Multiple Roots of Fruiting Body Formation in Amoebozoa

Multiple Roots of Fruiting Body Formation in Amoebozoa Establishment of multicellularity represents a major transition in eukaryote evolution. A subgroup of Amoebozoa, the dictyos- teliids, has evolved a relatively simple aggregative multicellular stage resulting in a fruiting body supported by a stalk. Protosteloid amoeba, which are scattered throughout the amoebozoan tree, differ by producing only one or few single stalked spores. Thus, one obvious difference in the developmental cycle of protosteliids and dictyosteliids seems to be the establishment of multi- cellularity. To separate spore development from multicellular interactions, we compared the genome and transcriptome of a Protostelium species (Protostelium aurantium var. fungivorum) with those of social and solitary members of the Amoebozoa. During fruiting body formation nearly 4,000 genes, corresponding to specific pathways required for differentiation processes, are upregulated. A comparison with genes involved in the development of dictyosteliids revealed conservation of >500 genes, but most of them are also present in Acanthamoeba castellanii for which fruiting bodies have not been documented. Moreover, expression regulation of those genes differs between P. aurantium and Dictyostelium discoideum. Within Amoebozoa differen- tiation to fruiting bodies is common, but our current genome analysis suggests that protosteliids and dictyosteliids used different routes to achieve this. Most remarkable is both the large repertoire and diversity between species in genes that mediate envi- ronmental sensing and signal processing. This likely reflects an immense adaptability of the single cell stage to varying environ- mental conditions. We surmise that this signaling repertoire provided sufficient building blocks to accommodate the relatively simple demands for cell–cell communication in the early multicellular forms. Key words: multicellular development, transcriptome, Protostelium, Dictyostelia, evolution of development, signaling, Amoebozoa. Introduction multicellular life styles may therefore have been relatively Multicellularity was independently acquired in different easy achieved compared with, for example, the rare event branches of eukaryotes. This transition from uni- to of organelle capture (Marin et al. 2005; Nowack et al. The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Genome Biol. Evol. 10(2):591–606. doi:10.1093/gbe/evy011 Advance Access publication January 25, 2018 591 Downloaded from https://academic.oup.com/gbe/article-abstract/10/2/591/4824916 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Hillmann et al. GBE 2011). It is unlikely that in each case a new way to this tran- 2013) and the syncytial slime mold Physarum polycephalum sition to multicellularity was invented. Rather, building blocks (Schaap et al. 2016). The latter organism alternates between already present in unicellular eukaryotes (and their last com- unicellular amoeboid or amoeboflagellate forms, which can mon ancestor; LCA) were likely recruited to this new task. either individually encyst or, after mating, transform into a During evolution, new layers of complexity (i.e., increase of multinuclear syncytium. This syncytium has the choice of form- the numbers of cell types) masked these building blocks ing a dehydrated but viable sclerotium, or fruiting bodies (fig. 1) (Hedges et al. 2004) so that we have difficulties to define with multiple spores and acellular stalks (Schaap et al. 2016). them. Genomes of the unicellular ancestors of Metazoa We previously investigated the extent to which genes that seem to have been already complex. Exaptations of genes are essential for multicellular development of D. discoideum were later used for the evolution of multicellular development are conserved throughout Dictyostelia and unicellular (Grau-Bove et al. 2017). Interestingly, Capsaspora owczarzaki, Amoebozoa. This study showed that genes involved in intra- which can transit to an aggregative state, employs genes for cellular signal processing were mostly common to both multi- this transition, the orthologs of which establish tissue archi- cellular and unicellular Amoebozoa, but that genes encoding tectures in Metazoa (Sebe-Pedros et al. 2013; Suga et al. membrane or secreted proteins were more unique to 2013). Thus, aggregative multicellularity seems to have the Dictyostelia. This suggested that novel adhesion proteins, sig- same roots as true multicellularity at least in this evolutionary nal, and signal sensors are prime requisites for evolving multi- branch. Moreover, regulatory changes rather than gene inno- cellularity. How these were acquired remains often elusive, vations seemed to have played a major role in the establish- but of the five known nonpeptide signals which induce cell ment of multicellularity in Metazoa (Sebe-Pedros et al. 2016). differentiation in D. discoideum, three genes encoding their There seems to be no significant genomic difference be- biosynthetic enzymes were likely to be acquired by tween the unicellular algae Chlamydomonas and the multi- Dictyostelia through lateral gene transfer (Glo ¨ ckner et al. cellular Volvox species (Umen and Olson 2012). However, 2016). A parallel study retracing genes with conserved upre- extracellular matrix proteins seem to be important for the gulation in multicellular development highlighted many con- establishment of multicellularity in this clade (Prochnik et al. served genes with likely roles in multicellular development 2010). that have not yet been functionally characterized (Schilde Each eukaryote branchor evenclosely relatedcladesseem et al. 2016). to follow its own route to multicellular development and have Multicellular development of Dictyostelia and other organ- established their own toolkit for this purpose (Niklas and isms depends not only on cell-type specialization but also on Newman 2013). coordination of cell movement and/or cell division to generate Multicellular organisms generally go through a unicellular form. This complicates identification of the mechanisms that spore or fertilized egg stage at least once, which then divides uniquely regulate cell differentiation. Comparison of repeatedly to generate the multicellular form. In animals, Dictyostelia with protosteloid amoebae, that lack this compli- plants, and fungi, the dividing cells remain attached to each cation, may uniquely identify the genes involved in differenti- other, representing clonal, multicellular organisms. However, ation processes. in many other multicellular organisms, dispersed cells can come Fruiting bodies of diverse morphologies are widespread together when food runs out to construct a fruiting body with among the Amoebozoa, and the protosteloid type of fruiting aerially borne spores. This type of aggregative multicellularity body with a stalked spore, or very few spores, but always was invented independently by Acrasis spp. in the eukaryote derived from one cell is the most common (fig. 1). A recent division Discoba (Brown, Silberman, et al. 2012; He et al. 2014), study presented a well resolved tree of Amoebozoa with life Fonticula alba in Holomycota (Brown et al. 2009), Guttulinopsis cycle characters mapped onto that tree (Kang et al. 2017). spp. in Rhizaria (Brown, Kolisko, et al. 2012), Sorodiplophrys Species displaying protosteloid fruiting bodies were found to stercorea in Stramenophiles (Tice, Silberman, et al. 2016), be scattered throughout the Amoebozoa phylogeny, but also Sorogena stoianovitchae in Alveolata (Lasek-Nesselquist and occur within the Eumycetozoa which include the Dictyostelia Katz 2001), and Copromyxa protea and Dictyostelium spp. in and Ph. polycephalum. Recently, single cell fruiting bodies Amoebozoa (Baldauf et al. 2000; Brown et al. 2011). were also found to occur among the more distant Among these organisms, the genetic model system Acanthamoebidae (Tice, Shadwick, et al. 2016). It is hypoth- Dictyostelium discoideum and its close relatives display the esized that fruiting body formation was already present in the most complex form of aggregative multicellularity with a freely last common ancestor of all Amoebozoa (Kang et al. 2017), migrating “slug” stage and four to five specialized cell types. Its but an independent origin of fruiting body formation in dif- genome and those of species representative of the five major ferent lineages is also possible (Cavalier-Smith et al. 2016). clades of dictyosteliids have been sequenced (Heidel et al. We here compare the genome content of Protostelium 2011; Glo ¨ ckner 2015; Glo ¨ ckner et al. 2016)and within aurantium var. fungivorum and Protostelium mycophagum Amoebozoa, sequenced genomes are also available for the with those of Dictyostelia, Ph. polycephalum,and strictly unicellular Acanthamoeba castellani (Clarke et al. Acanthamoeba castellani. We further analyze the 592 Genome Biol. Evol. 10(2):591–606 doi:10.1093/gbe/evy011 Advance Access publication January 25, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/2/591/4824916 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Multiple Roots of Fruiting Body Formation in Amoebozoa GBE FIG.1.—Amoebozoa fruiting bodies. Schematic view of the different “fruiting bodies” in the Amoebozoa. Depicted are only species where a genome sequence is available including the here presented Protostelium spp. genomes. The fruiting bodies are drawn to scale, the inset shows an enlargement of the tiny Protostelium fruiting body. No fruiting body is known for Acanthamoeba castellanii, indicated by a cross. However, other Acanthamoebae do form sporocarps, as was recently discovered for A. pyriformis (Tice, Shadwick, et al. 2016). transcriptome changes that occur in Protostelium aurantium sporocarps (fruiting body of a single cell) to new petri dishes. var. fungivorum (further addressed as P. aurantium) during its Liquid cultures of Protostelium sp. were carried out in petrid- transition from a feeding amoeba into a mature fruiting body. ishes filled with 2 mM PB buffer (pH 6.4) and R. mucilaginosa Our study suggests that fruiting body formation seemed to as the only food source. Cell numbers for both organisms have evolved analogously from the similar toolbox of genes. were determined either using a hemocytometer or an auto- Furthermore, despite their simple life style, Protostelium spp. matic cell counter (Casy TT Cell Counter, OLS Bio, Bremen, already display a large variety of cell signaling genes. Germany). Induction of the Developmental Cycle and Preparation of Materials and Methods Samples for RNA Isolation Species Isolation and Maintenance Protosteloid amoebae were cultivated in liquid cultures with Protosteloid amoebae were maintained as spore suspensions successive additions of R. mucilaginosa as a food source. For in 86% [v/v] glycerol at80 C. The type strain of Protostelium P. aurantium, the number of trophozoites reached confluency mycophaga Olive and Stoianovitch was obtained from the at 48- to 60-h postinoculation. At this time point, the growth Culture Collection of Algae and Protozoa (CCAP, Argyll, medium was aspirated and residual yeast cells were removed United Kingdom) and from the American Type Culture by two consecutive washes with PB. Adherent amoebae were Collection (ATCC, Manassas, VA). Protostelium aurantium scraped from the surface, resuspended in PB and transferred var. fungivorum wasisolatedasdescribed for protosteliids to a wMY-agar surface in 10 ml droplets. To obtain sufficient by Spiegel et al. (2006) with slight modifications. Briefly, material for RNA isolation, cells from 40 10-ml droplets were beechleaves were cut to1cm and centrally placed on the pooled for each time point in one of the three biological surface of buffered wMY agar (0.002 g l malt extract, replicates. Vegetative cells were harvested from plates directly 0.002 g l yeast extract, 2 mM potassium phosphate buffer after plating (timepoint 0 h). Starved cells were harvested after pH 6.4 [PB], and 15 g l agar) in petri dishes. The basidiomy- 1.5 h and nearly completed fruiting bodies were harvested cetous yeast Rhodotorula mucilaginosa DSM70404 was from plates incubated for 8 h. obtained from the Jena Microbial Resource collection and was streaked in a distance of 5 mm to the leaf pieces to pro- Nucleic Acids Isolation and Sequencing mote the outgrowth of fungivorous protosteloid amoeba. Plates were sealed with parafilm and incubated at 22 Cfor Chromosomal DNA was isolated from liquid cultures of pro- 10–14 days with daily microscopic inspections for trophozoite tosteloid amoebae. Adherent cells were washed in phosphate growth or the formation of fruiting bodies. Isolation was buffer and harvested by centrifugation at 200 x g which was achieved by four consecutive transfers of single amoeba or sufficient to deplete nearly all residual yeast cells. Trophozoites Genome Biol. Evol. 10(2):591–606 doi:10.1093/gbe/evy011 Advance Access publication January 25, 2018 593 Downloaded from https://academic.oup.com/gbe/article-abstract/10/2/591/4824916 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Hillmann et al. GBE were lysed in 50 mM Tris–HCl (pH 7.5) with 2% [w/v] of SDS then removed to yield a complete, closed circle mitochondrial and 0.5 mM Na EDTA, followed by a 1-h incubation at 60 C genome. with RNAase A (Sigma–Aldrich, Taufkirchen, Germany) and Proteinase K (Sigma–Aldrich) at final concentrations of 100 RNA Sequencing and 200 mgml , respectively. Further purification steps like Each of the three time points of the RNAseq experiment was extractions with phenol-chloroform and precipitation with covered by three biological replicates. The quality and amount isopropanol were carried out as described (Sambrook and of RNA was initially checked using the Agilent Bioanalyzer Russell 2001). 