The Dictyostelia present a splendid opportunity for studies of sociality and genetic conflicts of interest. These eukaryotic amoebae aggregate upon starvation to form a multicellular individual in which some formerly independent individuals die to form a stalk that lifts the others to a better place for sporulation and dispersal. Dictyostelia vary in their social organization and can be cultured from soil samples in nearly all parts of the world. Genome sequences are available for increasing numbers of species; many molecular pathways are known; experimental evolution is feasible. Who lives, who dies, and how sociality is structured are great questions that are easily addressed in this group.

Acrasids are amoebae with the capacity to form multicellular fruiting bodies in a process known as aggregative multicellularity (AGM). This makes acrasids the only known example of multicellularity among the earliest branches of eukaryotes (the former Excavata). Here, we report the Acrasis kona genome sequence plus transcriptomes from pre-, mid- and post-developmental stages. The genome is rich in novelty and genes with strong signatures of horizontal transfer, and multigene families encode nearly half of the amoeba's predicted proteome. Development in A. kona appears molecularly simple relative to the AGM model, Dictyostelium discoideum. However, the acrasid also differs from the dictyostelid in that it does not appear to be starving during development. Instead, developing A. kona appears to be very metabolically active, does not induce autophagy and does not up-regulate its proteasomal genes. Together, these observations strongly suggest that starvation is not essential for AGM development. Nonetheless, development in the two amoebae appears to employ remarkably similar pathways for signaling, motility and, potentially, construction of an extracellular matrix surrounding the developing cell mass. Much of this similarity is also shared with animal development, suggesting that much of the basic tool kit for multicellular development arose early in eukaryote evolution. Acrasis kona is a solitary amoeba which builds a multicellular fruiting body, despite being a distant relative of other multicellular eukaryotes. This study analysed A. kona's genome and developmental transcriptomes and find extensive similarity with common developmental pathways of other multicellular taxa.

Much of the higher-order phylogeny of eukaryotes is well resolved, but the root remains elusive. We assembled a dataset of 183 eukaryotic proteins of archaeal ancestry to test this root. The resulting phylogeny identifies four lineages of eukaryotes currently classified as "Excavata" branching separately at the base of the tree. Thus, Parabasalia appear as the first major branch of eukaryotes followed sequentially by Fornicata, Preaxostyla, and Discoba. All four excavate branch points receive full statistical support from analyses with commonly used evolutionary models, a protein structure partition model that we introduce here, and various controls for deep phylogeny artifacts. The absence of aerobic mitochondria in Parabasalia, Fornicata, and Preaxostyla suggests that modern eukaryotes arose under anoxic conditions, probably much earlier than expected, and without the benefit of mitochondrial respiration.

Acrasids are large, fast-moving, omnivorous amoebae. However, under certain conditions, they can also cooperate to form multicellular fruiting bodies in a process known as aggregative multicellularity (AGM). This makes acrasids the only known example of multicellularity among the earliest branches of eukaryotes (formerly superkingdom Excavata) and thus the outgroup to all other known multicellular eukaryotes. We have sequenced the genome of Acrasis kona , along with transcriptomes from cells in pre-, mid- and post-development. We find the A. kona genome to be rich in novelty, genes acquired by horizontal transfer and, especially, multigene families. The latter include nearly half of the amoeba’s protein coding capacity, and many of these families show differential expression among life cycle stages. Development in A. kona appears to be molecularly simple, requiring substantial upregulation of only 449 genes compared to 2762 in the only other AGM model, Dictyostelium discoideum. However, unlike the dictyostelid, developing A. kona also does not appear to be starving, being instead very metabolically active and inducing neither autophagy nor increasing ubiquitin-tagged proteolysis. Thus, contrary to current expectations, starvation does not appear to be essential for AGM development. Moreover, despite the ~ 2 billion years of evolution separating the two amoebae, their development appears to employ remarkably similar pathways for signaling, motility and construction of an extracellular matrix surrounding the developing cell mass. In addition, much of this similarity is shared with the clonal multicellularity of animals. This makes the acrasid something of a “bare bones” developmental model and suggests that much of the basic tool kit for multicellular development arose very early in eukaryotic evolution.

