Laura Wegener Parfrey; Erika Barbero; Elyse Lasser; Micah Dunthorn; Debashish Bhattacharya; David J Patterson; Laura A Katz

“ Perhaps the most critical aspect of the current state of eukaryotic systematics is the very limited taxonomic sampling to date. This is particularly problematic as the supergroup literature is often derived from a resampled pool of genes and taxa. More than 60 lineages of microbial eukaryotes have been identified by ultrastructure [7] , yet only about one-half of these have been included in molecular analyses. Furthermore, even when these lineages are included, they are generally represented by a single species. ”

“ We assess three aspects of each supergroup: (1) the stability of its taxonomy, (2) the support for monophyly (single evolutionary origin) in molecular analyses targeting a supergroup, and (3) the support for monophyly when a supergroup is included as an out-group in phylogenetic studies targeting other taxa. Our analysis demonstrates that supergroup taxonomies are unstable and that support for groups varies tremendously, indicating that the current classification scheme of eukaryotes is likely premature. ”

“ Many evolutionary innovations are found only in eukaryotes, some of which are present in all lineages (e.g., the cytoskeleton, nucleus) and others that are restricted to a few lineages (e.g., multicellularity, photosynthetic organelles [plastids] ) . These and other eukaryotic features evolved within microbial eukaryotes (protists) that thrived for hundreds of millions of years before they gave rise independently to multicellular eukaryotes, the familiar plants, animals, and fungi [1] . ”

“ Here the authors evaluate the stability of and support for the current classification system of eukaryotic cells (cells with nuclei) in which eukaryotes are divided into six kingdom level categories, or supergroups. These six supergroups unite diverse microbial and macrobial eukaryotic lineages, including the well-known groups of plants, animals, and fungi. The authors assess the stability of supergroup classifications through time and reveal a rapidly changing taxonomic landscape that is difficult to navigate for the specialist and generalist alike. ”

“ Rather, there is support for lower-level groups, such as the “Excavata” subgroups discussed above and perhaps also alveolates plus stramenopiles. This suggests that either there are no higher-level groupings to be found, or there is as yet inadequate data to resolve these clades. We believe that lack of taxon sampling is the key to resolution.
Further evidence against the six-supergroup view of eukaryotic diversity is the existence of “nomadic” taxa—lineages that do not have a consistent sister group, but instead wander between various weakly supported positions. ”

“ For each genealogy we evaluate the taxon sampling for the targeted supergroup (Membership; Figures 4–9) and the monophyly of all supergroups with at least two member taxa (Supergroup monophyly; Figures 4–9) . Monophyletic clades, those that include an ancestor and all of its descendants [45] , are scored (+; Figures 4–9) . We assess support for supergroups when they are targeted by specific studies and when they are included as out-groups in studies targeting other supergroups. A conservative measure of out-group monophyly was used because we required only two member lineages be present. ”

“ Robust taxa—those consistently supported by multiple datasets—are emerging and include the supergroup “Opisthokonta.” This group of animals, fungi, and their microbial relatives receives consistent support in molecular genealogies. This supergroup was monophyletic in 43 of 51 trees we examined (Figures 4–9) . “Opisthokonta” is also united by additional types of data: most members share a single posterior flagellum, contain plate-like cristae in mitochondria, and have an insertion within the Elongation Factor 1α gene [7] , [24] – [26] . ”

“ The “Alveolata” include ciliates, dinoflagellates, and apicomplexa, and its monophyly is well supported by morphology and molecules. “Chromista” was created as a kingdom to unite diverse microbial lineages with red algal plastids (and their nonphotosynthetic descendants) [ [5] , [16] , but no clear synapomorphy unites this clade.
The supergroup “Chromalveolata” includes microbes with critical roles in the environment and in human health. ”

“ We recommend increasing the lineages sampled and the number of diverse taxa within lineages. We are optimistic that as data become available from a greater diversity of taxa, eukaryotic phylogeny will become increasingly more resolved.
Multiple character sets.
We further anticipate that support for clades will increase as additional character sets are incorporated. Phylogenies based on single characters, whether genes, morphology, or ultrastructure, are subject to biases in the data and are not reliable by themselves. Hence, multiple character sets should be used to corroborate results. ”

“ Within the “Plantae” there have been numerous independent origins of multicellularity including: Volvox (Chlorophyta) [31] , the land plants, and red algae.
“Rhizaria” emerged from molecular data in 2002 to unite a heterogeneous group of flagellates and amoebae including: cercomonads, foraminifera, diverse testate amoebae, and former members of the polyphyletic radiolaria [32] . “Rhizaria” is an expansion of the “Cercozoa” [ [5] that was also recognized from molecular data [ [33] , [34] . ”

“ Cases of misidentification lead to inaccurate conclusions and require considerable effort to remedy. There is a rigorous standard for identifying microbial eukaryotes, but this standard is not always upheld. For example, the putative “Amoebozoa” species “Mastigamoeba invertens” that always branched outside the “Amoebozoa” clade [42] , [46] , [50] was misidentified [ [93] ; it has now been properly described as Breviata anathema and is not yet placed within any of the supergroups [93] . ”

“ The two photosynthetic clades “Chromalveolata” and “Plantae” receive differential support depending on the origin of the gene: high support in plastid genealogies but low in nuclear gene trees (Figures 5 and 8, see Results) . Molecular support for the “Excavata” as a whole is lacking from well-sampled gene trees (Figure 6) .
Although the six supergroups are not consistently supported by molecular genealogies, some nested clades are emerging as robust groups. For example, a sister relationship between Alveolata and Stramenopila is often recovered. ”

