Deep Phylogeny and Evolution of Slime Moulds (Mycetozoa)

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Protist, Vol. 161, 55–70, January 2010 http://www.elsevier.de/protis Published online date 5 August 2009

ORIGINAL PAPER

Deep Phylogeny and Evolution of Slime Moulds (Mycetozoa) Anna Maria Fiore-Donnoa,c,d,1, Sergey I. Nikolaevb,c, Michaela Nelsond, Jan Pawlowskic, Thomas Cavalier-Smitha, and Sandra L. Baldaufd a

University of Oxford, Department of Zoology, South Parks Road, Oxford OX1 3PS, UK CMU, Department of Genetic Medicine and Development, 1 rue Michel-Servet, 1211 Geneva 4, Switzerland c University of Geneva, Department of Zoology and Animal Biology, 30, Quai E-Ansermet, 1211 Geneva 4, Switzerland d University of York, Department of Biology, Box 373, Heslington, York YO10 5YW, UK b

Submitted December 1, 2008; Accepted May 2, 2009 Monitoring Editor: Herve Philippe

Mycetozoa, characterized by spore-bearing fruiting bodies, are the most diverse Amoebozoa. They traditionally comprise three taxa: Myxogastria, Dictyostelia and Protostelia. Myxogastria and Dictyostelia typically have multispored fruiting bodies, but controversy exists whether they are related or arose independently from different unicellular ancestors. Protostelid slime moulds, with single-spored fruiting bodies, are possible evolutionary intermediates between them and typical amoebae, but have received almost no molecular study. Protostelid morphology is so varied that they might not be monophyletic. We therefore provide 38 new 18S rRNA and/or EF-1a gene sequences from Mycetozoa and related species, including four protostelids and the enigmatic Ceratiomyxa fruticulosa. Phylogenetic analyses support the monophyly of Dictyostelia, Myxogastria, and Ceratiomyxa (here collectively called ‘‘macromycetozoa’’) and show that protostelids are Amoebozoa, mostly related to non-fruiting amoebae of the class Variosea, but may not be monophyletic; some phylogenetic relationships remain poorly resolved. Ceratiomyxa fruticulosa, originally regarded as a myxogastrid, but in recent decades included in Protostelia, is a deeply diverging sister to Myxogastria. The protostelids studied here plus varipodid amoebae and the flagellates Phalansterium and Multicilia together probably form the outgroup to macromycetozoa plus Archamoebae. Thus protostelids and Variosea are especially significant for understanding the evolutionary transition from solitary amoebae to macromycetozoa. & 2009 Elsevier GmbH. All rights reserved. Key words: Amoebozoa; Dictyostelia; macromycetozoa; Mycetozoa; Myxogastria; Protostelia.

Introduction One of the deep branches of the eukaryotic tree is composed of a collection of amoebal species, the phylum Amoebozoa, comprizing all naked and 1

Corresponding author; fax +44 1865 281 310. e-mail [email protected] (A.M. Fiore-Donno).

& 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.protis.2009.05.002

testate lobose amoebae, together with entamoebids, pelobionts, and mycetozoans (Bolivar et al. 2001; Cavalier-Smith 2003; Cavalier-Smith and Chao 1998; Cavalier-Smith et al. 2004; Fahrni et al. 2003; Nikolaev et al. 2005, 2006). They all share the characteristic of lobose or pointed

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pseudopodia (neither filopodial nor eruptive) and tubular mitochondrial cristae (if not amitochondriate) (Cavalier-Smith 1998; Dykstra 1977). Excluded from Amoebozoa are all ‘‘amoebae’’ with true filopodia (able to pull the cell forwards) most of which belong rather to Cercozoa (Cavalier-Smith and Chao 2003; Cavalier-Smith et al. 2004) and amoebae with discoid mitochondrial cristae and eruptive lobes (Naegleria, Vahlkampfia, Acrasis), which belong to Heterolobosea (Page and Blanton 1985). Despite its key position, the circumscription of Amoebozoa and its evolutionary affinities are only recently becoming clearer (Arisue et al. 2005; Bapteste et al. 2002; Cavalier-Smith et al. 2004; Fahrni et al. 2003; Kudryavtsev et al. 2005; Nikolaev et al. 2004, 2006). The organisms included in Amoebozoa are now broadly agreed upon, but there are currently two slightly different classifications of the main classes (Cavalier-Smith et al. 2004; Smirnov et al. 2005). Amoebozoa includes the subphylum Conosa (Cavalier-Smith 1998) (or class Conosea: Smirnov et al. 2005), comprizing two very different groups: Mycetozoa, the true slime-molds, free-living soil or aquatic amoebae and the amitochondrial Archamoebae (parasites like Entamoeba, and free-living Mastigamoeba and Pelomyxa). Flagellated species of Conosa are characterized by a cone of microtubules emanating from the often single centriole and subtending the nucleus (Cavalier-Smith 1998). Recent molecular analyses tend to confirm its monophyly, although it can be sensitive to taxon sampling (Nikolaev et al. 2006). One emerging new group, referred as Variosea (Cavalier-Smith et al. 2004) includes five genera: Filamoeba, Flamella (Kudryavtsev et al. 2009) – and related uncultured environmental genotypes, Multicilia, Phalansterium, and Acramoeba (formerly misidentified as the leptomyxid Gephyramoeba (Amaral Zettler et al. 2000; see Smirnov et al. 2008). Mycetozoa are characterized by their ability to form ‘‘fruiting bodies’’ bearing spores. However, ‘‘fruiting bodies’’ made by cell aggregation (as in Dictyostelia) also exist in several unrelated terrestrial organisms, and have therefore evolved more than once. These forms, probably resistant to drought, are found in groups as diverse as ciliates (Sorogena stoianovitchae), Heterolobosea (e.g. Acrasis and Pocheina), and even bacteria (Myxobacteria). True Mycetozoa traditionally comprise three groups: Myxogastria, Dictyostelia and Protostelia (Olive 1970) (Fig. 1). Their monophyly was challenged by the first ribosomal RNA trees, based only on Physarum polycephalum and Dictyostelium discoideum (for references see