2100 in combination with a RNA 6000 nano kit (both For the isolation of total RNA, cells were rapidly harvested Agilent technologies). The RIN (RNA integrity number) varies from the agar surface, resuspended in 2 ml of phosphate from 8.4 to 10 with an average of 9.44. Total RNA of 1 mg buffer, and centrifuged for 1 min at 6,000g. Pellets were was used for library preparation using Illumina’s TruSeq RNA shock-frozen in liquid N and stored80 C. RNA was prepared sample preparation kit v2 following the manufacturer’s de- by phenol extraction with subsequent additions of 500 mlof scription which included individual labeling by sequence barc- TRIsure (Bioline, Luckenwalde, Germany) and 200 ml of chloro- odes. The libraries were again quantified and quality checked form. Phase separation was achieved by centrifugation fol- using the Bioanalyzer 2100 and DNA 7500 kit. For sequencing, lowedby anadditionalextractionwith500 ml of chloroform. libraries were pooled, individually labeled, and loaded in two Precipitation was as for DNA. RNA quantity and impurities were lanes of a HiSeq 2500 running in single-end/51 cycle/high out- determined spectrophotometrically. Integrity of the isolated put mode. Data extraction was done in FastQ format using the RNA was checked by agarose gel electrophoresis tool bcl2FastQ v1.8.4 (provided by Illumina). Mapping of these reads to the genome was done with tophat2. The counts per gene were used to calculate differential expression using DNA Sequencing, Assembly, and Mapping DEseq2 (Love et al. 2014) with the Bioconductor package of R. Library preparation was done using Illumina’s TruSeq DNA PCR free library preparation kit following the manufacturer’s Gene Predictions description for 550-bp insert libraries. Library quality check and quantification was done as described earlier. For the annotation of the P. mycophagum genome, we used Sequencing was done in one lane of a HiSeq 2500 running GeneMarkES in self-training mode, which uses no apriori in paired-end/2 101 cycle/rapid mode. The reads derived knowledge on gene structures. For the annotation of the P. from genomic DNA were assembled using abyss-pe with a aurantium genome, we made use of the RNAseq data, which kmer size of 45–65 with step increments of 2. The assembly were mapped to the genome as described earlier. The sizes for the P. aurantium genome (excluding contaminants) mapped reads were used to define 52,801 splice sites. was in a range from 39 (kmer 45) to 68 Mb (kmer 65). The These data were used for the GeneMarkET program to enable kmer 45 assembly was chosen for further improvement after a better gene prediction. examination of contig lengths, coverage, and contribution of bacterial and fungal sequences to the assembly for both spe- ncRNAs cies. Specifically, the kmer45 assembly was the most contig- For prediction of nonprotein-coding RNAs (ncRNAs), we used uous of all assemblies and therefore represented the genomes GORAP (www.rna.uni-jena.de/en/software; last accessed best. The GC content of the protosteliid genomes was January 31, 2018), which searches for 2,474 known ncRNA <50.1% while contaminating DNA from other sources (bac- families from Rfam version 12.1 (Nawrocki et al. 2015). teria and the food fungus) was found to have a higher GC GORAP uses improved homology search strategies based on content. Thus, the GC content was a first indicator to readily sequence and secondary structures and was run with default discriminate between the Protostelium spp. genomes and settings. All predictions passed Rfam thresholds. All results contaminating sequences. Furthermore, all contigs were com- and files in fasta, gff, and stockholm format are available at pared with fungal and bacterial genomes to exclude contam- http://www.rna.uni-jena.de/supplements/protostelium;last inating sequences from further analysis. Here, we were using accessed January 31, 2018. BLAST analysis to identify fungal or bacterial sequences (BLASTn in case of bacteria and BLASTx for fungi). If a contig Orthology and Protein Annotation had a higher similarity to known bacterial or fungal sequences than to any Amoebozoa genome, it was excluded from the We defined the orthology between the two Protostelium analysis. Further gap closure was done with PAGIT (http:// genomes and to the dictyosteliid genomes using OrthoMCL www.sanger.ac.uk/science/tools/pagit; last accessed January (Li et al. 2003). The protein functions were evaluated by 31, 2018) using default values. For assembly of the mitochon- aligning the protein sequences to the protein sequence drial genome, we extended the contig using SSPACE (Boetzer database obtained from NCBI (version from 5/2016) using et al. 2011). The resulting overlap between contig ends was BLAST. Domains were searched using InterproScan 594 Genome Biol. Evol. 10(2):591–606 doi:10.1093/gbe/evy011 Advance Access publication January 25, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/2/591/4824916 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Multiple Roots of Fruiting Body Formation in Amoebozoa GBE FIG.2.—Phylogeny and mitochondrial organization. (A) Phylogeny of selected protosteloid amoeba together with selected other amoebozoa based on 18 S RNA sequences. The species of which the genome sequences were analyzed are given in bold letters. Superscript numbers refer to recently renamed 1 2 isolates of Protostelium aurantium with accession numbers FJ766461.1 and FJ766463.1. The alignment was cleaned of ambiguous positions using GBLOCKS (Talavera et al. 2007) reducing the observable distance between species and clades. A discrete Gamma distribution was used to model evolu- tionary rate differences among sites (5 categories [þG, parameter¼ 0.6214]). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Evolutionary analyses were conducted in MEGA6 (Tamura et al. 2013). (B) Mitochondrial synteny between selected Amoebozoa. Genes are drawn as boxes of equal length. Syntenic regions are connected by colored bands. (Jones et al. 2014). The biochemical pathways were analyzed frequencies, and growth was comparably slow, we decided using the KAAS server (Moriya et al. 2007). All programs were to isolate a wild strain by means of food choice selection, that run with default values. is, incubating environmental samples together with Rhodotorula cultures (Materials and Methods). This way we isolated a protosteloid amoeba in sensu Spiegel (Spiegel Phylogenetic Analysis of Adenylate and Guanylate et al. 1994, 2006; Shadwick et al. 2009) from a dead aerial Cyclases beech leaf in the Jena forest. The amoeba revealed striking The output of an Interproscan of the translated Protostelium similarities to the type strain of Protostelium mycophagum aurantium transcripts was scanned for proteins harboring as described previously (Olive and Stoianovitch 1960). Interpro domain IPR001054 for class III adenylate and guany- Phylogenic analysis based on its SSU rRNA (fig. 2A) as well late cyclases. The P. aurantium proteins harboring this domain as diagnostic morphological traits identified the isolate as a were alignedwiththe five Dictyostelium cyclases and a few strain of Protostelium aurantium as recently described by signature Ph. polycephalum and A. castellanii cyclases using Shadwick et al. (2017). Due to its rapid phagocytic feeding Clustal Omega with five combined iterations (Sievers and on the basidiomycetous yeast Rhodotorula mucilaginosa on Higgins 2014). After deletion of segments that did not align solid agar surfaces (supplementary data set S1, fig. S1A, unambiguously, alignments were subjected to Bayesian infer- Supplementary Material online), we coined this new isolate ence for phylogeny reconstruction using a mixed amino acid Protostelium aurantium var. fungivorum. A detailed taxo- model in MrBayes software (Ronquist and Huelsenbeck nomic description of this isolate is available within the sup- 2003). Analyses were run until convergence, and trees were plementary material of this article (supplementary data set rooted at midpoint (Hess and De Moares Russo 2007). For the S1, Description of Protostelium aurantium, Supplementary earliest diverging branches of each major clade, closest homo- Material online). logs were identified by BLASTp of all NCBI nonredundant Most characteristic for its identification as a protosteloid sequences. These protein sequences were aligned and in- amoeba was the abundant formation of globose fruiting bod- cluded in Bayesian inference of the final tree. ies carrying a single spore (fig. 3A–C). The sporocarp (fruiting body of a single cell) culminated on an acellular stalk fixed on the substratum (fig. 3D). As described for P. mycophagum, Results and Discussion asynchronous fruiting body formation occurred on solid sur- The Species faces in the absence of the fungal food source. The majority Protostelium mycophagum, the type strain (Olive and of cells initiated their full developmental cycle 2–4 h following Stoianovitch 1969) was maintained in culture together with the onset of starvation, passing through the formation the fungus Rhodotorula mucilaginosa. As fruiting body for- of immobile discs, cellular condensation, the formation of mation in this P. mycophagum strain occurred at lower the sporocarp, and stalk elevation, with each stage being Genome Biol. Evol. 10(2):591–606 doi:10.1093/gbe/evy011 Advance Access publication January 25, 2018 595 Downloaded from https://academic.oup.com/gbe/article-abstract/10/2/591/4824916 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Hillmann et al. GBE FIG.3.—Unicellular fruiting bodies of Protostelium aurantium. Light micrograph of a stalked fruiting body (A) and scanning electron micrographs of a tilted, but intact fruiting body (B), which will eventually open and liberate single spores (C), and the rigid base of the stalk (D). completed in 10–15 min (supplementary data set S1, fig. S2, P. aurantium lacks the genes rps7 and rpl14, located in the Supplementary Material online). rearranged segment. Comparisons to other Amoebozoa mi- tochondrial genomes show that these additionally lost genes lie in the same region where other ribosomal subunit genes Phylogeny of the Species and Their Genomes were lost in Protostelium spp. (fig. 2B). Thus, loss of such For phylogenetic reconstruction, we sampled 18 S RNA mitochondrial genes seems to be common in the sequences of Amoebozoa representatives from the NCBI Protostelium spp. analyzed. database, aligned them, and performed a maximum likeli- Based on the gene prediction, both nuclear haploid hood analysis (fig. 2A). We rooted our tree with the 18 S genomes are comparable in size and also encode a similar gene from Mus musculus. The tree readily identified our en- number of genes (table 1). For P. aurantium, this number vironmental isolate as a member of the Protostelium auran- was further supported by RNAseq data (supplementary data tium clade and revealed its distant relationship to Dictyostelia set S1, fig. S4, Supplementary Material online) using and Physarum. Despite their phylogenetic distance within the GeneMarkET (Borodovsky and Lomsadze 2011). A distin- Amoebozoa, all three species share the ability to construct a guishing hallmark of both genomes is the high number of fruiting body. As gene expression during fruiting body forma- introns per gene (supplementary data set S1, fig. S5, tion of dictyosteliids has been particularly well studied and Supplementary Material online). The median intron size is P. mycophagum did not sporulate readily, we focused mainly very small with 40–50 bases, but larger than the smallest in- on the P. aurantium genome andexpressiondata and its tron sizes reported so far (Slabodnick et al. 2017). Overall, the comparison to Dictyostelia. Protostelium spp. genomes analyzed here harbor circa 30% Based on our sequencing approach, we assembled the more genes than Dictyostelia. complete mitochondrial genomes and obtained draft nuclear In line with the extensive use of introns, we found not only genomes for both Protostelium species. The mitochondrial all small nuclear RNAs (snRNAs) of the major spliceosome but genomes are comparable with those in dictyosteliids with re- also the minor spliceosome to be present in both species. In spect to coding capacity and size. The order of genes differs total, a similar number of noncoding RNAs were annotated between the two species only for one segment of genes and a for P. mycophagum (590) and P. aurantium (525) (supplemen- few tRNA locus rearrangements (fig. 2B). Furthermore, tary data set S1, table S1, Supplementary Material online). 596 Genome Biol. Evol. 10(2):591–606 doi:10.1093/gbe/evy011 Advance Access publication January 25, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/2/591/4824916 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Multiple Roots of Fruiting Body Formation in Amoebozoa GBE Table 1 points (vegetative cells at 0 h, starvation and early differenti- Genomic Features of the Two Protostelium Species ation at 1.5 h, and the formation of prespores and mature Protostelium Protostelium fruiting bodies at 8 h). Although sporocarp formation was mycophagum aurantium asynchronous among the population, these time points Genome size in Mb (haploid) 39.7 38.3 were discernible via the predominant morphology (supple- Scaffolds 2,578 822 mentary data set S1, figs. S2 and S6A, Supplementary Mean GC content 42.2 47.9 Material online). We first assessed which genes are potentially Palindrome size (kb) 13.4 16.3 differentially expressed between any of those time points. The Mitochondrium (kb) 48.6 44.5 biological replicates showed little variation (supplementary Protein coding genes 15,691 17,172 data set S1, fig. S6B and C, Supplementary Material online). Mean size CDS/AA 1,386/462 1,512/504 The DEseq2 analysis (Love et al. 2014) implemented in the tRNAs 426 405 BioConductor R package revealed that there are 7,787 genes Introns 126,081 145,637 significantly downregulated and 3,980 genes significantly Mean intron length 83.2 63.4 upregulated. An example of an upregulated gene is given in Median intron length 50 46 supplementary data set S1, figure S7, Supplementary Material Introns/gene 8 8.5 Intron space (Mb) 10.5 9.2 online. A significance threshold of 0.001 reduces these num- CDS space (Mb) 21.8 26 bers to 6,579 and 2,685, respectively. We next analyzed which potential functions are encoded in this smaller gene set. This analysis revealed extensive reprogramming of sev- The amount of mobile elements in P. aurantium is esti- eral functions during the developmental cycle (fig. 4). A mated at 0.13% of the genome. Although several re- gene ontology (GO) term enrichment analysis of all upregu- verse transcriptase domains of both long terminal repeat lated genes using Gotermfinder (Boyle et al. 2004) showed (LTR) and non-LTR retrotransposons could be determined, that in the GO category “molecular function” ATP and GTP the corresponding elements are highly fragmented and binding proteins are strongly overrepresented (supplemen- mostly present in single copy. Only four LTR retrotranspo- tary data set S1, fig. S8, Supplementary Material online). sons could be analyzed in some detail, and they belong to However, no GO terms in the category “biological process” the Skipper and DGLT-A families of Ty3/gypsy type of LTR were significantly enriched. retrotransposons known from the dictyosteliid clade We next divided the genes into three categories (down- (Spaller et al. 2016). regulation, early upregulation, and late upregulation). This only roughly approximates the real transcript dynamics since we had only three distinct developmental stages in Gene Families and Orthology Relationships our time course. The KEGG pathway analysis (Moriya Both Protostelium spp. share among them 6,278 1:1 ortho- et al. 2007) revealed that up- and downregulated genes logs and 1,708 gene families with more than one member in can be part of the same pathway and concerted regulation at least one species. Protostelium mycophagum has 6,264 may affect only part of a pathway (fig. 5 and supplementary genes not shared with P. aurantium, whereas P. aurantium table S1, Supplementary Material online). Processes related has 5,942 such orphan genes, meaning that they were also to RNA generation, fatty acid synthesis, RNA and protein not found in other species. These numbers are somewhat transport, and oxidative phosphorylation were uniformly comparable to the 4,156 orphan genes reported for A. cas- downregulated. This downregulation is a likely hallmark of tellanii (Clarke et al. 2013). Interestingly, several of the largest the fact that the cells are experiencing starvation. Genes gene families in both species encode proteins with Leucine- that were upregulated early tended to be involved in repli- rich repeats, which are often involved in protein–protein inter- cation and repair, dNTP synthesis, and fatty acid degrada- actions. Other large gene families comprise Ankyrin repeat tion. These early events likely reflect the fact that the cell containing proteins, nucleotidyl cyclases, or kinases (supple- tries to recruit energy from fatty acids to complete the de- mentary data set S1, table S2, Supplementary Material velopmental cycle. In a later stage, P. aurantium devoted online). resources to the production of membrane components like glycerophospho- and sphingolipids, as well as chitin as Gene Regulation during Fruiting Body Formation a cell wall polysaccharide, indicative of the final stages of Upon starvation on a solid substratum, P. aurantium cells in- spore formation (fig. 5). dividually differentiate into a spore supported by an acellular We also found that signal transduction cascades that in- stalk. As P. aurantium displayed a higher frequency and duce cytoskeleton reorganization, survival, adhesion, and mi- abundancy of these structures relative to P. mycophagum, gration were upregulated early and this upregulation we chose P. aurantium to analyze the transcriptional changes increased further in differentiation. The second messenger during this differentiation process by RNAseq over three time cAMP seems to play a pivotal role here since its production Genome Biol. Evol. 10(2):591–606 doi:10.1093/gbe/evy011 Advance Access publication January 25, 2018 597 Downloaded from https://academic.oup.com/gbe/article-abstract/10/2/591/4824916 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Hillmann et al. GBE FIG.4.