Phylogenetic reconstruction using concatenated loci ("phylogenomics" or "supermatrix phylogeny") is a powerful tool for solving evolutionary splits that are poorly resolved in single gene/protein trees. However, recent phylogenomic attempts to resolve the eukaryote root have yielded conflicting results, along with claims of various artifacts hidden in the data. We have investigated these conflicts using two new methods for assessing phylogenetic conflict. ConJak uses whole marker (gene or protein) jackknifing to assess deviation from a central mean for each individual sequence, whereas ConWin uses a sliding window to screen for incongruent protein fragments (mosaics). Both methods allow selective masking of individual sequences or sequence fragments in order to minimize missing data, an important consideration for resolving deep splits with limited data. Analyses focused on a set of 76 eukaryotic proteins of bacterial ancestry previously used in various combinations to assess the branching order among the three major divisions of eukaryotes: Amorphea (mainly animals, fungi, and Amoebozoa), Diaphoretickes (most other well-known eukaryotes and nearly all algae) and Excavata, represented here by Discoba (Jakobida, Heterolobosea, and Euglenozoa). ConJak analyses found strong outliers to be concentrated in undersampled lineages, whereas ConWin analyses of Discoba, the most undersampled of the major lineages, detected potentially incongruent fragments scattered throughout. Phylogenetic analyses of the full data using an LG-gamma model support a Discoba sister scenario (neozoan-excavate root), which rises to 99-100% bootstrap support with data masked according to either protocol. However, analyses with two site-specific (CAT) mixture models yielded widely inconsistent results and a striking sensitivity to missing data. The neozoan-excavate root places Amorphea and Diaphoretickes as more closely related to each other than either is to Discoba, a fundamental relationship that should remain unaffected by additional taxa. [CAT-GTR; Discoba; eukaryote tree of life; HGT; jackknife; mixture models; mosaic genes; phylogenomics; sliding window; supermatrix.].

Phylogenomics uses multiple genetic loci to reconstruct evolutionary trees, under the stipulation that all combined loci share a common phylogenetic history, i.e., they are congruent. Congruence is primarily evaluated via single-gene trees, but these trees invariably lack sufficient signal to resolve deep nodes making it difficult to assess congruence at these levels. Two methods were developed to systematically assess congruence in multi-locus data. Protocol 1 uses gene jackknifing to measure deviation from a central mean to identify taxon-specific incongruencies in the form of persistent outliers. Protocol_2 assesses congruence at the sub-gene level using a sliding window. Both protocols were tested on a controversial data set of 76 mitochondrial proteins previously used in various combinations to assess the eukaryote root. Protocol_1 showed a concentration of outliers in under-sampled taxa, including the pivotal taxon Discoba. Further analysis of Discoba using Protocol_2 detected a surprising number of apparently exogenous gene fragments, some of which overlap with Protocol_1 outliers and others that do not. Phylogenetic analyses of the full data using the static LG-gamma evolutionary model support a neozoan-excavate root for eukaryotes (Discoba sister), which rises to 99-100% bootstrap support with data masked according to either Protocol_1 or Protocol_2. In contrast, site-heterogeneous (mixture) models perform inconsistently with these data, yielding all three possible roots depending on presence/absence/type of masking and/or extent of missing data. The neozoan-excavate root places Amorphea (including animals and fungi) and Diaphoretickes (including plants) as more closely related to each other than either is to Discoba (Jakobida, Heterolobosea, and Euglenozoa), regardless of the presence/absence of additional taxa.