Baldauf and Doolittle 1997; Philippe and Adoutte 1998). However, this now appears to be an artifact of accelerated evolution in P. polycephalum and its extreme and opposite nucleotide compositional bias (SSU rRNA C-G rich), compared with D. discoideum (A-T rich) (Steenkamp and Baldauf 2004). Elongation factor 1a (EF-1a) phylogeny robustly grouped one protostelid, Planoprotostelium aurantium, with D. discoideum and P. polycephalum (Baldauf and Doolittle 1997), but did not test the previously hypothesized monophyly of Mycetozoa (Olive 1975) as no other Amoebozoa were studied. Protostelia (Olive 1970) are of special evolutionary interest because they are simpler than, and perhaps transitional to, Dictyostelia and Myxogastria (Olive 1975). However, their main characteristic – a microscopic sporophore composed of a slender, tubular stalk bearing a single spore on its tip – accompanies a wide diversity of feeding stages, ranging from uninucleate amoebae or amoeboflagellates to multinucleate reticulate plasmodia (Fig. 1). Consequently, the monophyly of Protostelia has been questioned, although some monophyletic groups can be recognized (Spiegel 1990, 1991). One enigmatic species, the widespread and common Ceratiomyxa fruticulosa, was traditionally assigned to Myxogastria, but in a outlying position (Martin et al. 1983), as a separate subclass possessing ‘‘external’’ spores, but has also been treated as a protostelid instead (Olive 1975). More recently, it was assigned to an amended Myxogastrea, belonging to a sixth order, beside the five traditionally recognized ones (Cavalier-Smith et al. 2004). The spores are borne on slender stalks arising from the surface of coralloid gelatinous branches, from a prespore cell, reminiscent of Protostelia, in contrast to Myxogastria, where the spore mass is surrounded, even if temporarily, by a thin membranous peridium (Fig. 1). Ceratiomyxa fruticulosa, which can reach conspicuous dimensions, whitening logs for decimeters, has successfully been isolated only recently (Clark et al. 2004). To investigate evolutionary patterns and phylogenetic relationships within Conosa, significant for understanding the origins of multicellularity and syncytia in Mycetozoa, we sequenced 38 small subunit ribosomal RNA (SSU rRNA) and/or elongation factor 1a (EF-1a) genes from Myxogastria (10 species), Dictyostelia (5 species), Protostelia (4 species), Ceratiomyxa fruticulosa (3 strains), Filamoeba (2 species) and Acramoeba dendroida. Individual gene analyses led to mostly congruent, but not well-resolved trees. Concatenated

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Figure 1. Selected characteristics of Mycetozoa regarding plasmodium type, sporophore development, stalk and presence of sexuality. Characters common to more than one group are shaded in grey. Number of species according to Adl et al. (2007).

analysis of both genes increased support for the main branches, allowing more robust evolutionary interpretations.

Results Introns The SSU rRNA gene sequences of all protostelids and Ceratiomyxa fruticulosa were devoid of

introns. The Ceratiomyxa fruticulosa sequences were characterized by larger helices, making the whole gene longer than usual (42250 bp). Introns were found in four of the six Myxogastria species belonging to the bright-spored clade (C. vulgaris, C. cancellata, T. ferruginosa, A. stipata), in positions S529, S911, S956, S1199, of sizes ranging from 387 to 1110 bp. All these insertion sites for group I introns have already been reported in Myxogastria (positions indicated in Supplementary material 2) (Fiore-Donno et al.

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2008; Haugen et al. 2003; Lundblad et al. 2004; Wikmark et al. 2007). Nearly complete EF-1a nucleotide sequences range from 1184 to 2204 bp, varying according to spliceosomal introns, found in 17 distinct insertion sites. Intron-rich sequences were found in Ceratiomyxa fruticulosa, Echinostelium spp. and Schizoplasmodiopsis amoeboidea. All myxogastrian EF-1a sequences and Ceratiomyxa fruticulosa also possess an intron in a position previously described for Physarum polycephalum (Baldauf and Doolittle 1997; Fiore-Donno et al. 2005). It is absent in protostelids and Dictyostelia, but present in Filamoeba spp. and Phalansterium solitarium.

Single Gene Tree (SSU rRNA) with 76 Taxa (Amoebozoa) The well-sampled SSU rRNA gene was used to place the new protostelid sequences. We selected representatives of each major amoebozoan lineage (Lobosea, Acanthopodida, Thecamoebidae, Discosea, Conosa). As expected, the resulting tree (Fig. 2) shows some poorly resolved basal branches, but our results are concordant with previous publications (Brown et al. 2007; Nikolaev et al. 2006; Smirnov et al. 2008; Tekle et al. 2008), and Bayesian and ML topologies are nearly identical, differing only in Cavostelium apophysatum and Ceratiomyxa fruticulosa placement. Reasonable support is obtained for the clades Lobosea (bootstrap replicates reported on the best maximum likelihood tree: 99). Acanthopodida + Thecamoebidae (81); Conosa and Discosea are recovered, but not supported (23, 24). Ceratiomyxa fruticulosa appears as sister of Dictyostelia + Myxogastria, although with weak support (63). In this tree, all protostelids except Cavostelium apophysatum are intermingled with Variosea. Cavostelium apophysatum lies between Archamoebae and Ceratiomyxa, without support. Although not supported, the separation between Conosa and other Amoebozoa is consistently recovered, as in previous studies (Brown et al. 2007; Nikolaev et al. 2006), however with sometimes a different internal branching pattern (Tekle et al. 2008). To try to establish more firmly the

phylogeny of this group, a two-gene phylogeny has been conducted with a restricted sampling, including only representatives of Conosa, with Acanthamoeba spp. and Nuclearia simplex as outgroup, Acanthamoeba being the closest relative for which EF-1a sequences were also available.

Single Gene Trees (SSU rRNA and EF-1a) with 54 Taxa (Conosa) Both SSU rRNA and EF-1a trees (Fig. 3) show several well-supported terminal clades, including Myxogastria, Dictyostelia and Archamoebae, and, in general, poorly resolved basal branches. The most interesting result is the grouping of Myxogastria, Dictyostelia and Ceratiomyxa. This clade is retrieved in all obtained trees, but supported only in Bayesian analyses (Bayesian posterior probability, BPP), bootstrap replicates reported on the best maximum likelihood (BML) tree, bootstrap starting with a NJ tree (BNJ): 0.99; 53; 77). The respective positions of Myxogastria, Ceratiomyxa and Dictyostelia are unresolved in the SSU rRNA tree, while in the EF-1a tree, Ceratiomyxa is a sister group of Myxogastria (1.0; –; 83). Protostelids appear in two or three distinct clades, the composition and position of which differ between genes and is taxon-sampling dependent. In the SSU tree (Fig. 3), some protostelids branch as sisters to the clade Dictyostelia + Ceratiomyxa + Myxogastria, while the others form a weakly supported group with Filamoeba spp. and related genotypes. In the EF-1a tree, four protostelids form a clade, while P. aurantium branches next to Filamoeba. However, none of these clades is well supported in all analyses, except for the genus Schizoplasmodiopsis in the SSU tree. The fact that this genus appears paraphyletic in the EF-1a tree, is probably due to the limited informative sites present in this gene. To exclude possible laboratory errors, we sequenced the 50 parts of the EF-1a gene in those two species twice, from different PCRs, and our results were confirmed. Myxogastria are subdivided into two major clades, the dark-spored clade and the brightspored clade. The position of Echinostelium spp.

Figure 2. Small subunit (SSU) rRNA gene tree derived by maximum likelihood analysis of 1292 nucleotide positions of 75 representative Amoebozoa plus Nuclearia as outgroup. Results of 1000 ML bootstrap replicates 450 % are shown for each node, as percentages. A dot on the line indicates maximum support. Sequences in bold were obtained during this study. The scale bar indicates the fraction of substitutions per site. The ‘Arachnula’ sequence is from an unnamed filose varipodid amoeba that was misidentified, and not actually from Arachnula, which is a reticulose cercozoan (Bass et al. 2009).

