Abstract
Evolutionary relationships within Amoebozoa have been the subject of controversy for two reasons: 1) paucity of morphological characters in traditional surveys and 2) haphazard taxonomic sampling in modern molecular reconstructions. These along with other factors have prevented the erection of a definitive system that resolves confidently both higher and lower-level relationships. Additionally, the recent recognition that many protosteloid amoebae are in fact scattered throughout the Amoebozoa suggests that phylogenetic reconstructions have been excluding an extensive and integral group of organisms. Here we provide a comprehensive phylogenetic reconstruction based on 139 taxa using molecular information from both SSU-rDNA and actin genes. We provide molecular data for 13 of those taxa, 12 of which had not been previously characterized. We explored the dataset extensively by generating 18 alternative reconstructions that assess the effect of missing data, long-branched taxa, unstable taxa, fast evolving sites and inclusion of environmental sequences. We compared reconstructions with each other as well as against previously published phylogenies. Our analyses show that many of the morphologically established lower-level relationships (defined here as relationships roughly equivalent to Order level or below) are congruent with molecular data. However, the data are insufficient to corroborate or reject the large majority of proposed higher-level relationships (above the Order-level), with the exception of Tubulinea, Archamoebae and Myxogastrea, which are consistently recovered. Moreover, contrary to previous expectations, the inclusion of available environmental sequences does not significantly improve the Amoebozoa reconstruction. This is probably because key amoebozoan taxa are not easily amplified by environmental sequencing methodology due to high rates of molecular evolution and regular occurrence of large indels and introns. Finally, in an effort to facilitate future sampling of key amoebozoan taxa, we provide a novel methodology for genome amplification and cDNA extraction from single or a few cells, a method that is culture-independent and allows both photodocumentation and extraction of multiple genes from natural samples.
Highlights
Reconstructing relationships between amoeboid organisms is challenging. Both the perceived and intrinsic paucity of morphological characters when compared to macroscopic taxa, as well as difficulties in establishing homology, made deep inferences nearly impossible for the,200 years of studies based on microscopy
General topology The SSU-rDNA and actin genes for 13 lineages were sequenced (Figure 1, Table 1) and phylogenetic analyses were performed on a total of 139 taxa (Supplementary Table S1), using multiple reconstruction strategies (Figure 2, Table 2)
Topologies obtained in the 18 distinct phylogenetic reconstructions of concatenated SSU-rDNA and actin genes (Table 3, Figure S2) largely agree with previous analyses regarding the monophyletic status of lower-level relationships
Summary
Reconstructing relationships between amoeboid organisms is challenging. Both the perceived and intrinsic paucity of morphological characters when compared to macroscopic taxa, as well as difficulties in establishing homology, made deep inferences nearly impossible for the ,200 years of studies based on microscopy. With the advent of molecular techniques, amoeboid groups were found to be scattered across at least 30 lineages in the eukaryotic tree of life, with the amoebae producing lobose pseudopodia included in the Amoebozoa [5]. It was only in the early 2000s that the promise of molecular phylogenetic reconstruction reached the fine-grained relationships within Amoebozoa, with well-sampled analysis of SSU-rDNA and actin genes [6,7,8].
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