—Transcriptional reprogramming during the developmental cycle of Protostelium aurantium. Heat maps indicate higher (blue) and lower (red) expression of genes from major developmental and metabolic pathways when comparing early (0 min vs. 1.5 h, top line) or late stages (0 vs. 8 h, middle line, or 1.5 h vs. 8 h, bottom line) of development. Genes from each cluster are grouped in three temporal categories representing an upregulated expression either early, late, or throughout the developmental cycle. Heat maps illustrate the log expression ratio based on the mean RPKM values from three biological replicates for each time point. A more detailed table including gene accession numbers, numeric expression ratios, KEGG orthologies, and predicted protein functions are available as supplementary table S1, Supplementary Material online. started early in development (supplementary data set S1, fig. involved in common encystation processes in Amoebozoa S9, Supplementary Material online). Which of the numerous (Kawabe et al. 2015) is upregulated, presumably via cAMP. cyclase encoded in the genome of P. aurantium is responsible Throughout the differentiation process protein modification for this production or whether several cyclases are acting in a pathways (N-glycan biosynthesis) were enhanced. concerted way to achieve this, is currently unclear. However, Interestingly, N-Glycan biosynthesis plays also a role in D. dis- one of the cyclases (PROFUN_08491) is particularly upregu- coideum differentiation (Li et al. 2015). In P. aurantium,this lated during development (fig. 6) and therefore might be re- pathway may be involved in production of components of the sponsible for the early cAMP level rise. PKA activity, which is cell wall and the acellular stalk. 598 Genome Biol. Evol. 10(2):591–606 doi:10.1093/gbe/evy011 Advance Access publication January 25, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/2/591/4824916 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Multiple Roots of Fruiting Body Formation in Amoebozoa GBE FIG.5.—Developmentally regulated pathways in Protostelium aurantium based on RNAseq analysis. Red indicates functions represented by genes that are downregulated upon starvation, green and blue highlight functions that are upregulated in early and late development, respectively. Presumed Calcium upregulation is depicted with a yellow arrow. Functions associated with the nucleus are listed in a circle in the right lower corner, and those with the ribosome in the upper left corner. The cytoskeleton and exocytosis are influenced by the depicted pathways, but the impact of this is not clear. D. discoideum chemotaxis and stalk formation (Kawata 2011) Comparison between Social Amoebae and P. aurantium is upregulated in D. discoideum,whereas its P. aurantium Developmental Genes ortholog (PROFUN_03920) is slightly downregulated. Social amoebae have 60% orthologs between them as a Overall, not more than 75 genes were consistently identi- comparative study showed (Heidel et al. 2011). Since the evo- fied as important for development in D. discoideum by being lutionary distance between dictyosteliids and the protosteliids upregulated and essential during the developmental process analyzed here is even higher than within the dictyosteliids it is (Glo ¨ ckner et al. 2016; Schilde et al. 2016). However, for most not surprising that only 37% of the genes in P. aurantium of these, orthologous genes were found in the genomes of P. show similarities to genes in D. discoideum, as detected by aurantium (57), A. castellanii (47), and Ph. polycephalum (57) BLAST with a bit score threshold of 200. In two previous (table 2). It is therefore conceivable that these genes could studies, we have defined two sets of dictyosteliid genes which belong to the basic toolkit for the evolution of all differenti- are involved in the developmental cycle of social amoebae ated forms of development among the Amoebozoa. (Glo ¨ ckner et al. 2016; Schilde et al. 2016). The set of devel- The set of in total 233 D. discoideum DevUp and DevEs opmentally essential (DevEs) genes contains 374 genes that geneswithnohitsineither the P. aurantium, Ph. polycepha- upon knock-out cause a developmental defect (Glo¨ckner lum,and A. castellanii genomes is enriched in several GO et al. 2016). The set of developmentally upregulated terms, for example, “extracellular matrix organization” and (DevUp) genes contains 794 genes that are consistently de- “multicellular organismal process” (supplementary data set velopmentally upregulated across the Dictyostelium taxon S1, fig. S10, Supplementary Material online). We next groups (Schilde et al. 2016). In total, these two sets comprise searched for genes in the 1,168 gene set, which are shared 1,168 genes (table 2 and supplementary data set S1, fig. S11, exclusively between D. discoideum and P. aurantium and are Supplementary Material online). A higher percentage of not present in A. castellanii and Ph. polycephalum. This search DevEs than DevUp genes has identifiable counterparts in revealed only 13 genes with a range of putative functions the P. aurantium genome (76% vs. 48%). However, of the (supplementary data set S1, table S3, Supplementary in total 669 D. discoideum proteins with homologs in P. aur- antium, 172 have the same homolog, indicating gene family Material online). expansions in D. discoideum. Strikingly, the directionality of Three of the four genes of Dictyostelia (chlA, dgcA, dokA, developmental regulation of P. aurantium counterparts is of- iptA), which are likely derived from horizontal gene transfer ten opposite (table 2). For example, statA with a major role in from bacteria (Glo ¨ ckner et al. 2016) and which either sense Genome Biol. Evol. 10(2):591–606 doi:10.1093/gbe/evy011 Advance Access publication January 25, 2018 599 Downloaded from https://academic.oup.com/gbe/article-abstract/10/2/591/4824916 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Hillmann et al. GBE FIG.6.—Phylogeny of adenylate and guanylate cyclases. The phylogenetic tree was calculated with Bayesian inference (see Materials and Methods).The protein identifiers are color coded to reflect species names as indicated in the figure, and are annotated with the functional domain architecture of the proteins as determined by SMART (Schultz et al. 1998). PFAM domains are represented as colored to black graded rectangles. Posterior probabilities (BIPP) of tree nodes are indicated by colored dots. The heatmap indicates relative expression levels at 1.5 h (starvation) and 8 h (fruiting body formation) compared with the vegetative state. The cartoon illustrates the predominant cell types at these time points (see supplementary fig. S7, Supplementary Material online). (dokA) or synthesize developmental signals, are not present in Only two TFs which were previously identified in Dictyostelia the P. aurantium genome. However, two histidine kinases were slightly conserved only in P. aurantium (crtf and stkA) (PROFUN_01260 and PROFUN_15316) have the same do- but not found in Ph. polycephalum or A. castellanii. This indi- main structure as dokA (supplementary data set S1, fig. cates a common root for developmental regulation for only S13, Supplementary Material online) and therefore might en- few if any TFs and a likely loss in some lineages. Further ge- code similar functions even if they are not orthologs. nome sequences in the Amoebozoa are needed to trace the We also examined, whether transcription factors (TFs) im- origin of these TFs to the LCA. portant for the development in Dictyostelia are conserved in Overall, and somewhat contrary to expectations based on Protostelium spp. (table 3). Of a total of twelve TFs, ortholo- morphological similarities, the conservation of developmental gous proteins in P. aurantium were found for only seven and genes between Dictyostelia and P. aurantium is low and pos- none of them were highly expressed during development. sible not more than with other unicellular amoebozoan 600 Genome Biol. Evol. 10(2):591–606 doi:10.1093/gbe/evy011 Advance Access publication January 25, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/2/591/4824916 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Multiple Roots of Fruiting Body Formation in Amoebozoa GBE Table 2 Gene Regulation Differences between Social Amoebae, Protostelium aurantium, and Other Amoebozoa Gene Set Developmental Expression in Physarum Acanthamoeba P. aurantium Dictyostelium sp. Protostelium aurantium polycephalum castellanii Development Genes with %of Multiple Up Down Neutral Genes with Genes with Similarity Dictyostelium Hits Similarity Similarity (score>200) sp. Genes (score>200) (score>200) Upregulated during 783 377 48 96 128 199 50 370 310 development Essential for development 309 234 76 62 55 138 41 233 193 Upregulated and essential 75 57 78 13 21 21 15 57 47 All 1,167 668 57 171 204 358 106 660 550 NOTE.—About 1,167 genes of D. discoideum were identified previously as being either developmentally upregulated (Schilde et al. 2016), developmentally essential (Glo¨ ckner et al. 2016), or both (commonly identified in both studies). These genes were searched in the genomes of P. aurantium, Ph. polycephalum,and A. castellanii. To reduce spurious hits a BLAST similarity bit score of 200 was applied when comparing the encoded amino acid sequences. The column “Multiple hits” counts the occasions where different D. discoideum genes yielded the same P. aurantium hit. Table 3 Developmental TFs from Dictyostelium discoideum and Presence of Orthologous Proteins in Protostelium aurantium Gene ID TF Best Hit in P. aurantium Log Change in Comment Expression at 1.5 h, 8.5 h DDB_G0278077 crtf PROFUN_06017 0.42, 0.45 Expression of aggregation genes; Dictyostelia specific DDB_G0281387 srfA PROFUN_09032 1.13, n.d. Spore differentiation; MADS box; only box similar DDB_G0277589 gtaC PROFUN_10218 0.41, n.d. pstB cell sorting and basal disc formation; GATA zinc finger; also in Acanthamoeba DDB_G0278971 dimA n.d. – Development; bZIP transcription factor DDB_G0291372 dimB n.d. – Development; bZIP transcription factor DDB_G0279529 bzpF PROFUN_06600 n.d., 0.58 Spore viability; bZIP transcription factor; common TF DDB_G0281381 dstA PROFUN_03920 n.d., 0.29 Culmination, cudA expression; STAT transcription PROFUN_04291 n.d., n.d. factor; also in A. castellanii PROFUN_12834 0.64, n.d. PROFUN_01113 n.d., n.d. DDB_G0277147 stkA PROFUN_04849 n.d., n.d. Spore formation; GATA zinc finger; dictyosteliid specific PROFUN_12502 n.d., n.d. motif C terminal of zinc finger conserved DDB_G0284465 cudA PROFUN_14255 n.d., 0.82 Many prespore genes and for culmination; also in PROFUN_08418 n.d., n.d. A. castellanii DDB_G0281563 mybC n.d. – Culmination; homology to myb domains only DDB_G0275445 mybB n.d. – ACA expression; homology to myb domains only DDB_G0281969 mybE n.d. – Basal disc formation; homology to myb domains only NOTE.