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Molecular Phylogeny of Mycetozoa Nuclearia simplex AF484687 "Hartmannella" vermiformis AY680840 Echinamoeba exundans AF293895 E. thermarum AJ489262 99 Rhizamoeba saxonica AY121847 Leptomyxa reticulata AF293898 Lobosea (=Tubulinea) 94 Amoeba leningradensis AJ314605 Chaos nobile AJ314606 63 Heleopera sphagni AY848964 57 Hartmannella abertawensis DQ190241 Nolandella sp. EU273456 Acanthamoeba palestinensis L09599 A. culbertsoni AF019057 81 Acanthopodida + Thecamoebidae 96 Stenamoeba stenopodia AY294144 Sappinia diploidea DQ122380 Korotnevella stella AY183893 Vexillifera armata AY183891 64 Lingulamoeba leei AY183886 Discosea (=Flabellinea) 70 Vannella aberdonica AY121853 Vannella sp. AY929906 77 Platyamoeba plurinucleolus ATCC50745 AY121849 60 Multicilia marina AY268037 Phalansterium solitarium AF280078 Uncult. euk. clone Amb EF024087 Uncult. euk. clone WIM5 AM114801 CONOSA Soil amoeba AND16 AY965863 Soliformovum irregulare EF513181 Acramoeba dendroida AF293897 Filamoeba nolandi AF293896 Variosea 77 F. nolandi AY714368 F. sinensis AY714369 + Uncult. euk. clone WIM81 AM114809 Euk. clone Borok AY626163 Protostelia pro parte 81 Arachnula sp. EU273440 63 Uncult. euk. clone RT5iin44 AY082989 Protostelium mycophaga EU004603 Planoprotostelium aurantium EU004604 Schizoplasmodiopsis amoeboidea EF513179 99 S. vulgaris EF513180 Entamoeba polecki AF149913 E. gingivalis D28490 94 E. histolytica X65163 97 Endolimax nana AF149916 Archamoebae 95 Mastigamoeba simplex AF421218 95 Mastigamoeba sp. AF421220 Mastigella commutans AF421219 99 Mastigamoeba balamuthii L23799 Cavostelium apophysatum EF513172 Protostelia pro parte Ceratiomyxa fruticulosa F EF513175 C. fruticulosa CH1 EF513173 Ceratiomyxida 88 C. fruticulosa CH2 EF513174 Dictyostelium medusoides AM168088 D. fasciculatum AM168087 Acytostelium leptosomum AM168111 63 Dictyostelia A. subglobosum AM168110 79 Dictyostelium discoideum K02641 99 D. rhizopodium AM168063 Cribraria c ancellata EF513177 C. argillacea EF513176 M 54 97 C. vulgaris EF513178 97 y Tubifera ferruginosa EF513171 Arcyria denudata AY643825 x A. stipata EF513170 90 o Trichia persimilis AY643826 T. sordida EF513182 g Echinostelium arboreum AY842030 94 E. minutum AY842034 a E. coelocephalum AY842033 Stemonitis flavogenita AF239229 s Amaurochaete comata AY842031 t Comatricha nigricapillitia 3 AY643824 Lepidoderma tigrinum DQ903678 r Diderma globosum var. europaeum DQ903680 93 0.1 Physarum polycephalum X13160 i 97 92 Badhamia panicea var. nivalis DQ903680 a Physarum album DQ903681 88

59

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SSU rRNA Nuclearia simplex AY582827

Nuclearia simplex AF484687 Acanthamoeba culbertsoni AF019057 Acanthamoeba palestinensis L09599

Acanthamoeba culbertsoni AY582829 Acanthamoeba palestinensis

Phalansterium solitarium AF280078 0.83; -; 59 1.0; 93; 100 0.72; -; -

Acramoeba dendroida AF293897 Multicilia marina AY68037 Schizoplasmodiopsis amoeboidea EF513179 Schizoplasmodiopsis vulgaris EF513180

0.98; -; 1.0; -; 67 0.94; -; 59

0.99;64;85

Cavostelium apophysatum EF51318

Planoprotostelium aurantium EU004604

0.83; -; -

Schizoplasmodiopsis amoeboidea EF513197

soil amoeba AND16 AY965863 Acramoeba dendroida EF513194 unidentified eukaryote clone Borok AY626163 environmental clone RT5iin44 AY082989 PROTOSTELIA Planoprotostelium aurantium AF016240 Filamoeba sinensis AY714369 Filamoeba sinensis EF5131 Filamoeba nolandi AY714368 Filamoeba nolandi EF513192 0.59; 50; 57 Filamoeba nolandi AF293896 Entamoeba polecki AF149913 Entamoeba gingivalis D28490 Entamoeba histolytica X65163

0.72; -; 1.0; 88; 81

Endolimax nana AF149916 Mastigamoeba simplex AF421218 Mastigamoeba sp. AF421220 Mastigella commutans AF421219 Mastigamoeba balamuthi L23799

1.0; 88; 87 0.56; 53; 68 0.55; 54; 68 1.0; 99; 99 0.82; -; 55

0.64; -; 1.0; 72; 91

Entamoeba histolytica M92073

0.63; -; 1.0; -; 76

Mastigamoeba balamuthi

ARCHAMOEBAE

Cavostelium apophysatum EF513172 Soliformovum irregulare EF513181 PROTOSTELIA

0.96; -; 56

1.0; 85; 88 0.94; -; -

0.96; -; 1.0; 62; -

Schizoplasmodiospis vulgaris EF513198

Protostelium mycophaga EU004603

0.73; -; 1.0; 80; 86

PROTOSTELIA Soliformovum irregulare EF513199

1.0;98;97

Dictyostelium rhizopodium EU360787 Dictyostelium medusoides AM168087 D. discoideum X55973 Dictyostelium fasciculatum AM168088 Acytostelium leptosomum EU360789 Acytostelium leptosomum AM168111 Acytostelium subglobosum EU360788 Acytostelium subglobosum AM168110 D. fasciculatum EU360786 Dictyostelium rhizopodium AM168063 DICTYOSTELIA D. medusoides EU360785 Dictyostelium discoideum K02641

MYXOGASTREA Ceratiomyxa fruticulosa F EF513175 1.0; 87; 88 0.99; 53; 77

C. fruticulosa CH2 EF513174 C. fruticulosa CH1 EF513173 Ceratiomyxida

1.0;99;98 1.0;98;99

Ceratiomyxa fruticulosa F EF513188 C. fruticulosa CH1 EF513186

C. fruticulosa CH3 EU259001 C. fruticulosa CH2 EF513187 Cribraria cancellata EF513177 Arcyria stipata EF513183 C. vulgaris EF513178 A. denudata AY643820 C. argillacea EF513176 Cribraria cancellata AY643815

Tubifera ferruginosa EF513171 C. vulgaris EF513190 1.0;97;98 Trichia persimilis AY643826 bright-spored C. argillacea EF513189 T. sordida EF513182 clade Tubifera ferruginosa EF513201 1.0; 87; 88 Arcyria stipata EF513170 Trichia persimilis AY643821 A. denudata AY643825 T. sordida EF513200 Echinostelium arboreum AY842030 Echinostelium arboreum AY842032 1.0;80;76 E. coelocephalum AY842033 E. coelocephalum AY643813 Echinostelium E. minutum AY842034 E. minutum AY643814

