ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2017

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 1516

Genomics of Sorocarpic Amoebae

SANEA SHEIKH

ISSN 1651-6214ISBN 978-91-554-9913-6urn:nbn:se:uu:diva-320432

Dissertation presented at Uppsala University to be publicly examined in Lindhalsalen, Norbyvägen 18D, Uppsala, Friday, 9 June 2017 at 09:00 for the degree of Doctor of Philosophy. The examination will be conducted in English. Faculty examiner: Assistant professor Matthew W. Brown (Department of Biological Sciences, Mississippi State University, USA).

AbstractSheikh, S. 2017. Genomics of Sorocarpic Amoebae. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1516. 45 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-554-9913-6.

Sorocarpy is the aggregation of unicellular organisms to form multicellular fruiting bodies (sorocarps). This thesis is about the two best-known groups of sorocarpic amoebae, Dictyostelids and Acrasids.

Paper I describes assembly and analysis of a multigene dataset to identify the root of the dictyostelid tree. Phylogenetic analyses of 213 genes (conserved in all sequenced dictyostelid genomes and an outgroup) place the root between Groups 1+2 and 3+4 (now: Cavenderiaceae+ Acytosteliaceae and Raperosteliaceae + Dictyosteliaceae). Resolution of the dictyostelid root made it possible to proceed with a major taxonomic revision of the group.

Paper II focuses on the taxonomic revision of Dictyostelia based on molecular phylogeny and SSU ribosomal RNA sequence signatures. The two major divisions were treated at the rank of order as Acytosteliales ord. nov. and Dictyosteliales. The two major clades within each of these orders were given the rank of family. Twelve genera were recognized. This is the first revision of a major protist taxon using molecular signatures and offers guidelines for taxonomic revision of protist groups where morphology is insufficient.

Paper III presents the mitochondrial genome (mtDNA) of Acrasis kona. Over a quarter of the genome consists of novel open reading frames, while 16 genes present in the mtDNA of its relative, Naegleria gruberi, are missing. We identified many of these genes in the A. konanuclear DNA, and used phylogenetic analyses to show that most of these genes arose by transfer from mtDNA.

Paper IV presents the nuclear genome of A. kona, the second genome sequence of a free-living excavate. The 44 Mb genome has 15,868 open reading frames of which 4,987 are novel. A surprising number of genes are most similar to homologs in distant relatives, suggesting acquisition by horizontal gene transfer (HGT). Most HGT candidates are expressed and many constitute multi-gene families and/or have acquired introns and membrane targeting sequences. Strong HGT candidates include some genes essential to development and signaling in Dictyostelia. Flagellar motility and meiosis genes are also present and conserved, suggesting cryptic flagellar and sexual stages.

Sanea Sheikh, Department of Organismal Biology, Systematic Biology, Norbyv. 18 D, Uppsala University, SE-75236 Uppsala, Sweden.

© Sanea Sheikh 2017

ISSN 1651-6214ISBN 978-91-554-9913-6urn:nbn:se:uu:diva-320432 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-320432)

For my parents

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Sheikh, S., Glöeckner, G., Kuwayama, H., Schaap, P., Urushihara, H., Baldauf, S. L. (2015) Root of Dictyostelia based on 213 univer-sal proteins. Molecular Phylogenetics and Evolution, (92): 53–62.

II Sheikh, S., Thulin, M., Cavender, J., Escalante, R., Kawaka-mi, S., Lado, C., Landolt, J. C., Nanjundiah, V., Queller, D. C., Strassmann, J. E., Spiegel, F. W., Stephenson, S. L., Va-dell, E. M., Baldauf, S. L. A new classification of the Dicty-ostelids. Submitted.

III Fu, C-J., Sheikh, S., Miao, W., Andersson, S. G., Baldauf, S. L. (2014) Missing genes, multiple ORFs, and C-to-U type RNA editing in Acrasis kona (Heterolobosea, Excavata) mitochondrial DNA. Ge-nome Biology and Evolution, 6(9): 2240-57.

IV Sheikh, S*., Fu, C-J*., Miao, W., Baldauf, S. L. Multicellular-ity in the Excavata, the genome sequence of Acrasis kona. Manuscript.

Reprints were made with permission from the respective publishers.

Sanea Sheikh made primary contribution to the experimental design, data assembly and analyses in Paper I, II and IV. In Paper I she performed all the analyses, wrote the first draft of the manuscript and contributed to editing the final version. In Paper II she developed the protocols for identifying molecular signatures, performed all the analyses, prepared all the figures and helped write the manuscript. In Paper III she conducted phylogenetic anal-yses for the selected genes. In Paper IV she annotated the genome, per-formed all analyses except genome assembly and metabolic pathway anal-yses, wrote the first draft of the manuscript and did editing for the final ver-sion of the manuscript. * These authors contributed equally to this study.

Contents

Introduction ..................................................................................................... 9Protists ........................................................................................................ 9Amoebozoa ................................................................................................. 9Excavata .................................................................................................... 10Dictyostelids ............................................................................................. 11Acrasids .................................................................................................... 13Evolution of multicellularity .................................................................... 15Life cycle of sorocarpic amoebae ............................................................. 15Dictyostelid phylogeny and taxonomy ..................................................... 17Eukaryotic genome annotation ................................................................. 20The Acrasis kona genome ......................................................................... 21

Objectives ...................................................................................................... 22

Materials and Methods .................................................................................. 23Phylogenetic analyses ............................................................................... 23Genome annotation and analyses ............................................................. 23

Results and Discussion .................................................................................. 28Summary of Paper I .................................................................................. 28Summary of Paper II ................................................................................. 30Summary of Paper III ............................................................................... 32Summary of Paper IV ............................................................................... 34

Svensk Sammanfattning ................................................................................ 36

Acknowledgements ....................................................................................... 38

References ..................................................................................................... 42

Abbreviations

biPP: Bayesian inference posterior probability BLASTp: Basic local alignment search tool for proteins cAMP: Cyclic adenosine monophosphate EST: Expressed sequence tag mlBP: Maximum likelihood bootstrap percentages HGT: Horizontal gene transfer LBA: Long branch attraction LCA: Last common ancestor mtDNA: Mitochondrial DNA ncDNA: Nuclear DNA ORF: Open reading frame RNAseq: Whole transcriptome (RNA) shotgun sequencing SSU rRNA: Small subunit ribosomal RNA tBLASTn: Basic local alignment search tool for protein query against translated nucleotide databases

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Introduction

Protists

Protists, Protoctista (Hogg 1860) or Protista (Haeckel 1866; Scamardella 1999) are a diverse, paraphyletic assemblage of eukaryotes that lack “complex multicellularity” (i.e. all eukaryotes except plants, animals and fungi; Cava-lier-Smith 2003; Adl et al. 2005; 2007; 2012). Most protists are microbes, however the term has also been used to refer to organisms with simple multi-cellularity, such as diverse algae, or transient multicellularity, such as aggre-gating amoebae. The latter are characterized by a unicellular trophic (feeding) stage that alternates with an aggregative multicellular stage induced mainly by unfavorable environmental conditions such as scarcity of food. This thesis is about the most diverse and best studied of these aggregating amoebae, the Dictyostelids and Acrasids. These taxa were previously classified together due to similarities in their life cycles (van Tieghem 1880). However, both molecu-lar and morphological data now place them far apart with dictyostelids as-signed to supergroup Amoebozoa and acrasids to supergroup Excavata (Figure 1) (Page and Blanton 1985; Roger et al. 1996; Adl et al. 2005).

Amoebozoa

Amoebozoa is a major lineage of eukaryotes that is closely related to the ma-jor group including Metazoa and Fungi (Opisthokonta) (Figure 1). The cells of Amoebozoa have a fluid shape caused by dynamic protrusions known as pseudopodia (Schilde and Schaap 2013). These pseudopodia function both in locomotion and capture of prey. Amoebozoa consists mostly of heterotrophic amoebae, but the group also includes some amitochondriate parasites (e.g. Entamoeba), some flagellated cells (e.g. Multicilia), the plasmodial slime molds or giant amoebae (Myxogastria), and the cellular slime molds or social amoebae (Dictyostelia) (Burki 2014).