—n.d., orthologous protein or gene expression was not detected. genomes. Evolution of fruiting body formation could thus focused entirely on the morphogenetic process that protoste- have well been independent in the two species. However, liids and dictyosteliids do have in common, the differentiation analysis of further, phylogenetically more distant, protosteloid of spore(s), supported by a stalk. Although the stalk differs amoeba will help to resolve this question. from that of most Dictyostelia by not containing any cells, there is also a Dictyostelium clade, the acytosteliids, which lack cells in their stalk, but nevertheless have the same Spore and Stalk Genes stalk-specifying genes as the species with stalk cells The analysis described earlier comprised all known develop- (Urushihara et al. 2015). mentally essential and conserved developmentally upregu- About 92 genes with roles in spore and stalk formation lated genes, but many of these genes are typically involved have been identified in Dictyostelium discoideum (Glo¨ckner in multicellular morphogenesis which has never been ob- et al. 2016) and we investigated whether orthologs of these served for Protostelium sp. In the following analysis, we genes were also present in P. aurantium, Ph. polycephalum, Genome Biol. Evol. 10(2):591–606 doi:10.1093/gbe/evy011 Advance Access publication January 25, 2018 601 Downloaded from https://academic.oup.com/gbe/article-abstract/10/2/591/4824916 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Hillmann et al. GBE Table 4 Conserved Dictyostelium discoideum Spore and Stalk Genes in Three Amoebozoa Required for Dictyostelium discoideum Protostelium aurantium Physarum polycephalum Acanthamoeba castellani Sporulation 72 26 40 32 Stalk formation 20 2 6 2 and A. castellanii. Genes in the three species were validated as This compares rather poorly with Dictyostelium, A. castellanii, D. discoideum orthologs when 1) the D. discoideum and spe- and Ph. polycephalum, which have 55, 35, and 146 GPCRs, cies homologs were each other’s best bidirectional BLASTp hit respectively (table 5). Protostelium aurantium does have 9 (BBH), 2) they had the same or very similar functional domain heterotrimeric G-proteins, more similar to Dictyostelium, architectures, and 3) the species genes clustered with the which has 12 (supplementary data set S1, fig. S12, dictyosteliid orthologs in phylogenetic trees that also included Supplementary Material online). other close homologs (see supplementary data set S2, Supplementary Material online). All D. discoideum spore Sensor Histidine Kinases/Phosphatases and stalk genes and their best P. aurantium, Ph. polycepha- lum, and A. castellanii hits are listed in supplementary data set Histidine kinases/phosphatases with an attached sensor do- S1, table S4, Supplementary Material online, with likely ortho- main are used for detecting and processing a broad range of logs indicated in bold text. A summary of the analysis is listed chemical and physical stimuli in both pro- and eukaryotes. The in table 4. stimulus activates either phosphorylation or dephoshorylation Of the 92 Dictyostelium genes, 28 had orthologs in of a conserved histidine residue in the histidine kinase domain, P. aurantium, but a much larger number (46) had orthologs which respectively triggers forward or reverse histidine– in Ph. polycephalum. Even the nonsporulating A. castellanii aspartate–histidine relay of the phosphate to/from an aspar- has slightly more (34) spore and stalk genes in common with tate in a receiver domain. This then results in activation or Dictyostelium. Largely the same genes are shared between all inactivation of an attached effector, often an enzyme or TF four species, which suggests that they have a common role (Zschiedrich et al. 2016). Protostelium aurantium appears independent of sporulation. Their role in normal spore and to make up for its low number of GPCRs by having an ex- stalk formation in Dictyostelium may either result from a dic- tremely large number of 71 sensor histidine kinases/phospha- tyosteliid specific recruitment into such a role, or reflect a tases (SHKPs) (supplementary data set S1, fig. S13, pleiotropic effect of their loss on spore or stalk formation. Supplementary Material online). This is over four times more than D. discoideum andalsoexceeding Ph. polycephalum and A. castellanii (table 5). The P. aurantium SHKPs contain a va- Cell Signaling riety of sensor domains, among which is also a phytochrome Without undue emphasis on its development into fruiting domain. This domain was also found in two SHKPs of Ph. bodies, we also considered the cell signaling potential of polycephalum, where a phytochrome is known to mediate P. aurantium in its own right. Similar to most protists, P. aur- light-induced sporulation (Starostzik and Marwan 1995). antium amoebae need to sense a range of external stimuli in order to find prey, evade predators, and adapt to environ- mental change. To assess its sensory potential, we investi- Cyclic Nucleotide Signaling gated the presence of well-known categories of cell Many external stimuli exert their effect by modifying intracel- signaling genes. A more detailed description of such genes lular levels of the cyclic nucleotides (cNMPs) cAMP and cGMP. is provided in supplementary data set S1, pages 20–30, By binding to the conserved cyclic nucleotide binding domains Supplementary Material online. of protein kinases, ion channels, and other effector proteins, both molecules control a broad range of cellular responses. G-Protein Coupled Receptors cAMP is particularly important in the life cycle of D.discoideum Transmembrane receptors that interact with heterotrimeric where it acts both as an extracellular chemoattractant and as G-proteins are the most common sensors for external signals an intracellular messenger to regulate aggregation, morpho- in eukaryotes. They are subdivided into six families with family genesis, spore and stalk cell differentiation, and spore dor- 4 only being found in fungi. Protostelium aurantium has only mancy. In this organism, cAMP and cGMP are synthesized 17 G-protein coupled receptors (GPCRs), mostly belonging to by three adenylate- and two guanylate cyclases, respectively, the family 1 rhodopsin-like receptors, and completely lack the intracellularly detected by five cNMP binding proteins and family 3 metabotropic glutamate-like receptors (supplemen- hydrolyzed by seven cNMP phosphodiesterases (Saran et al. tary data set S1, fig. S12, Supplementary Material online). 2002; Bader et al. 2007). Query of the P. aurantium proteome 602 Genome Biol. Evol. 10(2):591–606 doi:10.1093/gbe/evy011 Advance Access publication January 25, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/2/591/4824916 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Multiple Roots of Fruiting Body Formation in Amoebozoa GBE Table 5 Cell Signaling Proteins in Amoebozoa Genomes Category Protostelium aurantium Dictyostelium discoideum Physarum polycephalum Acanthamoeba castellanii G-protein coupled receptors 17 55 146 35 Heterotrimeric G-proteins Alpha 9 12 26 6 Beta 1 1 1 n.d. Gamma 1 1 1 n.d. Histidine kinases/phosphatases 71 16 51 48 Cyclic nucleotide signaling Adenylate/guanylate cyclases 52 5 64 67 cNMP binding domains 27 5 28 7 cNMP phosphodiesterases 16 7 11 10 Protein kinases All (S/T, S/T/Y, Y) 827 295 447 377 Sensor tyrosine kinases (Y) 167 0 4 21 SH2 domain proteins 85 15 18 48 revealed a much larger repertoire of cyclases, binding pro- higher in P. aurantium than in the other Amoebozoa (supple- teins, and phosphodiesterases. mentary data set S1, fig. S15, Supplementary Material online). We detected 52 nucleotidyl cyclases in the P. aurantium genome (fig. 5), which is somewhat less than the 64 and 67 Serine/Threonine and Tyrosine Protein Kinases cyclases detected in the Ph. polycephalum and A. castellanii genomes, respectively (table 5). However, 66 of the A. castel- Protein kinases that phosphorylate other proteins on either lanii cyclases resulted from extensive duplication of a single serine/threonine (S/T) or tyrosine residues (Y) or both (S/T/Y) gene that harbors a cyclase domain flanked by two protein represent the major group of intracellular signal processing kinase domains (identifier ELR11792 in fig. 5), whereas the intermediates in eukaryotes with 518 members in humans remaining cyclase is a homolog of D. discoideum AcrA. The (Hanks 2003). Protostelium aurantium has no less than 827 kinase-flanked cyclases also represent about half of the Ph. proteins with S/T, Y, or S/T/Y kinase domains, vastly surpassing polycephalum cyclases, but are not present in P. aurantium. the other publically available amoebozoan genomes, as well Instead, P. aurantium has many mammalian-type cyclases as humans (table 5). Eukaryotes have in general many S/T or S/ with two cyclase domains and two sets of six transmembrane T/Y kinases, but the Y specific kinases were previously consid- domains. These cyclases are usually regulated by heterotri- ered to be present only in animals (Lim and Pawson 2010). meric G-proteins. Dictyostelium discoideum ACA and GCA Here, particularly the receptor tyrosine kinases play crucial also belong to this category. There are five homologs of the developmental roles as sensors for secreted and exposed pep- soluble D. discoideum guanylate cyclase SGC with two cyclase tides that act as growth factors, controlling cell division, or and one AAA-ATPase domain, which is implicated in chemo- differentiation inducing signals that control cell-type speciali- taxis (Saran et al. 2002). Although there are no obvious AcrA zation (McDonell et al. 2015). More recent sequencing of or ACG representatives, there is a large clade of 23 cyclases protozoan genomes revealed that tyrosine kinases with and with mostly two transmembrane domains and a single cyclase without intrinsic receptor domains are more widespread domain. These enzymes have closest homologs in Excavates (Manning et al. 2008; Suga et al. 2012; Clarke et al. 2013; and Prokaryotes with a similar domain configuration. The two Schaap et al. 2016). Around 167 tyrosine kinases were transmembrane domains may, as is the case for ACG (Saran detected in P. aurantium (supplementary data set S1, fig. et al. 2002), provide the cyclases with an external sensor S16, Supplementary Material online). Most are likely receptor domain. tyrosine kinases with either protein–protein interaction or With a total of 27 cyclic nucleotide binding proteins (cNBPs) polysaccharide binding domains in their extracellular regions, (supplementary data set S1, fig. S14, Supplementary Material suggesting roles in cell–cell recognition or adhesion. The phos- online), P. aurantium by far surpasses the 5 and 7 cNBPs of phorylated tyrosines in target proteins typically act as binding Dictyostelium and A. castellanii, respectively (table 5). Only Ph. sites for SH2 domains, causing proteins with SH2 domains to polycephalum has one more cNBP, but has less variety in ad- directly interact with the target protein. We detected 85 pro- ditional functional domains, which are the likely targets for teins with SH2 domains in the P. aurantium genome, also a 2- regulation by either cAMP or cGMP (Schaap et al. 2016). The to 5-fold increase in numbers compared with the other number of cyclic nucleotide phosphodiesterases (PDEs) is also Amoebozoa (table 5). Genome Biol. Evol. 10(2):591–606 doi:10.1093/gbe/evy011 Advance Access publication January 25, 2018 603 Downloaded from https://academic.oup.com/gbe/article-abstract/10/2/591/4824916 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Hillmann et al. GBE regulate the activity of the intracellular cAMP phosphodiester- Conclusions ase RegA and thereby the activity of PKA (Loomis 2014). All We sequenced the genomes of Protostelium aurantium and amoebae genomes also contain several to many adenylate Protostelium mycophagum as the first representatives of the cyclases for cAMP production to activate PKA (Clarke et al. morphologically similar but genetically diverse protosteliids, 2013; Schaap et al. 2016). In Dictyostelium,cAMP acting on which are characterized by forming a fruiting structure from PKA critically regulates the transition from growth to devel- a single amoeba consisting of a single spore or few spores on opment, the encapsulation of spore and stalk cells and the an acellular stalk. dormancy of spores (Loomis 2014). In both Dictyostelia, which With a size of 38 Mb and 17,000 genes, the P. aurantium have retained the unicellular survival strategy of encystation, genome is similar to that of A. castellanii (42 Mb, 15,000 and in A. castellanii, cAMP actingonPKA also mediate stress- genes), markedly smaller than the Ph. polycephalum genome induced encystation, with RegA antagonizing this process and (189 Mb, 29,000 genes) and larger than the Dictyostelium favoring the trophozoite stage (Du et al. 2014; Kawabe et al. genomes, which range from 34 Mb, 13,000 genes (D. discoi- 2015). Since similar to Dictyostelium (Brenner 1978), cAMP deum) to 23 Mb and 10,000 genes (D. lacteum). Although Ph. levels were also found to increase during P. aurantium devel- polycephalum’s large genome may reflect that this organism opment into fruiting bodies (supplementary data set S1, fig. displays many alternative morphologies and life cycle stages S9, Supplementary Material online), it is likely that this is one (amoeba, flagellate, cyst, sporulating syncytium, or sclerotic of the core processes that is conserved between Amoebozoa, syncytium), it is quite remarkable that the dictyosteliids with to mediate their transition into a walled dormant stage, when their complex multicellular life cycle and alternative abilities to experiencing stress. form either haploid or zygotic cysts have the smallest Another conclusion of our analyses is that despite its genomes of currently sequenced amoebozoa. Apparently, simple life cycle compared with Dictyostelium and Ph. pol- the multicellular life style did not require numerically more ycephalum, and having a five times smaller genome than genes. This is in agreement with analyses of the evolution Ph. polycephalum, P. aurantium has an extremely large of multicellularity in other eukaryote lineages (Niklas and repertoire of proteins for the detection and processing of Newman 2013; Nguyen et al. 2017). It is even conceivable external stimuli. This implies that P. aurantium has many that social amoebae were freed from an unknown selection more interactions with other organisms and/or with mem- pressure due to the evolution of their multicellularity. Thus, bers of its own species than is currently being realized. It is they might have been able to jettison surplus genes associated also remarkable that the multicellular Dictyostelium with with signaling cascades and reception. its complex life cycle has both a smaller number and less Because the single-celled P. aurantium fruiting body variety in its signal detection and processing proteins than might be considered as a prototype for the multicellular the unicellular Amoebozoa. Could this mean that the Dictyostelium fruiting body, we had expected that a signif- Dictyostelia evolved multicellularity, because they were icant number of Dictyostelium developmental genes origi- less adaptable than other Amoebozoa? Comparisons be- nated in the LCA of both lineages as was shown for tween genomes of related uni- and multicellular organisms developmental genes in Metazoa (Sebe-Pedros et al. in other eukaryote divisions might reveal whether this is a 2013). However, contrary to this expectation, the conserva- general trend. tion of developmentally essential Dictyostelium genes in Protostelium spp. is limited and actually not higher than for the evolutionary more distant A. castellanii. Our data Supplementary Material and the recent finding of a sporulating Acanthamoeba spe- Supplementary data are available at Genome Biology and cies (Tice, Shadwick, et al. 2016), makes it conceivable that Evolution online. the entire genus Acanthamoeba could comprise at least the genomic capacity for protosteloid fruiting. It is well possible that the lack of apparent conservation in Dictyostelium dis- Acknowledgments coideum could be due to the evolutionary distance between these taxa and that Dictyostelia have invented their own This study was supported in part by the Leibniz association toolbox for multicellularity. and a grant of the European Social Fund ESF—“Europe for Using RNAseq data from discernable time points during Thuringia” (to F.H.). S.N. was supported by the DFG-funded development, we were able to dissect early and late events excellence graduate school Jena School of Microbial in fruiting body formation. The same processes might be at Communication (JSMC). We thank Ivonne Go ¨ rlich and work in many other Amoebozoa, even if not true orthologous Cornelia Luge for their skillful laboratory assistance and but analogous proteins are being utilized. Emanuel Barth for his assistance in file conversion and data All free-living amoeba sequenced thus far do show a large processing. We also thank the Cologne Center for Genomics repertoire of sensor histidine kinases, which in Dictyostelium for sequencing data on P. aurantium. 604 Genome Biol. 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Abstract