0.1

0.92; -; 0.94; 54; 1.0; 99; 100 0.8; -; -

1.0; 87; 84 0.97; 77; 80 1.0; 97; 100 1.0; 79; 99 1.0; 98; 99 0.96; -; 57

1.0; 92; 98 1.0; -; 83 1.0; -; 91 0.74; -; 1.0; 62; 88 1.0; 97; 99 0.90; -; 60 0.57; -; 65 0.93; -; 69 1.0; 87; 86

Stemonitis flavogenita AF239229 bright -spored clade Amaurochaete comata AY842029 Comatricha nigricapillitia AY643824 1.0; -; 84 Stemonitis flavogenita AY643819 Amaurochaete comata AY842031 1.0; 66; 52 Comatricha nigricapillitia AY643818 0.85; -; Diderma globosum 0.99; 53; 89 Lepidoderma tigrinum EF513195 var. europaeum DQ903677 0.80; -; Diderma globosum Lepidoderma tigrinum DQ903678 1.0; 85; 99 1.0; 91; 89 var. europaeum EF513191 Physarum polycephalum X13160 Badhamia panicea 1.0; 67; 97 Badhamia panicea 1.0; 99; 99 var. nivalis EF513184 0.90; -; 52 1.0; 96; 95 var. nivalis DQ903680 Physarum polycephalum AF016243 0.79; -; 51 0.1 Physarum album DQ903681 Physarum album EF513196

Figure 3. Left: Small subunit (SSU) rRNA gene tree derived by Bayesian inference of 1234 nucleotide positions of 51 conosan taxa with Nucelaria and Acanthamoeba spp. as outgroup, and, right, Elongation factor 1A (EF1a) gene tree derived by Bayesian inference of 360 amino-acid positions of 42 taxa. The results of 1000 ML bootstrap replicates are shown for each node, as percentages, after the Bayesian posterior probability (BPP), in this order: bootstrap replicates reported on the best maximum likelihood tree (BML), bootstrap starting with a NJ tree (BNJ). A dot on the line indicates maximum support in all analyses, hyphens indicate bootstrap values o50%. Sequences in bold were obtained during this study. The scale bar indicates the fraction of substitutions per site.

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is unstable, sister to the dark-spored clade in the SSU tree, and in the bright-spored clade in the EF1a tree. In the dark-spored clade, the order Stemonitida (Stemonitis flavogenita, Comatricha nigricapillitia and Amaurochaete comata) appears as ancestral to Physarida. Holophyly of Physarida, on the other hand, is well supported (1.0; 91; 89) as also for families Physaridae (Physarum polycephalum, P. album and Badhamia panicea) (1.0; 99; 99) and Didymiidae (Diderma globosum, Lepidoderma tigrinum) (1.0; 100; 100). Of note in the bright-spored clade is the conflicting position of Arcyria spp., which appears basal in the EF1a tree (1.0; –; 91), and terminal in the SSU tree (1.0; 87; 88). The internal branching within Dictyostelia also differs between both trees, the

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SSU rRNA confirming previous results (see Schaap et al. 2006).

Combined Analyses (SSU rRNA þ EF1a) Discrepancies in the distal branch topology are attributed to the highly conserved nature of the EF1a gene (50% of constant sites), which normally does not contain enough variation to discriminate between closely related species; SSU rRNA is more suitable for this. However, neither gene alone satisfactorily resolved the basal topology of the tree; EF1a for the reason mentioned above, and SSU rRNA due to its high variability. As expected, combining both genes

Nuclearia simplex Acanthamoeba culbertsoni Acanthamoeba palestinensis Acramoeba dendroida

Acramoeba + PROTOSTELIA I

Soliformovum irregulare Planoprotostelium aurantium

0.97;-;0.91;-;67; 56; 0.98; 84; 85 77; 75;

Filamoeba + PROTOSTELIA II

Filamoeba sinensis Filamoeba nolandi

75; 69;

Cavostelium apophysatum

PROTOSTELIA III

Schizoplasmodiopsis vulgaris Schizoplasmodiopsis amoeboidea Entamoeba histolytica Mastigamoeba balamuthi

76; 68;

Archamoebae

Dictyostelium fasciculatum Dictyostelium medusoides

0.8;-;78; 79 97; 95

Group I

Acytostelium leptosomum Acytostelium subglobosum

Group II

Dictyostelium rhizopodium Dictyostelium discoideum

Group III + IV

Ceratiomyxa fruticulosa F Ceratiomyxa fruticulosa CH1

95; 99 88; 100

DICTYOSTELIA

Ceratiomyxida

Ceratiomyxa fruticulosa CH2 Cribraria cancellata

82; 82

C. vulgaris C. argillacea

99; 99

Liceida

Tubifera ferruginosa Trichia sordida Trichia persimilis

94; 92

Arcyria stipata Arcyria denudata Echinostelium arboreum Echinostelium coelocephalum Echinostelium minutum

80; 83

0.99; 66; 72 0.97;-; 61

95; 96 98; 97 90; 91

Stemonitis flavogenita Amaurochaete comata

Trichiida MYXOGASTREA Echinostelium Stemonitida

Comatricha nigricapillitia Lepidoderma tigrinum Diderma globosum var. europaeum Physarum polycephalum Physarum album Badhamia panicea var. nivalis

Physarida

M A C R O M Y C E T O Z O A

0.1

Figure 4. Bayesian phylogeny of Conosa inferred from concatenated alignments of small subunit (ssu) rRNA and elongation factor 1A (EF1a) genes, based on 1594 positions of 41 taxa. The results of 1000 ML nonparametric bootstrap replicates reported on the best ML tree obtained with RAxML (BML), and starting with a NJ tree (BNJ) are shown for each node, as percentages, after the Bayesian posterior probability (BPP). A dot on the line shows maximum support in all analyses, a thick line shows BPP=1.0 and BML or BNJo100%, hyphens indicate bootstrap values o50%. The new clade, macromycetozoa, composed of Dictyostelia, Ceratiomyxa fruticulosa and Myxogastria, is outlined by a grey box. Continuous vertical lines indicate monophyletic clades, dotted lines indicate paraphyletic assemblages. The scale bar indicates the fraction of substitutions per site.

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(analyzed with distinct models to take into account different rates of evolution) gives better overall resolution (Fig. 4). The grouping of Dictyostelia and Myxogastrea (sensu Cavalier-Smith et al. 2004, including Ceratiomyxa) is recovered (1.0; 99), and their respective relationships are well supported, with Dictyostelia as the earliest diverging branch (1.0; 82) and Ceratiomyxa as a sister to other Myxogastrea (1.0; 100) (Fig. 4). Archamoebae are the sister group of the previous clade (1.0; 76; 88), and Variosea þ Protostelia is recovered in Bayesian analyses (0.97 – not supported elsewhere). Protostelia appears to be divided into three groups: Soliformovum irregulare seems related with Acramoeba dendroida (1.0 – not supported elsewhere), although a long-branch attraction is not excluded. Planoprotostelium aurantium is associated with Filamoeba spp. (1.0; 75; 69), as also suggested in the EF1a analyses (1.0; 72; 91). Schizoplasmodiopsis spp. and Cavostelium apophysatum form a clade (1.0; 67; 56).