Ixodes ricinus (L.) (Acari: Ixodidae) is a major vector for the transmission of several important human pathogens. The aim of the present study was to evaluate the in vitro efficacy of different concentrations of essential oils (Eos) on I. ricinus tick nymphs. Oils were obtained from the leaves of three plants native to Libya: white wormwood (Artemisia herba alba Asso), marjoram (Origanum majorana L.) and Arar (Juniperus phoenicea L., English common name Phoenician juniper). Assays were done using the "open filter paper method". Two concentrations from each oil, 0.5 and 1 mu l/cm, were tested. The acaricidal effect was measured in terms of the lethal concentrations (LC50, LC95) and lethal time (LT50, LT95). Mortality rates were obtained by counting the surviving nymphs every 30 min for the first five hours and then at 24, 48 and 72 h. A mortality of 100% was recorded at the higher concentration of oils (1 mu l/cm(2)) from A. herba alba and J. phoenicea at the first 2 h of exposure. Exposure to O. majorana led to 100% mortality on the third day (72 h), and this effect decreased noticeably with 0.5 mu l/cm(2) oil at the same exposure time. However, 50% of ticks showed a paralysis effect and less movement after 2 h. The LC50 of mortality was reached within the first 24 h of exposure time at 0.5 mu l/cm(2) of O. majorana, which produced 60% tick's mortality. Chemical composition of the essential oils was elucidated by gas chromatography-mass spectrometry analyses. These results suggest that essential oils deserve further investigation as components of alternative approaches for I. ricinus tick control.

Essential oils extracted from the leaves of Libyan Rosemary ( Rosmarinus officinalis L.), and Miswak ( Salvadora persica L.) were evaluated for their acaricidal and repellent effects on Ixodes ricinus L. nymphs (Acari: Ixodidae) using a bioassay based on an ‘open filter paper method’. Rosmarinus officinalis leaf essential oil diluted to 0.5 and 1 µl/cm 2 in acetone exhibited, respectively, 20 and 100% tick mortality after about 5 h of exposure. A total of 50 and 95% of I. ricinus nymphs were killed by direct contact with the oil when exposed to lethal concentrations (LC) of 0.7 µl/cm 2 (LC 50 ) and 0.95 µl/cm 2 (LC 95 ), respectively. The LC 50 (0.5 µl/cm 2 ) was reached before the end of the first 24 h of exposure time (ET), as tick mortality at 24 h was 60%. Salvadora persica leaf essential oil at 1 µl/cm 2 showed a significant repellency effect against I. ricinus nymphs at 1.5 h ET. A 95% repellency was observed at a repellent concentration (RC 95 ) of 1 µl/cm 2 of S. persica , but no significant mortality was recorded at this dose of S. persica oil. Gas chromatography–mass spectrometry analyses showed that the main monoterpenes in both oils were 1,8-cineol, α-pinene, and β-pinene, although in markedly different proportions. These results suggest that essential oils have substantial potential as alternative approaches for I. ricinus tick control.

A distributional study of the dictyostelid cellular slime molds (dictyostelids) of South Africa was carried out during 2006 as part of the Global Biodiversity of Eumycetozoans project based at the University of Arkansas and funded by the National Science Foundation. Samples of soil/humus collected from 31 study sites yielded a total of 881 clones, with an average density of 210 clones/gram for all samples in which dictyostelids were detected. Eighteen different species were represented by one or more clones, and six of these could not be identified. These six species are described herein as new to science. In addition, information is provided on the ecological distribution of all of the species of dictyostelids now known to occur in South Africa.

Dictyostelia is a monophyletic group of transiently multicellular (sorocarpic) amoebae, whose study is currently limited to laboratory culture. This tends to favour faster growing species with robust sorocarps, while species with smaller more delicate sorocarps constitute most of the group's taxonomic breadth. The number of known species is also small (∼150) given Dictyostelia's molecular depth and apparent antiquity (>600 myr). Nonetheless, dictyostelid sequences are rarely recovered in culture independent sampling (ciPCR) surveys. We developed ciPCR primers to specifically target dictyostelid small subunit (SSU or 18S) rDNA and tested them on total DNAs extracted from a wide range of soils from five continents. The resulting clone libraries show mostly dictyostelid sequences (∼90%), and phylogenetic analyses of these sequences indicate novel lineages in all four dictyostelid families and most genera. This is especially true for the species-rich Heterostelium and Dictyosteliaceae but also the less species-rich Raperosteliaceae. However, the most novel deep branches are found in two very species-poor taxa, including the deepest branch yet seen in the highly divergent Cavenderiaceae. These results confirm a deep hidden diversity of Dictyostelia, potentially including novel morphologies and developmental schemes. The primers and protocols presented here should also enable more comprehensive studies of dictyostelid ecology.