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Figure 1. Eukaryotic tree of life. The tree shows the relationship between the three eukaryotic major groups: Amorphea (AMR), Diaphoretickes (SARP) and Excavates (Exc). The two organisms studied in this thesis are indicated in italics – Dictyostelia in Amoebozoa and Acrasis in Discoba. Figure modified from He et al. (2014).

Excavata

Excavata is probably molecularly least well characterized of the eukaryote supergroups. It is also the only one defined primarily based on a shared mor-phological character, a deeply excavated feeding groove (Simpson 2003). Excavates can be roughly divided into groups with and without aerobically functional mitochondria (Figure 1). Amitochondriate excavates (AME) live in anaerobic or microaerophilic habitats and include the Fornicata, Parabasalia, and Preaxostyla (Simpson 2003). It is still not clear whether AME is a mono-phyletic group (Liapounova et al. 2006). The mitochondriate excavates or Discoba are almost certainly monophyletic (He et al. 2014) with four major divisions: Jakobida, Heterolobosea, Euglenozoa and Tsukubamonadida (Adl et al. 2005; Yabuki et al. 2011; Kamikawa et al. 2014). The best studied exca-vates are Trypanosoma and its relatives (the Kinetoplastida), which are para-sites. Kinetoplastids cause a variety of diseases in humans and livestock such as sleeping sickness, Chagas disease and the wasting disease, Nagana (Daniels et al. 2010). The genome of the heterolobosean, Naegleria gruberi, is the only free-living excavate genome that has been annotated so far (Fritz-Laylin et al. 2010).

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Acrasidae is one of 4-5 major divisions within Heterolobosea and includes the genera Acrasis, Pocheina, Allovahlkampfia and Solumitrus (Page and Blanton 1985; Brown et al. 2012). Acrasis and Pochenia are both sorocarpic and very closely related. One member of their sister group Allocalhlkampfia has also been induced to form a sorocarp in the lab (Brown et al. 2012), but no soro-carp has been observed in Solumitrus spp. The morphologies of Acrasis and Allovhalkamfia amoebae have not been compared in detail, and it is possible that both Allovhalkamfia and Solumitrus are acrasids with unknown or re-duced multicellular stages (Pánek and Cepicka 2012). Acrasidae are the only known excavates that undergo aggregative multicellularity.

Dictyostelids

Dictyostelids are a large and ancient group of sorocarpic amoebae (Fiz-Palacios et al. 2013). All known species respond to starvation by aggregating by the hundreds to hundreds of thousands to form multicellular fruiting bodies (sorocarps). In most species, this process also involves a type of programmed cell death (not homologous to apoptosis), whereby a substantial fraction of the aggregating cells (~20% in Dictyostelium discoideum) are sacrificed to form an inert stalk for the fruiting body (Raper 1984). This raises all sorts of ques-tions such as – how do cells recognize each other, how closely do they need to be related in order to sacrifice themselves, etc. (Strassmann and Queller 2011). Aggregation and development require cells to communicate in various ways. A variety of secreted small molecules (“acrasins”) are used by different spe-cies for signaling during aggregation, including cAMP and small peptides such as glorin, folate and pterin. In addition, all dictyostelids appear to use cAMP after aggregation, in order to control cell differentiation during devel-opment. Kinship recognition uses a completely different system, the tgr genes, which encode rapidly evolving cell-surface proteins also involved in cell ad-hesion (Baldauf and Strassmann 2017). Dictyostelids were first isolated and described in 1869 (Brefeld 1869), and there are currently approximately 150 described species. Traditional taxonomy recognized three genera, which correspond to the three basic fruiting body morphologies - acytostelid, polysphondylid and dictyostelid (Figure 2). Acy-tostelids have small morphologically simple sorocarps with acellular stalks (no cell death). Polysphondylids have a cellular stalk with regularly spaced whorls of side branches, and dictyostelids are essentially all other species with a cellular stalk. Dictyostelids represent a wide variety of morphologies includ-ing simple sorocarps without side branches or various irregular patterns of side branches, and the sorocarps may be further clustered or even partially fused (Raper 1984; Kawakami et al. 2008) (Figure 2).

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Acytostelids were long considered to be the earliest branch of Dictyostelia, which, due to their small simple fruiting bodies and acellular stalk were thought to have diverged before invention of the cellular stalk. However, the first comprehensive molecular phylogeny (Schaap et al. 2006) did not show acytostelids as the deepest branch of Dictyostelia. Instead, they are found in the heterogeneous and newly circumscribed Acytosteliaceae (previously Group 2), which also includes species previously placed in the genera Poly-sphondylium and Dictyostelium (Figure 2 and Figure 3). In fact molecular, phylogeny rejects the monophyly of all three traditional genera. Instead Dic-tyostelia is divided into 4-5 major groups and 3 small complexes, none of which correspond to traditional genera (Romeralo et al. 2011). The acytoste-lids are also not monophyletic in the tree, suggesting that the cellular stalk was lost and then re-invented, polysphondylid morphology has evolved at least twice independently, and dictyostelids are scattered throughout the tree. To-gether these data indicate that Dictyostelia requires major taxonomic revision. However this was not practical until the root was firmly established, which was uncertain in the early single-gene trees (Schaap et al. 2006). The dicty-ostelid root and taxonomic revision are the subjects of Paper I and Paper II of this thesis.

Figure 2. The three basic dictyostelid morphotypes. Three general morphotypes form the basis of traditional dictyostelid taxonomy. These are acytostelids, characterized by an acellular stalk, polysphondylids with a cellular stalk and regular spaced whorls of side branches and dictyostelids with a cellular stalk and no or various irregular pat-terns of side branches. Figure modified from Swanson et al. (2002).

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Figure 3. Phylogeny of Dictyostelia. The tree shows the major divisions of Dictyoste-lia, the position of the root and the new names. The dotted line represents the position of parasitic sister taxa which could not be used as outgroups for Paper I. The names in shaded triangles show the old informal names of the molecular groups (Schaap et al. 2006). The names in red show the new names presented in Paper II.

Acrasids

Acrasids are the best-known non-dictyostelid sorocarpic amoebae. Like dicty-ostelids, they undergo aggregative multicellularity, but, unlike most dictyoste-lids, none of the acrasid cells comprising the sorocarp are sacrificed. Dicty-ostelids and acrasids were long considered sister taxa due to the similarity in their life cycles, with alternating single and multicellular stages (van Tieghem 1880). However, Olive pointed out that acrasids and dictyostelids show strik-ing differences in the morphology and behavior of their amoebae (Olive 1975; Page and Blanton 1985). Acrasid amoebae move much more rapidly and ab-ruptly than dictyostelid amoebae. Moreover, when they aggregate, acrasids migrate singly or in clumps rather than aligning themselves into the strikingly organized migration streams of many dictyostelids. Acrasid aggregates also do not migrate as a unit. Olive (1975) also noted that the stalk cells of most dic-tyostelids are dead, but the stalk cells in acrasids remain viable (Olive 1975).