Paired-site Tests To test the evolutionary hypotheses suggested by our trees, we compared different topologies by performing the approximately unbiased test (AU), considered to be the least-biased test available (Shimodaira 2002). More precisely, we tested the monophyly of macromycetozoa, alternative relationships in macromycetozoa and the possible monophyly of Protostelia. For this purpose, the best ML SSU rRNA þ EF1a tree obtained with Treefinder was compared with a set of alternative topologies, two by two (paired-sites test) (Fig. 5). Trees disrupting the monophyly of macromycetozoa are significantly rejected (P=0). In some SSU rRNA analyses, Ceratiomyxa appeared as the sister group of Myxogastria + Dictyostelia, or, more seldom, Ceratiomyxa + Dictyostelia appeared as a sister group of Myxogastria. Both topologies could not be excluded with confidence (P=0.1 and 0.05, respectively). Monophyly of Protostelia was rejected (P=0) (Fig. 5).

Discussion Phylogenetic Position of Ceratiomyxa and Emergence of Macromycetozoa Our results clearly show that Ceratiomyxa is more closely related to Dictyostelia and Myxogastria

Best Tree (Treefinder) macromycetozoa Cer Myx Myx = Myxogastria Cer = Ceratiomyxa Dic = Dictyostelia Arch = Archamoebae P = Protostelia A = Acramoeba dendroida F = Filamoeba spp.

Dic Arch P, A, F

A Log likelihood: - 27361.76 Monophyly of macromycetozoa disrupted: - by Archamoebae - by Protostelia Cer

Myx

Myx

Cer S. irregulare C. apophysatum

Arch Dic

Dic Arch

P, A, F

B Log likelihood: - 27399.32 Δ likelihood: 37.56 AU test, P = 0.

C Log likelihood: - 27400.43 Δ likelihood: 38.67 AU test, P = 0.

Alternative relationships in macromycetozoa Dic

Myx

Cer Myx

Cer Arch P, A, F

D Log likelihood: - 27368.64 Δ likelihood: 6.88 AU test, P = 0.0997

Dic

Arch P, A, F

E Log likelihood: - 27369.81 Δ likelihood: 8.05 AU test, P = 0.05

Figure 5. Alternative trees topologies tested with Approximately Unbiased test (AU). The best tree obtained with Treefinder (A) was compared with each of the altenative topologies (B to F).

(sensu Olive 1970, excluding Ceratiomyxa) than to any protostelid clade. Its position as a sister group to Myxogastria indicated by EF1a and combined data is in agreement with the presence of a second flagellum and acellular large plasmodium (Clark et al. 2004; Nelson and Scheetz 1975). In the light of our results, the presence of a second flagellum is an apomorphy for the clade composed of Myxogastria plus Ceratiomyxa. It is plausible that protostelid species possessing this characteristic may belong to this clade, i.e. Clastostelium recurvatum and Protosporangium

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spp. (Spiegel et al. 1986; Spiegel and Feldman 1988b). Ceratiomyxa fruticulosa differs from other Myxogastria and Protostelia by peculiarities of its flagellar apparatus (Spiegel 1981) and also in possessing quadrinucleate spores, germinating in a four-lobed structure, in which each lobe undergoes a mitosis and develops into a swarm cell (Nelson and Scheetz 1975). This, along with its peculiar fruiting-bodies bearing external spores, makes Ceratiomyxa a highly divergent myxogastrid, consistent with its position in our EF1a and combined trees. Adding Ceratiomyxa to our analyses reinforced the relationships between Myxogastria and Dictyostelia and led to the emergence of a new monophyletic clade of mycetozoans. We refer to it as ‘‘macromycetozoa’’ because its fruiting-bodies are generally visible to the naked eye, unlike those of the protostelids (Fig. 1). Although support for macromycetozoa is not always very strong, especially in the ML trees, the clade was consistently recovered in all analyses and alternative topologies were rejected by the AU tests (Fig. 5). This is in agreement with the monophyly of Dictyostelia and Myxogastria shown in our previous analyses (Nikolaev et al. 2006). Nevertheless, it contrasts with a recent study of Amoebozoa, in which the relationships among Conosa were unresolved, in spite of large taxon sampling (Tekle et al. 2008), but in which the stabilising effect on tree topology brought by Ceratiomyxa was lacking. Also, suppressing the fast-evolving sites may reduce the usually strong signal brought by the SSU ribosomal gene for related taxa, as the main part of this signal is contained in the variable helices. While the monophyly of macromycetozoa seems well established, the phylogenetic status of protostelids remains unclear. The six protostelid species sequenced until now, including four added in this study, branch in two or three weakly supported clades, sometimes in conflicting positions. There is insufficient resolution among the basal conosan lineages to establish whether Protostelia are polyphyletic, paraphyletic or even holophyletic (Figs 2–4). Much more extensive sampling of protostelid taxa and genes is needed to answer this question. If intermingling of Protostelia and Variosea as on our tree is confirmed by such studies, the question will arise whether this clade was ancestrally spore bearing and fruiting bodies were lost by Acramoeba and Filamoeba or whether fruiting bodies evolved polyphyletically. A third possibility is that Acramoeba and Filamoeba have undiscovered fruiting stages and the

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conditions for inducing them in culture have not been met. Whichever is correct, all examined protostelids most likely belong to Conosa (see also Brown et al. 2007), which was recently expanded to include a more narrowly circumscribed Variosea (Cavalier-Smith 2009), thus showing a relatively close relationship with macromycetozoa and Archamoebae. Our trees leave open the possibility that protostelids are paraphyletic ancestors or monophyletic sisters of macromycetozoa, but weakly suggest that the sister group of macromycetozoa may instead be the anaerobic Archamoebae, in which case Mycetozoa would be paraphyletic (if the conosan ancestor had fruiting bodies) or polyphyletic (if it lacked fruiting bodies).

Characters Common to Macromycetozoa and their Evolutionary Origin There are two characters that might have been present in ancestors of macromycetozoa. One is related to the sexual plasmodium, the second is the multispored sporophore. Paradoxically, the striking characteristic used to group Dictyostelia and typical Myxogastria – the fruiting bodies – may not be homologous. In Myxogastria, including Ceratiomyxa, the fruiting body arises from a typically diploid syncytial plasmodium whereas in Dictyostelia it arises from a haploid non-syncytial cell aggregate (Fig. 1). The large hyaline plasmodium of C. fruticulosa is similar to the plasmodia found in myxogastrid Stemonitida (Clark et al. 2004) and apparently homologous. In Myxogastria, the zygote is formed by the fusion of two compatible cells (in heterothallic strains) or identical cells (in homothallic strains). Meiosis, in Myxogastria, occurs while the spores are formed, thus they are genetically distinct and form a successive generation (in heterothallic strains). In contrast, the dictyostelian cellular pseudoplasmodia form by the aggregation of haploid amoebae, giving rise to fruiting bodies where sexuality is not involved. There is no succession of generations, the spores being genetically identical to the amoebae (Fig. 1). However, in some conditions, a sexual cycle occurs in Dictyostelia – at least it has been demonstrated and/or supposed in representatives of Groups II, III and IV (see Szabo et al. 1982 and references therein); it implies sexual maturation of haploid cells, which fuse in binucleate cells, eventually forming a zygote with a large single