Biotic and abiotic conditions in soil pose major constraints on growth and reproductive success of plants. Fungi are important agents in plant soil interactions but the belowground mycobiota associated with plants remains poorly understood. We grew one genotype each from Sweden and Italy of the widely-studied plant model Arabidopsis thaliana. Plants were grown under controlled conditions in organic topsoil local to the Swedish genotype, and harvested after ten weeks. Total DNA was extracted from three belowground compartments: endosphere (sonicated roots), rhizosphere and bulk soil, and fungal communities were characterized from each by amplification and sequencing of the fungal barcode region ITS2. Fungal species diversity was found to decrease from bulk soil to rhizosphere to endosphere. A significant effect of plant genotype on fungal community composition was detected only in the endosphere compartment. Despite A. thaliana being a non-mycorrhizal plant, it hosts a number of known mycorrhiza fungi in its endosphere compartment, which is also colonized by endophytic, pathogenic and saprotrophic fungi. Species in the Archaeorhizomycetes were most abundant in rhizosphere samples suggesting an adaptation to environments with high nutrient turnover for some of these species. We conclude that A. thaliana endosphere fungal communities represent a selected subset of fungi recruited from soil and that plant genotype has small but significant quantitative and qualitative effects on these communities.

Traditional morphology-based taxonomy of dictyostelids is rejected by molecular phylogeny. A new classification is presented based on monophyletic entities with consistent and strong molecular phylogenetic support and that are, as far as possible, morphologically recognizable. All newly named clades are diagnosed with small subunit ribosomal RNA (18S rRNA) sequence signatures plus morphological synapomorphies where possible. The two major molecular clades are given the rank of order, as Acytosteliales ord. nov. and Dictyosteliales. The two major clades within each of these orders are recognized and given the rank of family as, respectively, Acytosteliaceae and Cavenderiaceae fam. nov. in Acytosteliales, and Dictyosteliaceae and Raperosteliaceae fam. nov. in Dictyosteliales. Twelve genera are recognized: Cavenderia gen. nov. in Cavenderiaceae, Acytostelium, Rostrostelium gen. nov. and Heterostelium gen. nov. in Acytosteliaceae, Tieghemostelium gen. nov., Hagiwaraea gen. nov., Raperostelium gen. nov. and Speleostelium gen. nov. in Raperosteliaceae, and Dictyostelium and Polysphondylium in Dictyosteliaceae. The "polycephalum" complex is treated as Coremiostelium gen. nov. (not assigned to family) and the "polycarpum" complex as Synstelium gen. nov. (not assigned to order and family). Coenonia, which may not be a dictyostelid, is treated as a genus incertae sedis. Eighty-eight new combinations are made at species and variety level, and Dictyostelium ammophilum is validated.

David Caron (AC chair) University of Southern California, Los Angeles CA, USA Sandra Baldauf University of Uppsala, Uppsala, Sweden Sonya Dyhrman Columbia University, New York City NY, USA Laura Katz Smith College, Northampton MA, USA Connie Lovejoy Université Laval, Québec, Canada Alexandra Worden Monterey Bay Aquarium Research Institute, Moss Landing CA, USA John Archibald Dalhousie University, Halifax, Canada David Bass The Natural History Museum, London, UK Patrick Keeling University of British Columbia, Vancouver, Canada Jan Pawlowski University of Geneva, Geneva, Switzerland