Group 4

“violaceum”

Group 3C

Group 3B

Group 3A

Dictyostelium

Polysphondylium

Raperostelium

Hagiwaraea

Tieghemostelium

Group 2B Heterostelium

Group 2A Acytostelium

“polycarpum” Synstelium

“polycephalum” Coremiostelium

Rostrostelium

Dictyosteliales

Acytosteliales

Acytosteliaceae

Group 1 Cavenderia Cavenderiaceae

Dictyosteliaceae

Raperosteliaceae

Speleostelium

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Based on these observations, Olive (1975) separated class Acrasea from other sorocarpic amoebae. Page and Blanton (1985) then moved acrasids to Heter-olobosea based on similarities between their amoebae (Figure 1, Page and Blanton 1985). This classification was eventually confirmed with molecular data, which placed acrasids in Heterolobosea (Roger et al. 1996; Baldauf et al. 2000; Adl et al. 2005; Brown et al. 2010; Brown et al. 2012). Acrasis rosea has been used as a model organism for the study of cell ultra-structure until the late 1990s, because it is tractable in the lab and was also thought to be closely related to dictyostelids (Hellsten et al. 1998). In fact, A. rosea continued to be used as a model for the early evolution of dictyostelid multicellularity until the early 2000s. There are five described species of Acrasis, which are distinguished by the morphology of their sorocarps and confirmed by molecular data. These are Acrasis granulata (van Tieghem, 1880), Acrasis rosea (Olive et al. 1960), Acrasis kona, Acrasis takarsan and Acrasis helenhemmesae (Brown et al. 2010) (Figure 4). A. helenhemmesae is characterized by a simple spore chain, whereas A. takarsan and A. kona have branching spore masses and A. rosea has an amporphous spore mass (Figure 4). The differences in fruiting body morphology of A. kona and A. rosea and molecular phylogeny lead to their recognition and classification as separate species (Brown et al. 2012). Acrasids are assigned to the Heterolobosea, which are largely amoeboflagellates, and many are common in soil and other moist habitats. Most Heterolobosea can alternate between flagellate and amoeboid states, depending upon growth conditions, and this is probably the ancestral condition for the group (Harding et al. 2013). However, flagella have never been observed for most Acrasidae, including A. kona. Paper III and Paper IV in this thesis focus on the genome sequence of A. kona.

Figure 4. Fruiting body morphology of different Acrasis species. The various species of Acrasis are distinguished by the morphology of their fruiting bodies. These are consistently reproduced under laboratory conditions and the integrity of the species is confirmed by molecular phylogeny including multiple isolates of the different mor-phologies. Figure modified from Brown et al. (2012).

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Evolution of multicellularity

Multicellularity has arisen multiple times independently during the evolution of eukaryotes and even in some bacteria (Myxobacteria) (Lyons and Kolter 2015). There seem to be two fundamentally different pathways to multicellu-larity in eukaryotes; clonal (growth-based) multicellularity and aggregative multicellularity, where growth occurs only in the single-celled stage (Grosberg and Strathmann 2007; Duran-Nebreda and Solé 2014). Clonal multicellularity has arisen independently in more than 20 different lineages of eukaryotes in-cluding animals and fungi and multiple origins in plants (Archaeplastida) and stramenopile algae (King 2004; Parfrey and Lahr 2012). Recently, molecular phylogeny has shown that aggregative multicellularity has also evolved multi-ple times – at least twice in Amoebozoa (dictyostelids and copromyxids,), and at least once in Fungi (Fonticula alba), Alveolata (Sorogena stoinovitchae), Stramenopila (Sorodiplophrys stercorea), Rhizaria (Guttilinopsis) and Ex-cavata (Acrasis spp.) (Brown and Silberman 2013).

Life cycle of sorocarpic amoebae

The life cycle of aggregating amoebae is an alternation between an independ-ent single cell feeding-stage and a cooperative dispersal stage culminating in a multicellular fruiting body. Aggregating amoebae have been given a variety of names including social amoebae and cellular slime molds, but a more precise and now preferred term is sorocarpic amoeba. Life cycle of dictyostelids: The life cycle of the model organism D. discoideum can be divided into three parts: vegetative cycle, sexual cycle and develop-mental cycle (Figure 5). In the vegetative cycle, the amoebae feed on bacterial prey in the soil and divide by mitosis (asexual reproduction). Due to various reasons, mostly unfavorable environmental conditions, the amoebae can then enter either the developmental cycle or the sexual cycle. In the developmental cycle, dictyostelid amoebae aggregate in hundreds to hundreds of thousands to form a multicellular fruiting body that bears one or more spore heads consist-ing of dormant spores that is then dispersed as a unit (Figure 5). A variety of chemotactic agents or “acrasins” are employed for cell aggregation (e.g., glo-rin, folate and pterin), and different dictyostelids may respond to different acrasins. The most famous acrasin is cAMP, first discovered in D. discoideum. Dictyostelids prefer to aggregate with their kin and the recognition of kin ver-sus non-kin utilizes the tiger genes, tgrB1 and tgrB2, although genetically distinct clones of dictyostelids have been observed in the same fruiting body.

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This may reflect a balance between the risk of sacrificing oneself for non-kin and the advantage of joining with others to form an aggregate large enough to build a fruiting body (Strassmann and Queller 2011). In D. discoideum ~20% of the amoebae die in forming the stalk and evidence suggests that amoebae compete with each other to become spores. In the sexual cycle, amoebae of opposite mating types fuse to form a zygote, which then feeds on the remaining amoebae, eventually forming a thick-walled highly resistant macrocyst. The macrocyst is the site of recombination and meiosis, eventually germinating to release hundreds of haploid amoebae (Figure 5).

Figure 5. Generalized life cycle of Dictyostelia, based on Dictyostelium discoideum. The life cycle of Dictyostelia is divided into three inter-linked stages. During the vegetative cycle amoebae feed mainly on bacteria and divide asexually through mito-sis. The sexual and developmental cycles are responses to various stimuli, particularly scarcity of food. During the developmental cycle amoebae aggregate by hundreds of thousands to form a multicellular fruiting body (sorocarp) that bears one to several balls of spores. In the majority of species, ~20% of the cells making up the stalk of the sorocarp die during the process. During the sexual cycle, amoebae of complementary mating types fuse to form a zygote. The zygote devours the surrounding amoebae and forms a highly resilient macrocyst with a thick multilayered cell wall. The macrocyst eventually undergoes recombination and meiosis and releases hundreds of recombi-nant, haploid amoebae.

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Thus dictyostelids can form three kinds of resting stages – microcysts, macro-cysts and spores. These have different cell wall compositions and are formed under different conditions, although it is not entirely clear how cells decide whether to form microcysts, macrocysts or sorocarps. One possibility is the use of “quorum sensing”, which allows cells to detect the density and ratio of the surrounding amoebae and food (Du et al. 2015). If there is a higher density of other amoeba in the surroundings, the amoebae aggregate to form soro-carps; otherwise they form microcysts. Many species do not form macrocysts under laboratory conditions, but this may be due to a lack of a complementary mating type in laboratory strains. Microcysts are the only resting stage known for solitary amoebae, and it may be the ancestral pathway from which dicty-ostelid spores and macrocysts evolved (Du et al. 2014). Life cycle of Acrasis: The life cycle of Acrasis also includes the formation of a multicellular sorocarp through aggregation (Figure 6). Upon receiving signals from the environment, presumably reflecting unfavorable environmental condi-tions, the acrasid amoebae aggregate to form a ball of amoebae (sorogen). Once aggregation is complete, cells at the base of the sorogen begin to encyst and become stalk cells. The stalk continues to grow with additional cell encystment, while the remaining mass of amoebae sits on top of the growing stalk and rises with it. Once the stalk is fully formed, the remaining cells organize themselves into chains, which then encyst. The resulting mature A. kona sorocarp is a branched structure with multiple aerial spore chains. The acrasid spores are distinguished by the presence of raised hila at one or both ends forming spore-to-spore connections. These spores eventually germinate to release single-celled amoebae (Brown and Silberman 2013). Acrasid amoebae, like dictyostelid amoebae, can also form microcysts (Figure 6). The mechanisms for cell signal-ing and microcyst formation are unknown for Acrasis.

Dictyostelid phylogeny and taxonomy

Molecular phylogeny uses DNA and/or RNA sequences to try to reconstruct the evolutionary history of the molecules and, by inference, that of their hosts. The earliest molecular phylogenies utilized single genes or proteins to build trees. Single gene phylogenies are still usually used for investigating a new group of eukaryotes, for which there may be little or no pre-existing data and for which full genome sequencing is not possible. These initial investigations of new taxa usually rely on sequences of the small subunit ribosomal RNA gene (SSU rRNA), as this is the most widely taxonomically sequenced gene and is usually relatively easy to acquire from unknown taxa. It is also large (~2000 nucleotides), meaning it potentially contains a lot of taxonomic infor-mation, and well conserved, meaning it is easy to compare among even quite

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distantly related organisms. SSU rRNA is also multi-copy in probably all eu-karyotes, making it easy to amplify using standard well-developed laboratory procedures (polymerase chain reaction or PCR). Thus, the first molecular phy-logeny of Dictyostelia was based on SSU rRNA sequences (Schaap et al. 2006). This phylogeny showed that the traditional taxonomy of the group was deeply flawed and required major revision. One of the most critical points in a phylogenetic tree is its root. The root gives directionality to evolution within the tree and identifies the last common an-cestor of everything in the tree. Rooting a deep phylogenetic tree is one of the biggest challenges in phylogeny mainly due to the problems of long-branch attraction (LBA) and random rooting (Gribaldo and Philippe 2002; Huelsenbeck et al. 2002).