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nucleus (Blaskovics and Raper 1957; Szabo et al. 1982). The zygote secretes cAMP and acts as aggregation centre to attract the surrounding cells, which are engulfed and later digested (Lewis and O’Day 1994). The zygote is then surrounded by a three-layered wall, and becomes dormant. During germination, several rounds of cytoplasmic cleavages have been observed and nuclear divisions are hypothesized (Erdos et al. 1973). Occurrence of meiosis has been demonstrated by genetics (MacInnes and Francis 1974), in Dictyostelium mucoroides (a representative of Group IV), but its timing relative to the nuclear divisions is not known. We therefore hypothesize that the common feature in macromycetozoa could be the temporary suppression of cytoplasmic division following nuclear division in the diploid stage (plasmodium in Myxogastria and macrocyst in Dictyostelia). More data from the sexual cycle in Dictyostelia are needed; first about occurrence of the sexual cycle in representatives of Groups I, II, and III and then about the timing of meiosis. The contrast made historically between plasmodium (restricted to Myxogastria) and pseudoplasmodium (restricted to Dictyostelia) is misleading: in fact, Dictyostelia possess both. While the syncytial stage is common to both groups, only Dictyostelia have also developed a haploid cellular pseudoplasmodium leading to the formation of stalked fruiting-bodies bearing asexual spores. Cavalier-Smith et al. (2004) grouped Protostelia (excluding Ceratiomyxa) and Dictyostelia as a class Stelamoebea, based on the absence of a true plasmodium. Actually, in protostelids building a plasmodium (i.e. Schizoplasmodiopsis), sporulation is preceded by segmentation of the plasmodium into uninucleate or plurinucleate prespore cells (Olive 1975) (Fig. 1). Our results suggest that the plasmodium found in the protostelid Schizoplasmodiopsis has evolved independently, and may not be homologous to the macromycetozoan plasmodium, and that Stelamoebea is paraphyletic.

less spores (Echinostelium) genera. Physarida and Stemonitida have dark spores (black, dark-brown, violet-brown, purplish-grey), but Stemonitida lack the lime deposits in one or several parts of the fructification, the main characteristic of Physarida. Previous results, based on partial SSU rRNA and EF1a sequences already suggested the dichotomy between dark- and bright-spored groups but placed Echinostelium as the sister group to the four remaining orders (Fiore-Donno et al. 2005). Currently, in analyses conducted with complete SSU rRNA and EF1a sequences from a wider range of taxa, Echinostelium branches as the sister group of the dark-spored clade (although not in the EF1a tree, Fig. 3). In this dark-spored clade, Stemonitida are paraphyletic and basal to a monophyletic Physarida (Fiore-Donno et al. 2008) (Fig. 3), although this topology is not recovered when sampling is scarce (combined tree, Fig. 4). Our original contribution here resides in obtaining the first SSU rRNA gene sequences of Liceida-Cribraria spp. and Tubifera ferruginosa (which may be called Tubulifera arachnoidea  Jacq.) (Hernandez-Crespo and Lado 2005). Liceida are distinguished from Trichiida only by the absence of the capillitium (filaments in the sporophore) and the monophyly of the order has been questioned (Eliasson 1977). Cribraria are distinctive in many traits: always stalked, a net-like peridium surrounding the spore mass, and distinctive pigments (Iwata et al. 2003). It is intriguing that the most basal groups in both dark- and bright-spored clades (Cribraria and Echinostelium) are (nearly) always stalked. In Echinostelium, the stalk is secreted by the cytoplasm (Mims 1973; Spiegel and Feldman 1988a). If this were to be demonstrated also in Cribraria, as preliminary studies suggest (Garcia Sastre and Estrada-Torres 2005), then the ancestral state of Myxogastria must have possessed a secreted stalk, and perhaps a continuous evolutionary pathway could be drawn from Protostelia, to Ceratiomyxa and Myxogastria.

Cryptic Species in Ceratiomyxa fruticulosa Evolutionary Patterns in Myxogastria Olive’s classification of Myxogastria recognized five orders: Trichiida, Liceida, Physarida, Stemonitida and Echinostelida (Frederick 1990; Neubert et al. 1993; Olive 1970). Trichiida and Liceida are characterized by variously-colored (but never violet-brown or purplish-gray) spores, while Echinostelida present a mixing of darkspored (Clastoderma and Barbeyella) and color-

It has been suggested that Myxogastria morphospecies could be complexes of distinct biological species (Clark et al. 2004). The genetic distance between the four Ceratiomyxa fruticulosa isolates is very large, and some geographical pattern can be recognized: in the SSU rDNA tree, the two samples collected in Switzerland (CH1 and CH2), approximately 100 km apart, are more closely related than the sample collected in Southern

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France, at 400 km from CH2. CH3 was collected 20 km from CH2, and is sister to it in the EF1a tree. These may represent separate sibling species, possibly geographically restricted.

Conclusions Our study sheds new light on conosan classification. Of particular note is the consistentlysupported emergence of a monophyletic clade composed of Dictyostelia, Ceratiomyxa and Myxogastria, called macromycetozoa. Their common trait could be the temporary suppression, in the zygote, of cellular division following nuclear division. The ancestor of macromycetozoa could have already evolved a multispored fruiting body, as a multinucleate diploid zygote is present in all its members. Macromycetozoa exclude all other protostelids sampled in this study, thus questioning the validity of Mycetozoa. However, our data do not clearly deny or support its monophyly, making wider sampling of taxa and genes desirable. Our trees confirm that the mere absence/presence of a flagellum is not a satisfactory criterion for higher classification, as there were at least four independent losses in conosan evolution (in protostelids, Variosea and Dictyostelia). In contrast, the appearance of a second flagellum, with its characteristic basal body, is probably an apomorphic trait for Myxogastrea (sensu Cavalier-Smith et al. 2004). Myxogastria (sensu Olive 1970), with the widest sampling to date, including representatives of all five recognized orders for both genes, are clearly subdivided into two clades, the dark-spored clade (Stemonitida and Physarida) and the brightspored clade (Liceida and Trichiida), with Cribraria (Liceida) as an early diverging group, while the position of the colorless-spored genus Echinostelium remains to be tested with more genes.

Methods Cell cultures, DNA/RNA extraction, amplification, sequencing: Sporophores of Schizoplasmodiopsis vulgaris were isolated from dead plants collected in Switzerland (Geneva) (Table 1), cultured in weak-nutrient agar (Spiegel 1990) and fed with E. coli. Ceratiomyxa fruticulosa was collected from the field and DNA was extracted from fresh samples, using all material in the case of CH1 and CH2. Cultures of Filamoeba nolandi CCAP 1526 and F. sinensis CH26 (Dykova et al. 2005) were received encysted and revived, after providing E. coli as food, on non-nutrient saline agar (PAS, see www.ccap.ac.uk/ media/pdfrecipes.htm). Cultures of Schizoplasmodiopsis amoeboidea, Soliformovum irregulare and Cavostelium apophysatum were provided by Dr. F. Spiegel (Dept. Biol.