Establishment of multicellularity represents a major transition in eukaryote evolution. A subgroup of Amoebozoa, the dictyos- teliids, has evolved a relatively simple aggregative multicellular stage resulting in a fruiting body supported by a stalk. Protosteloid amoeba, which are scattered throughout the amoebozoan tree, differ by producing only one or few single stalked spores. Thus, one obvious difference in the developmental cycle of protosteliids and dictyosteliids seems to be the establishment of multi- cellularity. To separate spore development from multicellular interactions, we compared the genome and transcriptome of a Protostelium species (Protostelium aurantium var. fungivorum) with those of social and solitary members of the Amoebozoa. During fruiting body formation nearly 4,000 genes, corresponding to specific pathways required for differentiation processes, are upregulated. A comparison with genes involved in the development of dictyosteliids revealed conservation of >500 genes, but most of them are also present in Acanthamoeba castellanii for which fruiting bodies have not been documented. Moreover, expression regulation of those genes differs between P. aurantium and Dictyostelium discoideum. Within Amoebozoa differen- tiation to fruiting bodies is common, but our current genome analysis suggests that protosteliids and dictyosteliids used different routes to achieve this. Most remarkable is both the large repertoire and diversity between species in genes that mediate envi- ronmental sensing and signal processing. This likely reflects an immense adaptability of the single cell stage to varying environ- mental conditions. We surmise that this signaling repertoire provided sufficient building blocks to accommodate the relatively simple demands for cell–cell communication in the early multicellular forms. Key words: multicellular development, transcriptome, Protostelium, Dictyostelia, evolution of development, signaling, Amoebozoa. Introduction multicellular life styles may therefore have been relatively Multicellularity was independently acquired in different easy achieved compared with, for example, the rare event branches of eukaryotes. This transition from uni- to of organelle capture (Marin et al. 2005; Nowack et al. The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Genome Biol. Evol. 10(2):591–606. doi:10.1093/gbe/evy011 Advance Access publication January 25, 2018 591 Downloaded from https://academic.oup.com/gbe/article-abstract/10/2/591/4824916 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Hillmann et al. GBE 2011). It is unlikely that in each case a new way to this tran- 2013) and the syncytial slime mold Physarum polycephalum sition to multicellularity was invented. Rather, building blocks (Schaap et al. 2016). The latter organism alternates between already present in unicellular eukaryotes (and their last com- unicellular amoeboid or amoeboflagellate forms, which can mon ancestor; LCA) were likely recruited to this new task. either individually encyst or, after mating, transform into a During evolution, new layers of complexity (i.e., increase of multinuclear syncytium. This syncytium has the choice of form- the numbers of cell types) masked these building blocks ing a dehydrated but viable sclerotium, or fruiting bodies (fig. 1) (Hedges et al. 2004) so that we have difficulties to define with multiple spores and acellular stalks (Schaap et al. 2016). them. Genomes of the unicellular ancestors of Metazoa We previously investigated the extent to which genes that seem to have been already complex. Exaptations of genes are essential for multicellular development of D. discoideum were later used for the evolution of multicellular development are conserved throughout Dictyostelia and unicellular (Grau-Bove et al. 2017). Interestingly, Capsaspora owczarzaki, Amoebozoa. This study showed that genes involved in intra- which can transit to an aggregative state, employs genes for cellular signal processing were mostly common to both multi- this transition, the orthologs of which establish tissue archi- cellular and unicellular Amoebozoa, but that genes encoding tectures in Metazoa (Sebe-Pedros et al. 2013; Suga et al. membrane or secreted proteins were more unique to 2013). Thus, aggregative multicellularity seems to have the Dictyostelia. This suggested that novel adhesion proteins, sig- same roots as true multicellularity at least in this evolutionary nal, and signal sensors are prime requisites for evolving multi- branch. Moreover, regulatory changes rather than gene inno- cellularity. How these were acquired remains often elusive, vations seemed to have played a major role in the establish- but of the five known nonpeptide signals which induce cell ment of multicellularity in Metazoa (Sebe-Pedros et al. 2016). differentiation in D. discoideum, three genes encoding their There seems to be no significant genomic difference be- biosynthetic enzymes were likely to be acquired by tween the unicellular algae Chlamydomonas and the multi- Dictyostelia through lateral gene transfer (Glo ¨ ckner et al. cellular Volvox species (Umen and Olson 2012). However, 2016). A parallel study retracing genes with conserved upre- extracellular matrix proteins seem to be important for the gulation in multicellular development highlighted many con- establishment of multicellularity in this clade (Prochnik et al. served genes with likely roles in multicellular development 2010). that have not yet been functionally characterized (Schilde Each eukaryote branchor evenclosely relatedcladesseem et al. 2016). to follow its own route to multicellular development and have Multicellular development of Dictyostelia and other organ- established their own toolkit for this purpose (Niklas and isms depends not only on cell-type specialization but also on Newman 2013). coordination of cell movement and/or cell division to generate Multicellular organisms generally go through a unicellular form. This complicates identification of the mechanisms that spore or fertilized egg stage at least once, which then divides uniquely regulate cell differentiation. Comparison of repeatedly to generate the multicellular form. In animals, Dictyostelia with protosteloid amoebae, that lack this compli- plants, and fungi, the dividing cells remain attached to each cation, may uniquely identify the genes involved in differenti- other, representing clonal, multicellular organisms. However, ation processes. in many other multicellular organisms, dispersed cells can come Fruiting bodies of diverse morphologies are widespread together when food runs out to construct a fruiting body with among the Amoebozoa, and the protosteloid type of fruiting aerially borne spores. This type of aggregative multicellularity body with a stalked spore, or very few spores, but always was invented independently by Acrasis spp. in the eukaryote derived from one cell is the most common (fig. 1). A recent division Discoba (Brown, Silberman, et al. 2012; He et al. 2014), study presented a well resolved tree of Amoebozoa with life Fonticula alba in Holomycota (Brown et al. 2009), Guttulinopsis cycle characters mapped onto that tree (Kang et al. 2017). spp. in Rhizaria (Brown, Kolisko, et al. 2012), Sorodiplophrys Species displaying protosteloid fruiting bodies were found to stercorea in Stramenophiles (Tice, Silberman, et al. 2016), be scattered throughout the Amoebozoa phylogeny, but also Sorogena stoianovitchae in Alveolata (Lasek-Nesselquist and occur within the Eumycetozoa which include the Dictyostelia Katz 2001), and Copromyxa protea and Dictyostelium spp. in and Ph. polycephalum. Recently, single cell fruiting bodies Amoebozoa (Baldauf et al. 2000; Brown et al. 2011). were also found to occur among the more distant Among these organisms, the genetic model system Acanthamoebidae (Tice, Shadwick, et al. 2016). It is hypoth- Dictyostelium discoideum and its close relatives display the esized that fruiting body formation was already present in the most complex form of aggregative multicellularity with a freely last common ancestor of all Amoebozoa (Kang et al. 2017), migrating “slug” stage and four to five specialized cell types. Its but an independent origin of fruiting body formation in dif- genome and those of species representative of the five major ferent lineages is also possible (Cavalier-Smith et al. 2016). clades of dictyosteliids have been sequenced (Heidel et al. We here compare the genome content of Protostelium 2011; Glo ¨ ckner 2015; Glo ¨ ckner et al. 2016)and within aurantium var. fungivorum and Protostelium mycophagum Amoebozoa, sequenced genomes are also available for the with those of Dictyostelia, Ph. polycephalum,and strictly unicellular Acanthamoeba castellani (Clarke et al. Acanthamoeba castellani. We further analyze the 592 Genome Biol. Evol. 10(2):591–606 doi:10.1093/gbe/evy011 Advance Access publication January 25, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/2/591/4824916 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Multiple Roots of Fruiting Body Formation in Amoebozoa GBE FIG.1.—Amoebozoa fruiting bodies. Schematic view of the different “fruiting bodies” in the Amoebozoa. Depicted are only species where a genome sequence is available including the here presented Protostelium spp. genomes. The fruiting bodies are drawn to scale, the inset shows an enlargement of the tiny Protostelium fruiting body. No fruiting body is known for Acanthamoeba castellanii, indicated by a cross. However, other Acanthamoebae do form sporocarps, as was recently discovered for A. pyriformis (Tice, Shadwick, et al. 2016). transcriptome changes that occur in Protostelium aurantium sporocarps (fruiting body of a single cell) to new petri dishes. var. fungivorum (further addressed as P. aurantium) during its Liquid cultures of Protostelium sp. were carried out in petrid- transition from a feeding amoeba into a mature fruiting body. ishes filled with 2 mM PB buffer (pH 6.4) and R. mucilaginosa Our study suggests that fruiting body formation seemed to as the only food source. Cell numbers for both organisms have evolved analogously from the similar toolbox of genes. were determined either using a hemocytometer or an auto- Furthermore, despite their simple life style, Protostelium spp. matic cell counter (Casy TT Cell Counter, OLS Bio, Bremen, already display a large variety of cell signaling genes. Germany). Induction of the Developmental Cycle and Preparation of Materials and Methods Samples for RNA Isolation Species Isolation and Maintenance Protosteloid amoebae were cultivated in liquid cultures with Protosteloid amoebae were maintained as spore suspensions successive additions of R. mucilaginosa as a food source. For in 86% [v/v] glycerol at80 C. The type strain of Protostelium P. aurantium, the number of trophozoites reached confluency mycophaga Olive and Stoianovitch was obtained from the at 48- to 60-h postinoculation. At this time point, the growth Culture Collection of Algae and Protozoa (CCAP, Argyll, medium was aspirated and residual yeast cells were removed United Kingdom) and from the American Type Culture by two consecutive washes with PB. Adherent amoebae were Collection (ATCC, Manassas, VA). Protostelium aurantium scraped from the surface, resuspended in PB and transferred var. fungivorum wasisolatedasdescribed for protosteliids to a wMY-agar surface in 10 ml droplets. To obtain sufficient by Spiegel et al. (2006) with slight modifications. Briefly, material for RNA isolation, cells from 40 10-ml droplets were beechleaves were cut to1cm and centrally placed on the pooled for each time point in one of the three biological surface of buffered wMY agar (0.002 g l malt extract, replicates. Vegetative cells were harvested from plates directly 0.002 g l yeast extract, 2 mM potassium phosphate buffer after plating (timepoint 0 h). Starved cells were harvested after pH 6.4 [PB], and 15 g l agar) in petri dishes. The basidiomy- 1.5 h and nearly completed fruiting bodies were harvested cetous yeast Rhodotorula mucilaginosa DSM70404 was from plates incubated for 8 h. obtained from the Jena Microbial Resource collection and was streaked in a distance of 5 mm to the leaf pieces to pro- Nucleic Acids Isolation and Sequencing mote the outgrowth of fungivorous protosteloid amoeba. Plates were sealed with parafilm and incubated at 22 Cfor Chromosomal DNA was isolated from liquid cultures of pro- 10–14 days with daily microscopic inspections for trophozoite tosteloid amoebae. Adherent cells were washed in phosphate growth or the formation of fruiting bodies. Isolation was buffer and harvested by centrifugation at 200 x g which was achieved by four consecutive transfers of single amoeba or sufficient to deplete nearly all residual yeast cells. Trophozoites Genome Biol. Evol. 10(2):591–606 doi:10.1093/gbe/evy011 Advance Access publication January 25, 2018 593 Downloaded from https://academic.oup.com/gbe/article-abstract/10/2/591/4824916 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Hillmann et al. GBE were lysed in 50 mM Tris–HCl (pH 7.5) with 2% [w/v] of SDS then removed to yield a complete, closed circle mitochondrial and 0.5 mM Na EDTA, followed by a 1-h incubation at 60 C genome. with RNAase A (Sigma–Aldrich, Taufkirchen, Germany) and Proteinase K (Sigma–Aldrich) at final concentrations of 100 RNA Sequencing and 200 mgml , respectively. Further purification steps like Each of the three time points of the RNAseq experiment was extractions with phenol-chloroform and precipitation with covered by three biological replicates. The quality and amount isopropanol were carried out as described (Sambrook and of RNA was initially checked using the Agilent Bioanalyzer Russell 2001). 