Figure 6. Life cycle of Acrasis kona. Acrasid amoebae alternate between a free-living trophic stage and multicellular (sorocarpic) dispersal stage. Amoebae aggregate to form a mound usually in response to scarcity of food. After aggregation, the cells at the base of the mound encyst to become the stalk cells. The sorogen sits on top of the growing stalk while the stalk continues to grow with additional cell encystment. The cells in the sorogen then organize themselves into chains and then encyst to become spores. The mature sorocarp consists of multiple spore chains. These spores then ger-minate into amoebae. In an alternative response to adverse environmental conditions, individual amoebae can form microcysts, a resistant resting stage. The outer circle consists of microscopic images of A. kona in liquid medium (brown background) or on agar plates (pale or orange).

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LBA is the tendency of isolated long branches to group together in a phyloge-netic tree, regardless of their true evolutionary relationships. This is especially a problem in rooted trees, since outgroups tend to have long branches relative to the ingroup so that long ingroup branches tend to be drawn toward the base of the tree. If the ingroup and outgroup are separated by very large evolution-ary distances, there is also a potential problem of random rooting, where the root is simply placed on the longest branch in the tree (Graham et al. 2002). One of the ways to avoid such problems is to select slowly evolving, closely related outgroup taxa as well as breaking up long branches with intermediate-branching taxa, where possible. Using sequences from multiple genes is also a powerful tool for improving phylogenetic accuracy as together they include more phylogenetic signal, and most phylogenetic methods, under most condi-tions, are more accurate when given more data. One problem introduced by the first dictyostelid molecular phylogeny was the position of the root of the tree (Schaap et al. 2006). This was especially diffi-cult because Dictyostelia is an ancient group (~600 myr old; Fiz-Palacios et al. 2013) and its sister taxa (Myxogastria, Protostelids and Archamoebae) have highly divergent SSU rRNA sequences. However, genome sequences are now available from all major divisions of Dictyostelia as well as two sister taxa, Physarum polycephalum (Myxogastria; Pawlowski and Burki 2009; Barrantes et al. 2012), and the solitary amoeba, Acanthamoeba castellanii (Clarke et al. 2013). Paper I focuses on my work using these data in a multigene analysis to determine the position of the dictyostelid root. Molecular phylogeny of Dictyostelia shows that morphology is unreliable for taxonomic classification in the group. This is true for most protists, which often have relatively simple or a rapidly evolving morphology. This problem is becoming more apparent as many new protists are being discovered. How-ever, the rules of nomenclature (International Code of Nomenclature for algae, fungi, and plants (ICN)) require a description or diagnosis for valid publica-tion of new taxa. The descriptions and diagnoses have traditionally been based on morphology, but molecular identifiers have been used in a few instances, and guidelines for their use in taxonomy have recently been proposed (Tripp and Lendemer, 2014). Since there are few major clades within Dictyostelia that possess diagnostic morphological characters, we used SSU rRNA based molecular synapomorphies, together with morphological characters where possible, to revise the taxonomy of the dictyostelids following the guidelines of Tripp and Lendemer (2014) (Paper II and Figure 3).

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Eukaryotic genome annotation

As genome sequencing has become faster and cheaper it has also become more ambitious. Earlier genome projects focused on well-studied model or-ganisms, which made genome annotation less challenging due to the availabil-ity of large quantities of pre-existing data such as well characterized open reading frames (ORFs or gene models) supported by experimental data. Today it is feasible to sequence novel genomes that have little or no reference annota-tion available. Although this is very exciting, it also makes the process of ge-nome annotation challenging because novel genomes usually lack supporting data from the source organism, or, sometimes even from remotely related or-ganisms. However, gene prediction and annotation tools need to be optimized, trained and configured in order to accurately predicted genome features such as ORFs, introns, promoters etc. (Yandell et al. 2012). Fortunately, the lack of experimental data is compensated for by technological advances such as tran-scriptomics (RNAseq), high computing power, better software and specialist databases such as gene ontology, KOG, Expasy, and Pfam, and especially protein structure and domain databases. These databases also now include a wider taxon sampling, which makes them more useful for “orphan taxa”, such as Acrasis kona. However it is still challenging to find “elusive” features such as stage specific genes or low expression genes such as transcription factors. Excavata, the parent taxon of Acrasis kona, includes a number of important parasites (Trypanosoma, Giardia, Trichomonas etc.) whose genomes have been annotated. Such genomes tend to lack many genes important for free living organisms while many of the genes they retain tend to have fast evolu-tionary rates. This makes them unreliable for training gene predictors for non-parasite genomes. Within Discoba, there is only a single genome sequence available for a free-living organism, that of N. gruberi (Fritz-Laylin et al. 2010), although several transcriptomes have become available in the last year from more distantly related Heterolobosea and Euglenozoa (Keeling et al. 2014). Thus A. kona is uniquely important because it is only the second free-living excavate to be fully sequenced and a member of the only excavate group with aggregative multicellularity. Although A. kona genome annotation relies substantially on N. gruberi, the latter also still a distant relative (Roger et al. 1996; Pánek and Cepicka, 2012) and does not undergo aggregative mul-ticellularity. As a result, much of the gene prediction in A. kona has to be done for regions not supported by any external evidence.

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The Acrasis kona genome

The first Acrasis kona genome annotation project to be completed was the annotation of its mitochondrial genome (mtDNA, Paper III). This showed a number of novel features including extensive evolutionary gene transfer, two types of RNA editing, extreme AT bias, and a genome dominated by novel ORFs. My contribution to this work was to characterize genes missing from the A. kona mtDNA (relative to N. gruberi) but present in the A. kona nuclear DNA. Using phylogenetic trees, I was able to show that the nuclear copies of many of the missing genes were most closely related to their mtDNA homo-logs in N. gruberi. This indicates that the A. kona nuclear copies are derived by transfer (of RNA or DNA) from the mitochondrion to the nucleus (Paper III). The annotation of A. kona nuclear genome is the subject of Paper IV. The annotation is based on RNA and protein evidence. RNA data consists of 454-transcriptome data and pooled RNAseq data. Multiple automated approaches were used to predict the ORFs that were later curated manually. The genome size is 44 Mb and there are 15,868 predicted ORFs, roughly similar in number to N. gruberi (15,727 predicted ORFs). Of these genes, 4,987 have no homo-logs in the NCBI database. Two striking features of the A. kona genome are numerous genes shared uniquely with distantly related eukaryotes (especially Fungi, Amoebozoa and Metazoa) or Bacteria suggesting substantial horizontal gene transfer (HGT) between these and A. kona. The bacterial genes are espe-cially interesting because many seem to have originated in bacteria and ex-panded into multi-gene families in A. kona. The A. kona genome also has more than 80% of the genes previously identi-fied as being involved in flagellar motility. This is despite the fact that no fla-gellar stage is known from this species. A. kona also has a majority of the nec-essary genes for sexual recombination (meiosis), although cell fusion has nev-er been observed, and the genome assembly showed low levels of heterozy-gosity. Exhaustive searches for genes known to be important for signaling and development in D. discoideum (Glöckner et al. 2016) showed that a majority of them are present and well conserved in A. kona. In fact, a number of these genes are more similar between A. kona and D. discoideum than between D. fasciculatum and D. discoideum (Paper IV).