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Sci., Univ. Arkansas, Fayetteville, AR 72701. The type cultures of S. amoeboidea, S. irregulare and C. apophysatum are available from the American Type Culture Collection as strains ATCC 46943, ATCC 26826, and ATCC 38567, respectively. DNA was extracted from cultures by flooding and scraping the agar plates containing actively growing amoebae with either guanidine buffer (Chomczynski and Sacchi 1987) – for small amounts of cells (Protostelia) – or SDS buffer-for up to 108 cells (Filamoeba spp.) (Wikmark et al. 2007). DNA extraction of myxogastrians was performed using several dried (or fresh – C. fruticulosa CH1 and CH2) sporophores per sample (Table 1). Spores were ground with a plastic pestle in an Eppendorf tube with 200 ml of the lysis buffer of the DNeasy plant mini-kit (Qiagen, Hilden, Germany). The pestle was attached to an electric drill and the amount of broken spores was checked under the microscope. This was performed on ice to avoid melting the Eppendorf tube. DNA isolation then followed the manufacturer’s protocol. RNA was extracted and cDNA obtained from the fresh sample of Ceratiomyxa fruticulosa CH3 as described elsewhere (Nikolaev et al. 2004). Culturing and DNA extraction of Dictyostelia was as in Schaap et al. (2006). Protostelid sequences were obtained in two overlapping fragments, with the following combinations of primers: S1/ SR13, and S12m/RibB (Fiore-Donno et al. 2008), allowing to obtain a nearly complete SSU rRNA. Due to the high divergence of the SSU rRNA sequences of Myxogastria, interrupted by numerous introns, a large number of specific primers had to be used. In addition to those already published for the dark-spored Myxogastria clade (Fiore-Donno et al. 2008) we designed primers for Ceratiomyxa fruticulosa, Cribraria, Echinostelium, Liceida and Trichiida (Supplementary Material 1). Primers used to amplify the EF1a gene can be found in Baldauf and Doolittle (1997) and Fiore-Donno et al. (2005); in addition, we designed primers specific for Ceratiomyxa fruticulosa and Cribraria (Supplementary Material 1). Protostelids, Acramoeba dendroida and Filamoeba sequences were obtained in two overlapping fragments, with the following combinations of primers: first PCR with 1F-10R (amplifying nearly the whole gene) (Baldauf and Doolittle 1997), then semi-nested PCR using E800R (Fiore-Donno et al. 2005) and E700FN (Supplementary Material 1). Ceratiomyxa fruticulosa was amplified with a first run using Myx1F (FioreDonno et al. 2005) and Dic10R (Supplementary Material 1), and a second run using specific primers, designed on the cDNA sequence (Supplementary Material 1). Dictyostelia sequences were obtained with primers 1F-4R and 3F-10R (Baldauf and Doolittle 1997), and protocols as previously described (Baldauf and Doolittle 1997). Amplification parameters were adapted to the circumstances (elongation time according to the length of the expected product – 1 to 2 minutes), hybridization temperature according to the primers (50 to 53 1C). Cloning, when necessary, and sequencing followed the manufacturer’s protocols (pGEM-T-Easy Vector System 1, Promega). New sequences are deposited in the GenBank/EMBL databases under accession numbers EF513170-EF513201, EU259001 and EU360785–EU360789 (Fig. 3). Alignments are available as supplementary material. SSU rRNA data set: Sequences were manually aligned using BioEdit software version 7.0.5.3 (Hall 1999), following the secondary structure model proposed for Physarum polycephalum (Johansen et al. 1988). The extremely divergent sequence of Pelomyxa palustris AF320348 had to be

66 A.M. Fiore-Donno et al.

Table 1. Origin and identification of field-collected samples for this study. Species name CH1 CH2 CH3 F

¨ (O.F.Mull.) ¨ (O.F.Mull.) ¨ (O.F.Mull.) ¨ (O.F.Mull.)

T.Macbr. T.Macbr. T.Macbr. T.Macbr.

Date

Place of collection

Substratum

18/06/04 01/06/05 28/08/02 26/10/01

Switzerland, Fribourg Decaying log Switzerland, Geneva Decaying Betula log France, Ain Decaying log France, Var Pinus log

Coordinates

Voucher #

46.761N, 46.191N, 46.141N, 43.001N,

Not available Not available AMFD 101 MM 21983

08.111E 06.121E 05.881E 06.181E

PROTOSTELIA Schizoplasmodiopsis vulgaris

L.S.Olive & Stoian.

20/08/05 Switzerland, Geneva

Dead grass leaves

46.191N, 06.121E Not available

PHYSARALES Badhamia panicea var. nivalis Diderma globosum var. europaeum Lepidoderma tigrinum Physarum nutans

Meylan Buyck (Schrad.) Rostaf. Pers.

25/04/04 08/05/03 06/11/04 28/08/04

Twigs under melting snow Twigs under melting snow Broad-leaved log Leaf litter under Vinca

46.561N, 45.561N, 46.361N, 46.301N,

TRICHIALES Trichia sordida Arcyria stipata

Johannesen (Schwein.) Lister

10/05/02 Italy, Cuneo 22/10/05 Switzerland, Geneva

Log near melting snow Carpinus log

44.341N, 07.001E AMFD 81 46.211N, 06.051E AMFD 257

LICEALES Cribraria cancellata Cribraria vulgaris Cribraria argillacea Tubifera ferruginosa

(Batsch) Nann.-Bremek. 12/06/02 UK, Yorkshire Schrad. 26/06/02 UK, Yorkshire (Pers.ex J.F.Gmel.) Pers. 10/06/04 Switzerland, Geneva (Batsch) J.F.Gmel. 29/08/04 France, Haute-Savoie

Betula log Pinus log log Broad-leaved log

53.921N, 53.921N, 46.361N, 46.301N,

Switzerland, Jura France, Savoy Switzerland, Geneva Fance, Haute-Savoie

06.251E 06.421E 06.181E 06.261E

01.131W 00.851W 06.181E 06.261E

MM 29328 AMFD 110 AMFD 192 AMFD 168

AMFD AMFD AMFD AMFD

Vouchers origin: MM=Marianne Meyer herbarium, Le Bayet, 73730 Rognaix, France; AMFD=Anna Maria Fiore-Donno herbarium, Zoology Department, University of Oxford, South Parks Road, OX1 3PS, Oxford, UK.

