1 A 6 1C10 0 0 2 1 3 0 6 3 C0 4 5 0 1 1 C 1 A1 1 0 1 C 1 6 A 8 C 0 A 0 1 1 0 5 1 6 2 0 1 C A1 A 4 C C 1 1C 7 A 9 9 0 A C 1 A 1 3 A 2 C 0 0 1 A 3 A C C 0 A A 1 1 5 C 1 3 07 C C 1 0 1 1 1 1 0 A 1 0 1 1 6 1 0 0 1A3 3 6 0 1 6 1 7 A 1 1 1 5 1 2 1 1 0 4 0 1 1 A 2 C A 6 0 6 2 0 C 0 C 0 0 0 0 C C1 C 5 1 A 2 2 C C C A 0 6 A C A 6 1 4 2 1 5 2 1 0 A AC 11 6C 5 1 1 8 0 C 6 A A A 6 5 A C 666 C 7 5 4 0 0 1 A 0 A16 C 1 1 A 0 0 A A 1 1 0 6 22 AAA C 4 1 5 A C C0A6 1 0 1 0 1 1 66 6 A 1 1 C 0 A 48 A C CC 0 0 1 AA 1 1C C 6 1 9 C6 1 1 A 1 C 1 C 6 A CC 0 50C 1 111 A03 C 4A4 1 CA C 1 51 0 36 1 4A 0 6 1 0 00 C 1 A 0 30 01 0 0 C A01 1 A 6 0 30 105 18C 1 5 4 A 1 AC 5 5 10 1 C 7 9 6 0 071 1 6 C1 8 6 0 1 1 A 1 3 C A 610 A 3 0 A 4 A02 6 C 5 A 1 0 23 A C 93 C 1 1 C6 1 7 1 1 1 6 C 0 1 1 C 0 0 2 0 0 4 6A 1 0 A 1 3 66 6AC 0 4 0 1 C A C 4 5 CA 6A 1 8 A0 6 6 C AC10 8 54 6A A A6A 101 C105 0 C C1CC 301 1054 1 6A 10 01010 48 6A002 C C 5 42561A C656 A 6 160A 6 09C 6A A5 5 3 01 31C1 1 C 10C 5 2 1 6 03 6A 02 10 410 109 2 AC A 2 6 C 5 2 64 C4 10 54 C1 A 10 1 4 A0 C 01 036A C 0 5 1 A 1 0C 10 9 C 5 248 AC r 10 41 5A 0 2 5 st 613 0 1 5 00 C A 1 117 AC 1 1 5A C1 2 0050 5A130AC 17 02 54 20 11 00C005 040 7 0 51 CC 0A11 10C1 1 10C 5AA 0 1C05 C5A C A0455 2 5CA1 55A A C5 1 10 5AAC 00 55A C C 5 C1 5A 5A 5A 86 339150033 8 110AA07CC1 100314 AAC1C0553 85ACC1 7 A55C8 1045A 1058 5150A0C 5AC 5AC C10431023 10150506 5A C5A10C26 2A 5A5ACC 1041 5A 030 031 C1 1C0414046 5AC 1027 051A3C1 C150A3C21 011 5A5CA 5AC 5AC1 5A 2AC 5AC1042 1042A 5AC10095A5ACC10110950 2A 2 0 C1 5AC1025 C120A A2CAC 012 3C110 101202 2AC32 3 6 1023 5AC1056 A0C1 0213 2AC2A AC1 2AC1 1C051304 027 022 9 2A 2A2A2AC10 2AC105 C1C011083441 C10524 2AC 2AC12210A023C3A618C120A3C71008 A2CA1003359 2ACstr2AC1017 C1048 2AC10042AC1005 2AC10126AC10142AC1002 2ACC1013001 2AC2A12C0A11500 C1020 2AC10248 2A1021 045 1044 2 2AC11000157021AC1 25A5C046 22AACC1 C1100C61 22AAC 2A 100578 25 09 2AC1 C10C10 021A09 2A AC11052 22AC104 043 056 3AC1012 3AC1011 AC C1 C1 2 2A 2A 3AC1040 3AC1027 3AC1031A3C1030 3AC130A32C1031 3AC1009 3AC 3 3AC 3AC 3 1032A5ACC110015042 3 102A1C10 19 3AC1 41 33AC10 1052 A3CA10423 3A 3 4 3 C1062 C3 AC AC1 3 1A 3 10 3A3 056 0C AC 20 CA1C 341 1 3A3 041905 3A40 3A 3 04 AC3CA 3 AC4 C A 5 3A 10C3103A C16 133C 33AA C 0A110C000C1 10 0A3A10ACC 10 3130 028 055 0CAC 4 11 05 A15 4 3 0 8 1C31 8 0051 3 C A 6 0313A003 0170 A 10 C A7C39 3C1 02 1 3 C15 AC00 3 0 A3 103 14 A 2 CA 0 8 0 C1 2 1C36 17 0 3 43 3 01 3A 14A 10 3A 30 C C1 C C 41 str 01 4A 1 8 6 100 7 55 02 1 4 87 6 1 4 9 4 32 3C 0 0 0120 2 1A 15 00 54 6 1C 1 104 C2 1 0 41 C 0 A 0 8 0 A4A 1 C 4 1 C 1 C 4 C 0A 0 1 3 C A C 0 5 4A 32 04 A9 4 A 1 A 0 0 1 410 4 C465 4 1 C 0 C 00 4 0 A 15 7 A C 5 4 1 02 A3 01 1 1 A3 4 C14 4059 2 4 4 0 1 C 40 3 1 6 0 A AC0 13 5 C 1 6 1 4 1 9 0 0 0 1 4 0 5 C A C 0 C C C 4 811 8 1 A 4 4 6 2 0 A A 03 A3 1 C 7 4 4 A 3 C 0 4 A 4 0 1 C C 1 4 5 040 5 A 7 C 4 A C0 AA4 5 1 0 1 1 1 6 7 1 A 5 A 4 1 1 C 7 A1 4 3 0 1 A 2 70 7 C C 1 0 7 A C C 7 C 0 0 5 4 0 C 4C 8 0 0 0 A C 4 1 A 4 8 A 1 AA 1 0 A 0 A C 0 1 A 1 1 3 1 00 A 1 44 4 0 0 C 0 4 C0 10 4 A 1 1 CC 1 0 C 0 1 7 1 C 1 1 4 C 1 0 7 2 7 0 2 A C 1 7 A 50 C C 1 1 7 1 A C 1 7 4C A C A A 0 A 4 AC1 C 8 0 0 4 7 A 1 A A 0 7 9 A 4 A 7 C C 7 C A C 4 4 4 0 A 7 5 7 0 A A 5 4 C 4 7 C A 7 4 3 0 4 7 1 1 4 0 5