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Objectives

This thesis focuses on sorocarpic amoebae belonging to two distantly related eukaryotic supergroups, Amoebozoa and Excavata. This includes basic sys-tematic and taxonomic questions of the most widely studied sorocarpic amoe-bae, Dictyostelia (Amoebozoa) and annotation and analyses of the genome of Acrasis kona (Excavata; Discoba; Heterolobosea; Acrasidae). Specific objec-tives include:

1. Examine complete predicted proteome data for dictyostelids and available non-parasitic sister taxa to identify the position of the root of the dictyostelid tree and the consistency of the phylogenetic signal supporting it (Paper I).

2. Conduct the first taxonomic revision of a major group (Dictyostelia) using SSU rRNA molecular signatures to diagnose taxa (Paper II).

3. Annotate the complete mitochondrial genome of Acrasis kona and de-

termine the origin of nuclear homologs of missing mitochondrial genes (Paper III).

4. Annotate and analyze the nuclear genome of Acrasis kona, looking

particularly for genes potentially involved in development and cell-cell communication (Paper IV).

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Materials and Methods

Phylogenetic analyses

Throughout this thesis, various steps were carried out depending on the ques-tion to ensure the quality of the sequences used for the analyses. These con-trols mainly involved removal of illegitimate sequences (mainly paralogs), contamination and non-essential sequences giving extremely long branches.

All the sequences for phylogenetic analyses in each project were aligned using MUSCLE (Edgar 2004). The aligned sequences were trimmed using trimAl (Capella-Gutierrez et al. 2009) with the “automated1” algorithm. Any remain-ing insertions/deletions (indels) or misalignments were inspected by eye and either fixed or deleted. Appropriate substitution models for phylogenetic analysis were identified using RAxML (Stamatakis 2006). Perl, Python or bash scripts were developed to facilitate and pipeline the pro-cedures wherever needed. For Paper II, SSU rRNA sequences for all dictyostelids were downloaded from NCBI. For the identification of molecular signatures for each order, fam-ily and genus, the respective sequences were aligned using MUSCLE (Edgar 2004) and inspected by eye for identification of molecular synapomorphies. These correspond to sequences that are shared by all operational taxonomic units (OTUs) in the designated group and absent or substituted by other mo-lecular signatures in its sister group.

Genome annotation and analyses

The genome of A. kona was assembled using MIRA (Chevreux et al. 2004). Genome completeness assessments were made using CEGMA (Parra et al. 2007). Genes were predicted through a combination of approaches using SNAP (Korf 2004), Augustus (Stanke and Morgenstern 2005) and the Maker

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pipeline (Cantarel et al. 2007) to ensure reliability and consistency of the pre-dictions. All the predicted genes and supporting data (RNAseq, protein evi-dence from N. gruberi (Fritz-Laylin et al. 2010) and UniProt) were inspected manually to check for congruence between predictions. In case of incongru-ence between different predictions, the supporting data (mainly the RNAseq data) was checked and the most reliable gene model was selected. Functional annotation was carried out using the publically available Bioinformatics Infra-structure for Life Sciences (BILS) functional annotation pipeline and Inter-ProScan (Jones et al. 2014). All the annotations and supporting evidence was viewed in WebApollo (Lee et al. 2013). Presence/absence and conservation of A. kona genes was checked by BLASTp search of all A. kona predicted genes against a local database of all NCBI se-quences from Excavata, Metazoa, Fungi, Amoebozoa, Rhizaria, Stamenopila, Alveolata, Plants and Red algae (Archaeplastida), Bacteria and Archaea. An e-value cut-off of 1e-10 was used for all BLAST-related analyses. Separate BLASTp analyses against individual genomes were used to investigate specif-ic phenomena. These included presence/absence of dictyostelid developmen-tally essential genes, N. gruberi and dictyostelid signaling genes and genes involved in flagellar and amoeboid motility. Perl and bash scripts were devel-oped to pipeline the procedures. Detailed pipelines for phylogenetic analyses are described in Paper I and genome annotation procedure in Paper IV and are shown in Figure 7a-b and 8, respectively.

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Figure 7a. Bioinformatic pipeline used for data assembly for Paper I.

tblastn (1e-60)

trimAl

tblastn (1e-60)

MUSCLE

Filtering

tblastn (1e-60)

tblastn (1e-60)

D. fasciculatum P. pallidum D. discoideum D. purpureum

NCBI Proteome

NCBI Proteome

NCBI Proteome

NCBI Proteome

ProteinOrtho

Protein Clusters(2385 clusters)

Retained Clusters- less than 10 sequences per cluster- at least one sequence from each taxon

(1247 clusters)

Acanthamoeba castellanii ESTs

(NCBI)

D. lacteum Contigs(Schaap & Gloeckner)

Physarum polycephalum Contigs

(Genome Inst. UW)

Add new sequences to clustersDelete clusters without D.lacteum

Aligned clusters(223 clusters)

Full dataset! At least one outgroup per cluster! >120 aligned position! 223 total clusters

Trimmed dataset! Removed indel regions

(223 clusters)

Final filteringA. subglobosum

(Acytostelium Genome Consortium)

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Figure 7b. Pipeline for analyses of assembled data for Paper I.

CONSELCONSEL

PhyloBayes(site-specific CAT model)

MrBayes(mixed model, gamma)

MrBayes(mixed model, gamma)

Delete >70% root clusters

Delete >85% root clusters

Delete sites without outgroup data

Concatenate clusters: SeaView

RAxML(rapid bootstrap,

PROTGAMMALGF)

Delete/mask paralogs/extreme long branches

RAxML(rapid bootstrap, PROTGAMMALGF)

Single gene clusters(223 clusters)

Single Gene Trees (SGTs)

213 clusters

Full dataset(105351 sites)

Set 2(29475 sites)

Set 3(41575 sites)

Set 1(63424 sites)

Phylogenetic tree + nodal support (mlBP)

Phylogenetic trees + nodal support (mlBP, biPP)

RAxML(rapid bootstrap,

PROTGAMMALGF)

Hypothesis (AU) test of alternative roots

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Figure 8. Pipeline for nuclear genome annotation of Acrasis kona for Paper IV. The figure shows the software and methods used for nuclear genome annotation of A. kona. The green, orange and red boxes represent the three divisions of the annotation process: structural annotation (prediction of open reading frames), functional annota-tion and manual curation of the annotated predicted genes, respectively.

Information integeration(Annie)

"Evidence-based gene predictions"

"Ab-initio gene predictions"

Ab-initio+Evidence-based gene predictions

BLAST2GO

InterProScan

input

Manual curation (15,868 predicted ORFs)

Genome assembly (MIRA, SSPACE)

AUGUSTUSSNAP

Gene structure predictors

MAKER pipelinetraining output

train

input

Evidence

UniProt/Swiss-ProtNaegleria gruberi genome

454 transcriptome dataAssembled RNAseq data

(Trinity & Cufflinks)

"Evidence-based gene predictions"

"Ab-initio gene predictions"

Ab-initio+Evidence-based gene predictions

Visualization(WebApollo)

Structural Annotation Functional Annotation

Assembled RNAseq data (Cufflinks & Trinity)

PASA(862 selected gene models)

Gene predictor

Evidence

UniProt/Swiss-ProtNaegleria gruberi genome

454 transcriptome data AUGUSTUSSNAP

Gene structure predictors

"Ab-initio gene predictions"

train

train

input

train

Genome assembly (MIRA, SSPACE)

MAKER pipelinetraining output

input

Genome assembly (MIRA, SSPACE)

Evidence

UniProt/Swiss-ProtNaegleria gruberi genome

454 transcriptome dataAssembled RNAseq data

(Trinity & Cufflinks)