2100 in combination with a RNA 6000 nano kit (both For the isolation of total RNA, cells were rapidly harvested Agilent technologies). The RIN (RNA integrity number) varies from the agar surface, resuspended in 2 ml of phosphate from 8.4 to 10 with an average of 9.44. Total RNA of 1 mg buffer, and centrifuged for 1 min at 6,000g. Pellets were was used for library preparation using Illumina’s TruSeq RNA shock-frozen in liquid N and stored80 C. RNA was prepared sample preparation kit v2 following the manufacturer’s de- by phenol extraction with subsequent additions of 500 mlof scription which included individual labeling by sequence barc- TRIsure (Bioline, Luckenwalde, Germany) and 200 ml of chloro- odes. The libraries were again quantified and quality checked form. Phase separation was achieved by centrifugation fol- using the Bioanalyzer 2100 and DNA 7500 kit. For sequencing, lowedby anadditionalextractionwith500 ml of chloroform. libraries were pooled, individually labeled, and loaded in two Precipitation was as for DNA. RNA quantity and impurities were lanes of a HiSeq 2500 running in single-end/51 cycle/high out- determined spectrophotometrically. Integrity of the isolated put mode. Data extraction was done in FastQ format using the RNA was checked by agarose gel electrophoresis tool bcl2FastQ v1.8.4 (provided by Illumina). Mapping of these reads to the genome was done with tophat2. The counts per gene were used to calculate differential expression using DNA Sequencing, Assembly, and Mapping DEseq2 (Love et al. 2014) with the Bioconductor package of R. Library preparation was done using Illumina’s TruSeq DNA PCR free library preparation kit following the manufacturer’s Gene Predictions description for 550-bp insert libraries. Library quality check and quantification was done as described earlier. For the annotation of the P. mycophagum genome, we used Sequencing was done in one lane of a HiSeq 2500 running GeneMarkES in self-training mode, which uses no apriori in paired-end/2 101 cycle/rapid mode. The reads derived knowledge on gene structures. For the annotation of the P. from genomic DNA were assembled using abyss-pe with a aurantium genome, we made use of the RNAseq data, which kmer size of 45–65 with step increments of 2. The assembly were mapped to the genome as described earlier. The sizes for the P. aurantium genome (excluding contaminants) mapped reads were used to define 52,801 splice sites. was in a range from 39 (kmer 45) to 68 Mb (kmer 65). The These data were used for the GeneMarkET program to enable kmer 45 assembly was chosen for further improvement after a better gene prediction. examination of contig lengths, coverage, and contribution of bacterial and fungal sequences to the assembly for both spe- ncRNAs cies. Specifically, the kmer45 assembly was the most contig- For prediction of nonprotein-coding RNAs (ncRNAs), we used uous of all assemblies and therefore represented the genomes GORAP (www.rna.uni-jena.de/en/software; last accessed best. The GC content of the protosteliid genomes was January 31, 2018), which searches for 2,474 known ncRNA <50.1% while contaminating DNA from other sources (bac- families from Rfam version 12.1 (Nawrocki et al. 2015). teria and the food fungus) was found to have a higher GC GORAP uses improved homology search strategies based on content. Thus, the GC content was a first indicator to readily sequence and secondary structures and was run with default discriminate between the Protostelium spp. genomes and settings. All predictions passed Rfam thresholds. All results contaminating sequences. Furthermore, all contigs were com- and files in fasta, gff, and stockholm format are available at pared with fungal and bacterial genomes to exclude contam- http://www.rna.uni-jena.de/supplements/protostelium;last inating sequences from further analysis. Here, we were using accessed January 31, 2018. BLAST analysis to identify fungal or bacterial sequences (BLASTn in case of bacteria and BLASTx for fungi). If a contig Orthology and Protein Annotation had a higher similarity to known bacterial or fungal sequences than to any Amoebozoa genome, it was excluded from the We defined the orthology between the two Protostelium analysis. Further gap closure was done with PAGIT (http:// genomes and to the dictyosteliid genomes using OrthoMCL www.sanger.ac.uk/science/tools/pagit; last accessed January (Li et al. 2003). The protein functions were evaluated by 31, 2018) using default values. For assembly of the mitochon- aligning the protein sequences to the protein sequence drial genome, we extended the contig using SSPACE (Boetzer database obtained from NCBI (version from 5/2016) using et al. 2011). The resulting overlap between contig ends was BLAST. Domains were searched using InterproScan 594 Genome Biol. Evol. 10(2):591–606 doi:10.1093/gbe/evy011 Advance Access publication January 25, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/2/591/4824916 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Multiple Roots of Fruiting Body Formation in Amoebozoa GBE FIG.2.—Phylogeny and mitochondrial organization. (A) Phylogeny of selected protosteloid amoeba together with selected other amoebozoa based on 18 S RNA sequences. The species of which the genome sequences were analyzed are given in bold letters. Superscript numbers refer to recently renamed 1 2 isolates of Protostelium aurantium with accession numbers FJ766461.1 and FJ766463.1. The alignment was cleaned of ambiguous positions using GBLOCKS (Talavera et al. 2007) reducing the observable distance between species and clades. A discrete Gamma distribution was used to model evolu- tionary rate differences among sites (5 categories [þG, parameter¼ 0.6214]). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Evolutionary analyses were conducted in MEGA6 (Tamura et al. 2013). (B) Mitochondrial synteny between selected Amoebozoa. Genes are drawn as boxes of equal length. Syntenic regions are connected by colored bands. (Jones et al. 2014). The biochemical pathways were analyzed frequencies, and growth was comparably slow, we decided using the KAAS server (Moriya et al. 2007). All programs were to isolate a wild strain by means of food choice selection, that run with default values. is, incubating environmental samples together with Rhodotorula cultures (Materials and Methods). This way we isolated a protosteloid amoeba in sensu Spiegel (Spiegel Phylogenetic Analysis of Adenylate and Guanylate et al. 1994, 2006; Shadwick et al. 2009) from a dead aerial Cyclases beech leaf in the Jena forest. The amoeba revealed striking The output of an Interproscan of the translated Protostelium similarities to the type strain of Protostelium mycophagum aurantium transcripts was scanned for proteins harboring as described previously (Olive and Stoianovitch 1960). Interpro domain IPR001054 for class III adenylate and guany- Phylogenic analysis based on its SSU rRNA (fig. 2A) as well late cyclases. The P. aurantium proteins harboring this domain as diagnostic morphological traits identified the isolate as a were alignedwiththe five Dictyostelium cyclases and a few strain of Protostelium aurantium as recently described by signature Ph. polycephalum and A. castellanii cyclases using Shadwick et al. (2017). Due to its rapid phagocytic feeding Clustal Omega with five combined iterations (Sievers and on the basidiomycetous yeast Rhodotorula mucilaginosa on Higgins 2014). After deletion of segments that did not align solid agar surfaces (supplementary data set S1, fig. S1A, unambiguously, alignments were subjected to Bayesian infer- Supplementary Material online), we coined this new isolate ence for phylogeny reconstruction using a mixed amino acid Protostelium aurantium var. fungivorum. A detailed taxo- model in MrBayes software (Ronquist and Huelsenbeck nomic description of this isolate is available within the sup- 2003). Analyses were run until convergence, and trees were plementary material of this article (supplementary data set rooted at midpoint (Hess and De Moares Russo 2007). For the S1, Description of Protostelium aurantium, Supplementary earliest diverging branches of each major clade, closest homo- Material online). logs were identified by BLASTp of all NCBI nonredundant Most characteristic for its identification as a protosteloid sequences. These protein sequences were aligned and in- amoeba was the abundant formation of globose fruiting bod- cluded in Bayesian inference of the final tree. ies carrying a single spore (fig. 3A–C). The sporocarp (fruiting body of a single cell) culminated on an acellular stalk fixed on the substratum (fig. 3D). As described for P. mycophagum, Results and Discussion asynchronous fruiting body formation occurred on solid sur- The Species faces in the absence of the fungal food source. The majority Protostelium mycophagum, the type strain (Olive and of cells initiated their full developmental cycle 2–4 h following Stoianovitch 1969) was maintained in culture together with the onset of starvation, passing through the formation the fungus Rhodotorula mucilaginosa. As fruiting body for- of immobile discs, cellular condensation, the formation of mation in this P. mycophagum strain occurred at lower the sporocarp, and stalk elevation, with each stage being Genome Biol. Evol. 10(2):591–606 doi:10.1093/gbe/evy011 Advance Access publication January 25, 2018 595 Downloaded from https://academic.oup.com/gbe/article-abstract/10/2/591/4824916 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Hillmann et al. GBE FIG.3.—Unicellular fruiting bodies of Protostelium aurantium. Light micrograph of a stalked fruiting body (A) and scanning electron micrographs of a tilted, but intact fruiting body (B), which will eventually open and liberate single spores (C), and the rigid base of the stalk (D). completed in 10–15 min (supplementary data set S1, fig. S2, P. aurantium lacks the genes rps7 and rpl14, located in the Supplementary Material online). rearranged segment. Comparisons to other Amoebozoa mi- tochondrial genomes show that these additionally lost genes lie in the same region where other ribosomal subunit genes Phylogeny of the Species and Their Genomes were lost in Protostelium spp. (fig. 2B). Thus, loss of such For phylogenetic reconstruction, we sampled 18 S RNA mitochondrial genes seems to be common in the sequences of Amoebozoa representatives from the NCBI Protostelium spp. analyzed. database, aligned them, and performed a maximum likeli- Based on the gene prediction, both nuclear haploid hood analysis (fig. 2A). We rooted our tree with the 18 S genomes are comparable in size and also encode a similar gene from Mus musculus. The tree readily identified our en- number of genes (table 1). For P. aurantium, this number vironmental isolate as a member of the Protostelium auran- was further supported by RNAseq data (supplementary data tium clade and revealed its distant relationship to Dictyostelia set S1, fig. S4, Supplementary Material online) using and Physarum. Despite their phylogenetic distance within the GeneMarkET (Borodovsky and Lomsadze 2011). A distin- Amoebozoa, all three species share the ability to construct a guishing hallmark of both genomes is the high number of fruiting body. As gene expression during fruiting body forma- introns per gene (supplementary data set S1, fig. S5, tion of dictyosteliids has been particularly well studied and Supplementary Material online). The median intron size is P. mycophagum did not sporulate readily, we focused mainly very small with 40–50 bases, but larger than the smallest in- on the P. aurantium genome andexpressiondata and its tron sizes reported so far (Slabodnick et al. 2017). Overall, the comparison to Dictyostelia. Protostelium spp. genomes analyzed here harbor circa 30% Based on our sequencing approach, we assembled the more genes than Dictyostelia. complete mitochondrial genomes and obtained draft nuclear In line with the extensive use of introns, we found not only genomes for both Protostelium species. The mitochondrial all small nuclear RNAs (snRNAs) of the major spliceosome but genomes are comparable with those in dictyosteliids with re- also the minor spliceosome to be present in both species. In spect to coding capacity and size. The order of genes differs total, a similar number of noncoding RNAs were annotated between the two species only for one segment of genes and a for P. mycophagum (590) and P. aurantium (525) (supplemen- few tRNA locus rearrangements (fig. 2B). Furthermore, tary data set S1, table S1, Supplementary Material online). 596 Genome Biol. Evol. 10(2):591–606 doi:10.1093/gbe/evy011 Advance Access publication January 25, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/2/591/4824916 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Multiple Roots of Fruiting Body Formation in Amoebozoa GBE Table 1 points (vegetative cells at 0 h, starvation and early differenti- Genomic Features of the Two Protostelium Species ation at 1.5 h, and the formation of prespores and mature Protostelium Protostelium fruiting bodies at 8 h). Although sporocarp formation was mycophagum aurantium asynchronous among the population, these time points Genome size in Mb (haploid) 39.7 38.3 were discernible via the predominant morphology (supple- Scaffolds 2,578 822 mentary data set S1, figs. S2 and S6A, Supplementary Mean GC content 42.2 47.9 Material online). We first assessed which genes are potentially Palindrome size (kb) 13.4 16.3 differentially expressed between any of those time points. The Mitochondrium (kb) 48.6 44.5 biological replicates showed little variation (supplementary Protein coding genes 15,691 17,172 data set S1, fig. S6B and C, Supplementary Material online). Mean size CDS/AA 1,386/462 1,512/504 The DEseq2 analysis (Love et al. 2014) implemented in the tRNAs 426 405 BioConductor R package revealed that there are 7,787 genes Introns 126,081 145,637 significantly downregulated and 3,980 genes significantly Mean intron length 83.2 63.4 upregulated. An example of an upregulated gene is given in Median intron length 50 46 supplementary data set S1, figure S7, Supplementary Material Introns/gene 8 8.5 Intron space (Mb) 10.5 9.2 online. A significance threshold of 0.001 reduces these num- CDS space (Mb) 21.8 26 bers to 6,579 and 2,685, respectively. We next analyzed which potential functions are encoded in this smaller gene set. This analysis revealed extensive reprogramming of sev- The amount of mobile elements in P. aurantium is esti- eral functions during the developmental cycle (fig. 4). A mated at 0.13% of the genome. Although several re- gene ontology (GO) term enrichment analysis of all upregu- verse transcriptase domains of both long terminal repeat lated genes using Gotermfinder (Boyle et al. 2004) showed (LTR) and non-LTR retrotransposons could be determined, that in the GO category “molecular function” ATP and GTP the corresponding elements are highly fragmented and binding proteins are strongly overrepresented (supplemen- mostly present in single copy. Only four LTR retrotranspo- tary data set S1, fig. S8, Supplementary Material online). sons could be analyzed in some detail, and they belong to However, no GO terms in the category “biological process” the Skipper and DGLT-A families of Ty3/gypsy type of LTR were significantly enriched. retrotransposons known from the dictyosteliid clade We next divided the genes into three categories (down- (Spaller et al. 2016). regulation, early upregulation, and late upregulation). This only roughly approximates the real transcript dynamics since we had only three distinct developmental stages in Gene Families and Orthology Relationships our time course. The KEGG pathway analysis (Moriya Both Protostelium spp. share among them 6,278 1:1 ortho- et al. 2007) revealed that up- and downregulated genes logs and 1,708 gene families with more than one member in can be part of the same pathway and concerted regulation at least one species. Protostelium mycophagum has 6,264 may affect only part of a pathway (fig. 5 and supplementary genes not shared with P. aurantium, whereas P. aurantium table S1, Supplementary Material online). Processes related has 5,942 such orphan genes, meaning that they were also to RNA generation, fatty acid synthesis, RNA and protein not found in other species. These numbers are somewhat transport, and oxidative phosphorylation were uniformly comparable to the 4,156 orphan genes reported for A. cas- downregulated. This downregulation is a likely hallmark of tellanii (Clarke et al. 2013). Interestingly, several of the largest the fact that the cells are experiencing starvation. Genes gene families in both species encode proteins with Leucine- that were upregulated early tended to be involved in repli- rich repeats, which are often involved in protein–protein inter- cation and repair, dNTP synthesis, and fatty acid degrada- actions. Other large gene families comprise Ankyrin repeat tion. These early events likely reflect the fact that the cell containing proteins, nucleotidyl cyclases, or kinases (supple- tries to recruit energy from fatty acids to complete the de- mentary data set S1, table S2, Supplementary Material velopmental cycle. In a later stage, P. aurantium devoted online). resources to the production of membrane components like glycerophospho- and sphingolipids, as well as chitin as Gene Regulation during Fruiting Body Formation a cell wall polysaccharide, indicative of the final stages of Upon starvation on a solid substratum, P. aurantium cells in- spore formation (fig. 5). dividually differentiate into a spore supported by an acellular We also found that signal transduction cascades that in- stalk. As P. aurantium displayed a higher frequency and duce cytoskeleton reorganization, survival, adhesion, and mi- abundancy of these structures relative to P. mycophagum, gration were upregulated early and this upregulation we chose P. aurantium to analyze the transcriptional changes increased further in differentiation. The second messenger during this differentiation process by RNAseq over three time cAMP seems to play a pivotal role here since its production Genome Biol. Evol. 10(2):591–606 doi:10.1093/gbe/evy011 Advance Access publication January 25, 2018 597 Downloaded from https://academic.oup.com/gbe/article-abstract/10/2/591/4824916 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Hillmann et al. GBE FIG.4.