The most gene-rich and bacterial-like mitochondrial genomes known are those of Jakobida (Excavata). Of these, the most extreme example to date is the Andalucia godoyi mitochondrial DNA (mtDNA), including a cox15 gene encoding the respiratory enzyme heme A synthase (HAS), which is nuclear-encoded in nearly all other mitochondriate eukaryotes. Thus cox15 in eukaryotes appears to be a classic example of mitochondrion-to-nucleus (endosymbiotic) gene transfer, with A. godoyi uniquely retaining the ancestral state. However, our analyses reveal two highly distinct HAS types (encoded by cox15-1 and cox15-2 genes) and identify A. godoyi mitochondrial cox15-encoded HAS as type-1 and all other eukaryotic cox15-encoded HAS as type-2. Molecular phylogeny places the two HAS types in widely separated clades with eukaryotic type-2 HAS clustering with the bulk of α-proteobacteria (>670 sequences), whereas A. godoyi type-1 HAS clusters with an eclectic set of bacteria and archaea including two α-proteobacteria missing from the type-2 clade. This wide phylogenetic separation of the two HAS types is reinforced by unique features of their predicted protein structures. Meanwhile, RNA-sequencing and genomic analyses fail to detect either cox15 type in the nuclear genome of any jakobid including A. godoyi. This suggests that not only is cox15-1 a relatively recent acquisition unique to the Andalucia lineage but also the jakobid last common ancestor probably lacked both cox15 types. These results indicate that uptake of foreign genes by mtDNA is more taxonomically widespread than previously thought. They also caution against the assumption that all α-proteobacterial-like features of eukaryotes are ancient remnants of endosymbiosis.

Rhizaria is a major eukaryotic group of tremendous diversity, including amoebae with spectacular skeletons or tests (Radiolaria and Foraminifera), plasmodial parasites (Plasmodiophorida) and secondary endosymbionts (Chlorarachniophyta). Current phylogeny places Rhizaria in an unresolved trichotomy with Stramenopila and Alveolata (supergroup "SAR"). We assembled a 147-protein data set with extensive rhizarian coverage (M147), including the first transcriptomic data for a euglyphid amoeba. Phylogenetic pre-screening of individual proteins indicated potential problems with radically misplaced sequences due either to contamination of rhizarian sequences amplified from wild collected material and/or extremely long branches (xLBs). Therefore, two data subsets were extracted containing either all proteins consistently recovering rhizarian monophyly (M34) or excluding all proteins with ⩾3 xLBs (defined as ⩾2× the average terminal branch length for the tree). Phylogenetic analyses of M147 give conflicting results depending on the outgroup and method of analysis but strongly support an exclusive Rhizaria+Alveolata (R+A) clade with both data subsets (M34 and M37) regardless of phylogenetic method used. Support for an R+A clade is most consistent when a close outgroup is used and decreases with more distant outgroups, suggesting that support for alternative SAR topologies may reflect a long-branch attraction artifact. A survey of xLB distribution among taxa and protein functional category indicates that small "informational" proteins in particular have highly variable evolutionary rates with no consistent pattern among taxa.

•213 proteins were identified in all dictyostelid major groups and amoebozoan outgroups.•Each protein was low-copy number with >120 aligned positions.•Phylogenetic analyses of these data split Dictyostelia into two major divisions: Groups 1+2 and Groups 3+4.•All possible alternative roots are rejected with p<0.01.•Dictyostelia requires complete revision at the highest taxonomic levels.