AUGUSTUSSNAP

Gene structure predictors

"Evidence-based gene predictions"MAKER pipeline

training output

train

input

"Evidence-based gene predictions"

train

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Results and Discussion

Summary of Paper I

Sheikh, S., Glöeckner, G., Kuwayama, H., Schaap, P., Urushihara, H., Bal-dauf, S. L. (2015) Root of Dictyostelia based on 213 universal proteins. Mo-lecular Phylogenetics and Evolution, (92): 53-62. Dictyostelia are eukaryotic soil microbes found worldwide, most commonly in forest soils. They are best known for their response to starvation, where the cells aggregate by the hundreds of thousands to form multicellular fruiting bodies. Dictyostelids were previously classified in three genera corresponding to the three basic morphotypes: acytostelid (no cellular stalk), dictyostelid (cellular stalk and branching irregular where present) and polysphondylid (cellular stalk and regularly spaced whorls of side branches) (Raper 1984; Kawakami et al. 2008) (Figure 2). However, this long-standing (50-200+ years) traditional classification system was partly rejected based on morpho-logical data (Swanson et al. 2002) and then fully rejected by a comprehensive molecular phylogeny (Schaap et al. 2006). Molecular data further divide Dic-tyostelia into four major groups, informally referred to as “Groups 1-4” plus three smaller “complexes” whose relationships to the four major groups re-main largely uncertain (but see Singh et al. 2016). Most importantly, none of these groups correspond to any of the three traditional genera. The initial dictyostelid molecular phylogeny was provisionally rooted between “Group 1” and all other groups and complexes (1,2+3+4 root; Schaap et al. 2006). However, this root was very poorly supported and a root between “Groups 1+2” and “Group 3+4” (1+2,3+4 root) could not be rejected. Thus, a critical question in dictyostelid phylogeny has been the position of the root. This has important implications for the interpretation of larger evolutionary trends in the group. In Paper I, I sought to test the root of the Dictyostelia with a comprehensive data set including all large (>120 aligned positions), low or single copy number proteins that appear to have been ancestrally pre-sent and universally retained in Dictyostelia and their non-parasitic amoebo-zoan relatives. Automated clustering and phylogenetic screening identified

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681 universal low-to-single copy number protein clusters in Dictyostelia. Of these, 213 clusters were also found in at least one of the two available out-group taxa (Acanthamoeba castellanii and Physarum polycephalum) with substantial genome data available at the time. Phylogenetic analyses of a concatenation of the 213 clusters consistently sup-port the 1+2,3+4 root. Alternative hypothesis tests confirm this result, with all viable alternative hypotheses rejected with p<0.01. All protein clusters were also examined individually to determine their consistency with this root. A majority of proteins were found to strongly support the 1+2,3+4 root individu-ally. Removal of these “highly opinionated” clusters has no effect on support for this root or rejection of alternative hypotheses. Moreover, phylogenetic analysis of concatenations of protein clusters showing some support for the same alternative root recovered only the “1+2,3+4” root. Thus, other than a few isolated instances of what appear to be either horizontal gene transfer or contaminated data, the conservative core of the ancestral dictyostelid genome supports a single 1+2, 3+4 root. Most importantly, this resolution of the deep-est branches of Dictyostelia set the stage for full taxonomic revision of this important evolutionary model system.

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Summary of Paper II

Sheikh, S., Thulin, M., Cavender, J. C., Escalante, R., Kawakami, S., Lado, C., Landolt, J. C., Nanjundiah, V., Queller, D. C., Strassmann, J. E., Spiegel, F. W., Stephenson, S. L., Vadell, E. M., Baldauf, S. L. A new classification of the Dictyostelids. Submitted. It has been obvious for more than a decade that the taxonomy of Dictyostelia needs major revision. Like in many protist groups, morphological characters tend to be sparse and evolutionarily unstable and are therefore unreliable for taxonomy. However, the rules of nomenclature (ICN) require a description or diagnosis for the valid publication of new taxa. Recently, Tripp & Lendemer (2014) put forth some suggested guidelines for using molecular characters to diagnose taxa (Tripp and Lendemer 2014). This is a potential way to solve the problem of protist taxonomy although this requires comprehensive work that has not been attempted at a large scale before. In Paper II, we propose a taxonomic revision of the dictyostelids using mo-lecular diagnoses and morphological characters where possible. SSU rRNA sequences were used as these are the only molecular data for most taxa. All major clades with strong support from SSU rRNA data were revised. The only exception to this is the root, which requires multigene data to resolve. We used a conservative approach retaining as much of the old taxonomy as possi-ble. SSU rRNA alignments for all subgroups were inspected by eye to identify molecular signatures (synapomorphies) unique for one subgroup at a particu-lar position when compared with their respective sister taxon. Morphological data, from original species descriptions (mostly available online at http://www.discoverlife.org/ and or in Raper 1984), were also extracted for each subgroup. The combination of a few selected molecular signatures and morphological data was used to diagnose the dictyostelid subgroups, with additional molecular signatures provided in supplementary data. Like most protist groups, the major divisions of Dictyostelia were traditionally given a low taxonomic rank, which is inconsistent with the molecular depth and apparent antiquity of the group (Schaap et al 2006, Fiz-Palacios et al. 2013). Therefore, the two major dictyostelid clades defined by the root were treated at the rank of order, as Acytosteliales ord. nov. (previously Group 1+2) and Dictyosteliales (previously Group 3+4). Similarly the two major clades within each of these orders were named and treated at the rank of family, as Cavenderiaceae fam. nov. (Group 1) and Acytosteliaceae (Group 2) in Acyto-steliales (Group 1+2) and Raperosteliaceae fam. nov. (Group 3) and Dicty-

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osteliaceae (Group 4) in Dictyosteliales (Group 3+4). Twelve genera were recognized, nine of which are new. The recognized genera are Cavenderia gen. nov. in Cavenderiaceae, Acytostelium, Rostrostelium gen. nov. and Het-erostelium gen. nov. in Acytosteliaceae, Tieghemostelium gen. nov., Hagiwa-raea gen. nov., Raperostelium gen. nov. and Speleostelium gen. nov. in Raperosteliaceae, and Dictyostelium and Polysphondylium in Dictyosteliaceae. Two “complexes” not clearly resolved by SSU rRNA phylogeny were named but not assigned to family, “Polycephalum complex” (now Coremiostelium), or not assigned to family or order, “Polycarpum complex” (now Synstelium). A tree with the revised names is shown in Figure 3.

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Summary of Paper III

Fu, C.-J., Sheikh, S., Miao, W., Andersson, S. G., Baldauf, S. L. (2014) Miss-ing genes, multiple ORFs, and C-to-U type RNA editing in Acrasis kona (Het-erolobosea, Excavata) mitochondrial DNA. Genome Biology and Evolution, 6(9): 2240-57. Among the supergroups of eukaryotes, Excavata is by far the least well char-acterized (Adl et al. 2012; He et al. 2014). Within Excavata, Discoba is the only group to possess respiratory competent mitochondria and mitochondrial DNA (mtDNA) (Simpson et al. 2003). Discoba consists of major divisions - Jakobida, Euglenozoa, Heterolobosea, and Tsukubamonadida - and among them they exhibit a mtDNA diversity that is unsurpassed by any other major eukaryotic lineage (Gray et al. 2004). These range from the gene-rich bacteri-al-like mtDNAs of jakobids to the heavily encrypted and fragmented mtDNAs of Euglenozoa. However other than Naegleria little is yet known about the mtDNA of Heterolobosea. While annotating the genome sequence of the sorocarpic heterolobosean Acra-sis kona, we noted the presence of Naegleria mtDNA genes on A. kona nucle-ar contigs. Therefore, we investigated the mitochondrial genome of A. kona using PCR with Sanger sequencing to close all gaps. The genome consists of a 51.5 Kb molecule that is highly AT-rich (83.3%) including non-coding re-gions (89.8% AT), protein-coding genes (84% AT) and structural RNA genes (72.4% AT). A. kona mtDNA is missing many genes found in Naegleria mtDNA, but on the other hand, more than one-fourth (26.5%) of the A. kona mtDNA consists of novel ORFs. All of these ORFs are found on the sense DNA strand and in the same transcriptional orientation as the rest of the cod-ing content of the genome, suggesting they are transcriptionally active. We also identify C-to-U type editing in A. kona mtDNA, along with the presence of a DYW-type PPR editing protein encoded in the nucleus. The relocation of organelle genes to the nucleus is referred to as endosymbi-otic gene transfer (EGT). It was probably a major force in early mitochondrial (and chloroplast) evolution and is one of the main reasons why most organelle proteins are encoded in the nucleus and post-translationally imported into the organelles. Although EGT is thought to be an ongoing process in eukaryotes, the rate appears to be much slower, possibly due to the fact that there are few genes left in most mtDNAs, and there may be important barriers to transfer of those remaining few (Gray 2012). Therefore we investigated the genes miss-ing in A. kona mtDNA to see if they looked like EGTs. We were able to iden-tify 11 of the 16 missing Naegleria genes in a partial assembly of the A. kona nuclear genome. Molecular phylogeny confirmed that at least 10 of these 11