—Transcriptional reprogramming during the developmental cycle of Protostelium aurantium. Heat maps indicate higher (blue) and lower (red) expression of genes from major developmental and metabolic pathways when comparing early (0 min vs. 1.5 h, top line) or late stages (0 vs. 8 h, middle line, or 1.5 h vs. 8 h, bottom line) of development. Genes from each cluster are grouped in three temporal categories representing an upregulated expression either early, late, or throughout the developmental cycle. Heat maps illustrate the log expression ratio based on the mean RPKM values from three biological replicates for each time point. A more detailed table including gene accession numbers, numeric expression ratios, KEGG orthologies, and predicted protein functions are available as supplementary table S1, Supplementary Material online. started early in development (supplementary data set S1, fig. involved in common encystation processes in Amoebozoa S9, Supplementary Material online). Which of the numerous (Kawabe et al. 2015) is upregulated, presumably via cAMP. cyclase encoded in the genome of P. aurantium is responsible Throughout the differentiation process protein modification for this production or whether several cyclases are acting in a pathways (N-glycan biosynthesis) were enhanced. concerted way to achieve this, is currently unclear. However, Interestingly, N-Glycan biosynthesis plays also a role in D. dis- one of the cyclases (PROFUN_08491) is particularly upregu- coideum differentiation (Li et al. 2015). In P. aurantium,this lated during development (fig. 6) and therefore might be re- pathway may be involved in production of components of the sponsible for the early cAMP level rise. PKA activity, which is cell wall and the acellular stalk. 598 Genome Biol. Evol. 10(2):591–606 doi:10.1093/gbe/evy011 Advance Access publication January 25, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/2/591/4824916 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Multiple Roots of Fruiting Body Formation in Amoebozoa GBE FIG.5.—Developmentally regulated pathways in Protostelium aurantium based on RNAseq analysis. Red indicates functions represented by genes that are downregulated upon starvation, green and blue highlight functions that are upregulated in early and late development, respectively. Presumed Calcium upregulation is depicted with a yellow arrow. Functions associated with the nucleus are listed in a circle in the right lower corner, and those with the ribosome in the upper left corner. The cytoskeleton and exocytosis are influenced by the depicted pathways, but the impact of this is not clear. D. discoideum chemotaxis and stalk formation (Kawata 2011) Comparison between Social Amoebae and P. aurantium is upregulated in D. discoideum,whereas its P. aurantium Developmental Genes ortholog (PROFUN_03920) is slightly downregulated. Social amoebae have 60% orthologs between them as a Overall, not more than 75 genes were consistently identi- comparative study showed (Heidel et al. 2011). Since the evo- fied as important for development in D. discoideum by being lutionary distance between dictyosteliids and the protosteliids upregulated and essential during the developmental process analyzed here is even higher than within the dictyosteliids it is (Glo ¨ ckner et al. 2016; Schilde et al. 2016). However, for most not surprising that only 37% of the genes in P. aurantium of these, orthologous genes were found in the genomes of P. show similarities to genes in D. discoideum, as detected by aurantium (57), A. castellanii (47), and Ph. polycephalum (57) BLAST with a bit score threshold of 200. In two previous (table 2). It is therefore conceivable that these genes could studies, we have defined two sets of dictyosteliid genes which belong to the basic toolkit for the evolution of all differenti- are involved in the developmental cycle of social amoebae ated forms of development among the Amoebozoa. (Glo ¨ ckner et al. 2016; Schilde et al. 2016). The set of devel- The set of in total 233 D. discoideum DevUp and DevEs opmentally essential (DevEs) genes contains 374 genes that geneswithnohitsineither the P. aurantium, Ph. polycepha- upon knock-out cause a developmental defect (Glo¨ckner lum,and A. castellanii genomes is enriched in several GO et al. 2016). The set of developmentally upregulated terms, for example, “extracellular matrix organization” and (DevUp) genes contains 794 genes that are consistently de- “multicellular organismal process” (supplementary data set velopmentally upregulated across the Dictyostelium taxon S1, fig. S10, Supplementary Material online). We next groups (Schilde et al. 2016). In total, these two sets comprise searched for genes in the 1,168 gene set, which are shared 1,168 genes (table 2 and supplementary data set S1, fig. S11, exclusively between D. discoideum and P. aurantium and are Supplementary Material online). A higher percentage of not present in A. castellanii and Ph. polycephalum. This search DevEs than DevUp genes has identifiable counterparts in revealed only 13 genes with a range of putative functions the P. aurantium genome (76% vs. 48%). However, of the (supplementary data set S1, table S3, Supplementary in total 669 D. discoideum proteins with homologs in P. aur- antium, 172 have the same homolog, indicating gene family Material online). expansions in D. discoideum. Strikingly, the directionality of Three of the four genes of Dictyostelia (chlA, dgcA, dokA, developmental regulation of P. aurantium counterparts is of- iptA), which are likely derived from horizontal gene transfer ten opposite (table 2). For example, statA with a major role in from bacteria (Glo ¨ ckner et al. 2016) and which either sense Genome Biol. Evol. 10(2):591–606 doi:10.1093/gbe/evy011 Advance Access publication January 25, 2018 599 Downloaded from https://academic.oup.com/gbe/article-abstract/10/2/591/4824916 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Hillmann et al. GBE FIG.6.—Phylogeny of adenylate and guanylate cyclases. The phylogenetic tree was calculated with Bayesian inference (see Materials and Methods).The protein identifiers are color coded to reflect species names as indicated in the figure, and are annotated with the functional domain architecture of the proteins as determined by SMART (Schultz et al. 1998). PFAM domains are represented as colored to black graded rectangles. Posterior probabilities (BIPP) of tree nodes are indicated by colored dots. The heatmap indicates relative expression levels at 1.5 h (starvation) and 8 h (fruiting body formation) compared with the vegetative state. The cartoon illustrates the predominant cell types at these time points (see supplementary fig. S7, Supplementary Material online). (dokA) or synthesize developmental signals, are not present in Only two TFs which were previously identified in Dictyostelia the P. aurantium genome. However, two histidine kinases were slightly conserved only in P. aurantium (crtf and stkA) (PROFUN_01260 and PROFUN_15316) have the same do- but not found in Ph. polycephalum or A. castellanii. This indi- main structure as dokA (supplementary data set S1, fig. cates a common root for developmental regulation for only S13, Supplementary Material online) and therefore might en- few if any TFs and a likely loss in some lineages. Further ge- code similar functions even if they are not orthologs. nome sequences in the Amoebozoa are needed to trace the We also examined, whether transcription factors (TFs) im- origin of these TFs to the LCA. portant for the development in Dictyostelia are conserved in Overall, and somewhat contrary to expectations based on Protostelium spp. (table 3). Of a total of twelve TFs, ortholo- morphological similarities, the conservation of developmental gous proteins in P. aurantium were found for only seven and genes between Dictyostelia and P. aurantium is low and pos- none of them were highly expressed during development. sible not more than with other unicellular amoebozoan 600 Genome Biol. Evol. 10(2):591–606 doi:10.1093/gbe/evy011 Advance Access publication January 25, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/2/591/4824916 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Multiple Roots of Fruiting Body Formation in Amoebozoa GBE Table 2 Gene Regulation Differences between Social Amoebae, Protostelium aurantium, and Other Amoebozoa Gene Set Developmental Expression in Physarum Acanthamoeba P. aurantium Dictyostelium sp. Protostelium aurantium polycephalum castellanii Development Genes with %of Multiple Up Down Neutral Genes with Genes with Similarity Dictyostelium Hits Similarity Similarity (score>200) sp. Genes (score>200) (score>200) Upregulated during 783 377 48 96 128 199 50 370 310 development Essential for development 309 234 76 62 55 138 41 233 193 Upregulated and essential 75 57 78 13 21 21 15 57 47 All 1,167 668 57 171 204 358 106 660 550 NOTE.—About 1,167 genes of D. discoideum were identified previously as being either developmentally upregulated (Schilde et al. 2016), developmentally essential (Glo¨ ckner et al. 2016), or both (commonly identified in both studies). These genes were searched in the genomes of P. aurantium, Ph. polycephalum,and A. castellanii. To reduce spurious hits a BLAST similarity bit score of 200 was applied when comparing the encoded amino acid sequences. The column “Multiple hits” counts the occasions where different D. discoideum genes yielded the same P. aurantium hit. Table 3 Developmental TFs from Dictyostelium discoideum and Presence of Orthologous Proteins in Protostelium aurantium Gene ID TF Best Hit in P. aurantium Log Change in Comment Expression at 1.5 h, 8.5 h DDB_G0278077 crtf PROFUN_06017 0.42, 0.45 Expression of aggregation genes; Dictyostelia specific DDB_G0281387 srfA PROFUN_09032 1.13, n.d. Spore differentiation; MADS box; only box similar DDB_G0277589 gtaC PROFUN_10218 0.41, n.d. pstB cell sorting and basal disc formation; GATA zinc finger; also in Acanthamoeba DDB_G0278971 dimA n.d. – Development; bZIP transcription factor DDB_G0291372 dimB n.d. – Development; bZIP transcription factor DDB_G0279529 bzpF PROFUN_06600 n.d., 0.58 Spore viability; bZIP transcription factor; common TF DDB_G0281381 dstA PROFUN_03920 n.d., 0.29 Culmination, cudA expression; STAT transcription PROFUN_04291 n.d., n.d. factor; also in A. castellanii PROFUN_12834 0.64, n.d. PROFUN_01113 n.d., n.d. DDB_G0277147 stkA PROFUN_04849 n.d., n.d. Spore formation; GATA zinc finger; dictyosteliid specific PROFUN_12502 n.d., n.d. motif C terminal of zinc finger conserved DDB_G0284465 cudA PROFUN_14255 n.d., 0.82 Many prespore genes and for culmination; also in PROFUN_08418 n.d., n.d. A. castellanii DDB_G0281563 mybC n.d. – Culmination; homology to myb domains only DDB_G0275445 mybB n.d. – ACA expression; homology to myb domains only DDB_G0281969 mybE n.d. – Basal disc formation; homology to myb domains only NOTE.—n.d., orthologous protein or gene expression was not detected. genomes. Evolution of fruiting body formation could thus focused entirely on the morphogenetic process that protoste- have well been independent in the two species. However, liids and dictyosteliids do have in common, the differentiation analysis of further, phylogenetically more distant, protosteloid of spore(s), supported by a stalk. Although the stalk differs amoeba will help to resolve this question. from that of most Dictyostelia by not containing any cells, there is also a Dictyostelium clade, the acytosteliids, which lack cells in their stalk, but nevertheless have the same Spore and Stalk Genes stalk-specifying genes as the species with stalk cells The analysis described earlier comprised all known develop- (Urushihara et al. 2015). mentally essential and conserved developmentally upregu- About 92 genes with roles in spore and stalk formation lated genes, but many of these genes are typically involved have been identified in Dictyostelium discoideum (Glo¨ckner in multicellular morphogenesis which has never been ob- et al. 2016) and we investigated whether orthologs of these served for Protostelium sp. In the following analysis, we genes were also present in P. aurantium, Ph. polycephalum, Genome Biol. Evol. 10(2):591–606 doi:10.1093/gbe/evy011 Advance Access publication January 25, 2018 601 Downloaded from https://academic.oup.com/gbe/article-abstract/10/2/591/4824916 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Hillmann et al. GBE Table 4 Conserved Dictyostelium discoideum Spore and Stalk Genes in Three Amoebozoa Required for Dictyostelium discoideum Protostelium aurantium Physarum polycephalum Acanthamoeba castellani Sporulation 72 26 40 32 Stalk formation 20 2 6 2 and A. castellanii. Genes in the three species were validated as This compares rather poorly with Dictyostelium, A. castellanii, D. discoideum orthologs when 1) the D. discoideum and spe- and Ph. polycephalum, which have 55, 35, and 146 GPCRs, cies homologs were each other’s best bidirectional BLASTp hit respectively (table 5). Protostelium aurantium does have 9 (BBH), 2) they had the same or very similar functional domain heterotrimeric G-proteins, more similar to Dictyostelium, architectures, and 3) the species genes clustered with the which has 12 (supplementary data set S1, fig. S12, dictyosteliid orthologs in phylogenetic trees that also included Supplementary