There are ~12 supergroups of complex-celled organisms (eukaryotes), but relationships among them (including the root) remain elusive. For Paper I, I developed a dataset of 37 eukaryotic proteins of bacterial origin (euBac), representing the conservative protein core of the proto-mitochondrion. This gives a relatively short distance between ingroup (eukaryotes) and outgroup (mitochondrial progenitor), which is important for accurate rooting. The resulting phylogeny reconstructs three eukaryote megagroups and places one, Discoba (Excavata), as sister group to the other two (neozoa). This rejects the reigning “Unikont-Bikont” root and highlights the evolutionary importance of Excavata.For Paper II, I developed a 150-gene dataset to test relationships in supergroup SAR (Stramenopila, Alveolata, Rhizaria). Analyses of all 150-genes give different trees with different methods, but also reveal artifactual signal due to extremely long rhizarian branches and illegitimate sequences due to horizontal gene transfer (HGT) or contamination. Removing these artifacts leads to strong consistent support for Rhizaria+Alveolata. This breaks up the core of the chromalveolate hypothesis (Stramenopila+Alveolata), adding support to theories of multiple secondary endosymbiosis of chloroplasts.For Paper III, I studied the evolution of cox15, which encodes the essential mitochondrial protein Heme A synthase (HAS). HAS is nuclear encoded (nc-cox15) in all aerobic eukaryotes except Andalucia godoyi (Jakobida, Excavata), which encodes it in mitochondrial DNA (mtDNA) (mt-cox15). Thus the jakobid gene was postulated to represent the ancestral gene, which gave rise to nc-cox15 by endosymbiotic gene transfer. However, our phylogenetic and structure analyses demonstrate an independent origin of mt-cox15, providing the first strong evidence of bacteria to mtDNA HGT.Rickettsiales or SAR11 often appear as sister group to modern mitochondria. However these bacteria and mitochondria also have independently evolved AT-rich genomes. For Paper IV, I assembled a dataset of 55 mitochondrial proteins of clear α-proteobacterial origin (including 30 euBacs). Phylogenies from these data support mitochondria+Rickettsiales but disagree on the placement of SAR11. Reducing amino-acid compositional heterogeneity (resulting from AT-bias) stabilizes SAR11 but moves mitochondria to the base of α-proteobacteria. Signal heterogeneity supporting other alternative hypotheses is also detected using real and simulated data. This suggests a complex scenario for the origin of mitochondria.

There are ~12 supergroups of complex-celled organisms (eukaryotes), but relationships among them (including the root) remain elusive. For Paper I, I developed a dataset of 37 eukaryotic proteins of bacterial origin (euBac), representing the conservative protein core of the proto-mitochondrion. This gives a relatively short distance between ingroup (eukaryotes) and outgroup (mitochondrial progenitor), which is important for accurate rooting. The resulting phylogeny reconstructs three eukaryote megagroups and places one, Discoba (Excavata), as sister group to the other two (neozoa). This rejects the reigning “Unikont-Bikont” root and highlights the evolutionary importance of Excavata.For Paper II, I developed a 150-gene dataset to test relationships in supergroup SAR (Stramenopila, Alveolata, Rhizaria). Analyses of all 150-genes give different trees with different methods, but also reveal artifactual signal due to extremely long rhizarian branches and illegitimate sequences due to horizontal gene transfer (HGT) or contamination. Removing these artifacts leads to strong consistent support for Rhizaria+Alveolata. This breaks up the core of the chromalveolate hypothesis (Stramenopila+Alveolata), adding support to theories of multiple secondary endosymbiosis of chloroplasts.For Paper III, I studied the evolution of cox15, which encodes the essential mitochondrial protein Heme A synthase (HAS). HAS is nuclear encoded (nc-cox15) in all aerobic eukaryotes except Andalucia godoyi (Jakobida, Excavata), which encodes it in mitochondrial DNA (mtDNA) (mt-cox15). Thus the jakobid gene was postulated to represent the ancestral gene, which gave rise to nc-cox15 by endosymbiotic gene transfer. However, our phylogenetic and structure analyses demonstrate an independent origin of mt-cox15, providing the first strong evidence of bacteria to mtDNA HGT.Rickettsiales or SAR11 often appear as sister group to modern mitochondria. However these bacteria and mitochondria also have independently evolved AT-rich genomes. For Paper IV, I assembled a dataset of 55 mitochondrial proteins of clear α-proteobacterial origin (including 30 euBacs). Phylogenies from these data support mitochondria+Rickettsiales but disagree on the placement of SAR11. Reducing amino-acid compositional heterogeneity (resulting from AT-bias) stabilizes SAR11 but moves mitochondria to the base of α-proteobacteria. Signal heterogeneity supporting other alternative hypotheses is also detected using real and simulated data. This suggests a complex scenario for the origin of mitochondria.