“ Additionally, the authors find variable support for each of the supergroups in published analyses based on DNA sequence variation. The support for supergroups differs according to the taxonomic area under study and the origin of the genes (e.g., nuclear, plastid) used in the analysis. Encouragingly, combining a conservative approach to taxonomy with increased sampling of microbial eukaryotes and the use of multiple types of data is likely to produce a robust scaffold for the eukaryotic tree of life.
Introduction
Biological research is based on the shared history of living things. ”

“ Strikingly, the monophyly of both the original and expanded “Opisthokonta” members is strongly supported in all investigations targeting the group (ten of ten, Figure 7) . Two other supergroups are also well supported: “Rhizaria” monophyly is recovered in 11 of 14 studies focusing on this supergroup (Figure 9) and “Amoebozoa” retained in five of seven topologies (Figure 4) . However, support for these groups is expected, given that they were recognized from molecular gene trees [10] , [32] . ”

“ Further, phylogenetic relationships can be obscured by heterogeneous rates of evolution and divergent selection pressures. For example, genes in many parasitic lineages of eukaryotes experience elevated rates of evolution. If not properly accounted for, these fast lineages will group together due to long-branch attraction [79] , [80] . This was the case for Microsporidia, intracellular parasites of animals; early small subunit rDNA (SSU) genealogies placed the Microsporidia at the base of the tree with other amitochondriate taxa, including Giardia and Entamoeba [81] . ”

“ We highlight several trends contributing to the instability and discuss the requirements for establishing robust clades within the eukaryotic tree of life.==Abstract== SynopsisEvolutionary perspectives, including the classification of living organisms, provide the unifying scaffold on which biological knowledge is assembled. ”

“ Hurand, M & Nedelcu, AM (2006) , Life-history evolution and the origin of multicellularity, vol. 239, pp. 257–272, 16288782, pmid:16288782 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Cavalier-Smith, T (2002) , The phagotrophic origin of eukaryotes and phylogenetic classification of protozoa, vol. 52, pp. 297–354, 11931142, pmid:11931142 ↑ Cavalier-Smith, T & Chao, EE (1997) , Sarcomonad ribosomal RNA sequences, rhizopod phylogeny, and the origin of euglyphid amoebae, vol. ”

“ Supergroup definition based on Adl et al. 2005 [3] : A, Amoebozoa; C, Chromalveolata; E, Excavata; O, Opisthokonta; P, Plantae; R, Rhizaria. The position of Breviata, Br, was not considered when scoring the monophyly of Amoebozoa as this organism was misidentified and affiliations are unknown (see text) . Some nodes were constrained in reference [48] . References cited in this figure are [ [24] , [32] , [42] , [49] , [46] , [50] , [48] , [51] , [47] .] ] Results Taxonomic Instability
There is considerable instability in taxonomies of the six putative supergroups (Figure 3) . ”

“ The origin of gymnamoebae revisited, vol. 18, pp. 2306–2314, 11719580, pmid:11719580 ↑ Cavalier-Smith, T (1997) , Amoeboflagellates and mitochondrial cristae in eukaryote evolution: Megasystematics of the new protozoan subkingdoms Eozoa and Neozoa, vol. 147, pp. 237–258 1 2 Lühe, M & Lang, A (1913) , Erstes Urreich der Tiere, Jena: G. Fischer 1 2 Kudryavtsev, A; Bernhard, D; Schlegel, M; Chao, EEY & Cavalier-Smith, T (2005) , 18S ribosomal RNA gene sequences of Cochliopodium (Himatismenida) and the phylogeny of Amoebozoa, vol. ”

“ ASM Press ↑ Simpson, AGB & Patterson, DJ (1999) , The ultrastructure of Carpediemonas membranifera (Eukaryota) with reference to the “Excavate hypothesis.”, vol. 35, pp. 353–370 ↑ Kreier, J (1978) , Parasitic Protozoa Volume II: Intestinal flagellates, histomonads, trichomonads, amoeba, opalinids, and ciliates, New York: Academic Press ↑ Smith, P (1996) , Molecular biology of parasitic protozoa, Oxford: Oxford University Press 1 2 3 4 Wainright, PO ”

“ Opalinid phylogeny and classification, vol. 4, pp. 413–428 ↑ Karpov, S; Sogin, ML & Silberman, JD (2001) , Rootlet homology, taxonomy, and phylogeny of bicosoecids based on 18S rRNA gene sequences, vol. 2, pp. 34–47 ↑ Guillou, L; Chretinnot-Dinet, MJ; Boulben, S; Moon-Van der Staay, SY & Vaulot, D (1999) , Symbiomonas scintillans gen. et sp. nov. Picophagus flagellatus gen. et sp. nov. (Heterokonta) : Two new heterotrophic flagellates of picoplanctonic size, vol. ”

“ Endosymbiosis connects the dots, vol. 26, pp. 50–60, 14696040, pmid:14696040 ↑ McFadden, GI & van Dooren, GG (2004) , Evolution: Red algal genome affirms a common origin of all plastids, vol. 14, pp. R514–R516, 15242632, pmid:15242632 ↑ Weber, A; Linka, M & Bhattacharya, D (2006) , Single, ancient origin of a plastid metabolite translocator family in Plantae from an endomembranes-derived ancestor, vol. 5, pp. 609–612, 16524915, pmid:16524915 ↑ An update of the Angiosperm Phylogeny group classification for the orders and families of flowering plants, 2003, pp. ”

“ The authors have declared that no competing interests exist.A previous version of this article appeared as an Early Online Release on November 13, 2006 (doi: [10.1371/journal.pgen.0020220.eor 10.1371/journal.pgen.0020220.eor] ) .Author contributions. ”