A A 1 8 7 2 4 8 A 1 4 A 7 7 1 2 7 C 1 0 1 0 C 0 A 22 1 A 0 7 5 7 C C4 A 0 0 5 C 7 0 7 17 C 1 7 2 1 00 1 A 1 0 0 C 2 A A 0 C C A 1 A 3 C A 7 1 5 0 1 1 A 1 0

2 C 0 C 1 4 1 8 08 C C 1 A C 7 2 4 A C 0 1 3 1 C 7 7 CC 2 6 A 3 1 C A 3 1 A 2 C 0 7 C C 3 0 A 1 9 A 0 0 4 3 1 7 0 AA 0 0 A 7 1 4 C r A 5 0 t 1 5 0 A 3 1 44 A 6 3 0 0C1 C 4 1 0 7 7 1 s 4 4 1 5 1 0 7 4 0 C C 5 4 1 0A0 A 9 0 1 1 0 C 1 C 0 8 77 0 0 7 1 0 7 1 A 5 0 7 7 A 0 C C0 C 1 5 A AA A 0 1 5 0 A 1 7 A 4 1 4 0 4 2 A 1 1 3 0 7 7 7 0 A CC 9 C C C C C C 3 0 2 1 7 0 A 1 A 1 1 A 7 C 11 5 1 A A 1 C 0 0 46 C C A 00 7 C 3 0 7 1 5 4 3 1 2 12 A 1 2 0 A C 0 7 0 1 84 06 7 0 1 7 0 A 1 2 0 C 4 3 C 0 A 2 4 1 1 7 7 0

A 1 7 1 A C

C 1

0 s 1 t 7 r 4 A

6 0.2 Figure 14: FastTree with default settings of the 7 alpha concatenations. Colors are the same as in

Figure 4.

116

Figure 15: Same as Figure 14, but zoomed in to show only one of the 7 concatenations. Support values represent "SH-like local supports.”

117 6.4. Discussion

It was thought that concatenation might increase resolution and that adding additional taxa would break up long branches, allowing for the determination of the location of the deepest split, and therefore the root within Eukarya. Rooting the eukaryal tree will provide polarization to the tree, which will help future scientists choose between various evolutionary scenarios.

Rooting the tree will also help identify synapomorphies, which are needed to properly divide the

Eukarya into Kingdoms.

Unfortunately, there is a tradeoff between breaking up long branches and decreasing support values, because the more taxa that are added to an alignment, the lower the support values become. After exploring a myriad of options for using histone, tubulin, and proteasome concatenations to root LECA, there is not enough phylogenetic signal in any of these datasets to produce an answer with confidence. The analogy of throwing a dart across a football field seems to be holding true here and these datasets are not very good at throwing darts.

Given the lack of resolution in these phylogenetic trees and the Long Branch Attraction artifacts, no difference is observed between these trees and those previously published rooting the Eukarya. There do seem to be a number of supergroups that are being seen over and over again; it is just that where the root is within these groups changes. Except, that while I acknowledge a complete lack of trust in root placement, my competitors have thrown fewer darts and published on whichever rooting they happened to find.

The proteasome dataset is not fully exhausted. Taxa suffering from Long Branch

Attraction and those that have experienced Horizontal Gene Transfer could still be identified and thrown out. Or alternatively, a supermatrix approach could be used instead of gene 118 concatenation. But ultimately, that long Eukaryal stem branch leading to the outgroup is the biggest remaining problem. Identifying a gene duplication that occurred just prior to the diversification of LECA would be the ideal solution to this problem. Dr. Gogarten has suggested looking into the Chaperone family with this aim in mind; a task I will leave to incoming graduate students.

119 7 References

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