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nuclear genes are most closely related to homologs in N. gruberi mtDNA. This indicates that the A. kona nuclear genes arose by nucleic acid transfer (EGT) from mtDNA to ncDNA, sometime after Acrasis and Naegleria split from their last common ancestor. All but one of the 11 A. kona nuclear genes are also predicted to encode transit peptides, which are required for the import of most (but not all) mitochondrial proteins translated in the cytoplasm. Mapping of gene presence/absence onto a consensus phylogeny of Discoba reveals a sporadic pattern of gene loss and genome reorganization. In particu-lar, EGT and acquisition of novel ORFs is accelerated in the unique evolu-tionary lineage leading to A. kona. This contrasts sharply with its sister line-age, that of Naegleria, which shows remarkable genome conservation, with no predicted major changes since it split from its last common ancestor with Acrasis spp.

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Summary of Paper IV

Sheikh, S., Fu, C.-J., Miao, W., Baldauf, S. L. Multicellularity in the Excava-ta, the genome sequence of Acrasis kona. Manuscript. Heterolobosea is a morphologically and ecologically diverse group within Discoba (Excavata), mainly composed of amoeboflagellates. These are cells that can switch between amoeboid and flagellate forms, which is undoubtedly the ancestral condition in Heterolobosea (Harding et al. 2013). Most Heterolo-bosea can also form resting cysts, a common response of soil microbes to un-favorable environmental conditions. However, acrasids are unique among heteroloboseans in being able to form a multicellular dispersal stage. The lat-ter is a relatively complex process requiring cell-cell communication, aggrega-tion and cooperation among formerly free-living cells. There is very little mo-lecular data from any free-living member of Discoba, or indeed of Excavata, as most molecular study in this group has focused on parasites. To date, the only genome sequence from a free-living excavate is that of Naegleria gruberi. In Paper IV we report the nuclear genome sequence of a multicellular exca-vate, Acrasis kona. The genome of A. kona is estimated to be 44 Mb in size, with 15,868 predicted open reading frames, of which 31% (4,987) are novel. Amongst the remaining 10,881 A. kona genes, many are uniquely shared with various groups of non-excavate organisms. This suggests that these genes may have originated by horizontal transfer between these organisms and a relative-ly recent ancestor of the A. kona lineage. A more detailed investigation of the genes shared uniquely with Bacteria indicates that many constitute multi-gene families in A. kona. Many of these genes also encode signal sequences in A. kona, which target proteins to cellular membranes. In several cases, the por-tion of the gene encoding the signal sequence and the mature peptide are sepa-rated by an intron, suggesting that the signal sequence may have been ac-quired by exon shuffling. The A. kona genome also contains more than 80% of the genes predicted to be involved in flagellar motility (Fritz-Laylin et al. 2010). This suggests a recent loss of flagella or the presence of a cryptic flagellar stage. Recently it has been shown that some heterolobosean taxa previously thought to be strictly amoe-boid or flagellate can form the alternative state under certain conditions (Pánek et al. 2017). Therefore, it is possible that flagella can be induced in A. kona under conditions that have not yet been tested in the lab. The A. kona genome has complex signaling domains and pathways, some of which might be involved in the developmental stage of the organism. A com-

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parison of the genes involved in signaling and development shows that ap-proximately 77% of the genes necessary for development in Dictyostelium discoideum are also present in the A. kona genome, some of which may also be candidates for HGT. Comparison of signaling domains found in N. gruberi and other eukaryotes shows that A. kona has a comprehensive repertoire of genes potentially involved in a wide variety of functions related to signaling, recognition and development. Investigation of stage-specific gene expression (RNAseq) is in progress.

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Svensk Sammanfattning

Dictyostelider och acrasider är jordlevande amöbor med en ovanlig livscykel som kallas sorokarpi. När de äter och växer förekommer de som encelliga mikroorganismer, men när det blir brist på föda går de enskilda amöborna samman (aggregerar) till en flercellig fruktkropp. På grund av likheterna i livscykler antog man länge att dictyostelider och acrasider var nära släkt, men genom molekylära fylogenistudier 1996 visade man att så inte är fallet. Eu-karyoterna delas idag in i tre supergrupper där dictyosteliderna räknas till Amoebozoa medan acrasiderna räknas till Excavata (Figur 1).

Artikel I och II handlar om dictyostelidernas systematik. I artikel I använder jag 213 universella proteiner för att identifiera roten hos dictyostelidernas evolutionära släktträd. Genom att dela upp Dictyostelia i större undergrupper var det möjligt att göra en formell revidering av gruppens taxonomi, ett arbete som det länge har funnits ett behov av att göra. I artikel II föreslår vi en reviderad klassifikation av Dictyostelia. Vi diagnostiserar taxonomiska enheter med hjälp av molekylära signaturer (SSU rRNA), något som inte har gjorts tidigare för någon annan större organismgrupp. Eftersom morfologiska karaktärer inte kan användas på ett tillförlitligt sätt i taxonomiskt arbete med de flesta protister, utgör vårt arbete en viktig grund för vidare studier av andra eukaryota protistgrupper. Artikel III och IV fokuserar på annotering och analys av mitokondrie- och kärngenom hos Acrasis kona. Acrasiderna är den enda kända grupp inom Ex-cavata som periodvis är flercelliga. I artikel III analyserar vi mitokon-driegenomet (mtDNA) hos A. kona. En uppseendeväckande egenskap hos detta mitokondriegenom är att mer än en fjärdedel består av nyupptäckta ”öppna läsramar” (open reading frames, ORFs), samtidigt som40% av de pro-teinkodande mitokondriegenerna som finns hos den närmaste kända släktin-gen, Naegleria gruberi, saknas. Däremot kunde elva av dessa 16 ”saknade” gener identifieras i kärn-DNA:t hos A. kona. Genom molekylära fylogenier har jag visat att de flesta av dessa gener troligtvis har hamnat i kärnan genom att genetiskt material har överförts från mitokondrierna till kärnan. I artikel IV presenterar vi hela kärngenomet hos A. kona. Genomets storlek uppskattas till 44 Mb med 15 868 ORFs, varav 4987 är nyupptäckta. Av de andra 10 881

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är många bara kända från enstaka andra organismgrupper, vilket tyder på att A. kona stjäl (eller åtminstone ”byter”) gener från några av sina mest avlägsna släktingar. Den här processen kallas horisontell genöverföring (horizontal gene transfer, HGT). Några av de mest uppseendeväckande exemplen är gener som har erhållits från bakterier och sedan expanderat till multigenfamiljer hos A. kona. Vidare innehåller genomet hos A. kona över 80% av de gener som är kända för att vara inblandade i flagellrörelser och de flesta av generna som behövs för sexuell rekombination. Detta trots att flageller och sexuell rekom-bination aldrig har observerats hos den här organismen. Vi har också sett indi-kationer på gener involverade i identifiering av närbesläktade celler och cell-cell-kommunikation, något som kan ha betydelse för aggregering och utveck-lingen av flercellighet. För att förstå detta bättre behövs analyser av genuttryck för specifika utvecklingsstadier, ett arbete som vi har påbörjat.