Material online). other close homologs (see supplementary data set S2, Supplementary Material online). All D. discoideum spore Sensor Histidine Kinases/Phosphatases and stalk genes and their best P. aurantium, Ph. polycepha- lum, and A. castellanii hits are listed in supplementary data set Histidine kinases/phosphatases with an attached sensor do- S1, table S4, Supplementary Material online, with likely ortho- main are used for detecting and processing a broad range of logs indicated in bold text. A summary of the analysis is listed chemical and physical stimuli in both pro- and eukaryotes. The in table 4. stimulus activates either phosphorylation or dephoshorylation Of the 92 Dictyostelium genes, 28 had orthologs in of a conserved histidine residue in the histidine kinase domain, P. aurantium, but a much larger number (46) had orthologs which respectively triggers forward or reverse histidine– in Ph. polycephalum. Even the nonsporulating A. castellanii aspartate–histidine relay of the phosphate to/from an aspar- has slightly more (34) spore and stalk genes in common with tate in a receiver domain. This then results in activation or Dictyostelium. Largely the same genes are shared between all inactivation of an attached effector, often an enzyme or TF four species, which suggests that they have a common role (Zschiedrich et al. 2016). Protostelium aurantium appears independent of sporulation. Their role in normal spore and to make up for its low number of GPCRs by having an ex- stalk formation in Dictyostelium may either result from a dic- tremely large number of 71 sensor histidine kinases/phospha- tyosteliid specific recruitment into such a role, or reflect a tases (SHKPs) (supplementary data set S1, fig. S13, pleiotropic effect of their loss on spore or stalk formation. Supplementary Material online). This is over four times more than D. discoideum andalsoexceeding Ph. polycephalum and A. castellanii (table 5). The P. aurantium SHKPs contain a va- Cell Signaling riety of sensor domains, among which is also a phytochrome Without undue emphasis on its development into fruiting domain. This domain was also found in two SHKPs of Ph. bodies, we also considered the cell signaling potential of polycephalum, where a phytochrome is known to mediate P. aurantium in its own right. Similar to most protists, P. aur- light-induced sporulation (Starostzik and Marwan 1995). antium amoebae need to sense a range of external stimuli in order to find prey, evade predators, and adapt to environ- mental change. To assess its sensory potential, we investi- Cyclic Nucleotide Signaling gated the presence of well-known categories of cell Many external stimuli exert their effect by modifying intracel- signaling genes. A more detailed description of such genes lular levels of the cyclic nucleotides (cNMPs) cAMP and cGMP. is provided in supplementary data set S1, pages 20–30, By binding to the conserved cyclic nucleotide binding domains Supplementary Material online. of protein kinases, ion channels, and other effector proteins, both molecules control a broad range of cellular responses. G-Protein Coupled Receptors cAMP is particularly important in the life cycle of D.discoideum Transmembrane receptors that interact with heterotrimeric where it acts both as an extracellular chemoattractant and as G-proteins are the most common sensors for external signals an intracellular messenger to regulate aggregation, morpho- in eukaryotes. They are subdivided into six families with family genesis, spore and stalk cell differentiation, and spore dor- 4 only being found in fungi. Protostelium aurantium has only mancy. In this organism, cAMP and cGMP are synthesized 17 G-protein coupled receptors (GPCRs), mostly belonging to by three adenylate- and two guanylate cyclases, respectively, the family 1 rhodopsin-like receptors, and completely lack the intracellularly detected by five cNMP binding proteins and family 3 metabotropic glutamate-like receptors (supplemen- hydrolyzed by seven cNMP phosphodiesterases (Saran et al. tary data set S1, fig. S12, Supplementary Material online). 2002; Bader et al. 2007). Query of the P. aurantium proteome 602 Genome Biol. Evol. 10(2):591–606 doi:10.1093/gbe/evy011 Advance Access publication January 25, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/2/591/4824916 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Multiple Roots of Fruiting Body Formation in Amoebozoa GBE Table 5 Cell Signaling Proteins in Amoebozoa Genomes Category Protostelium aurantium Dictyostelium discoideum Physarum polycephalum Acanthamoeba castellanii G-protein coupled receptors 17 55 146 35 Heterotrimeric G-proteins Alpha 9 12 26 6 Beta 1 1 1 n.d. Gamma 1 1 1 n.d. Histidine kinases/phosphatases 71 16 51 48 Cyclic nucleotide signaling Adenylate/guanylate cyclases 52 5 64 67 cNMP binding domains 27 5 28 7 cNMP phosphodiesterases 16 7 11 10 Protein kinases All (S/T, S/T/Y, Y) 827 295 447 377 Sensor tyrosine kinases (Y) 167 0 4 21 SH2 domain proteins 85 15 18 48 revealed a much larger repertoire of cyclases, binding pro- higher in P. aurantium than in the other Amoebozoa (supple- teins, and phosphodiesterases. mentary data set S1, fig. S15, Supplementary Material online). We detected 52 nucleotidyl cyclases in the P. aurantium genome (fig. 5), which is somewhat less than the 64 and 67 Serine/Threonine and Tyrosine Protein Kinases cyclases detected in the Ph. polycephalum and A. castellanii genomes, respectively (table 5). However, 66 of the A. castel- Protein kinases that phosphorylate other proteins on either lanii cyclases resulted from extensive duplication of a single serine/threonine (S/T) or tyrosine residues (Y) or both (S/T/Y) gene that harbors a cyclase domain flanked by two protein represent the major group of intracellular signal processing kinase domains (identifier ELR11792 in fig. 5), whereas the intermediates in eukaryotes with 518 members in humans remaining cyclase is a homolog of D. discoideum AcrA. The (Hanks 2003). Protostelium aurantium has no less than 827 kinase-flanked cyclases also represent about half of the Ph. proteins with S/T, Y, or S/T/Y kinase domains, vastly surpassing polycephalum cyclases, but are not present in P. aurantium. the other publically available amoebozoan genomes, as well Instead, P. aurantium has many mammalian-type cyclases as humans (table 5). Eukaryotes have in general many S/T or S/ with two cyclase domains and two sets of six transmembrane T/Y kinases, but the Y specific kinases were previously consid- domains. These cyclases are usually regulated by heterotri- ered to be present only in animals (Lim and Pawson 2010). meric G-proteins. Dictyostelium discoideum ACA and GCA Here, particularly the receptor tyrosine kinases play crucial also belong to this category. There are five homologs of the developmental roles as sensors for secreted and exposed pep- soluble D. discoideum guanylate cyclase SGC with two cyclase tides that act as growth factors, controlling cell division, or and one AAA-ATPase domain, which is implicated in chemo- differentiation inducing signals that control cell-type speciali- taxis (Saran et al. 2002). Although there are no obvious AcrA zation (McDonell et al. 2015). More recent sequencing of or ACG representatives, there is a large clade of 23 cyclases protozoan genomes revealed that tyrosine kinases with and with mostly two transmembrane domains and a single cyclase without intrinsic receptor domains are more widespread domain. These enzymes have closest homologs in Excavates (Manning et al. 2008; Suga et al. 2012; Clarke et al. 2013; and Prokaryotes with a similar domain configuration. The two Schaap et al. 2016). Around 167 tyrosine kinases were transmembrane domains may, as is the case for ACG (Saran detected in P. aurantium (supplementary data set S1, fig. et al. 2002), provide the cyclases with an external sensor S16, Supplementary Material online). Most are likely receptor domain. tyrosine kinases with either protein–protein interaction or With a total of 27 cyclic nucleotide binding proteins (cNBPs) polysaccharide binding domains in their extracellular regions, (supplementary data set S1, fig. S14, Supplementary Material suggesting roles in cell–cell recognition or adhesion. The phos- online), P. aurantium by far surpasses the 5 and 7 cNBPs of phorylated tyrosines in target proteins typically act as binding Dictyostelium and A. castellanii, respectively (table 5). Only Ph. sites for SH2 domains, causing proteins with SH2 domains to polycephalum has one more cNBP, but has less variety in ad- directly interact with the target protein. We detected 85 pro- ditional functional domains, which are the likely targets for teins with SH2 domains in the P. aurantium genome, also a 2- regulation by either cAMP or cGMP (Schaap et al. 2016). The to 5-fold increase in numbers compared with the other number of cyclic nucleotide phosphodiesterases (PDEs) is also Amoebozoa (table 5). Genome Biol. Evol. 10(2):591–606 doi:10.1093/gbe/evy011 Advance Access publication January 25, 2018 603 Downloaded from https://academic.oup.com/gbe/article-abstract/10/2/591/4824916 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Hillmann et al. GBE regulate the activity of the intracellular cAMP phosphodiester- Conclusions ase RegA and thereby the activity of PKA (Loomis 2014). All We sequenced the genomes of Protostelium aurantium and amoebae genomes also contain several to many adenylate Protostelium mycophagum as the first representatives of the cyclases for cAMP production to activate PKA (Clarke et al. morphologically similar but genetically diverse protosteliids, 2013; Schaap et al. 2016). In Dictyostelium,cAMP acting on which are characterized by forming a fruiting structure from PKA critically regulates the transition from growth to devel- a single amoeba consisting of a single spore or few spores on opment, the encapsulation of spore and stalk cells and the an acellular stalk. dormancy of spores (Loomis 2014). In both Dictyostelia, which With a size of 38 Mb and 17,000 genes, the P. aurantium have retained the unicellular survival strategy of encystation, genome is similar to that of A. castellanii (42 Mb, 15,000 and in A. castellanii, cAMP actingonPKA also mediate stress- genes), markedly smaller than the Ph. polycephalum genome induced encystation, with RegA antagonizing this process and (189 Mb, 29,000 genes) and larger than the Dictyostelium favoring the trophozoite stage (Du et al. 2014; Kawabe et al. genomes, which range from 34 Mb, 13,000 genes (D. discoi- 2015). Since similar to Dictyostelium (Brenner 1978), cAMP deum) to 23 Mb and 10,000 genes (D. lacteum). Although Ph. levels were also found to increase during P. aurantium devel- polycephalum’s large genome may reflect that this organism opment into fruiting bodies (supplementary data set S1, fig. displays many alternative morphologies and life cycle stages S9, Supplementary Material online), it is likely that this is one (amoeba, flagellate, cyst, sporulating syncytium, or sclerotic of the core processes that is conserved between Amoebozoa, syncytium), it is quite remarkable that the dictyosteliids with to mediate their transition into a walled dormant stage, when their complex multicellular life cycle and alternative abilities to experiencing stress. form either haploid or zygotic cysts have the smallest Another conclusion of our analyses is that despite its genomes of currently sequenced amoebozoa. Apparently, simple life cycle compared with Dictyostelium and Ph. pol- the multicellular life style did not require numerically more ycephalum, and having a five times smaller genome than genes. This is in agreement with analyses of the evolution Ph. polycephalum, P. aurantium has an extremely large of multicellularity in other eukaryote lineages (Niklas and repertoire of proteins for the detection and processing of Newman 2013; Nguyen et al. 2017). It is even conceivable external stimuli. This implies that P. aurantium has many that social amoebae were freed from an unknown selection more interactions with other organisms and/or with mem- pressure due to the evolution of their multicellularity. Thus, bers of its own species than is currently being realized. It is they might have been able to jettison surplus genes associated also remarkable that the multicellular Dictyostelium with with signaling cascades and reception. its complex life cycle has both a smaller number and less Because the single-celled P. aurantium fruiting body variety in its signal detection and processing proteins than might be considered as a prototype for the multicellular the unicellular Amoebozoa. Could this mean that the Dictyostelium fruiting body, we had expected that a signif- Dictyostelia evolved multicellularity, because they were icant number of Dictyostelium developmental genes origi- less adaptable than other Amoebozoa? Comparisons be- nated in the LCA of both lineages as was shown for tween genomes of related uni- and multicellular organisms developmental genes in Metazoa (Sebe-Pedros et al. in other eukaryote divisions might reveal whether this is a 2013). However, contrary to this expectation, the conserva- general trend. tion of developmentally essential Dictyostelium genes in Protostelium spp. is limited and actually not higher than for the evolutionary more distant A. castellanii. Our data Supplementary Material and the recent finding of a sporulating Acanthamoeba spe- Supplementary data are available at Genome Biology and cies (Tice, Shadwick, et al. 2016), makes it conceivable that Evolution online. the entire genus Acanthamoeba could comprise at least the genomic capacity for protosteloid fruiting. It is well possible that the lack of apparent conservation in Dictyostelium dis- Acknowledgments coideum could be due to the evolutionary distance between these taxa and that Dictyostelia have invented their own This study was supported in part by the Leibniz association toolbox for multicellularity. and a grant of the European Social Fund ESF—“Europe for Using RNAseq data from discernable time points during Thuringia” (to F.H.). S.N. was supported by the DFG-funded development, we were able to dissect early and late events excellence graduate school Jena School of Microbial in fruiting body formation. The same processes might be at Communication (JSMC). We thank Ivonne Go ¨ rlich and work in many other Amoebozoa, even if not true orthologous Cornelia Luge for their skillful laboratory assistance and but analogous proteins are being utilized. Emanuel Barth for his assistance in file conversion and data All free-living amoeba sequenced thus far do show a large processing. We also thank the Cologne Center for Genomics repertoire of sensor histidine kinases, which in Dictyostelium for sequencing data on P. aurantium. 604 Genome Biol. 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Evol. 10(2):591–606 doi:10.1093/gbe/evy011 Advance Access publication January 25, 2018 Downloaded from https://academic.oup.com/gbe/article-abstract/10/2/591/4824916 by Ed 'DeepDyve' Gillespie user on 16 March 2018

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