Figure 1.
Brightfield light micrographs of standing protosteloid fruiting bodies. A) Protostelium mycophaga, B) Nematostelium ovatum, C) Ceratiomyxella tahitiensis, D) Soliformovum expulsum, E) Soliformovum irregularis, F) Cavostelium apophysatum, G) Schizoplasmodiopsis amoeboidea, H) Tychosporium acutostipes, I) Clastostelium recurvatum, J) Protosporangium articulatum, K) Protosteliopsis fimicola, L) isolate LHI05, M) Endostelium zonatum. Scale bar is 10 µm.
Figure 2.
Light micrographs of protosteloid amoebae. A) Protostelium mycophaga differential interference contrast microscopy (DIC), B) Nematostelium ovatum phase contrast microscopy (PC), C) Soliformovum expulsum DIC, D) Cavostelium apophysatum DIC, E) Schizoplasmodiopsis amoeboidea PC, F) Protosporangium articulatum PC, G) Protosteliopsis fimicola DIC, H) isolate LHI05 PC, I) Endostelium zonatum DIC. Scale bars are 10 µm except B which is 50 µm.
Table 1.
Characteristics of Protosteloid Amoeba SSU rRNA Gene Sequences.
Figure 3.
129 Taxa SSU Maximum Likelihood Tree of Protosteloid Amoebae, Other Amoebozoans and Eukaryotes as Outgroups.
Colored branches indicate lineages in which protosteloid fruiting occurs. Black branches highlight amoebozoan lineages and gray branches show other eukaryotes used as outgroups. Red, black, and gray fonts indicate species of amoebae with protosteloid fruiting, nonfruiting amoebozoans, and other eukaryotes used as outgroups, respectively. To allow the figure to fit legibly on a single page, and to conserve the long branch length, the long branch leading to Lindbladia has been broken and shifted above and left. One hundred twenty nine taxa and 1,169 aligned positions were used to infer the optimal maximum likelihood (ML) tree in RAxML 7.0.4 using the following model (GTR + Γ, α = 0.513834, 20 discrete rate categories). ML bootstrap values from analyses of 1,000 RAxML datasets and 1,000 GARLI 0.96 datasets are shown above and below the node respectively. ML bootstrap values: black oval = 90–100, white oval with black outline = 80–90, gray oval = 70–80, unmarked<20. Black circle highlights the support values for monophyly of Amoebozoa. For the GARLI analyses, the following model was used (GTR + Γ + I, α = 0.71950104, 4 discrete rate categories). The scale bar represents evolutionary distance in changes per site.
Figure 4.
SSU Maximum Likelihood Trees Assessing Placement of Protosteloid Amoebae within Selected Clades.
For all trees, the scale bars represent evolutionary distance in changes per site. Red font indicates protosteloid amoebae. ML bootstrap values from analyses of 1,000 datasets and Bayesian posterior probabilities are shown above and below the nodes respectively. A) Placement of Protosteliopsis fimicola among vannellids. ML tree of 9 SSU genes and 1,837 aligned positions inferred with a GTR + Γ + I (α = 0.5042, 4 discrete rate categories, and I = 0.3421) model of nucleotide substitution. For the Bayesian analyses two runs, each consisting of 4 MCMC chains, were run for 2,000,000 generations, sampling every 100th tree. The first 100 trees were discarded as burnin after assessing for convergence of parameters. Purple branches highlight Protosteliopsis fimicola clade. B) Placement of protosteloid Isolate LHI05 among acanthamoebids. ML tree of 18 ssu genes and 1,476 aligned positions inferred with a TrN + Γ, α = 0.2228, 4 discrete rate categories model of nucleotide substitution. For the Bayesian analyses two runs each consisting of 4 MCMC chains were run for 2,000,000 generations, sampling every 100th tree. The first 4,000 trees were discarded as burnin after assessing for convergence of parameters. C) Branching order of Schizoplasmodiids rooted with Phalansterium. Four taxa and 1,735 aligned positions were used to infer the optimal ML tree with a TrN + Γ, α = 0.3693, 4 discrete rate categories model. For the Bayesian analyses two runs each consisting of 4 MCMC chains were run for 5,000,000 generations, sampling every 100th tree. The first 5,000 trees were discarded as burnin after assessing for convergence of parameters.
Table 2.
P-Values for the AU Tests of Selected Hypotheses.
Table 3.
Protosteloid Amoeba Cultures Used.
Table 4.
SSU rDNA primers for protosteloid amoebae.