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Acknowledgements

I would like to start by thanking my supervisor, Sandie. There is a long list of things I want to thank you for but here are just a few of them. First of all, thank you for sending that email in 2010 when I inquired if you had any re-search projects for Masters students, it changed my life completely and made me do what I didn’t even know I loved doing. Thank you for giving me a chance to learn so much from you! Thank you for teaching me how to teach and for being so supportive and patient with me when I was struggling. It is only because of you that I started enjoying teaching. Thank you for the amaz-ing time that we had at all the conferences (and after the conferences) ☺ Thank you for taking time out to answer all my questions whenever I came to you no matter how busy you were. Thank you for meeting with me for hours even on weekends and during holidays to discuss my projects and for guiding me even when you had so much else to do. Thank you for teaching the way you teach, it made learning a lot more interesting and enjoyable! Thank you for always cracking the funniest jokes especially in the most frustrating situa-tions! It always made everything so much more fun! ☺ Thank you for coming to check how I was doing so many times a day towards the end. I think it was the only reason I didn’t panic (so much ☺). Thank you for believing in me and for encouraging me every step of the way. Thank you for granting me this opportunity to do my PhD and giving me a chance to enjoy every minute of it! You are an awesome supervisor and will forever have a very special place in my heart! ☺ I would also like to thank my other mentors, Siv, Henrik Lantz, Jacques Dainat, Doughlas and Henrik Viberg for being there to guide me and for making sure I got help whenever I needed it! ☺ Thank you Joan for being so kind and for all the fun in Spain! ☺ Thank you Mats being the most kind, helpful and patient person! Thank you for all the help with the taxonomy, it won’t have been possible without you! ☺ Thank you Marc for all the help with the annotation and for answering my two hundred billion thousand mil-lion hundred billion questions a day! I won’t have been able to do the annota-tion without your guidance! ☺ Thank you David Morrison for the great dis-cussions and for teaching me so much of what I know and for clearing so many of my concepts! ☺ Thank you Jan Andersson for being such a great opponent for my half-time defense. I learnt a lot from our discussion! ☺

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Thanks to everyone at Systematic biology! Thank you Inga for being such an inspiring woman ☺ Thank you Magnus for making sure the sun comes out every day and for all your comments for my thesis ☺ Thank you Mikael for helping me every time I had a question about teaching, for guiding me and for all the fun stories during fika ☺ Petra- You are such a strong and inspiring person! Thank you for bringing so much happiness and laughs with you every time you would come to our office! ☺ Thank you Hanna for always being so kind and cheerful! ☺ Thank you Thomas Jaenson for all the enjoyable ques-tions about Homeland and Pakistan ☺ Thank you Agneta for bringing so much color into the department ☺ Thank you Nahid for always being so car-ing ☺ Thank you Afsaneh for a charming greeting every time ☺ Thank you Leif and Sanja for bringing my little friend, Tove to work every now and then! It sure made those days a lot more fun! ☺ Thank you Martin for always being ready with your jokes! It is always a pleasure to talk to you ☺ Thank you Fabien for your helpful discussions and for your guidance every time I came to you with a question ☺ Thank you Baset for all the interesting stories about your research ☺ Thank you Paco for all the French deserts ☺ Jenni- Your laughs from the fika room have surely brightened up a lot of my days! Thank you! ☺ Thank you Diem and Anushree for all the fun conversations in the fika room and for your kind words during the last days of my PhD ☺ Sarina- Thank you for all the get-togethers, for all the motivations and for all the conversations! I am so glad to have gotten to know you! Thank you for all your help with my cover! ☺ Thank you Allison for always making me feel better when I had done something wrong! ☺ Thank you for always checking in on me and making sure I was doing ok! ☺ Stina-You are the most motivat-ed, hard working, organized person I know. I was hoping you would rub some of that off on me ☺ Thank you for being an awesome office mate and a great friend! ☺ Ioana- I am so glad we went to Czech Republic together and we got to know each other! I am really lucky to have such a “crazy” friend in my life! Thank you for all the “good” coffee! ☺ Karin- I really admire your positivity! I hope you always stay that way and I wish you all the best with your PhD ☺ Astrid- Thank you for all the fun fikas! Just so you know, you “occupy the good space in my life” ☺ Julia- I cannot count the number of times I was startled when I turned around and saw you were there! Thank you for being the silent and fun office-mate and a great friend! ☺ Raquel- It is always a pleasure spending time with you! Thank you for all the cakes, chocolates and candies! Best of luck with your PhD! ☺ Lore- I had the best time teaching with you! All the best with your PhD! ☺ Mahwash- Thank you for coming to see how I was doing every now and then! I wish you all the best with your PhD ☺ Ding- I won’t have known about this position if it wasn’t for you, thank you! Thank you for all the discussions and ideas ☺ CJ- Thank you for all the treats you brought back from home! Thank you for encouraging me ☺ Henrik- thank you for being the patient office-mate and for bearing with all the talking I used to do with everyone while you were trying to work! Wish

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you all the best for your PhD! ☺ Anders L.- I miss hearing your noise from the office next door! Thank you for making work fun (and startling, some-times) when you were here and thank you for all the scripts ☺ John- Here it is one more time- I am still fascinated by your presentation on your defense. It was amazing! Thank you for all the fun stories about your experiences with Pakistani food! ☺ Hugo and Anneleen- thank you for being such great office mates. I still remember how you both would ask if I had been married off eve-ry time I would come back from Pakistan and it still makes me laugh out loud ☺ Sara- I really hope you and I become Swedish pros soon! It is always a pleasure to talk to you ☺ Mayank- I had a great time teaching with you and enjoyed meeting up with you for lunch to catch up! Best of luck with your PhD! ☺ Thank you Feifei, Anders, Joran, Dani, Jesper for being amazing colleagues! I had a great time teaching with you! ☺ Åsa, Mohammad, Cécile, Magdalena, Aaron, Vasily, Veera, Seol-Jong, Yingzi, Juma, Stella, Brendan, Markus, Caesar, Petr, Iker, Kristiina, Jürgen, Anneli and Katerina- your presence has made all this time so much better! Thank you! ☺ I wish you all the happiness in the world ☺ Shahina- I had a great time when you were here and I miss you a lot now that you aren’t ☺ Omama, Samia, Mubah- I miss you guys every single day! Thank you for always being just a message away ☺ Zauq- Thank you for doing so much for me and for always being there ☺ Shirin- thank you for being my family in Sweden! ☺ Berivan, Claudia, Sebastian, Javeria, Saad, Akif, Cansu, Marina and Arusjak- Thank you for being such great friends! I wish you all the best in life! ☺ Thanks to all the funding, grants and stipends that I got over the years that have allowed me to travel for all the conference and workshops that I have been to. For my family… Abu, Amma- Thank you for making me the person I am today! Thank you for letting me find my own path and for supporting me through everything, for worrying about me every single minute I have been away but still letting me know that you are happy if I am happy. I am the luckiest person to have par-ents like you! ☺ Rabbiya baji- my beautiful sister! Life was unjustly short for her but not a day goes by when she is not thought of. She taught me how to stay strong and have a positive attitude towards everything in life! I will for-ever be grateful for that! ☺ Sumair bhai, Muniza, Yahya and Alizeh- Thank you bhai for always being there and for everything that you have done for me! I am so lucky to have a brother like you! I wish you all the happiness in the world! The three of you are a source of inspiration to me every single day. Thank you bhai for bringing up such beautiful kids! I love you Yahya and

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Alizeh! ☺ Muniza- It is always a pleasure to talk to you and I am looking forward to spending a lot more time with you! ☺ Umer bhai, Trish baji, Zak and Sophie- Thank you bhai for all your support. I couldn’t have done this if you hadn’t been there for me ☺ Thank you baji for having the kindest heart! Thank you for always being so positive towards everything! I have learnt so much from you! Thank you for making me feel I can be as crazy as I want and it still won’t be too much ☺ ☺ Thank you for raising such amazing kids. I love you Zak and Sophie! ☺ Tehman- Not enough space to thank you for all you have done for me mf! Thank you for always looking out for me and for all the fun times! I won’t have had a life that I am so happy in if it wasn’t for you! I love you mf! Thank you so much for pushing me when you did just so I could be where I am today! Shikhands mf! I owe it all to you my beautiful family!! ☺

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