94 98 146 196

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Ceratiomyxa Ceratiomyxa fruticulosa Ceratiomyxa fruticulosa Ceratiomyxa fruticulosa Ceratiomyxa fruticulosa

Authors

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Molecular Phylogeny of Mycetozoa excluded, and only representative species were selected for Dictyostelia and Myxogastria. A first dataset, including the 13 new nearly complete SSU rRNA sequences obtained in this study and representatives of all amoebozoan major clades was used to infer the position of the protostelids and Ceratiomyxa fruticulosa (1292 positions, 76 taxa) (Supplementary Material 3). More detailed phylogenetic relationships within Conosa were then assessed using 1234 unambiguously aligned positions for 54 taxa (Supplementary Material 4). Nuclearia simplex, Acanthamoeba culbertsoni and A. palestinensis were used as outgroups. Only unambiguously alignable sites were kept for analyses, excluding the major part of the variable regions V2, V4 and V7, due to their high divergence in Ceratiomyxa fruticulosa, Cribraria spp. and Echinostelium. Two clades showed an unbalanced nucleotide composition: entamoebae, (very high A=34%, very low C=16%, GC content=39%), and Trichiida, with high GC content. The number of variable and very variable sites is very high in both alignments (947 and 848 site patterns, respectively), with only 22% and 28.9% constant sites. Gaps and completely undetermined characters account for less than 0.5%. The General Time Reversible model taking into account invariant characters and a gamma-distributed rate heterogeneity among sites (GTR þ l þ gamma) (Lanave et al. 1984; Rodriguez et al. 1990), was chosen using Modeltest 3.7 (Posada and Crandall 1998). Maximum likelihood analyses were run using RAxML (Stamatakis et al. 2005) for both datasets with this substitution model and 25 rate categories. The best-scoring maximum likelihood tree was inferred, under the GTRMIX model, from 100 randomized starting maximum parsimony trees. The best-scoring tree was used to report the confidence values as percentages (below called BML) obtained through 1000 non-parametric bootstraps under the GTRCAT model. For the second dataset, additional analyses were run using Treefinder (Jobb 2007), with the same substitution model and 4 rate categories. A thousand nonparametric bootstrap replicates were run, using a BIONJ distance-based starting tree (below called BNJ). For both datasets, Bayesian analyses were performed with MrBayes, version 3.1.1 (Huelsenbeck and Ronquist), using the aforementioned model of substitution, the gamma distribution being approximated by 8 categories. Two runs starting from different random trees were performed and sampled every 10 generations, with 8 simultaneous chains, for 5 million generations. Convergence of the two runs, evaluated by a standard deviation of split frequencies o0.01, was not realized in the first dataset, or after nearly 4 millions generation in the second dataset (burn-in: 380000). To try to reach convergence in the first dataset, the frequency at which the trees samples from different runs are compared has been increased ten times (from every 1000 to every 100 generations), with only 10% samples discarded from the cold chain (default setting=25%), without better results. Reducing the number of categories to four did not allow convergence either. For the first dataset, the trees sampled before each chain reached stationarity, as seen in a graphic plotting likelihood values, were discarded as burn-in (1000). The remaining trees were assembled in a consensus tree (results not shown). The topology obtained was identical to that of the ML tree, except for the protostelid Cavostelium apophysatum (associated with Filamoeba spp. with a very low posterior probability, 0.54) and Ceratiomyxa (sister to Myxogastria, PP=0.54). On the whole, the unsupported branches in the ML tree show also a low posterior probability in the Bayesian analyses. For the second

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dataset, a consensus tree showing Bayesian posterior probality values (BPP) for each clade is provided. EF1a data set: All available Myxogastria, Dictyostelia (D. discoideum), Archamoebae (E. histolytica) and Protostelia (P. aurantium) EF1a sequences retrieved from GenBank were included in an alignment, along with our 25 new sequences, and with Nuclearia simplex, Acanthamoeba palestinensis and A. culbertsoni as outgroups. Mastigamoeba balamuthii sequence was retrieved from EST sequences from the Lambda ZAP II Library available in GenBank. The longest fragment, BE646534, was corrected in 7 positions according to BE636646 and BE636546. In subsequent analyses, 360 unambiguously aligned amino-acid positions for 42 taxa were used (Supplementary Material 5). The alignment contained only a few informative sites: Paup 4.0b10 (Swofford 2003) was used to determine the number of constant characters (180, 50%) and parsimony-informative sites (only 135). Gaps and missing characters accounted for 1.17%, mainly due to the missing 50 half of C. fruticulosa CH2 (41% of the aligned positions missing). Bayesian analyses were were run with the GTR+gamma substitution model, with 4 rate categories (no estimation of invariant sites), and the evolutionary model Rtrev (Dimmic et al. 2002) (chosen by MrBayes V.3.1.1). One million generations were run, the first 15000 trees before convergence was reached being discarded as burn-in. Maximum likelihood analyses were run using the same programs mentioned above, with respectively 25 (RAxML) and 4 (Treefinder) rates categories. Analyses with RAxML were as above, except that the models were PROTMIXRTREV to infer the best tree, and PROTCATRTREV to generate the 1000 bootstrap trees. Combined SSU rRNAþEF1a data set: An alignment of 1594 positions, where the EF1a amino-acids accounted for 22.6% and the SSU nucleotides for 77.4%, was made for 41 taxa (Supplementary Material 6). Only C. fruticulosa CH2 had an incomplete EF1a sequence. Bayesian analyses were conducted estimating all parameters independently for each gene, with the same parameters and models as for individual genes. One million generations were run, the first 48000 trees before convergence was reached being discarded as burn-in. Maximum likelihood analyses were conducted with Treefinder, allowing separate models for each gene (EF1a: rtREV with gamma optimized, 4 rates categories; SSU rRNA: GTR with gamma optimized, 4 rates categories). Another ML analysis was run with RAxML 7.0.4, using a distinct model for each gene, with joint branch lenght optimization, using the same models and practice as for individual genes. Tests: Alternative tree topologies were evaluated using the approximately unbiased test (AU) (Shimodaira 2002) under Treefinder (Jobb 2007). In this paired-sites test, two alternatives trees, according to the hypotheses to be tested, were used as input file, along with the combined alignment (SSU rRNA þ EF1a), with the parameters described above. 100000 replicates were run.

Acknowledgements We thank I. Dykova for cultures of Filamoeba sinensis and F. nolandi, A. Smirnov for providing DNA from Acramoeba dendroida, Marianne Meyer (France, Savoy) for two samples and Dmitriy Leontiev (Ukraine) for photos of Ceratiomyxa. We also thank an anonymous reviewer for detailed

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and pertinent remarks. This work was supported by grant PBSKA—110567 from the Swiss National Science Foundation to the first author and by the Ernst and Lucie Schmidheiny Foundation, Geneva, and by Leverhulme Trust research grant R1008101.

Cavalier-Smith T (2003) Protist phylogeny and the high-level classification of Protozoa. Eur J Protistol 39:338–348

Appendix A. Supporting Information

Cavalier-Smith T, Chao EE-Y (2003) Phylogeny and classification of phylum Cercozoa (Protozoa). Protist 154:341–358

Supplementary data associated with this article can be found in the online version at doi:10.1016/ j.protis.2009.05.002.

Cavalier-Smith T, Chao EE-Y, Oates B (2004) Molecular phylogeny of Amoebozoa and the evolutionary significance of the unikont Phalansterium. Eur J Protistol 40:21–48

Cavalier-Smith T (2009) Megaphylogeny, cell body plans, adaptive zones: causes and timing of eukaryote basal radiations. J Eukaryot Microbiol 56:26–33 Cavalier-Smith T, Chao EE-Y (1998) Hyperamoeba rRNA phylogeny and the classification of the phylum Amoebozoa. J Eukaryot Microbiol 46:5A

Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159

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